Secondary literature sources for the SH3 domain

For each of the hand selected primary papers associated with the SH3 domain, 100 neighbouring papers are retrieved from PubMed. If one of these neighbouring papers is referenced by more than two primary papers, it is shown in the table below.

Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain.

Zurdo J, Guijarro JI, Jimenez JL, Saibil HR, Dobson CM
J Mol Biol. 2001; 311: 325-40

The formation of amyloid fibrils by the SH3 domain of the alpha-subunit of bovine phosphatidylinositol-3'-kinase (PI3-SH3) has been investigated under carefully controlled solution conditions. NMR and CD characterisation of the denatured states from which fibrils form at low pH show that their properties can be correlated with the nature of the resulting aggregates defined by EM and FTIR spectroscopy. Compact partially folded states, favoured by the addition of anions, are prone to precipitate rapidly into amorphous species, whilst well-defined fibrillar structures are formed slowly from more expanded denatured states. Kinetic data obtained by a variety of techniques show a clear lag phase in the formation of amyloid fibrils. NMR spectroscopy shows no evidence for a significant population of small oligomers in solution during or after this lag phase. EM and FTIR indicate the presence of amorphous aggregates (protofibrils) rich in beta-structure after the lag phase but prior to the development of well-defined amyloid fibrils. These observations strongly suggest a nucleation and growth mechanism for the formation of the ordered aggregates. The morphologies of the fibrillar structures were found to be highly sensitive to the pH at which the protein solutions are incubated. This can be attributed to the effect of small perturbations in the electrostatic interactions that stabilise the contacts between the protofilaments forming the amyloid fibrils. Moreover, different hydrogen bonding patterns related to the various aggregate morphologies can be distinguished by FTIR analysis. Copyright 2001 Academic Press.

c-ABL tyrosine kinase activity is regulated by association with a novel SH3-domain-binding protein.

Zhu J, Shore SK
Mol Cell Biol. 1996; 16: 7054-62

The c-ABL tyrosine kinase is activated following either the loss or mutation of its Src homology domain 3 (SH3), resulting in both increased autophosphorylation and phosphorylation of cellular substrates and cellular transformation. This suggests that the SH3 domain negatively regulates c-ABL kinase activity. For several reasons this regulation is thought to involve a cellular protein that binds to the SH3 domain. Hyperexpression of c-ABL results in an activation of its kinase, the kinase activity of purified c-ABL protein in the absence of cellular proteins is independent of either the presence or absence of a SH3 domain, and point mutations and deletions within the SH3 domain are sufficient to activate c-ABL transforming ability. To identify proteins that interact with the c-ABL SH3 domain, we screened a cDNA library by the yeast two-hybrid system, using the c-ABL SH3SH2 domains as bait. We identified a novel protein, AAP1 (ABL-associated protein 1), that associates with these c-ABL domains and fails to bind to the SH3 domain in the activated oncoprotein BCRABL. Kinase experiments demonstrated that in the presence of AAP1, the ability of c-ABL to phosphorylate either glutathione S-transferase-CRK or enolase was inhibited. In contrast, AAP1 had little effect on the phosphorylation of glutathione S-transferase-CRK by the activated ABL oncoproteins v-ABL and BCRABL. We conclude that AAP1 inhibits c-ABL tyrosine kinase activity but has little effect on the tyrosine kinase activities of oncogenic BCRABL or v-ABL protein and propose that AAP1 functions as a trans regulator of c-ABL kinase. Our data also indicate that loss of susceptibility to AAP1 regulation correlates with oncogenicity of the activated forms of c-ABL.

Recognition and specificity in protein tyrosine kinase-mediated signalling.

Zhou S, Cantley LC
Trends Biochem Sci. 1995; 20: 470-5

There are several factors that contribute to the specificities of protein tyrosine kinases (PTKs) in signal transduction pathways. While protein-protein interaction domains, such as the Src homology (SH2 and SH3) domains, regulate the cellular localization of PTKs and their substrates, the specificities of PTKs are ultimately determined by their catalytic domains. The use of peptide libraries has revealed the substrate specificities of SH2 domains and PTK catalytic domains, and has suggested cross-talk between these domains.

SH2 domain specificity determination using oriented phosphopeptide library.

Zhou S, Cantley LC
Methods Enzymol. 1995; 254: 523-35

Secondary structures of lipid-associating peptides: a Fourier transform infrared study.

Zhong Q, Clark-Lewis I, Cushley RJ
Pept Res. 1994; 7: 99-106

Four peptides from 20 to 28 residues in length were studied by Fourier transform infrared (FTIR) spectroscopy in solution and in complexes with dimyristoylphosphatidylcholine (DMPC). The four peptides included the 20-residue lipid-associating peptide, LAP-20, which was predicted to form an amphipathic helical structure in the presence of lipids, and three other peptides whose sequences had less amphipathic helix-forming properties. The complexes were shown by electron microscopy to be discoidal in shape with mean diameters of 21-27 nm. At the concentrations used for IR, the peptides appeared to form oligomers consisting of intermolecular beta-sheets. In the presence of lipids, the amount of beta-structure decreased; however, amounts of beta-structure were still approximately equal to amounts of alpha-helix. The IR results for LAP-20 contradicted previous circular dichroism results that predicted 50%-90% alpha-helix in DMPC complexes. Convex constraint analysis (CCA) deconvolution of the circular dicroism (CD) spectrum to estimate secondary structures predicted amounts of helix similar to those predicted by IR, but there was still substantial disagreement between IR and CD estimates of other secondary structures. For LAP-20 in complexes, CD predicted random structure. Possible physiological consequences of partial disordering of peptide structures are discussed.

Stimulation by phospholipids of a protein-tyrosine-phosphatase containing two src homology 2 domains.

Zhao Z, Shen SH, Fischer EH
Proc Natl Acad Sci U S A. 1993; 90: 4251-5

PTP1C, a protein-tyrosine-phosphatase (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) containing two src homology 2 domains, is poorly active when assayed with various protein substrates in vitro. Its activity is stimulated > 1000-fold by anionic phospholipids when myelin basic protein or mitogen-activated protein kinase is used as substrate but reduced in the presence of several other substrates. Data are presented to indicate a direct interaction of the enzyme with phospholipids. Enzyme stimulation directed only toward certain specific substrates is interpreted by assuming that these compounds also bind to the phospholipid vesicles where they will be subjected to rapid enzymatic attack. A possible regulation of PTP1C by its translocation to the cell membrane is hypothesized.

Three-dimensional structure of the Hck SH2 domain in solution.

Zhang W, Smithgall TE, Gmeiner WH
J Biomol NMR. 1997; 10: 263-72

The hematopoietic cellular kinase (Hck) is a member of the Src family of non-receptor protein-tyrosine kinases that is expressed predominantly in granulocytes, monocytes and macrophages. Recent observations suggest that Hck may be activated in HIV-infected macrophages and in chronic myelogenous leukemia cells that express Bcr-Abl. In order to increase our understanding of the structural basis for regulation of Hck activity under normal and pathological conditions, we have solved the solution structure of the uncomplexed Hck SH2 domain using NMR spectroscopy. A novel procedure that uses intraresidue HN-H alpha distances as references for converting NOE intensities into distance restraints has been described. A total of 1757 significant experimental restraints were derived from NMR spectroscopic data including 238 medium-range and 487 long-range distance restraints and 177 torsion angle restraints. These restraints were used in a simulated annealing procedure to generate 20 structures with the program DYANA. Superimposition of residues 5-104 upon the mean coordinate set yielded an average atomic rmsd values of 0.42 +/- 0.08 A for the N,C alpha,C' atoms and 0.81 +/- 0.08 A for all heavy atoms. Rmsd values for those residues in the regions of ordered secondary structure were 0.27 +/- 0.04 A for the N,C alpha,C' atoms and 0.73 +/- 0.06 A for all heavy atoms.

Sequential assignment and secondary structure determination for the Src homology 2 domain of hematopoietic cellular kinase.

Zhang W, Smithgall TE, Gmeiner WH
FEBS Lett. 1997; 406: 131-5

The hematopoietic cellular kinase (Hck) is a member of the Src family of non-receptor protein-tyrosine kinases and participates in signal transduction events regulating the growth, differentiation and function of phagocytes. The secondary structure of the SH2 domain for Hck was determined for a 13C/15N-enriched sample using multi-dimensional NMR spectroscopy. The secondary structure for the domain was determined from chemical shift indices [1H alpha, 13C alpha and 13C'], sequential NOEs [d(alphaN)(i, i+1) and d(NN)(i, i+1)], and 3J(alphaN) scalar coupling constants. The Hck SH2 domain consists of two alpha-helices and seven beta-strands. Complementary strands of beta-sheets were identified from long-range NOEs using a novel 3D, 13C/15N-edited HMQC-NOESY-(HCACO)NH experiment that correlated 1H alpha resonances between beta-strands. The secondary structure for Hck SH2 is similar to that predicted from the sequence alignment of the Src-family protein tyrosine kinases.

NMR studies of unfolded states of an SH3 domain in aqueous solution and denaturing conditions.

Zhang O, Forman-Kay JD
Biochemistry. 1997; 36: 3959-70

The isolated N-terminal SH3 domain of the Drosophila adapter protein drk (drkN SH3 domain) exists in a dynamic equilibrium between a folded (F(exch)) and an unfolded (U(exch)) state under native-like buffer conditions [Zhang, O., & Forman-Kay, J. D. (1995) Biochemistry 34, 6784-6794]. The effect of binding a proline-rich peptide derived from the protein Sos, a biological target of the drkN SH3 domain, on this equilibrium has been investigated. The stabilization of the F(exch) folded state upon binding provides an example of the link between binding and protein folding or stabilization. We have compared NMR parameters of the U(exch) state with those of a denatured state in 2 M guanidine hydrochloride (U(Gdn)). Variable-temperature experiments demonstrate that interactions in a region encompassing residues Gln 23-Leu 28 in the U(exch) state are destabilized upon addition of guanidine hydrochloride. Data from an 15N HSQC-NOESY-HSQC experiment as well as recently developed methods provide more unambiguous structural information than described previously, showing the presence of preferential structure in both unfolded states. Backbone NOEs observed in both unfolded states as well as chemical shifts and coupling constants suggest a rapid equilibrium between extended structure and turn-like structures which may play a role in initiation of protein folding. However, differences in detailed structural features between the two unfolded states argue that caution is needed in interpretation of results from structural characterization of protein conformational states generated using denaturing conditions.

Structural characterization of folded and unfolded states of an SH3 domain in equilibrium in aqueous buffer.

Zhang O, Forman-Kay JD
Biochemistry. 1995; 34: 6784-94

The isolated N-terminal Src homology 3 (SH3) domain of Drosophila drk exists in equilibrium between folded and unfolded states in aqueous buffer near neutral pH. Nuclear magnetic resonance spectra recorded on both states simultaneously exhibit an approximate 1:1 ratio of protein conformations. The folded form is similar to other known SH3 structures, especially the N-terminal SH3 domain of the mammalian homologue GRB2. A stretch of sequential amide-amide nuclear Overhauser effect cross-peaks for resonances of the unfolded state is observed in a region corresponding to beta-strands in the folded state. The results suggest that turn-like conformations may be preferentially sampled in the folding pathway for this predominantly beta-structured SH3 domain. In addition, a stable turn at Leu-28 is observed in the unfolded but not in the folded state. Comparison of this unfolded form with a denatured state in 2 M guanidine hydrochloride shows that, while both are highly disordered, these states are not identical and more residual structure is present under nondenaturing conditions.

Solution structure of Grb2 reveals extensive flexibility necessary for target recognition.

Yuzawa S, Yokochi M, Hatanaka H, Ogura K, Kataoka M, Miura K, Mandiyan V, Schlessinger J, Inagaki F
J Mol Biol. 2001; 306: 527-37

Grb2 is an adaptor protein composed of a single SH2 domain flanked by two SH3 domains. Grb2 functions as an important evolutionary conserved link between a variety of cell membrane receptors and the Ras/MAP kinase-signaling cascade. Here, we describe the solution structure of Grb2 as revealed by NMR and small angle X-ray scattering measurements. We demonstrate that Grb2 is a flexible protein in which the C-terminal SH3 domain is connected to the SH2 domain via a flexible linker. This is in contrast to the previously described Grb2 crystal structure, which showed a compact structure with intramolecular contact between two SH3 domains. Binding experiments on Grb2 and peptides containing two different proline-rich sequences indicate that Grb2 adapts the relative position and orientation of the two SH3 domains to bind bivalently to the target peptide sequences.

Regulation of the p85/p110alpha phosphatidylinositol 3'-kinase. Distinct roles for the n-terminal and c-terminal SH2 domains.

Yu J, Wjasow C, Backer JM
J Biol Chem. 1998; 273: 30199-203

Our previous studies on the p85/p110alpha phosphatidylinositol 3-kinase showed that the p85 regulatory subunit inhibits the p110alpha catalytic subunit, and that phosphopeptide activation of p85/p110alpha dimers reflects a disinhibition of p110alpha (Yu, J., Zhang, Y., McIlroy, J., Rordorf-Nikolic, T., Orr, G. A., and Backer, J. M. (1998) Mol. Cell. Biol. 18, 1379-1387). We now define the domains of p85 required for inhibition of p110alpha. The iSH2 domain of p85 is sufficient to bind p110alpha but does not inhibit it. Inhibition of p110alpha requires the presence of the nSH2 domain linked to the iSH2 domain. Phosphopeptides increase the activity of nSH2/iSH2-p110alpha dimers, demonstrating that the nSH2 domain mediates both inhibition of p110alpha and disinhibition by phosphopeptides. In contrast, phosphopeptides did not increase the activity of iSH2/cSH2-p110alpha dimers, or dimers composed of p110alpha and an nSH2/iSH2/cSH2 construct containing a mutant nSH2 domain. Phosphopeptide binding to the cSH2 domain increased p110alpha activity only in the context of an intact p85 containing both the nSH2 domain and residues 1-322 (the SH3, proline-rich and breakpoint cluster region-homolgy domains). These data suggest that the nSH2 domain of p85 is a direct regulator of p110alpha activity. Regulation of p110alpha by phosphopeptide binding to the cSH2 domain occurs by a mechanism that requires the additional presence of the nSH2 domain and residues 1-322 of p85.

Dynamic coupling between the SH2 and SH3 domains of c-Src and Hck underlies their inactivation by C-terminal tyrosine phosphorylation.

Young MA, Gonfloni S, Superti-Furga G, Roux B, Kuriyan J
Cell. 2001; 105: 115-26

The effect of C-terminal tyrosine phosphorylation on molecular motions in the Src kinases Hck and c-Src is investigated by molecular dynamics simulations. The SH2 and SH3 domains of the inactive kinases are seen to be tightly coupled by the connector between them, impeding activation. Dephosphorylation of the tail reduces the coupling between the SH2 and SH3 domains in the simulations, as does replacement of connector residues with glycine. A mutational analysis of c-Src expressed in Schizosaccharomyces pombe demonstrates that replacement of residues in the SH2-SH3 connector with glycine activates c-Src. The SH2-SH3 connector appears to be an inducible "snap lock" that clamps the SH2 and SH3 domains upon tail phosphorylation, but which allows flexibility when the tail is released.

Genetic analysis of a phosphatidylinositol 3-kinase SH2 domain reveals determinants of specificity.

Yoakim M, Hou W, Songyang Z, Liu Y, Cantley L, Schaffhausen B
Mol Cell Biol. 1994; 14: 5929-38

Phosphatidylinositol 3-kinase is an important element in both normal and oncogenic signal transduction. Polyomavirus middle T antigen transforms cells in a manner depending on association of its tyrosine 315 phosphorylation site with Src homology 2 (SH2) domains on the p85 subunit of the phosphatidylinositol 3-kinase. Both nonselective and site-directed mutagenesis have been used to probe the interaction of middle T with the N-terminal SH2 domain of p85. Most of the 24 mutants obtained showed reduced middle T binding. However, mutations that showed increased binding were also found. Comparison of middle T binding to that of the platelet-derived growth factor receptor showed that some mutations altered the specificity of recognition by the SH2 domain. Mutations altering S-393, D-394, and P-395 were shown to affect the ability of the SH2 domain to select peptides from a degenerate phosphopeptide library. These results focus attention on the role of the EF loop in the SH2 domain in determining binding selectivity at the third position after the phosphotyrosine.

Prediction and structural characterization of an independently folding substructure in the src SH3 domain.

Yi Q, Bystroff C, Rajagopal P, Klevit RE, Baker D
J Mol Biol. 1998; 283: 293-300

Previous studies of the conformations of peptides spanning the length of the alpha-spectrin SH3 domain suggested that SH3 domains lack independently folding substructures. Using a local structure prediction method based on the I-sites library of sequence-structure motifs, we identified a seven residue peptide in the src SH3 domain predicted to adopt a native-like structure, a type II beta-turn bridging unpaired beta-strands, that was not contained intact in any of the SH3 domain peptides studied earlier. NMR characterization confirmed that the isolated peptide, FKKGERL, adopts a structure similar to that adopted in the native protein: the NOE and 3JNHalpha coupling constant patterns were indicative of a type II beta-turn, and NOEs between the Phe and the Leu side-chains suggest that they are juxtaposed as in the prediction and the native structure. These results support the idea that high-confidence I-sites predictions identify protein segments that are likely to form native-like structures early in folding.

Binding of Vav to Grb2 through dimerization of Src homology 3 domains.

Ye ZS, Baltimore D
Proc Natl Acad Sci U S A. 1994; 91: 12629-33

The protooncogenic protein Vav has the structure of an intracellular signal transducer. It is exclusively expressed in cells of hematopoietic lineage and plays a crucial role in hematopoietic cell differentiation. Here we report that both in cell extracts and within intact mammalian cells Vav binds to Grb2 (Sem-5/ASH/Drk), an adaptor molecule which plays a key role in Ras activation. The interaction became evident from a yeast two-hybrid screen and its specificity was demonstrated by in vitro binding assays. It is mediated by an unusual protein-protein binding reaction: dimerization of specific intact Src homology 3 domains of each of the partners. Signaling during hematopoietic lineage differentiation may therefore involve the tissue-specific signal transducer Vav linking into the ubiquitous pathway involving Grb2 and ultimately Ras.

The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop.

Yasukawa H, Misawa H, Sakamoto H, Masuhara M, Sasaki A, Wakioka T, Ohtsuka S, Imaizumi T, Matsuda T, Ihle JN, Yoshimura A
EMBO J. 1999; 18: 1309-20

The Janus family of protein tyrosine kinases (JAKs) regulate cellular processes involved in cell growth, differentiation and transformation through their association with cytokine receptors. However, compared with other kinases, little is known about cellular regulators of the JAKs. We have recently identified a JAK-binding protein (JAB) that inhibits JAK signaling in cells. In the studies presented here we demonstrate that JAB specifically binds to the tyrosine residue (Y1007) in the activation loop of JAK2, whose phosphorylation is required for activation of kinase activity. Binding to the phosphorylated activation loop requires the JAB SH2 domain and an additional N-terminal 12 amino acids (extended SH2 subdomain) containing two residues (Ile68 and Leu75) that are conserved in JAB-related proteins. An additional N-terminal 12-amino-acid region (kinase inhibitory region) of JAB also contributes to high-affinity binding to the JAK2 tyrosine kinase domain and is required for inhibition of JAK2 signaling and kinase activity. Our studies define a novel type of regulation of tyrosine kinases and might provide a basis for the design of specific tyrosine kinase inhibitors.

A novel PF/PN motif inhibits nuclear localization and DNA binding activity of the ESX1 homeoprotein.

Yan YT, Stein SM, Ding J, Shen MM, Abate-Shen C
Mol Cell Biol. 2000; 20: 661-71

Despite their significance for mammalian embryogenesis, the molecular mechanisms that regulate placental growth and development have not been well defined. The Esx1 homeobox gene is of particular interest because it is among the few regulatory genes that have specific expression and function in the placenta during murine development. In addition, the ESX1 protein contains several notable features that are not often associated with homeoproteins, including an atypical homeodomain of the paired-like class, a proline-rich region that contains an SH3 binding motif, and a novel repeat region consisting of prolines alternating with phenylalanines or asparagines that we term the PF/PN motif. We have found that the ESX1 protein is expressed in the labyrinth layer of the placenta in vivo, where its subcellular localization is primarily cytoplasmic. Our results suggest that this unexpected subcellular localization is conferred by the PF/PN motif, which inhibits nuclear localization of ESX1 in cell culture, as well as its DNA binding activity in vitro. Finally, we show that the proline-rich region of ESX1 mediates interactions in vitro with the c-abl SH3 domain as well as with certain WW domains. We propose that the PF/PN motif provides a novel mechanism for regulating nuclear entry and that the essential function of ESX1 during placental development is mediated by its ability to couple cytoplasmic signal transduction events with transcriptional regulation in the nucleus.

An SH3-binding site conserved in Bruton's tyrosine kinase and related tyrosine kinases mediates specific protein interactions in vitro and in vivo.

Yang W, Malek SN, Desiderio S
J Biol Chem. 1995; 270: 20832-40

Mutations in Bruton's tyrosine kinase (Btk) have been associated with immunodeficiencies in man and in the mouse. Btk and two related proteins, Itk and Tec, are members of a distinct family of tyrosine kinases. These kinases are believed to function in various receptor-mediated signaling pathways, but their specific functions are as yet undefined. Btk and its homologues share extensive sequence similarity, including a conserved region, the Tec-homology (TH) domain, that has been proposed to mediate specific intermolecular or intramolecular interactions. The TH region of Btk contains a functional SH3-binding site at residues 189-192. SH3 binding is selective: Btk is retained by the SH3 domain of Fyn but not by that of Blk, another Src-type kinase. TH-SH3 binding in vitro is abolished by specific, single amino acid substitutions within the Btk TH domain or the Fyn SH3 domain. We provide two lines of evidence that the SH3-binding site in the Btk TH domain mediates protein interactions in intact cells. First, treatment of cells with pervanadate induces an increase in the phosphotyrosine content of kinase-inactive Btk; this response is substantially reduced by a mutation that inactivates the SH3-binding site in the Btk TH domain. Second, in cell lysates Btk is found in association with an as yet unidentified 72-kDa phosphotyrosine-containing protein; this interaction requires a functional SH3-binding site in the TH domain. The TH domain may therefore interact in vivo with other proteins that regulate the phosphorylation state of Btk.

Differential interactions of human Sos1 and Sos2 with Grb2.

Yang SS, Van Aelst L, Bar-Sagi D
J Biol Chem. 1995; 270: 18212-5

The guanine nucleotide exchange factor Son of sevenless (Sos) performs a crucial step in the coupling of receptor tyrosine kinases to Ras activation. Mammalian cells contain two related but distinct Sos proteins, Sos1 and Sos2. Although they share a high degree of overall similarity, it is not known to what extent their biological and biochemical properties overlap. In the present study, we have compared the interactions of the two human homologues of Sos, hSos1 and hSos2, with the adaptor protein Grb2. We show that hSos2 interacts with Grb2 via its proline-rich COOH-terminal domain and that this interaction is dependent on the SH3 domains of Grb2. In general, these characteristics are similar to the ones reported previously for the interaction of hSos1 with Grb2. However, the apparent binding affinity of hSos2 for Grb2 is significantly higher relative to that of hSos1 both in vitro and in vivo. The region conferring this higher binding affinity has been mapped to residues 1126-1242 of the hSos2 COOH-terminal domain. These results suggest that Sos1 and Sos2 may differentially contribute to receptor-mediated Ras activation.

SH3 domain-mediated interaction of dystroglycan and Grb2.

Yang B, Jung D, Motto D, Meyer J, Koretzky G, Campbell KP
J Biol Chem. 1995; 270: 11711-4

Dystroglycan is a novel laminin receptor that links the extracellular matrix and sarcolemma in skeletal muscle. The dystroglycan complex containing alpha- and beta-dystroglycan also serves as an agrin receptor in muscle, where it may regulate agrin-induced acetylcholine receptor clustering at the neuromuscular junction. beta-Dystroglycan has now been expressed in vitro and shown to directly interact with Grb2, an adapter protein involved in signal transduction and cytoskeletal organization. Protein binding assays with two Grb2 mutants, Grb2/P49L and Grb2/G203R, which correspond to the loss-of-function mutants in the Caenorhabditis elegans sem-5, demonstrated that the dystroglycan-Grb2 association is through beta-dystroglycan C-terminal proline-rich domains and Grb2 Src homology 3 domains. Affinity chromatography has also shown endogenous skeletal muscle Grb2 interacts with beta-dystroglycan. Immunoprecipitation experiments have demonstrated that Grb2 associates with alpha/beta-dystroglycan in vivo in both skeletal muscle and brain. The specific dystroglycan-Grb2 interaction may play an important role in extracellular matrix-mediated signal transduction and/or cytoskeleton organization in skeletal muscle that may be essential for muscle cell viability.

Deletion of Src homology 3 domain results in constitutive activation of Tec protein-tyrosine kinase.

Yamashita Y, Miyazato A, Ohya K, Ikeda U, Shimada K, Miura Y, Ozawa K, Mano H
Jpn J Cancer Res. 1996; 87: 1106-10

Tec protein-tyrosine kinase (PTK) is the prototype of a new subfamily of non-receptor type PTKs, and is abundantly expressed in hematopoietic tissues. We have revealed that Tec is inducibly tyrosine-phosphorylated and activated by stimulation with a wide range of cytokines. To get more insight into the signaling mechanism through Tec, we have generated a constitutively active form of Tec PTK. Deletion of the Src homology (SH) 3 domain gave rise to a hyperphosphorylated and activated Tec kinase (Tec deltaSH3). The activity of Tec deltaSH3 was confirmed in 293 cells, as well as in cytokine-dependent hematopoietic cells (BA/F3). Tec deltaSH3 should be a useful tool to study the in vivo substrates of Tec PTK.

Identification of the Abl- and rasGAP-associated 62 kDa protein as a docking protein, Dok.

Yamanashi Y, Baltimore D
Cell. 1997; 88: 205-11

A 62 kDa protein is highly phosphorylated in many cells containing activated tyrosine kinases. This protein, characterized mainly by its avid association with rasGAP, has proved elusive. Anti-phosphotyrosine antibody was used to purify p62. From peptide sequence, molecular cloning revealed a cDNA encoding a novel protein, p62dok, with little homology to others but with a prominent set of tyrosines and nearby sequences suggestive of SH2 binding sites. In cells, v-Abl tyrosine kinase binds and strongly phosphorylates p62dok, which then binds rasGAP. A monoclonal antibody, 2C4, to the rasGAP-associated p62 reacts with p62dok. Thus, p62dok appears to be the long-sought major substrate of many tyrosine kinases.

Examining the specificity of Src homology 3 domain--ligand interactions with alkaline phosphatase fusion proteins.

Yamabhai M, Kay BK
Anal Biochem. 1997; 247: 143-51

Sixteen-amino-acid-long peptides, corresponding to the optimal ligand preferences of the Src homology 3 (SH3) domains of Abl, Cortactin, Crk, p53BP2, and Src, were fused to the N-terminus of Escherichia coli alkaline phosphatase (AP). These secreted fusion proteins have been used as one-step detection probes of peptide ligand-SH3 domain interactions on microtiter plates and membranes. The binding of both the class I and II SH3 ligand-AP fusion proteins to their targets is robust and specific in comparison to chemically synthesized biotinylated peptides, used either in monovalent or tetravalent formats. p53BP2 and Cortactin SH3 ligand-AP fusions have been used to screen a mouse embryo lambda cDNA expression library and resulted in the cloning of p53BP2 and several known proteins with SH3 domains similar to that of Cortactin, respectively. In addition, the approximately 60-amino-acid-long SH3 domains of Src and Abl were fused to AP and the resulting fusion proteins were found to bind specifically to their respective peptide ligands in microtiter plates and proteins containing proline-rich regions in screens of a lambda cDNA expression library. Thus, SH3 peptide ligand- and SH3 domain-AP fusion proteins are convenient and sensitive reagents for examining the specificity of SH3 domain-ligand interactions, identifying potentially interacting proteins, and establishing high-throughput screens of combinatorial chemical libraries.

Intersectin, a novel adaptor protein with two Eps15 homology and five Src homology 3 domains.

Yamabhai M, Hoffman NG, Hardison NL, McPherson PS, Castagnoli L, Cesareni G, Kay BK
J Biol Chem. 1998; 273: 31401-7

We screened a Xenopus laevis oocyte cDNA expression library with a Src homology 3 (SH3) class II peptide ligand and identified a 1270-amino acid-long protein containing two Eps15 homology (EH) domains, a central coiled-coil region, and five SH3 domains. We named this protein Intersectin, because it potentially brings together EH and SH3 domain-binding proteins into a macromolecular complex. The ligand preference of the EH domains were deduced to be asparajine-proline-phenylalanine (NPF) or cyclized NPF (CX1-2NPFXXC), depending on the type of phage-displayed combinatorial peptide library used. Screens of a mouse embryo cDNA library with the EH domains of Intersectin yielded clones for the Rev-associated binding/Rev-interacting protein (RAB/Rip) and two novel proteins, which we named Intersectin-binding proteins (Ibps) 1 and 2. All three proteins contain internal and C-terminal NPF peptide sequences, and Ibp1 and Ibp2 also contain putative clathrin-binding sites. Deletion of the C-terminal sequence, NPFL-COOH, from RAB/Rip eliminated EH domain binding, whereas fusion of the same peptide sequence to glutathione S-transferase generated strong binding to the EH domains of Intersectin. Several experiments support the conclusion that the free carboxylate group contributes to binding of the NPFL motif at the C terminus of RAB/Rip to the EH domains of Intersectin. Finally, affinity selection experiments with the SH3 domains of Intersectin identified two endocytic proteins, dynamin and synaptojanin, as potential interacting proteins. We propose that Intersectin is a component of the endocytic machinery.

A motif-based profile scanning approach for genome-wide prediction of signaling pathways.

Yaffe MB, Leparc GG, Lai J, Obata T, Volinia S, Cantley LC
Nat Biotechnol. 2001; 19: 348-53

The rapid increase in genomic information requires new techniques to infer protein function and predict protein-protein interactions. Bioinformatics identifies modular signaling domains within protein sequences with a high degree of accuracy. In contrast, little success has been achieved in predicting short linear sequence motifs within proteins targeted by these domains to form complex signaling networks. Here we describe a peptide library-based searching algorithm, accessible over the World Wide Web, that identifies sequence motifs likely to bind to specific protein domains such as 14-3-3, SH2, and SH3 domains, or likely to be phosphorylated by specific protein kinases such as Src and AKT. Predictions from database searches for proteins containing motifs matching two different domains in a common signaling pathway provides a much higher success rate. This technology facilitates prediction of cell signaling networks within proteomes, and could aid in the identification of drug targets for the treatment of human diseases.

Disabled-2 (Dab2) is an SH3 domain-binding partner of Grb2.

Xu XX, Yi T, Tang B, Lambeth JD
Oncogene. 1998; 16: 1561-9

Disabled-2 (Dab2), a mammalian structural homolog of Drosophila Disabled (Dab), is a mitogen-responsive phosphoprotein. It has been speculated to be a negative regulator of growth since its expression is lost in ovarian carcinomas. Dab2 contains a C-terminal proline-rich domain with sequences similar to those found in Sos, a guanine nucleotide exchange factor for Ras. The proline-rich sequences of Sos mediate the interaction of Sos with Grb2, an adaptor protein which coupled tyrosine kinase receptors to Sos. Herein, we have investigated the possibility that Dab2 interacts with Grb2. In experiments of co-immunoprecipitation from BAC1.2F5 macrophage cell lysates, significant quantities of Grb2 were associated with both Sos and Dab2, although Dab2 and Sos were not present in the same complex. Transfection of Dab2 into a Dab2-negative cell line (293 cells) decreased the amount of Grb2 associated with Sos, suggesting that Dab2 competes with Sos for binding to Grb2. Proline-rich peptides corresponding to Dab2 (#661-669) and to Sos (#1146-1161) inhibited the binding of Dab2 to Grb2, but were less effective in disrupting the Grb2-Sos complex. The expressed proline-rich domain of Dab2 (#600-730) bound Grb2, but other regions of Dab2 failed to bind Grb2. Both of the individual SH3 domains of Grb2 bound to Sos (N-terminal SH3 domain >> C-terminal SH3 domain), but binding to Dab2 required the intact Grb2, suggesting cooperative binding using both SH3 domains of Grb2. These data indicate that Dab2 binds to the SH3 domains of Grb2 via its C-terminal proline-rich sequences. Dab2 may modulate growth factor/Ras pathways by competing with Sos for binding to Grb2.

Crystal structures of c-Src reveal features of its autoinhibitory mechanism.

Xu W, Doshi A, Lei M, Eck MJ, Harrison SC
Mol Cell. 1999; 3: 629-38

Src family kinases are maintained in an assembled, inactive conformation by intramolecular interactions of their SH2 and SH3 domains. Full catalytic activity requires release of these restraints as well as phosphorylation of Tyr-416 in the activation loop. In previous structures of inactive Src kinases, Tyr-416 and flanking residues are disordered. We report here four additional c-Src structures in which this segment adopts an ordered but inhibitory conformation. The ordered activation loop forms an alpha helix that stabilizes the inactive conformation of the kinase domain, blocks the peptide substrate-binding site, and prevents Tyr-416 phosphorylation. Disassembly of the regulatory domains, induced by SH2 or SH3 ligands, or by dephosphorylation of Tyr-527, could lead to exposure and phosphorylation of Tyr-416.

Triple resonance-based assignment for Abl SH(32) and its complex with a consolidated ligand.

Xu R, Cahill S, Cowburn D
J Biomol NMR. 1999; 14: 187-8

Chemical ligation of folded recombinant proteins: segmental isotopic labeling of domains for NMR studies.

Xu R, Ayers B, Cowburn D, Muir TW
Proc Natl Acad Sci U S A. 1999; 96: 388-93

A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N. Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

Flexibility of interdomain contacts revealed by topological isomers of bivalent consolidated ligands to the dual Src homology domain SH(32) of abelson.

Xu Q, Zheng J, Xu R, Barany G, Cowburn D
Biochemistry. 1999; 38: 3491-7

Src homology (SH)2 and SH3 domains are found in a variety of proteins involved in the control of cellular signaling and architecture. The possible interrelationships between domains are not easily investigated, even though several cases of multiple domain-containing constructs have been studied structurally. As a complement to direct structural methods, we have developed consolidated ligands and tested their binding to the Abl SH(32) complex. Consolidated ligands combine in the same molecule peptide sequences recognized by SH2 and SH3 domains, i.e., Pro-Val-pTyr-Glu-Asn-Val and Pro-Pro-Ala-Tyr-Pro-Pro-Pro-Pro-Val-Pro, respectively; these are joined by oligoglycyl linkers. Four types of ligands were chemically synthesized, representing all the possible relative orientations of ligands. Their affinities were found to vary with binding portion topologies and linker lengths. Two of these types were shown to bind to both SH2 and SH3 dual domains with high affinities and specificities, showing increases of one order of magnitude, as compared to the most strongly bound monovalent equivalent. These results suggest that the relative orientation of SH2 and SH3 in Abl SH(32) is not fixed, and this synthetic approach may be generally useful for determining the structures of ligated complexes and for developing reagents with high affinities and specificities.

Monoclonal antibodies to alphaI spectrin Src homology 3 domain associate with macropinocytic vesicles in nonerythroid cells.

Xu J, Ziemnicka D, Scalia J, Kotula L
Brain Res. 2001; 898: 171-7

Spectrins represent a family of membrane-associated proteins responsible for membrane flexibility and cell shape in erythrocytes, and probably in most nonerythroid cells. Spectrin functions as a tetramer consisting of two heterodimers each containing two subunits termed alpha and beta. In humans, alphaI and alphaII spectrins but not beta spectrins are characterized by the presence of an Src homology 3 (SH3) domain. As a tool to investigate the function of spectrin SH3 domains we derived several monoclonal antibodies (mAb) to the recombinant human alphaI or alphaII spectrin SH3 domain. Immunostaining using these monoclonal antibodies indicated expression of alphaI spectrin in cell bodies and alphaII spectrin in neurites of granule neurons in mouse primary cerebellar cultures. Monoclonal antibodies reactive to alphaI spectrin SH3 domain indicated expression of a protein(s) containing an alphaI-like SH3 domain in cytoplasmic vesicular-like structures in GFAP-positive cells in these cultures. In NIH 3T3 fibroblasts, these antibodies label macropinocytic vesicles. Together, these data and Western blotting results suggest expression of at least three spectrin-SH3 domain antibody-reactive proteins.

Src homology domains of v-Src stabilize an active conformation of the tyrosine kinase catalytic domain.

Xu B, Miller WT
Mol Cell Biochem. 1996; 158: 57-63

To examine the interactions between Src homology domains and the tyrosine kinase catalytic domain of v-Src, various combinations of domains have been expressed in bacteria as fusion proteins. Constructs containing the isolated catalytic domain, SH2 + catalytic domain, and SH3 + SH2 + catalytic domains were active in autophosphorylation assays. For the catalytic domain of v-Src, but not for v-Abl, addition of exogenous Src SH3-SH2 domains stimulated the autophosphorylation activity. In contrast to results for autophosphorylation, constructs containing Src homology domains were more active towards a synthetic peptide substrate than the isolated catalytic domain. The ability of the SH2 and SH3 domains of v-Src to stabilize an active enzyme conformation was also confirmed by refolding after denaturation in guanidinium hydrochloride. Collectively the data suggest that, in addition to their roles in intermolecular protein-protein interactions, the Src homology regions of v-Src exert a positive influence on tyrosine kinase function, potentially by maintaining an active conformation of the catalytic domain.

Differential activation and redistribution of c-Src and Fyn in platelets, assessed by MoAb specific for C-terminal tyrosine-dephosphorylated c-Src and Fyn.

Wu Y, Ozaki Y, Inoue K, Satoh K, Ohmori T, Yatomi Y, Owadab K
Biochim Biophys Acta. 2000; 1497: 27-36

Tyrosine kinases, c-Src and Fyn, in their active form, have their C-terminal tyrosine residue dephosphorylated. In this study, we used clone 28, a monoclonal antibody (MoAb) that recognizes dephosphorylated C-terminal tyrosine of c-Src and Fyn, to investigate the mode of activation and mobilization of these kinases. Independently of integrin alphaIIbbeta3 signaling, the Fyn activity increased by 8.3-fold 5 s after stimulation with 20 microM TRAP (thrombin receptor agonist peptide), while that of c-Src increased only by 2.9-fold 15 s after stimulation. Both c-Src and Fyn translocated to the Triton-insoluble cytoskeletal fraction in an aggregation-dependent manner. Five minutes after TRAP-stimulation, 85% of Fyn translocated to the cytoskeleton, while only about 20% of c-Src was recovered in this fraction. The Triton-insoluble fraction was further fractionated by RIPA (radioimmunoprecipitation assay) buffer containing 0.1% SDS. While active c-Src was predominantly present in the Triton-insoluble/RIPA-insoluble fraction, clone 28-negative c-Src was present in the Triton-insoluble/RIPA-soluble fraction. On the other hand, Fyn was present only in the Triton-insoluble/RIPA-insoluble fraction. These findings suggest that the mode of activation and redistribution into the cytoskeleton differs between c-Src and Fyn, and that clone 28 provides a useful tool for investigating the activation and mobilization of Src family tyrosine kinases.

Proteolytic activation of ETK/Bmx tyrosine kinase by caspases.

Wu YM, Huang CL, Kung HJ, Huang CY
J Biol Chem. 2001; 276: 17672-8

Etk/Bmx is a member of the Btk/Tec family of kinases, which are characterized by having a pleckstrin homology domain at the N terminus, in addition to the Src homology 3 (SH3), SH2, and the catalytic domains, shared with the Src family kinases. Etk, or Btk kinases in general, has been implicated in the regulation of apoptosis. To test whether Etk is the substrate for caspases during apoptosis, in vitro translated [(35)S]methionine-labeled Etk was incubated with different apoptotic extracts and recombinant caspases, respectively. Results showed that Etk was proteolyzed in all conditions tested with identical cleavage patterns. Caspase-mediated cleavage of Etk generated a C-terminal fragment, containing the complete SH2 and tyrosine kinase domains, but without intact pleckstrin homology and SH3 domains. This fragment has 4-fold higher kinase activity than that of the full-length Etk. Ectopic expression of the C-terminal fragment of Etk sensitized the PC3 prostate cancer cells to apoptosis in response to apoptosis-inducing stimuli. The finding, together with an earlier report that Etk is potentially antiapoptotic, suggests that Etk may serve as an apoptotic switch, depending on the forms of Etk existing inside the cells. To our knowledge, this is the first case where the activity of a tyrosine kinase is induced by caspase cleavage.

Structural basis for the specific interaction of lysine-containing proline-rich peptides with the N-terminal SH3 domain of c-Crk.

Wu X, Knudsen B, Feller SM, Zheng J, Sali A, Cowburn D, Hanafusa H, Kuriyan J
Structure. 1995; 3: 215-26

BACKGROUND: Proline-rich segments in the guanine nucleotide exchange factor C3G bind much more strongly to the N-terminal Src homology 3 domain (SH3-N) of the proto-oncogene product c-Crk than to other SH3 domains. The presence of a lysine instead of an arginine in the peptides derived from C3G appears to be crucial for this specificity towards c-Crk. RESULTS: In order to understand the chemical basis of this specificity we have determined the crystal structure of Crk SH3-N in complex with a high affinity peptide from C3G (PPPALPPKKR, Kd approximately 2 microM) at 1.5 A resolution. The peptide adopts a polyproline type II helix that binds, as dictated by electrostatic complementarity, in reversed orientation relative to the orientation seen in the earliest structures of SH3-peptide complexes. A lysine in the C3G peptide is tightly coordinated by three acidic residues in the SH3 domain. In contrast, the co-crystal structure of c-Crk SH3-N and a peptide containing an arginine at the equivalent position (determined at 1.9 A resolution) reveals non-optimal geometry for the arginine and increased disorder. CONCLUSIONS: The c-Crk SH3 domain engages in an unusual lysine-specific interaction that is rarely seen in protein structures, and which appears to be a key determinant of its unique ability to bind the C3G peptides with high affinity.

Characterization of interactions of Nck with Sos and dynamin.

Wunderlich L, Farago A, Buday L
Cell Signal. 1999; 11: 25-9

One of the adaptor proteins, Nck, comprises a single SH2 domain and three SH3 domains that are important in protein-protein interactions. The in vivo association of Nck with the guanine nucleotide exchange factor Sos has been well documented; however, the precise nature of the interaction is unclear. To determine which SH3 domains are involved in the Nck-Sos interaction, individual SH3 domains of Nck were generated as glutathione S-transferase fusion proteins. We found that exclusively the third (C-terminal) SH3 domain of Nck has the ability to bind to Sos. In addition, in [35S]methionine labelled K562 cells, a 100,000 Mr protein was found to be associated with the third SH3 domain of Nck. This protein was identified as dynamin, a GTP-binding protein that has been implicated in clathrin-coated vesicle formation. Dynamin and Nck co-precipitated when cell lysates were immunoprecipitated with anti-Nck antibody. These data suggest that Nck may contribute to Ras activation and the function of dynamin in membrane trafficking through its third SH3 domain.

Design and synthesis of SH3 domain binding ligands: modifications of the consensus sequence XPpXP.

Witter DJ, Famiglietti SJ, Cambier JC, Castelhano AL
Bioorg Med Chem Lett. 1998; 8: 3137-42

Spirolactam-based Pro-Pro mimetics incorporated in the consensus sequence XPpXP, lead to effective nonpeptide ligands of SH3 domains.

Solution structure of the Grb2 N-terminal SH3 domain complexed with a ten-residue peptide derived from SOS: direct refinement against NOEs, J-couplings and 1H and 13C chemical shifts.

Wittekind M, Mapelli C, Lee V, Goldfarb V, Friedrichs MS, Meyers CA, Mueller L
J Mol Biol. 1997; 267: 933-52

Refined ensembles of solution structures have been calculated for the N-terminal SH3 domain of Grb2 (N-SH3) complexed with the ac-VPPPVPPRRR-nh2 peptide derived from residues 1135 to 1144 of the mouse SOS-1 sequence. NMR spectra obtained from different combinations of both 13C-15N-labeled and unlabeled N-SH3 and SOS peptide fragment were used to obtain stereo-assignments for pro-chiral groups of the peptide, angle restraints via heteronuclear coupling constants, and complete 1H, 13C, and 15N resonance assignments for both molecules. One ensemble of structures was calculated using conventional methods while a second ensemble was generated by including additional direct refinements against both 1H and 13C(alpha)/13C(beta) chemical shifts. In both ensembles, the protein:peptide interface is highly resolved, reflecting the inclusion of 110 inter-molecular nuclear Overhauser enhancement (NOE) distance restraints. The first and second peptide-binding sub-sites of N-SH3 interact with structurally well-defined portions of the peptide. These interactions include hydrogen bonds and extensive hydrophobic contacts. In the third highly acidic sub-site, the conformation of the peptide Arg8 side-chain is partially ordered by a set of NOE restraints to the Trp36 ring protons. Overall, several lines of evidence point to dynamical averaging of peptide and N-SH3 side-chain conformations in the third subsite. These conformations are characterized by transient charge stabilized hydrogen bond interactions between the peptide arginine side-chain hydrogen bond donors and either single, or possibly multiple, acceptor(s) in the third peptide-binding sub-site.

1H NMR study on the binding of Pin1 Trp-Trp domain with phosphothreonine peptides.

Wintjens R, Wieruszeski JM, Drobecq H, Rousselot-Pailley P, Buee L, Lippens G, Landrieu I
J Biol Chem. 2001; 276: 25150-6

The recent crystal structure of Pin1 protein bound to a doubly phosphorylated peptide from the C-terminal domain of RNA polymerase II revealed that binding interactions between Pin1 and its substrate take place through its Trp-Trp (WW) domain at the level of the loop Ser(11)-Arg(12) and the aromatic pair Tyr(18)-Trp(29), and showed a trans conformation for both pSer-Pro peptide bonds. However, the orientation of the ligand in the aromatic recognition groove still could be sequence-specific, as previously observed in SH3 domains complexed by peptide ligands or for different class of WW domains (Zarrinpar, A., and Lim, W. A. (2000) Nat. Struct. Biol. 7, 611-613). Because the bound peptide conformation could also differ as observed for peptide ligands bound to the 14-3-3 domain, ligand orientation and conformation for two other biologically relevant monophosphate substrates, one derived from the Cdc25 phosphatase of Xenopus laevis (EQPLpTPVTDL) and another from the human tau protein (KVSVVRpTPPKSPS) in complex with the WW domain are here studied by solution NMR methods. First, the proton resonance perturbations on the WW domain upon complexation with both peptide ligands were determined to be essentially located in the positively charged beta-hairpin Ser(11)-Gly(15) and around the aromatic Trp(29). Dissociation equilibrium constants of 117 and 230 microm for Cdc25 and tau peptides, respectively, were found. Several intermolecular nuclear Overhauser effects between WW domain and substrates were obtained from a ligand-saturated solution and were used to determine the structures of the complexes in solution. We found a similar N to C orientation as the one observed in the crystal complex structure of Pin1 and a trans conformation for the pThr-Pro peptidic bond in both peptide ligands, thereby indicating a unique binding scheme for the Pin1 WW domain to its multiple substrates.

Cooperative self-assembly of SH2 domain fragments restores phosphopeptide binding.

Williams KP, Shoelson SE
Biochemistry. 1993; 32: 11279-84

Multifunctional proteins frequently can be subdivided into discrete functional domains. Selected cytoplasmic proteins involved in signal transduction contain catalytic domains in addition to protein binding modules termed Src homology (SH) domains; SH2 domains bind phosphotyrosyl peptide sequences. Even as isolated modules, SH2 domains have the intrinsic capacity to fold properly and retain sequence selectivity for binding. Following limited digestion with trypsin, the 14-kDa SH2 domains of Src and PI 3-kinase p85 were split at a lysine within the flexible, phosphotyrosine-binding (BC) loop into 5- and 9-kDa fragments. Whereas the purified fragments did not exhibit cooperative unfolding or phosphopeptide binding, when combined they spontaneously reassembled to restore specific phosphopeptide binding and the unique spectroscopic signatures of bound and free intact SH2 domains. Like fragments of intact proteins, we now show that fragments of SH2 domains, and therefore protein modules, possess the intrinsic capacity for self-assembly with restoration of function. Analyses of fragment structures may provide insights into pathways of module folding, which will facilitate a more global understanding of how complex, multifunctional proteins fold.

A photoaffinity scan maps regions of the p85 SH2 domain involved in phosphoprotein binding.

Williams KP, Shoelson SE
J Biol Chem. 1993; 268: 5361-4

Src homology 2 (SH2) domains are modular phosphotyrosine binding pockets found within a wide variety of cytoplasmic signaling molecules. Here we develop a new approach to analyzing protein-protein interfaces termed photoaffinity scanning, and apply the method to map regions of the phosphatidylinositol 3-kinase p85 SH2 domain that participate in phospho-protein binding. Each residue except phosphotyrosine (pY) within a tightly binding, IRS-1-derived phosphopeptide (GNGDpYMPMSPKS) was substituted with the photoactive amino acid, benzoylphenylalanine (Bpa). Whereas most substitutions had little effect on binding affinity, Bpa substitution of either Met (+1 and +3 with respect to pY) reduced affinity 50-100-fold to confirm their importance in the pYMXM recognition motif. In three cases photolysis of SH2 domain/Bpa phosphopeptide complexes led to cross-linking of > 50% of the SH2 domain; cross-link positions were identified by microsequence, amino acid composition, and electrospray mass spectrometric analyses. Bpa-1 cross-links within alpha-helix I, whereas Bpa+1 and Bpa+4 cross-link the SH2 domain within the flexible loop C-terminal to alpha-helix II. Moreover, cross-linking at any position prevents SH2 domain cleavage at a trypsin-sensitive site within the flexible loop between beta-strands 1 and 2. Therefore, at least three distinct SH2 regions in addition to the beta-sheet participate in phosphoprotein binding; the loop cross-linked by phosphopeptide residues C-terminal to pY appears to confer specificity to the phosphoprotein/SH2 domain interaction.

The 2.35 A crystal structure of the inactivated form of chicken Src: a dynamic molecule with multiple regulatory interactions.

Williams JC, Weijland A, Gonfloni S, Thompson A, Courtneidge SA, Superti-Furga G, Wierenga RK
J Mol Biol. 1997; 274: 757-75

The Src protein tyrosine kinase plays a critical role in a variety of signal transduction pathways. Strict regulation of its activity is necessary for proper signalling. We present here the crystal structure of chicken Src which is phosphorylated at Tyr527 and represents its least active form. Our structure, similar to the recently reported human Hck and Src structures, contains the SH3, SH2 and the kinase domains and the C-terminal regulatory tail but not the N-terminal unique domain. The SH3 domain uses its hydrophobic surface to coordinate the SH2-kinase linker such that residues Gln251 and Leu255 specifically interact with side chains in the beta2-beta3 and the alphaC-beta4 loops of the N-terminal lobe opposite of the kinase active site. This position of the SH3 domain and the coordination of the SH2-kinase linker also optimally places the SH2 domain such that the phosphorylated Tyr527 in the C-terminal tail interacts with the SH2 binding pocket. Analogous to Cdk2 kinase, the position of the Src alphaC-helix in the N-terminal lobe is swung out disrupting the position of the active site residues. Superposition of other protein kinases including human Hck and Src onto chicken Src indicate that the alphaC-helix position is affected by the relative position of the N-terminal lobe with respect to the C-terminal lobe of the kinase and that the presence of the SH3/SH2-kinase linker/N-terminal lobe interactions restricts the kinase lobes and alphaC-helix access to the active conformation. These superpositions also suggest that the highly conserved alphaC-beta4 loop restricts the conformational freedom of the N-terminal lobe by anchoring it to the C-terminal lobe. Finally, based on sequence alignments and conservation of hydrophobic residues in the Src SH2-kinase linker as well as in the alphaC-beta4 and beta2-beta3 loops, we propose that the Src-related kinases, Abl, Btk and Csk, share the same quaternary structure.

Accommodating structurally diverse peptides in proteins.

Wilkinson AJ
Chem Biol. 1996; 3: 519-24

Many peptide-binding proteins must bind numerous ligands that differ in size, sequence and sometimes orientation. A variety of strategies for coping with structurally diverse peptide ligands have been revealed by biochemical and structural studies of proteins with roles in immunity, transport and signal transduction.

The PAG gene product, a stress-induced protein with antioxidant properties, is an Abl SH3-binding protein and a physiological inhibitor of c-Abl tyrosine kinase activity.

Wen ST, Van Etten RA
Genes Dev. 1997; 11: 2456-67

Biochemical and genetic evidence suggests that the tyrosine kinase activity of c-Abl is tightly regulated in vivo by a cellular factor binding to the Src homology 3 (SH3) domain of Abl. We used the yeast two-hybrid system to identify a gene, PAG, whose protein product (Pag) interacts specifically with the Abl SH3 domain. Pag, also known as macrophage 23-kD stress protein (MSP23), is a member of a novel family of proteins with antioxidant activity implicated in the cellular response to oxidative stress and in control of cell proliferation and differentiation. In a co-expression assay, Pag associates with c-Abl in vivo and inhibits tyrosine phosphorylation induced by overexpression of c-Abl. Inhibition requires the Abl SH3 and kinase domains and is not observed with other Abl SH3-binding proteins. Expression of Pag also inhibits the in vitro kinase activity of c-Abl, but not SH3-mutated Abl or v-Abl. When transfected in NIH-3T3 cells, Pag is localized to nucleus and cytoplasm and rescues the cytostatic effect induced by c-Abl. These observations suggest Pag is a physiological inhibitor of c-Abl in vivo.

Identification of Src, Fyn, and Lyn SH3-binding proteins: implications for a function of SH3 domains.

Weng Z, Thomas SM, Rickles RJ, Taylor JA, Brauer AW, Seidel-Dugan C, Michael WM, Dreyfuss G, Brugge JS
Mol Cell Biol. 1994; 14: 4509-21

Src homology 3 (SH3) domains mediate protein-protein interactions necessary for the coupling of cellular proteins involved in intracellular signal transduction. We previously established solution-binding conditions that allow affinity isolation of Src SH3-binding proteins from cellular extracts (Z. Weng, J. A. Taylor, C. E. Turner, J. S. Brugge, and C. Seidel-Dugan, J. Biol. Chem. 268:14956-14963, 1993). In this report, we identified three of these proteins: Shc, a signaling protein that couples membrane tyrosine kinases with Ras; p62, a protein which can bind to p21rasGAP; and heterogeneous nuclear ribonucleoprotein K, a pre-mRNA-binding protein. All of these proteins contain proline-rich peptide motifs that could serve as SH3 domain ligands, and the binding of these proteins to the Src SH3 domain was inhibited with a proline-rich Src SH3 peptide ligand. These three proteins, as well as most of the other Src SH3 ligands, also bound to the SH3 domains of the closely related protein tyrosine kinases Fyn and Lyn. However, Src- and Lyn-specific SH3-binding proteins were also detected, suggesting subtle differences in the binding specificity of the SH3 domains from these related proteins. Several Src SH3-binding proteins were phosphorylated in Src-transformed cells. The phosphorylation of these proteins was not detected in cells transformed by a mutant variant of Src lacking the SH3 domain, while there was little change in tyrosine phosphorylation of other Src-induced phosphoproteins. In addition, the coprecipitation of v-Src with two tyrosyl-phosphorylated proteins with M(r)s of 62,000 and 130,000 was inhibited by incubation with a Src SH3 peptide ligand, suggesting that the binding of these substrate proteins is dependent on interactions with the SH3 domain. These results strongly suggest a role for the Src SH3 domain in the recruitment of substrates to this protein tyrosine kinase, either through direct interaction with the SH3 domain or indirectly through interactions with proteins that bind to the SH3 domain.

Detection of Src homology 3-binding proteins, including paxillin, in normal and v-Src-transformed Balb/c 3T3 cells.

Weng Z, Taylor JA, Turner CE, Brugge JS, Seidel-Dugan C
J Biol Chem. 1993; 268: 14956-63

The Src homology 3 (SH3) domain, located in the amino-terminal, noncatalytic half of pp60src, is highly conserved among members of the Src family of tyrosine kinases. SH3 domains have also been identified in a variety of proteins otherwise unrelated to protein-tyrosine kinases. The presence of SH3 domains in proteins with diverse functions suggests this domain may be important for directing protein-protein interactions necessary for protein function or cellular localization. To explore possible interactions between the SH3 domain and cellular proteins, we have established conditions for the isolation of proteins that bind in solution to the Src SH3 domain. A 67-amino acid fragment of c-Src containing either the entire glutathione S-transferase-SH3 domain (GST-SH3) or the SH3 domain from the neuronal form of c-Src (GST-SH3+) was expressed as a glutathione S-transferase fusion protein. The GST fusion proteins were incubated with lysates from [35S]methionine-labeled Balb/c 3T3 cells or v-Src-transformed Balb/c 3T3 cells. We found that GST-SH3, but not wild-type GST, specifically interacted with multiple cellular proteins, whereas GST-SH3+ only weakly associated with a small subset of these proteins. The majority of the SH3-binding proteins were found in particulate and detergent-insoluble cell fractions. Anti-phosphotyrosine immunoblots of the SH3-binding proteins revealed that several of the SH3-binding proteins are phosphorylated on tyrosine in v-Src-transformed cells. In addition, a number of the SH3-binding proteins were phosphorylated on serine and/or threonine in in vitro kinase assays, suggesting that one or more of the SH3-binding proteins has kinase activity. We identified paxillin, a vinculin-binding protein, as one of the Src SH3-binding proteins. This finding strongly supports the hypothesis that SH3 domains may be involved in subcellular localization of proteins to cytoskeleton and/or cellular membranes.

Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions.

Weng Z, Rickles RJ, Feng S, Richard S, Shaw AS, Schreiber SL, Brugge JS
Mol Cell Biol. 1995; 15: 5627-34

SH3 domains mediate intracellular protein-protein interactions through the recognition of proline-rich sequence motifs on cellular proteins. Structural analysis of the Src SH3 domain (Src SH3) complexed with proline-rich peptide ligands revealed three binding sites involved in this interaction: two hydrophobic interactions (between aliphatic proline dipeptides in the SH3 ligand and highly conserved aromatic residues on the surface of the SH3 domain), and one salt bridge (between Asp-99 of Src and an Arg three residues upstream of the conserved Pro-X-X-Pro motif in the ligand). We examined the importance of the arginine binding site of SH3 domains by comparing the binding properties of wild-type Src SH3 and Abl SH3 with those of a Src SH3 mutant containing a mutated arginine binding site (D99N) and Abl SH3 mutant constructs engineered to contain an arginine binding site (T98D and T98D/F91Y). We found that the D99N mutation diminished binding to most Src SH3-binding proteins in whole cell extracts; however, there was only a moderate reduction in binding to a small subset of Src SH3-binding proteins (including the Src substrate p68). p68 was shown to contain two Arg-containing Asp-99-dependent binding sites and one Asp-99-independent binding site which lacks an Arg. Moreover, substitution of Asp for Thr-98 in Abl SH3 changed the binding specificity of this domain and conferred the ability to recognize Arg-containing ligands.(ABSTRACT TRUNCATED AT 250 WORDS)

Shb is a ubiquitously expressed Src homology 2 protein.

Welsh M, Mares J, Karlsson T, Lavergne C, Breant B, Claesson-Welsh L
Oncogene. 1994; 9: 19-27

To identify serum-inducible genes in the insulin-producing cell line beta TC-1, a library subtraction screening procedure was performed on serum-deprived (G0) and serum-restimulated (G1) insulin-producing beta TC-1 cells. A cDNA containing a motif with strong homology to Src homology 2 (SH2) domains was found using this procedure and called Shb. The Shb cDNA contains two methionine codons in its N-terminus and thus may code for two proteins of 67 and 56 kDa, each with one SH2 domain in its C-terminus. No other structural similarity to proteins with catalytic activity could be detected, suggesting that Shb is a so called adaptor. Shb contains the proline-rich sequence PPPGPGR between the two proposed initiator methionines which resembles a sequence for binding to Src homology 3 (SH3) domains. A second proline-rich sequence was detected after the second methionine codon. The Shb cDNA hybridized to a similar or identical mRNA of 3.1 kb expressed in mouse brain, liver, kidney, heart, NIH3T3 fibroblasts and beta TC-1 cells. Western blot analysis of the same tissues using an antiserum directed against a synthetic peptide corresponding to a part of the SH2 domain of Shb, revealed reactivity with two proteins of 56 and 67 kDa. In addition, a third reactive component of 40 kDa was detected in most tissues. Transfection and transient expression of the Shb cDNA in COS-1 cells yielded increased expression of the 67, 56 and 40 kDa proteins. Transfection and stable expression of the Shb cDNA in pig aortic endothelial cells showed increased expression primarily of the 67 kDa protein. A fusion protein consisting of the SH2 domain of Shb linked to glutathione S-transferase showed increased binding to glycoproteins of cells stimulated with platelet-derived growth factor (PDGF-BB). Furthermore, the autophosphorylated PDGF beta-receptor but not the autophosphorylated epidermal growth factor (EGF) receptor bound specifically to immobilized fusion protein. It is concluded that Shb is a novel SH2-containing protein with proline-rich domains and therefore probably involved in the signal-transduction of some ligand-activated tyrosine kinase receptors.

An analysis of the interactions between the Sem-5 SH3 domain and its ligands using molecular dynamics, free energy calculations, and sequence analysis.

Wang W, Lim WA, Jakalian A, Wang J, Wang J, Luo R, Bayly CI, Kollman PA
J Am Chem Soc. 2001; 123: 3986-94

The Src-homology-3 (SH3) domain of the Caenorhabditis elegans protein Sem-5 binds proline-rich sequences. It is reported that the SH3 domains broadly accept amide N-substituted residues instead of only recognizing prolines on the basis of side chain shape or rigidity. We have studied the interactions between Sem-5 and its ligands using molecular dynamics (MD), free energy calculations, and sequence analysis. Relative binding free energies, estimated by a method called MM/PBSA, between different substitutions at sites -1, 0, and +2 of the peptide are consistent with the experimental data. A new method to calculate atomic partial charges, AM1-BCC method, is also used in the binding free energy calculations for different N-substitutions at site -1. The results are very similar to those obtained from widely used RESP charges in the AMBER force field. AM1-BCC charges can be calculated more rapidly for any organic molecule than can the RESP charges. Therefore, their use can enable a broader and more efficient application of the MM/PBSA method in drug design. Examination of each component of the free energy leads to the construction of van der Waals interaction energy profiles for each ligand as well as for wild-type and mutant Sem-5 proteins. The profiles and free energy calculations indicate that the van der Waals interactions between the ligands and the receptor determine whether an N- or a Calpha-substituted residue is favored at each site. A VC value (defined as a product of the conservation percentage of each residue and its van der Waals interaction energy with the ligand) is used to identify several residues on the receptor that are critical for specificity and binding affinity. This VC value may have a potential use in identifying crucial residues for any ligand-protein or protein-protein system. Mutations at two of those crucial residues, N190 and N206, are examined. One mutation, N190I, is predicted to reduce the selectivity of the N-substituted residue at site -1 of the ligand and is shown to bind similarly with N- and Calpha-substituted residues at that site.

c-src structure in human cancers with elevated pp60c-src activity.

Wang P, Fromowitz F, Koslow M, Hagag N, Johnson B, Viola M
Br J Cancer. 1991; 64: 531-3

We used RNAase protection and restriction fragment length polymorphism assays to detect activating mutations of c-src in a spectrum of human tumours. No mutations were detected at codons 98, 381, 444, and 530. We conclude that mutational activation is not the mechanism of enhancement of pp60c-src-specific kinase activity found in a number of human cancer types.

Molecular determinants for Csk-catalyzed tyrosine phosphorylation of the Src tail.

Wang D, Huang XY, Cole PA
Biochemistry. 2001; 40: 2004-10

Phosphorylation of a critical tail tyrosine residue in Src modulates its three-dimensional structure and protein tyrosine kinase activity. The protein tyrosine kinase Csk is responsible for catalyzing the phosphorylation of this key Src tyrosine residue, but the detailed molecular basis for Src recognition and catalysis is poorly understood. In this study, we investigate this phosphorylation event using purified recombinant Csk and Src proteins and mutants. It was shown that the apparent k(cat) and K(m) values for Csk phosphorylation of catalytically impaired Src (dSrc) are similar to the parameters for Csk-catalyzed phosphorylation of the Src family member Lck. The SH3 (Src homology 3) and SH2 (Src homology 2) domains of dSrc were fully dispensable with respect to rapid phosphorylation, indicating that the catalytic domain and tail of dSrc are sufficient for the high efficiency of dSrc as a substrate. Of the eight Src tail residues examined, only the fully conserved Glu (Y-3 position) and Gln (Y-1 position) investigated by alanine scanning mutagenesis caused large reductions (10--40-fold) in dSrc substrate efficiency. The Y-3 Glu requirement was stringent as conservative replacements with Asp or Gln were no better than Ala whereas replacement of the Y-1 Gln with Ile was readily tolerated. Interestingly, en bloc replacement of the tail with a seven amino acid consensus sequence derived from a peptide library analysis was no better than the wild-type sequence. Surprisingly, the dSrc Y527F protein, although not a Csk substrate, enhanced Csk-catalyzed phosphorylation of dSrc. These results and other data suggest that Src dimerization (or higher order oligomerization) is important for high-efficiency Csk-catalyzed phosphorylation of the Src tail.

Proline-rich sequences mediate the interaction of the Arg protein tyrosine kinase with Crk.

Wang B, Mysliwiec T, Feller SM, Knudsen B, Hanafusa H, Kruh GD
Oncogene. 1996; 13: 1379-85

Arg is a ubiquitously expressed member of the Abelson family of nonreceptor protein-tyrosine kinases. Defining the Arg sequences that mediate its interaction with other proteins is essential to elucidating its role in cellular signaling. In this report we demonstrate that Arg associates with c-Crk, an adaptor protein composed of an SH2 domain and two SH3 domains, and examine the molecular mechanism of the interaction. In vitro experiments revealed that three proline-rich sequences with distinct specificities for SH3 domains are located in the Arg C-terminal domain, just C-terminal to the kinase domain, and that two of these sequences bind to the Crk N-terminal SH3 domain. These two sequences conform to the PxLPxK/R motif that has been observed in other proteins that bind the Crk N-terminal SH3 domain. The interaction of Arg with c-Crk in living cells was confirmed by the detection of coimmunoprecipitation in coinfected Sf9 cells. In addition, increased phosphorylation of c-Crk was observed in cotransfected COS cells, indicating that Crk is an Arg substrate. The site of c-Crk phosphorylation by Arg was identified as tyrosine 221, a residue whose modification has been shown to result in an intramolecular SH2 interaction and a folded conformation. These experiments extend the known Arg protein interacting motifs to include SH3 binding sites and suggest that Arg may function as an effector as well as a regulator of Crk activity.

SH2 domains: a question of independence.

Wandless TJ
Curr Biol. 1996; 6: 125-7

The three-dimensional structures of parts of two enzymes that contain tandem Src homology 2 (SH2) domains have recently been determined. The structures suggest how the SH2 domains function in concert to regulate enzymatic activity and localization.

Crystal structure of the phosphotyrosine recognition domain SH2 of the Src oncogene product complexed with tyrosine-phosphorylated peptides.

Waksman G
Cell Mol Biol (Noisy-le-grand). 1994; 40: 611-8

SH2 domains are protein modules specialized in recognizing phosphorylated tyrosine residues and their sequence contexts. The mechanism of phosphotyrosine recognition and context recognition was elucidated in a series of X-ray crystallographic experiments which are discussed below.

Drosophila src family proteins.

Wadsworth SC
Comp Biochem Physiol B. 1990; 97: 403-6

The beta D-sheet residues of the Lck-derived SH2 domain determine specificity of the interaction with tyrosine-phosphorylated ligands in Ramos B cells.

von Bonin A, Wienands J, Manning U, Zuber JF, Baumann G
J Biol Chem. 1994; 269: 33035-41

Src homology 2 (SH2) domains are noncatalytic regions that are conserved among a group of cellular signaling proteins. SH2 domains share the common property of binding phosphotyrosine-containing peptides. Previously, we showed that SH2 domains expressed as recombinant glutathione S-transferase-fusion proteins (GST-SH2) from GTPase-activating protein, Shc, zeta-chain-associated protein tyrosine kinase Zap-70, and Src-like tyrosine kinases precipitated distinct sets of phospho-proteins from activated B cells. To determine the intrinsic structural motifs responsible for the binding specificity within the different SH2 domains, we created chimeric SH2 domains especially focusing on crystal structure-defined contact residues. Recombinant SH2 domains of Lck, Zap-70, and Shc were tested in Ramos B cell lysates for phosphotyrosine-dependent protein binding. Biomolecular interaction analysis (BIAcore) was used to characterize the interaction between the various recombinant SH2 molecules and defined phosphorylated peptides. In agreement with the crystal structure data from the Src and the Lck SH2 domains, our results show that most of the "specificity information" of the Lck SH2 domain is provided by the beta D-sheet, located downstream of the SH2 conserved consensus motif GTFLVRES. In addition, the overall affinity is critically influenced by residues located at the N terminus of the SH2 domain.

Interactions between SH2 and SH3 domains.

Vihinen M, Smith CI
Biochem Biophys Res Commun. 1998; 242: 351-6

Src homology 2 (SH2) and SH3 domains are abundant protein and peptide binding modules in signalling molecules. Certain SH2 and SH3 domains have been shown to form functional and physical interactions. The structural basis of dimer formation was studied by docking three dimensional structures of the domains and by analysing structural and functional properties of the dimers. The experimentally verified dimers were noticed to have very large buried surfaces, extensive hydrogen bonding networks, and complementary surfaces, properties which are characteristic for protein-protein interactions. The number of hydrogen bonds between the domains is exceptionally high for interacting protein pairs. Also the buried accessible surface is large, especially when considering the small size of the domains. The dimer results were used to describe mutation information in structural terms and to discuss regulation of protein tyrosine kinases.

Structural basis of SH2 domain mutations in X-linked agammaglobulinemia.

Vihinen M, Nilsson L, Smith CI
Biochem Biophys Res Commun. 1994; 205: 1270-7

The three-dimensional structure of Bruton's agammaglobulinemia tyrosine kinase (Btk) SH2 domain was modeled based on v-Src. Btk SH2 is presumably very related to the other SH2 structures consisting of two beta-sheets surrounded by two alpha-helices, with a well conserved hydrophobic core and phoshotyrosyl peptide binding site. The model was used to predict the recognition sequence of the target protein, which probably is YEXI/L. Mutations in the Btk sequence can cause the human disease X-linked agammaglobulinemia and reasons for the disease in Btk SH2 mutations were inferred from the model.

Bergerac-SH3: "frustation" induced by stabilizing the folding nucleus.

Viguera AR, Serrano L
J Mol Biol. 2001; 311: 357-71

The influence of an inserted exogenous independent folding element on the thermodynamics and folding properties of SH3 domain from alpha-spectrin has been investigated by creating a fused form between this small all-beta domain and a stable beta-hairpin (BH19). NMR analysis of synthetic peptides shows that insertion of BH19 nucleates formation of the original natural beta-hairpin (distal loop) that is part of the SH3 folding nucleus. The resulting protein (Bergerac-SHH) is more stable, folds faster and contains an elongated hairpin protruding from the globular domain as determined by 2D-NMR. "Protein engineering" analysis of the inserted region shows that it is folded in the transition state. Interestingly, stabilisation by insertion of the distal loop region results in the appearance of a compact intermediate revealed by a curved chevron plot at low denaturant concentration. This effect is eliminated at low salt concentrations by a single mutation of a hydrophobic residue within BH19 sequence, which is most probably involved in non-native interactions. Local stabilisation by enlargement and reinforcement of the folding nucleus, global compaction by the addition of salt and non-native interactions are shown to contribute to the observed deviation from the two-state behaviour. Copyright 2001 Academic Press.

Characterization of the interaction of natural proline-rich peptides with five different SH3 domains.

Viguera AR, Arrondo JL, Musacchio A, Saraste M, Serrano L
Biochemistry. 1994; 33: 10925-33

The interaction of six different proline-rich peptides with five SH3 domains has been investigated by using spectroscopic techniques. These peptides correspond to natural sequences and have been implicated in the interaction of some SH3 domains with other proteins. We have determined the Kd values for all of the possible combinations between the peptides and the SH3 domains. Low specificity and low affinity (> 5 microM) are the most remarkable conclusions from these studies. None of the peptides tested here were found to bind with significant affinity to spectrin-SH3 or n-src-SH3. Abl-SH3 seems to be the most selective of the domains analyzed here, while Fyn-SH3 is the most promiscuous. CD and FTIR studies indicate that these peptides adopt to different extents a PPII-like structure in aqueous solution. However, analysis of the SH3 domain complexes with these peptides suggests that proline-rich peptides do not necessarily adopt an overall PPII structure over their entire length upon binding to the different SH3 domains.

Structural similarity of two T cell signaling regulators suggests a conserved and interactive mechanism of immunosuppression.

Vieira A
Mol Immunol. 1998; 35: 881-4

A primary structural element is identified which is present in the Nef protein of immunodeficiency viruses and in the cytoplasmic domain of a regulatory T cell protein, CTLA-4. In both proteins, conserved residues that lie within this element have been shown to be important for the regulation of cellular signaling and endocytic trafficking. The structural similarity identified suggests conserved and, possibly, interactive mechanisms for Nef- and CTLA-4-dependent modulation of T cell function.

Molecular and cellular analysis of Grb2 SH3 domain mutants: interaction with Sos and dynamin.

Vidal M, Goudreau N, Cornille F, Cussac D, Gincel E, Garbay C
J Mol Biol. 1999; 290: 717-30

Quantitative analysis of Grb2/dynamin interaction through plasmon resonance analysis (BIAcore) using Grb2 mutants showed that the high affinity measured between Grb2 and dynamin is essentially mediated by the N-SH3 domain of Grb2. In order to study the interactions between Grb2 and either dynamin or Sos in more detail, Grb2 N-SH3 domains containing different mutations have been analysed. Two mutations were located on the hydrophobic platform binding proline-rich peptides (Y7V and P49L) and one (E40T) located in a region that we had previously shown to be essential for Grb2/dynamin interactions. Through NMR analysis, we have clearly demonstrated that the structure of the P49L mutant is not folded, while the other E40T and Y7V mutants adopt folded structures that are quite similar to that described for the reference domain. Nevertheless, these point mutations were shown to alter the overall stability of these domains by inducing an equilibrium between a folded and an unfolded form. The complex formed between the peptide VPPPVPPRRR, derived from Sos, and the E40T mutant was shown to have the same 3D structure as that described for the wild-type SH3 domain. However, the VPPPVPPRRR peptide adopts a slightly different orientation when it is complexed with the Y7V mutant. Finally, the affinity of the proline-rich peptide GPPPQVPSRPNR, derived from dynamin, for the Grb2 N-SH3 domain was too low to be analyzed by NMR. Thus, the interaction between either Sos or dynamin and the SH3 mutants were tested on a cellular homogenate by means of a far-Western blot analysis. In these conditions, the P49L mutant was shown to be devoid of affinity for Sos as well as for dynamin. The Y7V SH3 mutant displayed a decrease of affinity for both Sos and dynamin, while the E40T mutant exhibited a decrease of affinity only for dynamin. These results support the existence of two binding sites between dynamin and the Grb2 N-SH3 domain.

Function, location, and regulation of the src protein-tyrosine kinase.

Varmus H, Hirai H, Morgan D, Kaplan J, Bishop JM
Princess Takamatsu Symp. 1989; 20: 63-70

The physiological roles, precise locations, and relevant targets of the 60 kD protein-tyrosine kinase encoded by viral and cellular src genes p60src are not known, despite intensive study. We describe recent work that bears upon these unresolved problems: (i) p60c-src is phosphorylated during mitosis on threonine and serine residues by the protein kinase encoded by the mammalian homologue of cdc2, suggesting that c-src may contribute to the phenotype of mitotic cells; (ii) multiple regions in the amino-terminal portion of p60src are required for its proper intracellular localization--a short signal for myristylation and signals for association with cytoplasmic granules and with perinuclear and plasma membranes; and (iii) regions (called SH3 and SH2) upstream of the kinase domain modulate the behavior of p60src in complex ways, with some mutations in SH2 rendering p60 host-dependent for transformation. The latter mutants may prove to be powerful tools for identifying proteins that modify or serve as targets for src-encoded protein-tyrosine kinases.

The K protein domain that recruits the interleukin 1-responsive K protein kinase lies adjacent to a cluster of c-Src and Vav SH3-binding sites. Implications that K protein acts as a docking platform.

Van Seuningen I, Ostrowski J, Bustelo XR, Sleath PR, Bomsztyk K
J Biol Chem. 1995; 270: 26976-85

The heterogeneous ribonucleoprotein particle (hnRNP) K protein interacts with multiple molecular partners including DNA, RNA, serine/threonine, and tyrosine kinases and the product of the proto-oncogene, Vav. The K protein is phosphorylated in vivo and in vitro on serine/threonine residues by an interleukin 1 (IL-1)-responsive kinase with which it forms a complex. In this study we set out to map the K protein domains that bind kinases. We demonstrate that the K protein contains a cluster of at least three SH3-binding sites (P1, PPGRGGRPMPPSRR, amino acids 265-278; P2, PRRGPPPPPPGRG, 285-297; and P3, RARNLPLPPPPPPRGG, 303-318) and that each one of these sites is capable of selectively engaging c-Src and Vav SH3 domains but not SH3 domains of Abl, p85 phosphatidylinositol 3-kinase, Grb-2, and Csk. We demonstrate that the K protein domain that recruits and is phosphorylated in an RNA-dependent manner by the IL-1-responsive kinase, designated KPK for K protein kinase, is contained within the 338-425-amino acid stretch and thus is contiguous but does not include the cluster of the SH3-binding sites. K protein and KPK co-immunoprecipitate from cell extracts with either c-Src or Vav, suggesting that K protein-KPK-c-Src and K protein-KPK-Vav complexes exist in vivo. Furthermore, in the context of K protein, c-Src can reactivate KPK in vitro. The succession of kinase-binding sites contained within the K protein that allow it to form multienzyme complexes and facilitate kinase cross-talk suggest that K protein may serve as a docking platform that promotes molecular interactions occurring during signal transduction.

Introduction of a loss-of-function point mutation from the SH3 region of the Caenorhabditis elegans sem-5 gene activates the transforming ability of c-abl in vivo and abolishes binding of proline-rich ligands in vitro.

Van Etten RA, Debnath J, Zhou H, Casasnovas JM
Oncogene. 1995; 10: 1977-88

We have introduced two loss-of-function point mutations from highly conserved regions of the src homology 3 (SH3) domains of the Caenorhabditis elegans sem-5 gene into the SH3 domain of the murine type IV c-abl tyrosine kinase proto-oncogene. One of the mutations, P131L, activated abl to transform fibroblasts while the other, G128R, did not. When combined with independent activating mutations in the c-abl kinase domain or NH2-terminus, the G128R mutation blocked transformation by the double mutant, suggesting that the G128R mutant was unable to transform cells for trivial reasons. The c-Abl G128R mutant, like wild type c-Abl protein, was localized to the nucleus and actin cytoskeleton and had normal tyrosine kinase activity in vitro, while the transforming c-Abl P131L protein was localized exclusively to the cytoplasm and exhibited decreased in vitro kinase activity. By real-time biospecific interaction analysis, the wild type Abl SH3 domain bound to two proteins containing proline-rich motifs with dissociation constants of 0.2 and 17 microM; the G128R mutant bound with 50-fold lower affinity, and no binding was detected by the P131L mutant. Both mutations completely abolished binding of the Abl SH3 domain to proline-rich target proteins in a filter-binding assay. These results suggest that the transforming activity of Abl is regulated in vivo by an inhibitor protein which associates with the SH3 domain via a proline-rich sequence.

Competitive binding assay of src homology domain 3 interactions between 5-lipoxygenase and growth factor receptor binding protein 2.

VanderNoot VA, Fitzpatrick FA
Anal Biochem. 1995; 230: 108-14

c-src homology 3 domains (SH3) modulate the formation of a number of protein complexes that are important in cell signaling and cytoskeletal organization. The SH3 domain is recognized by short conserved proline-rich motifs which adopt left-handed polyproline helices on binding. In order to examine molecular determinants of the proline motif:SH3 interaction, an enzyme-linked immunosorbent assay was developed to observe binding of 5-lipoxygenase to SH3 domains of growth factor receptor binding protein 2 (Grb2). The assay makes use of glutathione S-transferase fusion proteins of Grb2 and fragments of Grb2 immobilized onto wells of standard 96-well microtiter plates. Equilibrium binding is monitored colorimetrically and the measured absorbance is proportional to 5-LO concentration. The interactions is specific for the Grb2 portion of the fusion proteins, and 5-LO binds preferentially to Grb2 fragments containing an SH3 domain. Competitive binding assays with a synthetic peptide which mimicked the proline-rich region of 5-LO yielded results that are consistent with previous estimates. Binding was examined in the presence of a number of peptides containing the consensus sequence -PXXP-, in the presence of enzyme activity mediators and in the presence of plant lipoxygenases that lack the proline-rich binding motif. Results suggest that the specificity of the Grb2:5-LO interaction is high.

Initial events of myelination involve Fyn tyrosine kinase signalling.

Umemori H, Sato S, Yagi T, Aizawa S, Yamamoto T
Nature. 1994; 367: 572-6

Myelin is the lipoprotein multimembrane that functions as an insulator preventing the flow of ion currents across the axonal membrane and facilitating the conduction of nerve impulses. It is synthesized by oligodendrocytes in the central nervous system at about the time of birth in mammals. During the initial stages of myelination, several proteins are phosphorylated on tyrosine. Among these proteins, we identified Fyn tyrosine kinase, one of the non-receptor-type tyrosine kinases of the Src family. Here we report that Fyn tyrosine kinase is activated during the initial stages of myelination and that it is associated with the large myelin-associated glycoprotein (MAG), an adhesion molecule that has been implicated in myelinogenesis. The Fyn-large MAG association requires amino-terminal domains of Fyn that include SH2 and SH3 (Src homology domains 2 and 3). Crosslinking of large MAG with antibody induces a rapid increase in the specific activity of Fyn kinase. These results indicate that Fyn participates in the initial events of myelination as a signalling molecule downstream of large MAG; indeed, we find that fyn-deficient mice exhibit impaired myelination.

Solution structure of the human BTK SH3 domain complexed with a proline-rich peptide from p120cbl.

Tzeng SR, Lou YC, Pai MT, Jain ML, Cheng JW
J Biomol NMR. 2000; 16: 303-12

X-linked agammaglobulinemia (XLA), an inherited disease, is caused by mutations in the Bruton's tyrosine kinase (BTK). The absence of functional BTK leads to failure of B cell differentiation which incapacitates antibody production in XLA patients leading to, sometimes lethal, bacterial infections. Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in one patient family. To understand the role of BTK in B cell development, we have determined the solution structure of BTK SH3 domain complexed with a proline-rich peptide from the protein product of c-cbl protooncogene (p120cbl). Like other SH3 domains, BTK SH3 domain consists of five beta-strands packed in two beta-sheets forming a beta-barrel-like structure. The rmsd calculated from the averaged coordinates for the BTK SH3 domain residues 218-271 and the p120cbl peptide residues 6-12 of the complex was 0.87 A (+/-0.16 A) for the backbone heavy atoms (N, C, and Calpha) and 1.64 A (+/-0.16 A) for all heavy atoms. Based on chemical shift changes and inter-molecular NOEs, we have found that the residues located in the RT loop, n-Src loop and helix-like loop between beta4 and beta5 of BTK SH3 domain are involved in ligand binding. We have also determined that the proline-rich peptide from p120cbl binds to BTK SH3 domain in a class I orientation. These results correlate well with our earlier observation that the truncated BTK SH3 domain (deletion of beta4, beta5 and the helix-like loop) exhibits weaker affinity for the p120cbl peptide. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context and hence the weaker binding. These results delineate the importance of the C-terminus in the binding of SH3 domains and also indicate that improper folding and the altered binding behavior of mutant BTK SH3 domain likely lead to XLA.

Identification and kinetic analysis of the interaction between Nck-2 and DOCK180.

Tu Y, Kucik DF, Wu C
FEBS Lett. 2001; 491: 193-9

Nck-2 is a newly identified adapter protein comprising three N-terminal SH3 domains and one C-terminal SH2 domain. We have identified in a yeast two-hybrid screen DOCK180, a signaling protein implicated in the regulation of membrane ruffling and migration, as a binding protein for Nck-2. Surface plasmon resonance analyses reveal that the second and the third SH3 domains interact with the C-terminal region of DOCK180. The interactions mediated by the individual SH3 domains, however, are much weaker than that of the full length Nck-2. Furthermore, a point mutation that inactivates the second or the third SH3 domain dramatically reduced the interaction of Nck-2 with DOCK180, suggesting that both SH3 domains contribute to the DOCK180 binding. A major Nck-2 binding site, which is recognized primarily by the third SH3 domain, has been mapped to residues 1819-1836 of DOCK180. Two additional, albeit much weaker, Nck-2 SH3 binding sites are located to DOCK180 residues 1793-1810 and 1835-1852 respectively. Consistent with the mutational studies, kinetic analyses by surface plasmon resonance suggest that two binding events with equilibrium dissociation constants of 4.15+/-1.9x10(-7) M and 3.24+/-1.9x10(-9) M mediate the binding of GST-Nck-2 to GST fusion protein containing the C-terminal region of DOCK180. These studies identify a novel interaction between Nck-2 and DOCK180. Furthermore, they provide a detailed analysis of a protein complex formation mediated by multiple SH3 domains revealing that tandem SH3 domains significantly enhance the weak interactions mediated by each individual SH3 domain.

Characterization and functional allotment of proto-oncogenes belonging to the src family.

Toyoshima K, Yamanashi Y, Katagiri T, Inoue K, Semba K, Yamamoto T
Princess Takamatsu Symp. 1989; 20: 111-7

Eight proto-oncogenes encode cytoplasmic protein tyrosine kinases whose primary structures are closely related to that of p60proto-src. This group of related genes is called the src-family. Their products share common structures: namely, a glycine residue at position 2 from the N terminus, a unique domain, a modulatory domain (SH2, SH3), a kinase (catalytic) domain, and a regulatory domain. Oncogenic activation of these genes may occur by various mutations such as 1) deletion or substitution of the tyrosine at the C-terminal position, 2) mutation at a defined position in the kinase domain or 3) deletion or mutation in the modulatory domain. The expressions of the proto-oncogenes of this family are tissue specific and are unique for each gene, suggesting functional allotment of these gene products. In the hematopoietic system, the genes appear to be expressed in specific cell lineages. In this paper, the functional allotments of these genes in the hematopoietic system are considered with special reference to the functions of lck, lyn, and fyn in cells of T and B lymphocyte lineages.

Autophosphorylation activity and association with Src family kinase of Sky receptor tyrosine kinase.

Toshima J, Ohashi K, Iwashita S, Mizuno K
Biochem Biophys Res Commun. 1995; 209: 656-63

"Sky" is a putative receptor tyrosine kinase predominantly expressed in the brain. Sky, like Axl/Ufo/Ark and c-Eyk, has an extracellular domain composed of two immunoglobulin-like domains and two fibronectin type III domains. Immunoblot analysis using an antibody raised against a C-terminal peptide of Sky identified a 98-kDa Sky protein in COS cells transfected with sky cDNA (COS/sky cells). A 98-kDa protein in the immunoprecipitates with anti-Sky antibody was autophosphorylated on tyrosine, by in vitro kinase reaction. When the lysates of COS/sky cells were immunoprecipitated with anti-Sky antibody and immunoblotted with an anti-phosphotyrosine antibody, a 60-kDa phosphotyrosine-containing protein, in addition to the tyrosine-phosphorylated Sky, was detected. Using the anti-Src antibody, which is reactive to Src, Fyn and Yes, we obtained evidence for an association between the Src family tyrosine kinase and the tyrosine-phosphorylated Sky receptor. These results suggest that the Src family kinase may play an important role in signal transduction of the Sky receptor.

Cloning, expression and genetic mapping of the mouse SH3 domain protein, SH3D2B.

Torrey T, Kim W, Morse HC 3rd, Kozak CA
Mamm Genome. 1998; 9: 74-5

Carboxymethyl-phenylalanine as a replacement for phosphotyrosine in SH2 domain binding.

Tong L, Warren TC, Lukas S, Schembri-King J, Betageri R, Proudfoot JR, Jakes S
J Biol Chem. 1998; 273: 20238-42

The crystal structure of human p56(lck) SH2 domain in complex with an inhibitor containing the singly charged p-(carboxymethyl)phenylalanine residue (cmF) as a phosphotyrosine (Tyr(P) or pY) replacement has been determined at 1.8 A resolution. The binding mode of the acetyl-cmF-Glu-Glu-Ile (cmFEEI) inhibitor is very similar to that of the pYEEI inhibitor, confirming that the cmFEEI inhibitor has a similar mechanism of SH2 domain inhibition despite its significantly reduced potency. Observed conformational differences in the side chain of the cmF residue can be interpreted in terms of maintaining similar interactions with the SH2 domain as the Tyr(P) residue. The crystal structure of the free p56(lck) SH2 domain has been determined at 1.9 A resolution and shows an open conformation for the BC loop and an open phosphotyrosine binding pocket, in contrast to earlier studies on the src SH2 domain that showed mostly closed conformation. The structural information presented here suggests that the carboxymethyl-phenylalanine residue may be a viable Tyr(P) replacement and represents an attractive starting point for the design and development of SH2 domain inhibitors with better pharmaceutical profiles.

Inhibition of human immunodeficiency virus type 1 virion entry by dominant-negative Hck.

Tokunaga K, Kiyokawa E, Nakaya M, Otsuka N, Kojima A, Kurata T, Matsuda M
J Virol. 1998; 72: 6257-9

To study the role of Src family tyrosine kinases in infection with human immunodeficiency virus type 1 (HIV-1), we constructed an Hck mutant, HckN, that hinders signaling from wild-type Hck. HIV-1 produced in HckN-expressing cells was significantly less infectious to HeLa-CD4-LTR-beta-gal (MAGI) cells than HIV-1 produced in mock-transfected cells. The inhibitory effect of HckN was compensated for by the expression of vesicular stomatitis virus G protein. Finally, we found that the HIV-1 produced in the HckN-expressing cells entered into the cells less efficiently than did the control HIV-1. These results suggest that the Src family tyrosine kinases regulate entry of HIV-1 into target cells.

The cellular kinase binding motifs (PxxP and RR) in human immunodeficiency virus type 1 Nef protein are dispensable for producer-cell-dependent enhancement of viral entry.

Tokunaga K, Ikuta K, Adachi A, Matsuda M, Kurata T, Kojima A
Virology. 1999; 257: 285-9

We have recently demonstrated that human immunodeficiency virus type 1 (HIV-1) Nef is required for enhancing viral infectivity by increasing the efficiency of viral entry in a producer-cell-dependent manner, suggesting the possible involvement of a cellular factor(s) in the enhancement of viral entry. Moreover, it has been reported that a proline-rich (PxxP) motif and an Arg-Arg (RR) motif in HIV-1 Nef bind to the SH3 domain of the Src-family tyrosine kinase Hck and to a serine/threonine kinase, respectively. To address whether these cellular kinase binding motifs, PxxP and RR, could be involved in virus producer-cell-dependent enhancement of viral entry, we constructed two nef mutant proviral clones in which these motifs were mutated. The results show that the HIV-1 Nef PxxP motif, which significantly influenced viral infectivity, and the RR motif, which modestly affected viral infectivity, were both dispensable for enhanced viral entry, thus suggesting that another interaction of Nef with a cellular factor(s) is involved in the efficiency of viral entry.

Multiple features of the p59fyn src homology 4 domain define a motif for immune-receptor tyrosine-based activation motif (ITAM) binding and for plasma membrane localization.

Timson Gauen LK, Linder ME, Shaw AS
J Cell Biol. 1996; 133: 1007-15

The src family tyrosine kinase p59fyn binds to a signaling motif contained in subunits of the TCR known as the immune-receptor tyrosine-based activation motif (ITAM). This is a specific property of p59fyn because two related src family kinases, p60src and p56lck, do not bind to ITAMs. In this study, we identify the residues of p59fyn that are required for binding to ITAMs. We previously demonstrated that the first 10 residues of p59fyn direct its association with the ITAM. Because this region of src family kinases also directs their fatty acylation and membrane association (Resh, M.D. 1993, Biochim. Biophys. Acta 1155:307-322; Resh, M.D. 1994. Cell. 76:411-413), we determined whether fatty acylation and membrane association of p59fyn correlates with its ability to bind ITAMs. Four residues (Gly2, Cys3, Lys7, and Lys9) were required for efficient binding of p59fyn to the TCR. Interestingly, the same four residues are present in p56lyn, the other src family tyrosine kinase known to bind to the ITAM, suggesting that this set of residues constitutes an ITAM recognition motif. These residues were also required for efficient fatty acylation (myristoylation at Gly2 and palmitoylation at Cys3), and plasma membrane targeting of p59fyn. Thus, the signals that direct p59fyn fatty acylation and plasma membrane targeting also direct its specific ability to bind to TCR proteins.

Engineering the substrate specificity of the Abl tyrosine kinase.

Till JH, Chan PM, Miller WT
J Biol Chem. 1999; 274: 4995-5003

c-Abl is a non-receptor tyrosine kinase that is involved in a variety of signaling pathways. Activated forms of c-Abl are associated with some forms of human leukemia. Presently, no high resolution structure of the tyrosine kinase domain of Abl is available. We have developed a structural homology model of the catalytic domain of Abl based on the crystal structure of the insulin receptor tyrosine kinase. Using this model as a guide, we selected residues near the active site predicted to play a role in peptide/protein substrate recognition. We expressed and purified 15 mutant forms of Abl with single amino acid substitutions at these positions and tested their peptide substrate specificity. We report here the identification of seven residues involved in recognition of the P-1, P+1, and P+3 positions of bound peptide substrate. Mutations in these residues cause distinct changes in substrate specificity. The results suggest features of Abl substrate recognition that may be relevant to related tyrosine kinases.

SH2- and SH3-mediated interactions between focal adhesion kinase and Src.

Thomas JW, Ellis B, Boerner RJ, Knight WB, White GC 2nd, Schaller MD
J Biol Chem. 1998; 273: 577-83

Intramolecular SH2 and SH3 interactions mediate enzymatic repression of the Src kinases. One mechanism of activation is disruption of these interactions by the formation of higher affinity SH2 and SH3 interactions with specific ligands. We show that a consensus Src SH3-binding site residing upstream of the Src SH2-binding site in FAK can function as a ligand for the Src SH3 domain. Surface plasmon resonance experiments indicate that a FAK peptide containing both the Src SH2- and SH3-binding sites exhibits increased affinity for Src. Furthermore, the presence of both sites in vitro more potently activates c-Src. A FAK mutant (FAKPro-2) with substitutions destroying the SH3-binding site shows reduced binding to Src in vivo. This mutation also reduces Src-dependent tyrosine phosphorylation on the mutant itself and downstream substrates, such as paxillin. These observations suggest that an SH3-mediated interaction between Src-like kinases and FAK may be important for complex formation and downstream signaling in vivo.

Heteronuclear NMR studies of the combined Src homology domains 2 and 3 of pp60 c-Src: effects of phosphopeptide binding.

Tessari M, Gentile LN, Taylor SJ, Shalloway DI, Nicholson LK, Vuister GW
Biochemistry. 1997; 36: 14561-71

The results of heteronuclear NMR studies on the combined Src homology domains 2 and 3 (SH3-SH2) of pp60 c-Src are presented. Resonance assignments were obtained using heteronuclear triple-resonance experiments in conjunction with 15N-separated nuclear Overhauser effect spectroscopy (NOESY) data. A modified three-dimensional 13CO-15N-1H spectral correlation experiment [(HACA)CO(CA)-NH] with improved sensitivity is presented that provided additional sequential information and resolved several ambiguities. Chemical shifts and sequential- and medium-range NOE cross peaks indicate that the structures of both the SH3 and SH2 portions of the polypeptide are very similar to those of the isolated SH3 and SH2 domains. Binding of a high-affinity phosphopeptide, EPQpYEEIPIYL, induces large chemical shift changes at several locations in the SH2 domain. Comparison with known results for peptide binding to SH2 domains shows that the residues displaying the largest effects are all involved in peptide binding or undergo significant conformational changes upon binding. However, subtle changes of both 1H and 15N chemical shifts are observed for residues within the SH3 domain and the connecting linker region, indicating possible cross-domain communication.

Structure of the N-terminal SH3 domain of GRB2 complexed with a peptide from the guanine nucleotide releasing factor Sos.

Terasawa H, Kohda D, Hatanaka H, Tsuchiya S, Ogura K, Nagata K, Ishii S, Mandiyan V, Ullrich A, Schlessinger J, et al.
Nat Struct Biol. 1994; 1: 891-7

Src-homology 3 (SH3) domains mediate signal transduction by binding to proline-rich motifs in target proteins. We have determined the high-resolution NMR structure of the complex between the amino-terminal SH3 domain of GRB2 and a ten amino acid peptide derived from the guanine nucleotide releasing factor Sos. The NMR data show that the peptide adopts the conformation of a left-handed polyproline type II helix and interacts with three major sites on the SH3 domain. The orientation of the bound peptide is opposite to that of proline-rich peptides bound to the SH3 domains of Abl, Fyn and p85.

The cell cycle and c-Src.

Taylor SJ, Shalloway D
Curr Opin Genet Dev. 1993; 3: 26-34

The activity of the proto-oncogene encoded c-Src product is tightly regulated in vivo. In recent years, a model has emerged of how this regulation is achieved. In particular, protein kinases and phosphatases that are potential regulators of c-Src activity in the cell cycle have been identified and characterized.

Src and the control of cell division.

Taylor SJ, Shalloway D
Bioessays. 1996; 18: 9-11

The finely tuned mechanisms that control cell cycle progression go awry in cancer, pointing to proto-oncogene products as important players in cell-cycle regulation. One such proto-oncoprotein, c-Src, has previously been directly implicated, based on its requirement for growth factor-stimulated DNA synthesis. Roche et al. have now shown that c-Src or its close relatives are also required for cell division to occur. The demonstration of essential functions for the Src family at multiple points in the cell cycle raises important questions about the normal and transforming activities of these and other proto-oncoproteins.

An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis.

Taylor SJ, Shalloway D
Nature. 1994; 368: 867-71

The tyrosine kinase activity of c-Src is stimulated during mitosis by dephosphorylation of its regulatory tyrosine residue. This is associated with increased accessibility of its Src homology-2 (SH2) domain for binding a phosphotyrosine-containing peptide. But physiological targets of activated c-Src in mitosis have not yet been identified. Here we report that a 68K protein (p68) becomes tyrosine-phosphorylated and physically associates with Src during mitosis in mouse fibroblasts. p68 independently binds the Src SH2 and SH3 domains in vitro and both domains are required for p68 phosphorylation and binding in vivo. p68 is closely related to the p62 protein that is associated with the Ras GTPase-activating protein (GAP) and selectively binds, directly or indirectly, polyribonucleotides. Because the Src SH3 domain also binds heterogeneous nuclear ribonucleoprotein K, these results raise the intriguing possibility that c-Src may regulate the processing, trafficking or translation of RNA in a cell-cycle-dependent manner.

Functional interaction between c-Src and its mitotic target, Sam 68.

Taylor SJ, Anafi M, Pawson T, Shalloway D
J Biol Chem. 1995; 270: 10120-4

The c-Src tyrosine kinase phosphorylates and binds to a 68-kDa RNA-binding protein in mitotic cells. We have examined the mechanism and functional consequence of the interaction of c-Src with this protein, Sam 68 (Src associated in mitosis, 68 kDa). In whole cell homogenates, Sam 68 was the predominant substrate and binding partner of overexpressed c-Src. Mitotic, tyrosine-phosphorylated Sam 68 bound selectively to recombinant SH2 domains with significantly different affinities (c-Src approximately Ras GTPase activating protein > p85 alpha (amino-terminal) > Grb2 >> p85 alpha (COOH-terminal)). In vitro translated Sam 68 also bound selectively to recombinant SH3 domains, with the highest affinity for the Src and p85 alpha SH3 domains. SH3 binding was inhibited by specific Sam 68 peptides. In vitro translated Sam 68 bound directly to immobilized poly(U), and this was inhibited by binding of Src and p85 SH3 domains to Sam 68. The results suggest that the selection of Sam 68 as a mitotic target by c-Src is the result of highly specific interaction with SH2 and SH3 domains and that this interaction may modulate the RNA binding activity of Sam 68.

C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of CRK and GRB2/ASH proteins.

Tanaka S, Morishita T, Hashimoto Y, Hattori S, Nakamura S, Shibuya M, Matuoka K, Takenawa T, Kurata T, Nagashima K, et al.
Proc Natl Acad Sci U S A. 1994; 91: 3443-7

CRK protein, together with GRB2/ASH and Nck proteins, belongs to the adaptor-type Src homology (SH)2-containing molecules, which transduce signals from tyrosine kinases. Here another guanine nucleotide-releasing protein (GNRP), C3G, has been identified as a CRK SH3-binding protein. The nucleotide sequence of a 4.1-kb C3G cDNA contains a 3.2-kb open reading frame encoding a 121-kDa protein, and antibodies against C3G have been shown to detect a protein of 130-140 kDa. The carboxyl terminus of C3G has a peptide sequence homologous to GNRPs for Ras, and the expression of this carboxyl terminus region suppresses the loss of CDC25 function in the yeast Saccharomyces cerevisiae. The C3G protein expressed in Escherichia coli binds to CRK and GRB2/ASH proteins. Mutational analysis of C3G assigns the SH3 binding region to a 50-amino acid region containing a proline-rich sequence. The mRNAs of both the C3G and CRK proteins are expressed ubiquitously in human adult and fetal tissues. The results of these studies suggest that the complex of CRK and C3G, or GRB2/ASH and C3G, may transduce the signals from tyrosine kinases to Ras in a number of different tissues.

Structure of 85 kDa subunit of human phosphatidylinositol 3-kinase analyzed by using monoclonal antibodies.

Tanaka S, Matsuda M, Nagata S, Kurata T, Nagashima K, Shizawa Y, Fukui Y
Jpn J Cancer Res. 1993; 84: 279-89

An 85 kDa subunit (p85 alpha) of phosphatidylinositol 3-kinase (PI-3K) has one SH3 and two SH2 regions [SH2(N) and SH2(C)], which direct protein-protein interaction. We have established eighteen hybridomas producing monoclonal antibodies against p85 alpha to study the structure-function relationship of this protein. Epitope mapping using a series of deletion mutants expressed in E. coli showed that the monoclonal antibodies bound to at least 5 distinct epitope regions, which were well dispersed on p85 alpha except for its carboxyl-terminus. Monoclonal antibodies against amino-terminal regions and polyclonal antibodies against carboxyl-terminal regions immunoprecipitated p85 alpha expressed in human cells and in E. coli. On the other hand, monoclonal antibodies against the central part of p85 alpha failed to immunoprecipitate p85 alpha efficiently; however, they could immunoprecipitate p85 alpha mutants with deletion of either the amino- or the carboxyl-terminal region. Similar results were obtained by immunocytochemistry using confocal microscopy. These results suggested that steric hindrance prevents binding of monoclonal antibodies to the central part of p85 alpha where SH2(N) is located. The SH2(N) may have a distinct function from SH2(C), which is located at the carboxyl-terminal region and has been shown to mediate the binding of PI-3K to activated growth factor receptors.

DNA sequence encoding the amino-terminal region of the human c-src protein: implications of sequence divergence among src-type kinase oncogenes.

Tanaka A, Gibbs CP, Arthur RR, Anderson SK, Kung HJ, Fujita DJ
Mol Cell Biol. 1987; 7: 1978-83

We sequenced the 5'-coding region of the human c-src gene, exons 2 through 5, corresponding to one-third of the human c-src protein consisting of 536 amino acids. Sequence analysis of the src type of protein kinases revealed that the amino-terminal region encoded by exon 2 contains sequences specific for the src proteins and raised the possibility that this region is involved in the recognition of a src-specific substrate(s) or receptor(s).

Signal transductions of SH2/SH3: Ash/Grb-2 downstream signaling.

Takenawa T, Miura K, Miki H, Watanabe K
Adv Pharmacol. 1996; 36: 139-53

Distinct binding patterns of HS1 to the Src SH2 and SH3 domains reflect possible mechanisms of recruitment and activation of downstream molecules.

Takemoto Y, Sato M, Furuta M, Hashimoto Y
Int Immunol. 1996; 8: 1699-705

We previously identified a gene, LckBP1, which encodes a protein that binds to the Lck SH3 domain and is identical to murine SH1. Using unstimulated T lymphocytes, we further demonstrated that Lck binds to HS1 in vivo and that HS1 is tyrosine phosphorylated upon TCR stimulation. In the present report, we analyzed the binding pattern of several src kinases and HS1 in greater detail. The Lck SH3 domain binds to HS1 constitutively, while the Lck SH2 domain associates with HS1 only upon TCR stimulation. A similar binding pattern was observed with Lyn and HS1, but not with Fyn and HS1, in which the Fyn SH2 region associates with HS1 upon TCR stimulation but the Fyn SH3 region does not associate with HS1 regardless of TCR stimulation. Such distinct binding patterns of the src kinase SH2 and SH3 domains to HS1 may represent a mechanism by which src family kinases select substrates and activate particular downstream signaling pathways.

The effect of transient global ischemia on the interaction of Src and Fyn with the N-methyl-D-aspartate receptor and postsynaptic densities: possible involvement of Src homology 2 domains.

Takagi N, Cheung HH, Bissoon N, Teves L, Wallace MC, Gurd JW
J Cereb Blood Flow Metab. 1999; 19: 880-8

Transient ischemia increases tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) receptor subunits NR2A and NR2B in the rat hippocampus. The authors investigated the effects of this increase on the ability of the receptor subunits to bind to the Src homology 2 (SH2) domains of Src and Fyn expressed as glutathione-S-transferase-SH2 fusion proteins. The NR2A and NR2B bound to each of the SH2 domains and binding was increased approximately twofold after ischemia and reperfusion. Binding was prevented by prior incubation of hippocampal homogenates with a protein tyrosine phosphatase or by a competing peptide for the Src SH2 domain. Ischemia induced a marked increase in the tyrosine phosphorylation of several proteins in the postsynaptic density (PSD), including NR2A and NR2B, but had no effect on the amounts of individual NMDA receptor subunits in the PSD. The level of Src and Fyn in PSDs, but not in other subcellular fractions, was increased after ischemia. The ischemia-induced increase in the interaction of NR2A and NR2B with the SH2 domains of Src and Fyn suggests a possible mechanism for the recruitment of signaling proteins to the PSD and may contribute to altered signal transduction in the postischemic hippocampus.

Differences in binding of PI 3-kinase to the src-homology domains 2 and 3 of p56 lck and p59 fyn tyrosine kinases.

Susa M, Rohner D, Bichsel S
Biochem Biophys Res Commun. 1996; 220: 729-34

Two T cell-specific src-family tyrosine kinases, p56 lck (lck) and p59 fyn (fyn), are implicated in regulating PI 3-kinase activity in response to interleukin-2 (IL-2), a cytokine that induces T cell proliferation. The src- homology domains 3 (SH3) of src-family kinases can directly interact with the PI 3-kinase regulatory subunit p85 and this may be a mechanism to regulate PI 3-kinase activity. In order to understand the mode of PI 3-kinase activation by the IL-2 receptor, we examined the association of PI 3-kinase to SH2 and SH3 domains of lck and fyn in IL-2-dependent kit 225 cells. The fyn SH3 domain bound more PI 3-kinase and its p85 subunit than the lck SH3 domain, while the lck SH2 domain bound more PI 3-kinase than the fyn SH2 domain. None of these interactions were regulated by IL-2. Low binding of PI 3-kinase to the lck SH3 domain was not observed in IL-2-independent Jurkat T cells. Thus, SH3 and SH2 domains of lck and fyn bound different amounts of PI 3-kinase, a feature that was dependent on a T cell type, but was not influenced by IL-2.

Picture story. An SH2 domain in disguise.

Surridge C
Nat Struct Biol. 1999; 6: 211-211

Csk inhibition of c-Src activity requires both the SH2 and SH3 domains of Src.

Superti-Furga G, Fumagalli S, Koegl M, Courtneidge SA, Draetta G
EMBO J. 1993; 12: 2625-34

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its carboxy-terminal tail. A kinase that phosphorylates Tyr527, called Csk, has recently been identified. We expressed c-Src in yeast to test the role of the SH2 and SH3 domains of Src in the negative regulation exerted by Tyr527 phosphorylation. Inducible expression of c-Src in Schizosaccharomyces pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in either the SH2 or SH3 domain were as lethal as wild type c-Src, but were insensitive to Csk, even though they were substrates for Csk in vivo. Peptide binding experiments revealed that Src proteins with mutant SH3 domains adopted a conformation in which the SH2 domain was not interacting with the tail. These data support the model of an SH2 domain-phosphorylated tail interaction repressing c-Src activity, but expand it to include a role for the SH3 domain. We propose that the SH3 domain contributes to the maintenance of the folded, inactive configuration of the Src molecule by stabilizing the SH2 domain-phosphorylated tail interaction. Moreover, the system we describe here allows for further study of the regulation of tyrosine kinases in a neutral background and in an organism amenable to genetic analysis.

Structure-function relationships in Src family and related protein tyrosine kinases.

Superti-Furga G, Courtneidge SA
Bioessays. 1995; 17: 321-30

There is increasing evidence to suggest that cytoplasmic tyrosine kinases of the Src family have a pivotal role in the regulation of a number of cellular processes. Members of this family have been implicated in cellular responses to a variety of extracellular signals, such as those arising from growth factors and cell-cell interactions, as well as in differentiative and developmental processes in both vertebrates and invertebrates. A better understanding of the regulation and of the structure-function relationships of these enzymes might aid in the development of specific ways to interfere with their action, as well as serving as a paradigm for regulation of other protein tyrosine kinases that have SH2 and SH3 domains. In this review we will first discuss the regulation of Src family protein tyrosine kinases, with particular emphasis on their SH2 and SH3 domains. We will then briefly review other non-receptor protein tyrosine kinases that have SH2 and SH3 domains.

Regulation of the Src protein tyrosine kinase.

Superti-Furga G
FEBS Lett. 1995; 369: 62-6

Members of the Src family of protein tyrosine kinases are involved in a variety of cellular processes, including cell growth, cell differentiation and neuronal signalling. N-terminal to the catalytic domain, Src family members contain a Src homology 2 (SH2) domain, a Src homology 3 (SH3) domain, and a unique domain, all capable of protein-protein interactions. Negative regulation by phosphorylation of a conserved tyrosine residue at the C-terminal tail of the molecules is characteristic of this family of enzymes. Phosphorylation of this residue causes the intramolecular interactions of the SH2 domain with the tail, and of the SH3 domain with an as yet undefined region, probably within the catalytic domain. Enzymatically active Src family kinases, on the other hand, are phosphorylated at a tyrosine in the middle of the catalytic domain and phosphorylation of this residue is a prerequisite for high activity. Regulators of these enzymes may thus act by altering the phosphorylation state of the two key tyrosine residues or by interfering with the regulatory intramolecular interactions, either by direct binding or by modification of the interfaces involved.

c-Abl tyrosine kinase binds and phosphorylates phospholipid scramblase 1.

Sun J, Zhao J, Schwartz MA, Wang JY, Wiedmer T, Sims PJ
J Biol Chem. 2001; 276: 28984-90

Phospholipid scramblase 1 (PLSCR1) is a plasma membrane protein that has been proposed to play a role in the transbilayer movement of plasma membrane phospholipids. PLSCR1 contains multiple proline-rich motifs resembling Src homology 3 (SH3) domain-binding sites. An initial screen against 13 different SH3 domains revealed a marked specificity of PLSCR1 for binding to the Abl SH3 domain. Binding between intracellular PLSCR1 and c-Abl was demonstrated by co-immunoprecipitation of both proteins from several cell lines. Deletion of the proline-rich segment in PLSCR1 (residues 1--118) abolished its binding to the Abl SH3 domain. PLSCR1 was Tyr-phosphorylated by c-Abl in vitro. Phosphorylation was abolished by mutation of Tyr residues Tyr(69)/Tyr(74) within the tandem repeat sequence (68)VYNQPVYNQP(77) of PLSCR1, implying that these residues are the likely sites of phosphorylation. Cellular PLSCR1 was found to be constitutively Tyr-phosphorylated in several cell lines. The Tyr phosphorylation of PLSCR1 was increased upon overexpression of c-Abl and significantly reduced either upon cell treatment with the Abl kinase inhibitor STI571, or in Abl-/- mouse fibroblasts, suggesting that cellular PLSCR1 is a normal substrate of c-Abl. Cell treatment with the DNA-damaging agent cisplatin activated c-Abl kinase and increased Tyr phosphorylation of PLSCR1. The cisplatin-induced phosphorylation of PLSCR1 was inhibited by STI571 and was not observed in Abl-/- fibroblasts. These findings indicate that c-Abl binds and phosphorylates PLSCR1, and raise the possibility that an interaction between c-Abl and plasma membrane PLSCR1 might contribute to the cellular response to genotoxic stress.

Affinity purification of Csk protein tyrosine kinase based on its catalytic requirement for divalent metal cations.

Sun G, Budde RJ
Protein Expr Purif. 2001; 21: 8-12

Protein tyrosine kinase Csk requires two Mg2+ ions for activity: one magnesium is part of the ATP-Mg complex, and the second free Mg2+ ion is required as an essential activator. Zn2+ can bind to this site to replace Mg2+, which inhibits Csk kinase activity. The binding is reversible and removal of Zn2+ results in an active Csk apoenzyme. In this communication, we report that this tight binding can be used as a mechanism for affinity purification of Csk. When bacterial cell lysate containing overexpressed GST-Csk was applied to a column of Zn2+-iminodiacetic acid immobilized to agarose, Csk was specifically retained by the column. Since the binding of Csk to Zn2+ is not affected by up to 200 mM NaCl, high ionic strength conditions were used in the purification procedure, minimizing nonspecific binding due to ionic interactions. Washing the column with 200 mM NaCl and 50 mM imidazole removed virtually all other proteins from the column while Csk remained bound. The retained Csk enzyme was eluted with 1 M imidazole. The 1 M imidazole-eluted fraction contained pure Csk that had a specific activity similar to the enzyme purified by a glutathione-agarose affinity column. Copyright 2001 Academic Press.

The SH3 and SH2 domains are capable of directing specificity in protein interactions between the non-receptor tyrosine kinases cSrc and cYes.

Summy JM, Guappone AC, Sudol M, Flynn DC
Oncogene. 2000; 19: 155-60

The c-src and c-yes proto-oncogenes encode 60 000 and 62 000 Dalton non-receptor tyrosine kinases of the Src family, pp60c-src and pp62c-yes, respectively. These kinases are over 80% homologous outside of their unique amino termini, yet several studies suggest that differences exist in the regulation, activation, and function of cSrc and cYes. The determinants of specificity in signaling between these proteins, however, remain unclear. In order to investigate the roles of the Src Homology (SH) 3 and 2 domains in mediating signaling specificity between cSrc and cYes, chimeras were created in which the SH3 and/or SH2 domains of cSrc or the fully activated variant Src527F were replaced by the corresponding domains of cYes. These constructs were used to assess the effects of the Yes SH3 and SH2 domains on the ability of Src to form stable complexes with and induce tyrosine phosphorylation of Src SH3 and SH2 domain binding partners in vivo. Both the Yes SH3 and SH2 domains were found to alter the capacity of Src to form stable associations with heterologous proteins. The Yes SH3 domain was unable to affinity absorb the Src SH3/SH2 binding partner AFAP-110 from COS-1 cell lysates, and chimeric constructs of Src527F containing the cYes SH3 domain were unable to efficiently co-immunoprecipitate with AFAP-110 from chicken embryo fibroblasts. Interactions with the Src SH2 domain binding partner pp130cas were unaffected. Additionally, only chimeras containing the cYes SH2 domain were able to co-immunoprecipitate with an unidentified 87 kDa tyrosine-phosphorylated protein. These results indicate that the SH3 and SH2 domains are capable of directing specificity in substrate binding between Src and Yes, suggesting potential mechanisms for generating specificity in signaling between these two highly related non-receptor tyrosine kinases.

Structure and function of the WW domain.

Sudol M
Prog Biophys Mol Biol. 1996; 65: 113-32

Additional member of the protein-tyrosine kinase family: the src- and lck-related protooncogene c-tkl.

Strebhardt K, Mullins JI, Bruck C, Rubsamen-Waigmann H
Proc Natl Acad Sci U S A. 1987; 84: 8778-82

We report the isolation and nucleotide sequence of a 3.7-kilobase (kb) cDNA clone from chicken spleen corresponding to a previously undescribed member of the src family of protooncogenes. It encodes a protein with a C-terminal domain related to the src family of protein-tyrosine kinases (EC 2.7.1.112) and, among these, has most significant homology to the lck gene isolated from a murine leukemia virus-induced thymoma cell line. The gene is therefore referred to as c-tkl for cellular tyrosine kinase related to lck. Analysis of genomic DNA reveals that c-tkl is a chromosomal locus distinct from c-src and c-lck. Furthermore, the size of c-tkl mRNA as well as its pattern of expression indicates that it is not the chicken homologue of lck but a different gene. A 3.8-kb transcript of the c-tkl gene, identical to the size determined for c-src mRNA, was observed in cultured chicken embryo fibroblasts and in chicken spleen and brain. In contrast, detection of a definite c-src mRNA signal with mRNA from spleen was not possible under the hybridization conditions employed when the 5' end of v-src was used as the probe, and none of the 11 clones obtained from the cDNA library corresponded to a c-src transcript. Thus previous studies of c-src mRNA expression in spleen may have actually detected c-tkl transcripts.

Inhibition of Lyn function in mast cell activation by SH3 domain binding peptides.

Stauffer TP, Martenson CH, Rider JE, Kay BK, Meyer T
Biochemistry. 1997; 36: 9388-94

While Lyn tyrosine kinase has been shown to be necessary for IgE-receptor (FcepsilonRI)-mediated mast cell activation, the mechanism of Lyn activation is not yet understood. Using a micro-electroporation technique to quantitatively introduce peptides into the cytosol of tumor mast cells, we show that proline-rich peptides that preferentially bind Src family SH3 domains block receptor-induced repetitive calcium spikes in a concentration dependent manner. The Src family member Lyn was the likely target, since a series of phage displaying derived peptides with increased Lyn SH3 domain binding specificity inhibited FcepsilonRI-mediated calcium signaling at concentrations consistent with binding to Lyn rather than other Src-type kinases. Furthermore, SH3 binding peptides prevented the plasma membrane translocation of a fluorescently labeled Syk tandem SH2 domain, which binds to phosphorylated FcepsilonRI, suggesting that the peptides specifically block the Lyn-mediated step by which FcepsilonRI cross-linking leads to receptor phosphorylation. Our study suggests that the binding of proline-rich peptides, or corresponding cellular interaction partners, to Lyn SH3 domain suppresses the Lyn-mediated phosphorylatation of FcepsilonRI and calcium signaling.

Distinct ligand preferences of Src homology 3 domains from Src, Yes, Abl, Cortactin, p53bp2, PLCgamma, Crk, and Grb2.

Sparks AB, Rider JE, Hoffman NG, Fowlkes DM, Quillam LA, Kay BK
Proc Natl Acad Sci U S A. 1996; 93: 1540-4

Src homology 3 (SH3) domains are conserved protein modules 50-70 amino acids long found in a variety of proteins with important roles in signal transduction. These domains have been shown to mediate protein-protein interactions by binding short proline-rich regions in ligand proteins. However, the ligand preferences of most SH3 domains and the role of these preferences in regulating SH3-mediated protein-protein interactions remain poorly defined. We have used a phage-displayed library of peptides of the form X6PXXPX6 to identify ligands for eight different SH3 domains. Using this approach, we have determined that each SH3 domain prefers peptide ligands with distinct sequence characteristics. Specifically, we have found that the Src SH3 domain selects peptides sharing the consensus motif LXXRPLPXpsiP, whereas Yes SH3 selects psiXXRPLPXLP, Abl SH3 selects PPXthetaXPPPpsiP, Cortactin SH3 selects +PPpsiPXKPXWL, p53bp2 SH3 selects RPXpsiPpsiR+SXP, PLCgamma SH3 selects PPVPPRPXXTL, Crk N-terminal SH3 selects psiPpsiLPpsiK, and Grb2 N-terminal SH3 selects +thetaDXPLPXLP (where psi, theta, and + represent aliphatic, aromatic, and basic residues, respectively). Furthermore, we have compared the binding of phage expressing peptides related to each consensus motif to a panel of 12 SH3 domains. Results from these experiments support the ligand preferences identified in the peptide library screen and evince the ability of SH3 domains to discern subtle differences in the primary structure of potential ligands. Finally, we have found that most known SH3-binding proteins contain proline-rich regions conforming to the ligand preferences of their respective SH3 targets.

Identification and characterization of Src SH3 ligands from phage-displayed random peptide libraries.

Sparks AB, Quilliam LA, Thorn JM, Der CJ, Kay BK
J Biol Chem. 1994; 269: 23853-6

We have used the Src homology 3 (SH3) domain to screen two phage-displayed random peptide libraries, each containing 2 x 10(8) unique members, and have identified a series of high affinity peptide ligands. The peptides possess similar proline-rich regions, which yield a consensus Src SH3-binding motif of RPLPPLP. We have confirmed this motif by screening a phage-displayed peptide library biased for SH3 ligands and identifying the same consensus sequence. Binding studies using synthetic peptides suggest that the RPLPPLP motif is important for SH3 binding and confers specificity for the Src SH3 domain, and that residues which flank the motif may also contribute to binding. Peptides that contain the RPLPPLP motif compete Src, but not Abl or phospholipase C gamma, SH3 interactions with SH3-binding proteins from cell lysates (IC50 = 1-5 microM). Furthermore, RPLPPLP-related peptides are able to accelerate progesterone-induced maturation of Xenopus laevis oocytes. A similar acceleration has been observed in oocytes treated with activated, but not normal, Xenopus Src, suggesting the possibility that the peptides are able to antagonize the negative regulation of Src activity by Src SH3 in vivo.

Screening phage-displayed random peptide libraries for SH3 ligands.

Sparks AB, Adey NB, Quilliam LA, Thorn JM, Kay BK
Methods Enzymol. 1995; 255: 498-509

Autophosphorylation induces autoactivation and a decrease in the Src homology 2 domain accessibility of the Lyn protein kinase.

Sotirellis N, Johnson TM, Hibbs ML, Stanley IJ, Stanley E, Dunn AR, Cheng HC
J Biol Chem. 1995; 270: 29773-80

Lyn is a member of the Src family of protein-tyrosine kinases that can readily undergo autophosphorylation in vitro. The site of autophosphorylation is Tyr397 which corresponds to the consensus autophosphorylation site of other Src family tyrosine kinases. The rate of autophosphorylation is concentration-dependent, indicating that the reaction follows an intermolecular mechanism. Autophosphorylation results in a 17-fold increase in protein-tyrosine kinase activity. Kinetic analysis demonstrates that phosphorylation of a substrate peptide by Lyn following autophosphorylation occurs with a 63-fold decrease in Km but no significant change in Vmax, suggesting that autophosphorylation relieves the conformational constraint that prevents binding of the substrate peptide to the active site of the kinase. Using a phosphotyrosine-containing peptide (pYEEI) that has previously been shown to bind to the Src homology 2 (SH2) domain of Src family tyrosine kinases with high affinity, we found that autophosphorylation results in a significant decrease in accessibility of the Lyn SH2 domain, indicating that conformational changes in the protein kinase domain induced by autophosphorylation can be propagated to the SH2 domain. Our study suggests that autophosphorylation plays an important role in regulating Lyn by modulating both its kinase activity and its interaction with other phosphotyrosine-containing molecules.

SH2 domains recognize specific phosphopeptide sequences.

Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, et al.
Cell. 1993; 72: 767-78

A phosphopeptide library was used to determine the sequence specificity of the peptide-binding sites of SH2 domains. One group of SH2 domains (Src, Fyn, Lck, Fgr, Abl, Crk, and Nck) preferred sequences with the general motif pTyr-hydrophilic-hydrophilic-Ile/Pro while another group (SH2 domains of p85, phospholipase C-gamma, and SHPTP2) selected the general motif pTyr-hydrophobic-X-hydrophobic. Individual members of these groups selected unique sequences, except the Src subfamily (Src, Fyn, Lck, and Fgr), which all selected the sequence pTyr-Glu-Glu-Ile. The variability in SH2 domain sequences at likely sites of contact provides a structural basis for the phosphopeptide selectivity of these families. Possible in vivo binding sites of the SH2 domains are discussed.

A single point mutation switches the specificity of group III Src homology (SH) 2 domains to that of group I SH2 domains.

Songyang Z, Gish G, Mbamalu G, Pawson T, Cantley LC
J Biol Chem. 1995; 270: 26029-32

Src homology 2 (SH2) domains recognize phosphotyrosine-containing sequences, and thereby mediate the association of specific signaling proteins in response to tyrosine phosphorylation (Pawson, T., and Schlessinger, J. (1993) Curr. Biol. 3, 434-442). We have shown that specific binding of SH2 domains to tyrosine-phosphorylated sites is determined by sequences adjacent to the phosphotyrosine. Based on the phosphopeptide specificity and crystal structures, SH2 domains were classified into four different groups (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., R. B. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778). The beta D5 residues of SH2 domains were predicted to be critical in distinguishing these groups (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., R. B. B., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778; Eck, M. J., Shoelson, S. E., and Harrison, S. C. (1993) Nature 362, 87-91). We report here that replacing the aliphatic residues at the beta D5 positions of two Group III SH2 domains (phosphoinositide 3-kinase N-terminal SH2 domain and phospholipase C-gamma C-terminal SH2 domain) with Tyr (as found in Group I SH2 domains) results in a switch in phosphopeptide selectivity, consistent with the specificities of Group I SH2 domains. These results establish the importance of the beta D5 residue in determining specificities of SH2 domains.

Domain interactions in protein tyrosine kinase Csk.

Sondhi D, Cole PA
Biochemistry. 1999; 38: 11147-55

Csk (C-terminal Src kinase) is a protein tyrosine kinase that phosphorylates Src family member C-terminal tails, resulting in downregulation of Src family members. It is composed of three principal domains: an SH3 (Src homology 3) domain, an SH2 (Src homology 2) domain, and a catalytic domain. The impact of the noncatalytic domains on kinase catalysis was investigated. The Csk catalytic domain was expressed in Escherichia coli as a recombinant glutathione S-transferase-fusion protein and demonstrated to have 100-fold reduced catalytic efficiency. Production of the catalytic domain by proteolysis of full-length Csk afforded a similar rate reduction. This suggested that the reduction in catalytic efficiency of the recombinant catalytic domain was intrinsic to the sequence and not an artifact related to faulty expression. This rate reduction was similar for peptide and protein substrates and was due almost entirely to a reduced k(cat) rather than to effects on substrate K(m)s. Viscosity experiments on the catalytic fragment kinase reaction demonstrated that the chemical (phosphoryl transfer) step had a reduced rate. While the Csk SH2 domain had no intermolecular effect on the kinase activity of the Csk catalytic domain, the SH3 domain and SH3-SH2 fragment led to a partial rescue (4-5-fold) of the lost kinase activity. This rescue was not achieved with two other SH3 domains (lymphoid cell kinase, Abelson kinase). The extrapolated K(d) of interaction for the Csk catalytic domain with the Csk SH3 domain was 2.2 microM and that of the Csk catalytic domain with the Csk SH3-SH2 fragment was 8.8 microM. Taken together, these findings suggest that there is likely an intramolecular interaction between the catalytic and SH3 domains in full-length Csk that is important for efficient catalysis. By employing a Csk SH3 specific type II polyproline helix peptide and carrying out site-directed mutagenesis, it was established that the SH3 surface that interacts with the catalytic domain was distinct from the surface that binds type II polyproline helix peptides. This finding suggests a novel mode of protein-protein interaction for an SH3 domain. The implications for Csk substrate selectivity, regulation, and function are discussed.

EEN encodes for a member of a new family of proteins containing an Src homology 3 domain and is the third gene located on chromosome 19p13 that fuses to MLL in human leukemia.

So CW, Caldas C, Liu MM, Chen SJ, Huang QH, Gu LJ, Sham MH, Wiedemann LM, Chan LC
Proc Natl Acad Sci U S A. 1997; 94: 2563-8

The MLL gene, the closest human homologue to the Drosophila trithorax gene, undergoes chromosomal translocation with a large number of different partner genes in both acute lymphoid and acute myeloid leukemias. We have identified a new partner gene, EEN, fused to MLL in a case of acute myeloid leukemia. The gene is located on chromosome 19p13, where two other MLL partner genes, ENL and ELL/MEN have also been identified. The deduced protein of 368 aa contains a central alpha-helical region and a C-terminal Src homology 3 (SH3) domain most similar to the C-terminal SH3 domain found in the Grb2/Sem-5/Drk family of genes. Sequence analysis of the fusion MLL/EEN transcript in our patient reveals that exon 6 of MLL is fused to the N-terminal end of EEN, a fusion that would create a chimeric protein that includes the major functional domain of EEN. EEN is expressed in a variety of tissue types and encodes a protein of approximately 46 kDa. The EEN protein is the human homologue of a member of a recently described murine SH3 domain-containing protein family. It is also highly related to a putative gene identified in Caenorhabditis elegans, and a number of similar sequences are present in the EST databases of several species.

Activation of the Abl tyrosine kinase in vivo by Src homology 3 domains from the Src homology 2/Src homology 3 adaptor Nck.

Smith JM, Katz S, Mayer BJ
J Biol Chem. 1999; 274: 27956-62

The nonreceptor tyrosine kinase c-Abl is tightly regulated in vivo, but the mechanisms that normally repress its activity are not well understood. We find that a construct encoding the first two Src homology 3 (SH3) domains of the Src homology 2/SH3 adaptor protein Nck can activate c-Abl in human 293T cells. A myristoylated Nck SH3 domain construct, which is expected to localize to membranes, potently activated Abl when expressed at low levels. An unmyristoylated Nck SH3 domain construct, which localizes to the cytosol and nucleus, also activated Abl but only at high levels of expression. Activation by both myristoylated and unmyristoylated Nck constructs required the C terminus of Abl; a C-terminally truncated form of Abl was not activated, although this construct could still be activated by deletion of its SH3 domain. Activation did not require the major binding sites in the Abl C terminus for Nck SH3 domains, however, suggesting that the mechanism of activation does not require direct binding to the C terminus. Activation of c-Abl by Nck SH3 domains provides a robust experimental system for analyzing the mechanisms that normally repress Abl activity and how that normal regulation can be perturbed.

Grb2 SH3 binding to peptides from Sos: evaluation of a general model for SH3-ligand interactions.

Simon JA, Schreiber SL
Chem Biol. 1995; 2: 53-60

BACKGROUND: Grb2 acts as an adaptor protein in the transduction of signals from receptor tyrosine kinases to Ras. It binds to phosphotyrosine on the cytoplasmic tail of cell-surface receptors via its central SH2 domain, and to its immediate downstream target, Sos, via two SH3 domains. The basis of the Grb2-Sos interaction is not fully understood. We previously proposed a model for SH3 domain binding specificity, based on two solution structures of the Src SH3 domain complexed with high-affinity ligands, in which the ligands are bound in a polyproline type II conformation in two distinct orientations, class I and class II. Here, we have used this model to predict the identity and orientation of Grb2 SH3 ligands in the human Sos protein. RESULTS: Six contiguous fragments from the carboxy-terminal portion of hSos (amino acids 1000-1333), each containing a single potential SH3 binding site, were expressed in E. coli as GST fusion proteins. Four of these proteins were predicted to associate with SH3 domains. The amino-terminal Grb2 SH3 domain was shown to bind strongly to only these four fragments. CONCLUSIONS: We have used a general model for SH3-ligand interactions to predict the nature of Grb2 SH3 interactions with the hSos protein. Comparison of the four hSos sequences that bind Grb2 revealed a preference for the PXXPXR motif, consistent with the predicted class II-type binding interaction. The interaction between Grb2 and hSos peptides is predominantly via the amino-terminal SH3 domain, although the carboxy-terminal SH3 domain may increase the overall stability of the Grb2-hSos complex.

Amino-terminal basic residues of Src mediate membrane binding through electrostatic interaction with acidic phospholipids.

Sigal CT, Zhou W, Buser CA, McLaughlin S, Resh MD
Proc Natl Acad Sci U S A. 1994; 91: 12253-7

Membrane targeting of pp60src (Src) is mediated by its myristoylated amino terminus. We demonstrate that, in addition to myristate, six basic residues in the amino terminus are essential for high-affinity binding to the lipid bilayer via electrostatic interaction with acidic phospholipids. Specifically, c-Src was shown to bind 2500-fold more strongly to vesicles composed of the physiological ratio of 2:1 phosphatidylcholine (PC)/phosphatidylserine (PS) than to neutral PC bilayer vesicles. The apparent Kd for binding of c-Src to the PC/PS bilayer was 6 x 10(-7) M. This interaction is sufficiently strong to account for c-Src membrane targeting. Mutants of c-Src in which the amino-terminal basic residues were replaced by neutral asparagine residues exhibited binding isotherms approaching that of wild-type binding to neutral bilayers (apparent Kd of 2 x 10(-3) M). The transforming v-Src and activated c-Src (Y527F) proteins also bound more strongly to PC/PS bilayers (apparent Kd of approximately 1 x 10(-5) M) than to neutral PC bilayers. In vivo experiments with Src mutants confirmed the role of positive charge in mediating membrane binding and cellular transformation.

Common in vitro substrate specificity and differential Src homology 2 domain accessibility displayed by two members of the Src family of protein-tyrosine kinases, c-Src and Hck.

Sicilia RJ, Hibbs ML, Bello PA, Bjorge JD, Fujita DJ, Stanley IJ, Dunn AR, Cheng HC
J Biol Chem. 1998; 273: 16756-63

Hck and Src are members of the Src family of protein- tyrosine kinases that carry out distinct and overlapping functions in vivo (Lowell, C. A., Niwa, M., Soriano, P., and Varmus, H. E. (1996) Blood 87, 1780-1792). In an attempt to understand how Hck and Src can function both independently and in concert, we have compared 1) their in vitro substrate specificity and 2) the accessibility of their Src homology 2 (SH2) domain. Using several synthetic peptides, we have demonstrated that Hck and Src recognize similar structural features in the substrate peptides, suggesting that both kinases have the intrinsic ability to carry out overlapping cellular functions by phosphorylating similar cellular proteins in vivo. Using a phosphotyrosine-containing peptide that has previously been shown to bind the SH2 domain of Src family kinases with high affinity, we found that although Src could bind to the phosphopeptide, Hck showed no interaction. The inability of Hck to bind the phosphopeptide was not a result of a stable intramolecular interaction between its SH2 domain and C-terminal regulatory phosphotyrosine residue (Tyr-520), as most Hck molecules in the purified Hck preparation were not tyrosine-phosphorylated. In contrast to intact Hck, a recombinant truncation analog of Hck was able to bind the phosphopeptide with an affinity similar to that of the Src SH2 domain, suggesting that conformational constraints are imposed on intact Hck that limit accessibility of its SH2 domain to the phosphopeptide. Furthermore, the difference in SH2 domain accessibility is a potential mechanism that enables Src and Hck to perform their respective unique functions by 1) targeting them to different subcellular compartments, whereupon they phosphorylate different cellular proteins, and/or 2) facilitating direct binding to their cellular substrates.

Structures of Src-family tyrosine kinases.

Sicheri F, Kuriyan J
Curr Opin Struct Biol. 1997; 7: 777-85

The crystal structures of three Src-family tyrosine kinases have been determined recently. The structure of the catalytic domain of Lck has been determined in the active autophosphorylated state. The structures of larger constructs of c-Src and Hck, containing the SH3, SH2 and catalytic domains, as well as a C-terminal regulatory tail, have been determined in the down-regulated state, phosphorylated in the C-terminal tail. A comparison of these structures leads to an unanticipated mechanism for the regulation of catalytic activity by cooperative interactions between the SH2, SH3 and catalytic domains.

The kinase-deficient Src acts as a suppressor of the Abl kinase for Cbl phosphorylation.

Shishido T, Akagi T, Ouchi T, Georgescu MM, Langdon WY, Hanafusa H
Proc Natl Acad Sci U S A. 2000; 97: 6439-44

The kinase activity of Abl is known to be regulated by a putative trans-acting inhibitor molecule interacting with the Src homology (SH) 3 domain of Abl. Here we report that the kinase-deficient Src (SrcKD) directly inhibits the tyrosine phosphorylation of Cbl and other cellular proteins by Abl. We found that both the SH2 and SH3 domains of SrcKD are necessary for the suppressor activity toward the Abl kinase phosphorylating Cbl. To suppress the Cbl phosphorylation by Abl, the interaction between the SH3 domain of SrcKD and Cbl is required. This interaction between SrcKD and Cbl is regulated by a closed structure of Cbl. The binding of Abl to the extreme carboxyl-terminal region of Cbl unmasks the binding site of SrcKD to Cbl. This results in a ternary complex that inhibits the Abl-mediated phosphorylation of Cbl by steric hindrance. These results illustrate a mechanism by which the enzymatically inactive Src can exert a biological function in vivo.

Evidence for SH3 domain directed binding and phosphorylation of Sam68 by Src.

Shen Z, Batzer A, Koehler JA, Polakis P, Schlessinger J, Lydon NB, Moran MF
Oncogene. 1999; 18: 4647-53

Sam68 is a 68 kDa protein that associates with and is phosphorylated by the c-Src kinase at mitosis. It contains a KH domain implicated in RNA binding and several proline-rich motifs that resemble known SH3 binding sites. The SH3 domains of c-Src, phosphatidylinositol 3-OH kinase, phospholipase C-gamma and Grb2 protein (containing two SH3 domains), but not other SH3 domains tested, were capable of binding Sam68 in vitro. Synthetic peptides corresponding to the proline motifs of Sam68 inhibited with different efficiencies the binding of SH3 domains to Sam68 suggesting that the proline motifs of Sam68 function as specific SH3 domain binding sites. Mutation of Sam68 SH3 binding sites further indicated that the SRC SH3 domain mediates binding of Src to unphosphorylated Sam68. Phosphorylation of Sam68 by Src kinase was inhibited when the Src SH3 binding site of Sam68 was mutated or when corresponding peptides were added to in vitro kinase reactions indicating that binding of the Src SH3 domain to a specific site near the amino-terminus of Sam68 (including residues 38 - 45: PPLPHRSR) facilitates phosphorylation of Sam68 by the Src kinase domain. Sam68-based proline peptides had no effect on the phosphorylation of another in vitro substrate of Src, enolase. These results suggest that Src effectively mounts Sam68 through its SH3 domain, possibly as a mechanism to position the kinase domain close to substrate tyrosine residues in the carboxyl-half of the protein.

Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae.

Shenoy-Scaria AM, Dietzen DJ, Kwong J, Link DC, Lublin DM
J Cell Biol. 1994; 126: 353-63

Recent work has demonstrated that p56lck, a member of the Src family of protein tyrosine kinases (PTKs), is modified by palmitoylation of a cysteine residue(s) within the first 10 amino acids of the protein (in addition to amino-terminal myristoylation that is a common modification of the Src family of PTKs). This is now extended to three other members of this family by showing incorporation of [3H]palmitate into p59fyn, p55fgr, and p56hck, but not into p60src. The [3H]palmitate was released by treatment with neutral hydroxylamine, indicating a thioester linkage to the protein. Individual replacement of the two cysteine residues within the first 10 amino acids of p59fyn and p56lck with serine indicated that Cys3 was the major determinant of palmitoylation, as well as association of the PTK with glycosyl-phosphatidylinositol-anchored proteins. Introduction of Cys3 into p60src led to its palmitoylation. p59fyn but not p60src partitioned into Triton-insoluble complexes that contain caveolae, microinvaginations of the plasma membrane. Mapping of the requirement for partitioning into caveolae demonstrated that the amino-terminal sequence Met-Gly-Cys is both necessary and sufficient within the context of a Src family PTK to confer localization into caveolae. Palmitoylation of this motif in p59fyn also modestly increased its overall avidity for membranes. These results highlight the role of the amino-terminal motif Met-Gly-Cys in determining the structure and properties of members of the Src family of PTKs.

Structure-based design of an osteoclast-selective, nonpeptide src homology 2 inhibitor with in vivo antiresorptive activity.

Shakespeare W, Yang M, Bohacek R, Cerasoli F, Stebbins K, Sundaramoorthi R, Azimioara M, Vu C, Pradeepan S, Metcalf C 3rd, Haraldson C, Merry T, Dalgarno D, Narula S, Hatada M, Lu X, van Schravendijk MR, Adams S, Violette S, Smith J, Guan W, Bartlett C, Herson J, Iuliucci J, Weigele M, Sawyer T
Proc Natl Acad Sci U S A. 2000; 97: 9373-8

Targeted disruption of the pp60(src) (Src) gene has implicated this tyrosine kinase in osteoclast-mediated bone resorption and as a therapeutic target for the treatment of osteoporosis and other bone-related diseases. Herein we describe the discovery of a nonpeptide inhibitor (AP22408) of Src that demonstrates in vivo antiresorptive activity. Based on a cocrystal structure of the noncatalytic Src homology 2 (SH2) domain of Src complexed with citrate [in the phosphotyrosine (pTyr) binding pocket], we designed 3',4'-diphosphonophenylalanine (Dpp) as a pTyr mimic. In addition to its design to bind Src SH2, the Dpp moiety exhibits bone-targeting properties that confer osteoclast selectivity, hence minimizing possible undesired effects on other cells that have Src-dependent activities. The chemical structure AP22408 also illustrates a bicyclic template to replace the post-pTyr sequence of cognate Src SH2 phosphopeptides such as Ac-pTyr-Glu-Glu-Ile (1). An x-ray structure of AP22408 complexed with Lck (S164C) SH2 confirmed molecular interactions of both the Dpp and bicyclic template of AP22408 as predicted from molecular modeling. Relative to the cognate phosphopeptide, AP22408 exhibits significantly increased Src SH2 binding affinity (IC(50) = 0.30 microM for AP22408 and 5.5 microM for 1). Furthermore, AP22408 inhibits rabbit osteoclast-mediated resorption of dentine in a cellular assay, exhibits bone-targeting properties based on a hydroxyapatite adsorption assay, and demonstrates in vivo antiresorptive activity in a parathyroid hormone-induced rat model.

Dynamin binds to SH3 domains of phospholipase C gamma and GRB-2.

Seedorf K, Kostka G, Lammers R, Bashkin P, Daly R, Burgess WH, van der Bliek AM, Schlessinger J, Ullrich A
J Biol Chem. 1994; 269: 16009-14

Src homology 3 (SH3) domains are found in a variety of proteins that are involved in signal transduction or represent components of the cytoskeleton. These domains are thought to serve as modules that mediate specific protein-protein interactions that include proline-rich sequences on the target protein. We have identified proteins of 110, 80, 65, and 43 kDa in human embryonic fibroblasts that bind specifically to the SH3 domain of phospholipase C gamma, a primary substrate of receptor tyrosine kinases, and characterized the 110-kDa band as the microtubule-activated GTPase dynamin. In addition, dynamin binds the son of sevenless adaptor protein GRB-2 with even higher affinity. This interaction does not require the dynamin GTPase function and involves a proline-rich target sequence between residues 812 and 820 of dynamin.

The structure of crystalline profilin-beta-actin.

Schutt CE, Myslik JC, Rozycki MD, Goonesekere NC, Lindberg U
Nature. 1993; 365: 810-6

The three-dimensional structure of bovine profilin-beta-actin has been solved to 2.55 A resolution by X-ray crystallography. There are several significant local changes in the structure of beta-actin compared with alpha-actin as well as an overall 5 degrees rotation between its two major domains. Actin molecules in the crystal are organized into ribbons through intermolecular contacts like those found in oligomeric protein assemblies. Profilin forms two extensive contacts with the actin ribbon, one of which appears to correspond to the solution contact in vitro.

Identification of D-peptide ligands through mirror-image phage display.

Schumacher TN, Mayr LM, Minor DL Jr, Milhollen MA, Burgess MW, Kim PS
Science. 1996; 271: 1854-7

Genetically encoded libraries of peptides and oligonucleotides are well suited for the identification of ligands for many macromolecules. A major drawback of these techniques is that the resultant ligands are subject to degradation by naturally occurring enzymes. Here, a method is described that uses a biologically encoded library for the identification of D-peptide ligands, which should be resistant to proteolytic degradation. In this approach, a protein is synthesized in the D-amino acid configuration and used to select peptides from a phage display library expressing random L-amino acid peptides. For reasons of symmetry, the mirror images of these phage-displayed peptides interact with the target protein of the natural handedness. The value of this approach was demonstrated by the identification of a cyclic D-peptide that interacts with the Src homology 3 domain of c- SRC. Nuclear magnetic resonance studies indicate that the binding site for this D-peptide partially overlaps the site for the physiological ligands of this domain.

The SH3 domain of Crk binds specifically to a conserved proline-rich motif in Eps15 and Eps15R.

Schumacher C, Knudsen BS, Ohuchi T, Di Fiore PP, Glassman RH, Hanafusa H
J Biol Chem. 1995; 270: 15341-7

The Crk protein belongs to the family of proteins consisting of mainly Src homology 2 and 3 (SH2 and SH3) domains. These proteins are thought to transduce signals from tyrosine kinases to downstream effectors. In order to understand the specificity and effector function of the SH3 domain of Crk, we screened an expression library for binding proteins. We isolated Eps15, a substrate of the epidermal growth factor receptor (EGFR) tyrosine kinase, and Eps15R, a novel protein with high sequence homology to the carboxyl-terminal domain of Eps15. Antibodies raised against a fragment of the Eps15R gene product immunoprecipitated a protein of 145 kDa. Eps15 and Eps15R bound specifically to the amino-terminal SH3 domain of Crk and coprecipitated equivalently with both c-Crk and v-Crk from cell lysates. The amino acid sequences of Eps15 and Eps15R featured several proline-rich regions as putative binding motifs for SH3 domains. In both Eps15 and Eps15R, we identified one proline-rich motif which accounts for their interaction with the Crk SH3 domain. Each binding motif contains the sequence P-X-L-P-X-K, an amino acid stretch that is highly conserved in all proteins known to interact specifically with the first SH3 domain of Crk. Furthermore, we found that immunoprecipitates of activated EGFR-kinase stably bound in vitro-translated Eps15 only in the presence of in vitro-translated v-Crk. Crk might therefore be involved in Eps15-mediated signal transduction through the EGFR.

SH2/SH3 signaling proteins.

Schlessinger J
Curr Opin Genet Dev. 1994; 4: 25-30

SH2 and SH3 domains are small protein modules that mediate protein-protein interactions in signal transduction pathways that are activated by protein tyrosine kinases. SH2 domains bind to short phosphotyrosine-containing sequences in growth factor receptors and other phosphoproteins. SH3 domains bind to target proteins through sequences containing proline and hydrophobic amino acids. SH2 and SH3 domain containing proteins, such as Grb2 and phospholipase C gamma, utilize these modules in order to link receptor and cytoplasmic protein tyrosine kinases to the Ras signaling pathway and to phosphatidylinositol hydrolysis, respectively. The three-dimensional structures of several SH2 and SH3 domains have been determined by NMR and X-ray crystallography, and the molecular basis of their specificity is beginning to be unveiled.

Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor.

Schindler T, Sicheri F, Pico A, Gazit A, Levitzki A, Kuriyan J
Mol Cell. 1999; 3: 639-48

The crystal structure of the autoinhibited form of Hck has been determined at 2.0 A resolution, in complex with a specific pyrazolo pyrimidine-type inhibitor, PP1. The activation segment, a key regulatory component of the catalytic domain, is unphosphorylated and is visualized in its entirety. Tyr-416, the site of activating autophosphorylation in the Src family kinases, is positioned such that access to the catalytic machinery is blocked. PP1 is bound at the ATP-binding site of the kinase, and a methylphenyl group on PP1 is inserted into an adjacent hydrophobic pocket. The enlargement of this pocket in autoinhibited Src kinases suggests a route toward the development of inhibitors that are specific for the inactive forms of these proteins.

Structural mechanism for STI-571 inhibition of abelson tyrosine kinase.

Schindler T, Bornmann W, Pellicena P, Miller WT, Clarkson B, Kuriyan J
Science. 2000; 289: 1938-42

The inadvertent activation of the Abelson tyrosine kinase (Abl) causes chronic myelogenous leukemia (CML). A small-molecule inhibitor of Abl (STI-571) is effective in the treatment of CML. We report the crystal structure of the catalytic domain of Abl, complexed to a variant of STI-571. Critical to the binding of STI-571 is the adoption by the kinase of an inactive conformation, in which a centrally located "activation loop" is not phosphorylated. The conformation of this loop is distinct from that in active protein kinases, as well as in the inactive form of the closely related Src kinases. These results suggest that compounds that exploit the distinctive inactivation mechanisms of individual protein kinases can achieve both high affinity and high specificity.

Dimer formation through domain swapping in the crystal structure of the Grb2-SH2-Ac-pYVNV complex.

Schiering N, Casale E, Caccia P, Giordano P, Battistini C
Biochemistry. 2000; 39: 13376-82

Src homology 2 (SH2) domains are key modules in intracellular signal transduction. They link activated cell surface receptors to downstream targets by binding to phosphotyrosine-containing sequence motifs. The crystal structure of a Grb2-SH2 domain-phosphopeptide complex was determined at 2.4 A resolution. The asymmetric unit contains four polypeptide chains. There is an unexpected domain swap so that individual chains do not adopt a closed SH2 fold. Instead, reorganization of the EF loop leads to an open, nonglobular fold, which associates with an equivalent partner to generate an intertwined dimer. As in previously reported crystal structures of canonical Grb2-SH2 domain-peptide complexes, each of the four hybrid SH2 domains in the two domain-swapped dimers binds the phosphopeptide in a type I beta-turn conformation. This report is the first to describe domain swapping for an SH2 domain. While in vivo evidence of dimerization of Grb2 exists, our SH2 dimer is metastable and a physiological role of this new form of dimer formation remains to be demonstrated.

SH2 domain structure and function.

Schaffhausen B
Biochim Biophys Acta. 1995; 1242: 61-75

An emerging theme in both the biology of signal transduction and the biochemistry of proteins has been the modular function of small protein domains. In some cases these can directly regulate catalytic activity. In others, they serve to interconnect important regulatory proteins. SH2 (src homology 2) domains represent some of the best studied models. Originally identified on the basis of homology in src and fps [1], SH2s are elements that ordinarily respond to tyrosine phosphorylation by binding the phosphorylated sequence. As such, they are key elements in tyrosine kinase regulation of cellular processes. Because SH2 interactions result from phosphorylation, such elements provide a regulatable circuitry along which signals can be transmitted in a timely manner. Because the regulation is based on a common mechanism, signal generators can target several different proteins coordinately. The PDGF receptor (PDGFr), for example, may interact with as many as ten different elements [2,3]. There are a number of excellent reviews on SH2 domains available [4-11]. This discussion will try to show how genetic, biochemical and biophysical results can be integrated in a satisfying way.

Bacterial expression of an active tyrosine kinase from a protein A/truncated c-src fusion protein.

Saya H, Lee PS, Nishi T, Izawa I, Nakajima M, Gallick GE, Levin VA
FEBS Lett. 1993; 327: 224-30

The carboxy-terminal half of the c-src protein fused to the protein A moiety was expressed in bacteria. The protein A/truncated c-src fusion protein, which does not have SH2 and SH3 domains, is found in the periplasmic space allowing for a simple one-step purification and demonstrated high efficiency in autophosphorylation and exogeneous substrate phosphorylation. The missense mutation at codon 294 (Ile-->Thr), which is located in the ATP-binding domain of the c-src, resulted in dramatic reduction of tyrosine kinase activity of the fusion protein. Using the fusion protein, we also revealed that staurosporin, a well-known kinase inhibitor, directly affects autophosphorylation of the C-terminal half of the c-src protein. This truncated c-src expression system provides a good source of enzyme for diverse experiments and is an ideal model for understanding the implication of structural alterations in the catalytic activity of the c-src kinase by site-directed mutagenesis experiments.

Src homology-2 domains: structure, mechanisms, and drug discovery.

Sawyer TK
Biopolymers. 1998; 47: 243-61

Src homology-2 (SH2) domains and their associated catalytic or noncatalytic proteins constitute critical signal transduction targets for drug discovery. Such SH2 proteins are found in the regulation of a number of cellular processes, including growth, mitogenesis, motility, metabolism, immune response, and gene transcription. From the relationship of tyrosine phosphorylation and intracellular regulation by protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPs), the dynamic and reversible binding interactions of SH2 domain containing proteins with their cognate phosphotyrosine (pTyr) containing proteins provide a third dimensionality to the orchestration of signal transduction pathways that exist as a result of pTyr formation, degradation, and molecular recognition events. This review highlights several key research achievements impacting our current understanding of SH2 structure, mechanisms, and drug discovery that underlie the role(s) of SH2 domains in signal transduction processes, cellular functions, and disease states.

Quantitative analysis of Grb2-Sos1 interaction: the N-terminal SH3 domain of Grb2 mediates affinity.

Sastry L, Lin W, Wong WT, Di Fiore PP, Scoppa CA, King CR
Oncogene. 1995; 11: 1107-12

Grb2 is an adaptor protein that links receptor and cytoplasmic tyrosine kinases to the Ras signalling pathway by binding the Ras-specific guanine nucleotide exchange factor, Sos1, through its SH3 domains. The Grb2-SH3 domain binding has been localized to the carboxy-terminal two hundred amino acids of Sos1 (Sos1-c). By using real time biospecific interaction analysis (BIAcore), we studied the kinetic parameters and binding affinity of the Grb2-Sos1-c interaction. The binding of Grb2 to Sos1-c is a high affinity interaction with a moderate association rate (9.45 x 10(4) per M per s), a slow dissociation rate (13.8 x 10(-5) s), and an affinity constant of 1.48 nM. BIAcore measurements on isolated N-terminal and C-terminal SH3 domains (NSH3 and CSH3) further indicate that the high affinity Grb2-Sos1-c interaction is primarily mediated through the NSH3 domain (Kd = 1.68 nM). The CSH3 domain shows substantially reduced binding to Sos1-c in these measurements. Inhibition studies with BIAcore using proline rich peptides derived from the C-terminus of Sos1 show that there is a single major binding site for Grb2 in Sos1. This binding site is contained within the peptide N20, which corresponds to amino acids 1143-1162 of Sos1. This peptide completely blocks the Grb2-Sos1-c and NSH3-Sos1-c interactions with IC50 values of 8 microM and 4 microM respectively. The discrete interaction between the NSH3 domain and the N20 peptide may be amenable for drug discovery through screening or peptidomimetic approaches.

Substrate specificity for normal but not mutationally activated variants of src family kinases.

Sartor O, Robbins KC
J Biol Chem. 1993; 268: 21014-20

Although structural features and expression patterns of the src family of tyrosine kinases have been extensively analyzed, there are no direct comparative studies of the putative protein substrates that are tyrosine-phosphorylated by the normal cellular versions of these enzymes. In this report, we have expressed normal and enzymatically activated versions of the fyn, fgr, and src translational products by transfection of appropriate cDNAs into mouse fibroblasts. Because the same parental cell line was used for all transfections, each enzyme was expressed in a similar milieu of potential in vivo substrates. After verification of appropriate expression from each transfected cDNA and assessment of relative transforming potency, a series of putative protein substrates was specifically assayed for expression and tyrosine phosphorylation. Our data indicate that the normal src family kinases display some degree of substrate specificity but that specificity is diminished when these enzymes are constitutively activated. In the course of these studies, tyrosine-phosphorylated proteins were noted to coimmunoprecipitate with some of these putative in vivo substrates. Some of these coimmunoprecipitating proteins have been reported previously, whereas others, such as the presence of p59fyn in anti-p80/85 immunoprecipitates, are heretofore undescribed.

Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4.

Saksela K, Cheng G, Baltimore D
EMBO J. 1995; 14: 484-91

Human immunodeficiency virus (HIV) and simian immunodeficiency virus Nef proteins contain a conserved motif with the minimal consensus (PxxP) site for Src homology region 3 (SH3)-mediated protein-protein interactions. Nef PxxP motifs show specific binding to biotinylated SH3 domains of Hck and Lyn, but not to those of other tested Src family kinases or less related proteins. A unique cooperative role of a distant proline is also observed. Endogenous Hck of monocytic U937 cells can be specifically precipitated by matrix-bound HIV-1 Nef, but not by mutant protein lacking PxxP. Intact Nef PxxP motifs are dispensable for Nef-induced CD4 down-regulation, but are required for the higher in vitro replicative potential of Nef+ viruses. Thus, CD4 down-regulation and promotion of viral growth are two distinct functions of Nef, and the latter is mediated via SH3 binding.

Chemical synthesis and applications of phosphopeptides.

Sakaguchi K, Roller PP, Appella E
Genet Eng (N Y). 1996; 18: 249-78

Structural similarities in the noncatalytic domains of phenylalanyl-tRNA and biotin synthetases.

Safro M, Mosyak L
Protein Sci. 1995; 4: 2429-32

Detailed comparison between the structures of the Escherichia coli biotin synthetase/repressor protein (BirA) and the recently solved Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) reveals significant similarities outside their respective catalytic domains. These comprise a DNA-binding alpha+beta domain and an Src-homology 3 (SH3)-like domain that were observed in both enzymes. This similarity provides a novel example in which all domains of one multidomain protein appear to be constituents of the other multidomain protein and supports a concept of a common ancestor for two different synthetase families.

Molecular cloning and expression of chicken C-terminal Src kinase: lack of stable association with c-Src protein.

Sabe H, Knudsen B, Okada M, Nada S, Nakagawa H, Hanafusa H
Proc Natl Acad Sci U S A. 1992; 89: 2190-4

Cloning and sequencing of chicken C-terminal Src kinase (CSK), a tyrosine kinase that phosphorylates the regulatory C-terminal tyrosine residue present on cytoplasmic tyrosine kinases of the Src family, demonstrated a high degree of interspecies conservation as well as src homology 2 and 3 domains N-terminal to the kinase domain. The lack of autophosphorylation sites distinguishes CSK from other tyrosine kinases. CSK is unique and does not belong to a gene family, suggesting that it may phosphorylate other members of the Src family of tyrosine kinases in addition to c-Src. Since complex formation between c-Src and CSK seemed a likely regulatory step in the control of c-Src kinase activity, such an association was investigated by immunoprecipitation and Western blotting as well as intracellular localization studies. Although some portions of CSK were found in a membrane fraction, no complex formation between CSK and c-Src was observed, suggesting that the src homology 2 domain of CSK does not play a role in the direct interaction of c-Src.

Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Src.

Sabe H, Hata A, Okada M, Nakagawa H, Hanafusa H
Proc Natl Acad Sci U S A. 1994; 91: 3984-8

Csk (C-terminal Src kinase), a protein-tyrosine kinase, bearing the Src homology 2 and 3 (SH2 and SH3) domains, has been implicated in phosphorylation of c-Src Tyr-527, resulting in suppression of c-Src kinase activity. We found that mutations in the SH2 or SH3 domain of Csk, though they did not affect its kinase activity, resulted in a loss of suppression of c-Src activity in fibroblasts. In normal fibroblasts, tyrosine-phosphorylated paxillin and focal adhesion kinase pp125FAK, which colocalize at focal adhesion plaques, were the major proteins to which the Csk SH2 domain bound. Loss of binding to these proteins by the Csk SH2 mutants correlated with loss of the activity to suppress c-Src. Consistent with this observation, the levels of tyrosine phosphorylation of paxillin and pp125FAK were greatly reduced during mitosis, whereas the kinase activity of c-Src was elevated. We suggest that the SH2 domain is required for Csk to suppress c-Src, perhaps in combination with the SH3 domain, by anchoring Csk to a particular subcellular location where c-Src may exist. Our data also indicate that a certain fraction of the Csk and Src family kinases function at the focal adhesion plaques. The activity of the c-Src kinase localized at the focal adhesion plaques appears to be regulated by cell adhesion to the extracellular matrix.

Regulation of c-Fgr protein kinase by c-Src kinase (CSK) and by polycationic effectors.

Ruzzene M, James P, Brunati AM, Donella-Deana A, Pinna LA
J Biol Chem. 1994; 269: 15885-91

The protein tyrosine kinase expressed by the protooncogene c-fgr is phosphorylated and down-regulated in vitro by the c-Src kinase (CSK). CSK catalyzed phosphorylation affects Tyr-511 of c-Fgr, homologous to Tyr-527 of c-Src and it prevents the autophosphorylation normally occurring at c-Fgr Tyr-400, homologous to c-Src Tyr-416. Polylysine, histones H1 and H2A and other polycationic proteins on the other hand stimulate c-Fgr activity while promoting enhanced autophosphorylation of both Tyr-400 and Tyr-511. Once phosphorylated at Tyr-511 and down-regulated by CSK, c-Fgr is no more susceptible to polylysine stimulation. Previous autophosphorylation (at Tyr-400) reduces c-Fgr susceptibility to down-regulation by CSK, although Tyr-511 can be still phosphorylated by it. If a more exhaustive autophosphorylation (of both Tyr-400 and Tyr-511) is performed in the presence of polylysine, c-Fgr becomes totally insensitive to CSK down-regulation. These data support the concept that down-regulation of c-Fgr by Tyr-511 phosphorylation is prevented if Tyr-400 is also phosphorylated and they are consistent with an outcompetition of phospho-Tyr-511 from the Src homology 2 domain by phospho-Tyr-400, which, in c-Fgr, is surrounded by an amino acid sequence divergent from that of the other Src-related protein tyrosine kinases.

Linear and cyclic synthetic peptides related to the main autophosphorylation site of the Src tyrosine kinases as substrates and inhibitors of Lyn.

Ruzza P, Calderan A, Filippi B, Biondi B, Deana AD, Cesaro L, Pinna LA, Borin G
Int J Pept Protein Res. 1995; 45: 529-39

Tyrosine protein kinases (TPKs) of the src family contain two major phosphoacceptor sites which are homologous to the Tyr 416 and Tyr 527 of pp60c-src. The former represents the main autophosphorylation sites of these enzymes, and its phosphorylation correlates with increased kinase activity. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a high affinity toward the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH, which reproduces the main autophosphorylation site of the Src family enzymes [Donella-Deana, A., Marin, O., Brunati, A.M. & Pinna, L.A. (1992) Eur. J. Biochem. 204, 1159-1163]. Our study was addressed to the synthesis of some derivatives of this sequence in order to obtain both peptide substrates suitable for the detection of the Src-like tyrosine kinase activity and active site-directed inhibitors specific for this class of enzymes. For this purpose we synthesized by classical solution methods the heptapeptide and its dimeric form. Moreover, in order to improve the proteolytic resistance of these peptides we also synthesized their cyclic derivatives and their N-terminal acetylated and C-terminal amidated analogs. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as Lyn substrates was examined. The kinetic data obtained indicate that the extent of the peptide phosphorylation varies considerably depending on the flexibility and length of the analogs. While the cyclization and the C-terminal amidation of the heptapeptide are detrimental for the Lyn activity, dimeric derivatives display very favourable kinetic constants. In particular the cyclic dimer is an especially suitable substrate for the tyrosine kinase and a powerful inhibitor of both the phosphorylation activity of Lyn and the enzyme autophosphorylation.

Conservation analysis and structure prediction of the SH2 family of phosphotyrosine binding domains.

Russell RB, Breed J, Barton GJ
FEBS Lett. 1992; 304: 15-20

Src homology 2 (SH2) regions are short (approximately 100 amino acids), non-catalytic domains conserved among a wide variety of proteins involved in cytoplasmic signaling induced by growth factors. It is thought that SH2 domains play an important role in the intracellular response to growth factor stimulation by binding to phosphotyrosine containing proteins. In this paper we apply the techniques of multiple sequence alignment, secondary structure prediction and conservation analysis to 67 SH2 domain amino acid sequences. This combined approach predicts seven core secondary structure regions with the pattern beta-alpha-beta-beta-beta-beta-alpha, identifies those residues most likely to be buried in the hydrophobic core of the native SH2 domain, and highlights patterns of conservation indicative of secondary structural elements. Residues likely to be involved in phosphotyrosine binding are shown and orientations of the predicted secondary structures suggested which could enable such residues to cooperate in phosphate binding. We propose a consensus pattern that encapsulates the principal conserved features of the SH2 domains. Comparison of the proposed SH2 domain of akt to this pattern shows only 12/40 matches, suggesting that this domain may not exhibit SH2-like properties.

The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

Rozakis-Adcock M, Fernley R, Wade J, Pawson T, Bowtell D
Nature. 1993; 363: 83-5

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

Structure of the human lck gene: differences in genomic organisation within src-related genes affect only N-terminal exons.

Rouer E, Van Huynh T, Lavareda de Souza S, Lang MC, Fischer S, Benarous R
Gene. 1989; 84: 105-13

Although cDNA sequences coding for several Rous sarcoma virus Src-related protein tyrosine kinases (PTKs) have been reported for several years, knowledge of the structure and organisation of genes of the src family is still limited. In this work, a detailed structure and organisation of the human lck gene is reported. A 17-kb genomic clone encoding human p56 Lck, a lymphocyte-specific PTK of the Src-related subfamily, has been isolated. The human lck gene is organized in 13 exons, one more than in the human cellular (c)-src gene. The twelve coding exons are located in this clone, whereas the putative 5'-noncoding exon is probably located very far upstream from the second exon. Splicing sites for exons 4 to 12, which encode both conserved phospholipase-C-like and catalytic domains of the Src-like PTKs, arise exactly at the same position for the human lck, human c-src and c-fgr genes. The only differences concern the splice sites of exons 1' and 2, which encode the unique N-terminal domain of human Lck. These results give further evidence that the different PTKs of the Src-like family have probably evolved through the mechanism of exon shuffling.

Jury returns on structure prediction.

Rost B, Sander C
Nature. 1992; 360: 540-540

Direct demonstration of an intramolecular SH2-phosphotyrosine interaction in the Crk protein.

Rosen MK, Yamazaki T, Gish GD, Kay CM, Pawson T, Kay LE
Nature. 1995; 374: 477-9

Many signal transduction processes are mediated by the binding of Scr-homology-2 (SH2) domains to phosphotyrosine (pTyr)-containing proteins. Although most SH2-pTyr interactions occur between two different types of molecules, some appear to involve only a single molecular type. It has been proposed that the enzymatic activity and substrate recognition of the Src-family kinases, and the protein-binding and transforming activity of Crk-family adaptor proteins, are regulated by intramolecular SH2-pTyr interactions. In addition, the DNA-binding activity of Stat transcription factors seems to be regulated by SH2-mediated homodimerization. Here we examine the phosphorylated and non-phosphorylated forms of murine Crk II (p-mCrk and mCrk, respectively) using a combination of physical techniques. The Crk protein contains a single SH2 domain and two SH3 domains in the order SH2-SH3-SH3. There is a tyrosine-phosphorylation site between the two SH3 domains at residue 221 which is phosphorylated in vivo by the Abl tyrosine kinase. Using NMR spectroscopic analysis, we show here that the SH2 domain of purified p-mCrk is bound to pTyr, and by hydrodynamic measurements that the phosphorylated protein is monomeric. These results provide direct demonstration of an intramolecular SH2-pTyr interaction in a signalling molecule.

Requirement for Src family protein tyrosine kinases in G2 for fibroblast cell division.

Roche S, Fumagalli S, Courtneidge SA
Science. 1995; 269: 1567-9

The protein tyrosine kinase c-Src is transiently activated at the transition from the G2 phase to mitosis in the cell cycle of mammalian fibroblasts. Fyn and Yes, the other members of the Src family present in fibroblasts, were also found to be activated at mitosis. In cells microinjected with a neutralizing antibody specific for Src, Fyn, and Yes (anti-cst.1) during G2, cell division was inhibited by 75 percent. The block occurred before nuclear envelope breakdown. Antibodies specific for phosphatidylinositol-3 kinase alpha and phospholipase C-gamma 1 had no effect. Microinjection of the Src homology 2 (SH2) domain of Fyn was also inhibitory. Functional redundancy between members of the Src family was observed; a Src-specific antibody had no effect in NIH 3T3 cells but inhibited cell division in fibroblasts in which the only functional Src family kinase was Src itself. Thus, Src family kinases and proteins associating with their SH2 domains are required for entry into mitosis.

Physical association between Src homology 3 elements and the protein product of the c-cbl proto-oncogene.

Rivero-Lezcano OM, Sameshima JH, Marcilla A, Robbins KC
J Biol Chem. 1994; 269: 17363-6

To investigate the nature of proteins recognized by Src homology 3 (SH3) domains, a cDNA expression library was prepared from macrophages and screened with a probe representing the three SH3 domains of p47nck. Two clones were isolated, and one, designated SAKAP I (for Src A box Nck-associated protein I), contained the carboxyl-terminal half of the cbl proto-oncogene product. Studies in vitro demonstrated reactivity between SAKAP I and SH3 domains derived from a variety of molecules. Wide variations in this assay suggested a high degree of specificity inherent in SAKAP I binding. Moreover, it was possible to demonstrate an in vivo association between p47nck and p120c-cbl in HL60 cells. These findings suggest that proteins containing SH3 elements regulate Cbl function.

Specificity of protein interactions with highly related SRC homology (SH) domains of FGR and FYN protein-tyrosine kinases.

Rivero-Lezcano OM, Robbins KC
FEBS Lett. 1994; 338: 183-6

As an approach toward identification and isolation of cellular proteins that may act as substrates or effectors of the SRC-family of protein-tyrosine kinases, fusion proteins containing noncatalytic elements of two highly related SRC-family members were tested for their ability to recognize distinct molecules present in lysates of cells known to normally express both enzymes. Our results demonstrate differences of protein binding between the SH2 elements of FYN and FGR kinases, but do not discriminate proteins binding to their SH3 domains.

Mutations in the non-catalytic domains of Fyn and Fgr tyrosine kinases reveal differences in mechanisms of their regulation.

Rivero-Lezcano OM, Marcilla A, Robbins KC
Oncogene. 1995; 11: 2675-9

Non-catalytic domains of tyrosine kinases from the Src family are believed to be regulated intra- and intermolecularly through protein-protein interactions. We have deleted the SH2 and SH3 domains from Fyn and Fgr and have generated two point mutations in residues completely conserved in all members of the Src family. The dramatically different biological effects of these mutations suggest that non-catalytic domains regulate Src family kinase activities through distinctly different mechanisms.

PACSIN 2, a novel member of the PACSIN family of cytoplasmic adapter proteins.

Ritter B, Modregger J, Paulsson M, Plomann M
FEBS Lett. 1999; 454: 356-62

The PACSIN-related proteins are cytoplasmic adapter proteins with a common arrangement of domains and conserved regions. Here we report the cloning, sequencing, and expression of PACSIN 2, a novel member of the PACSIN protein family and accordingly rename the original PACSIN to PACSIN 1. The sequences of the murine and human cDNAs reveal an open reading frame encoding a putative protein of 486 residues. Despite its high sequence similarity to PACSIN 1, PACSIN 2 is encoded by distinct transcripts in human and mouse, in particular displaying a ubiquitous expression pattern. Immunofluorescence microscopy of PACSIN 2-transfected NIH3T3 fibroblasts reveal a broad, vesicle-like cytoplasmic staining. In contrast to FAP52, another PACSIN-related protein derived from chicken brain, PACSIN 2 could not be detected at focal contacts. Taken together, these findings suggest that PACSIN 2 is a novel PACSIN isoform with similar domain and motif arrangement, but an unrestricted expression pattern, which may participate in the organization of the actin cytoskeleton and the regulation of vesicular traffic.

Functional rapidly folding proteins from simplified amino acid sequences.

Riddle DS, Santiago JV, Bray-Hall ST, Doshi N, Grantcharova VP, Yi Q, Baker D
Nat Struct Biol. 1997; 4: 805-9

Early protein synthesis is thought to have involved a reduced amino acid alphabet. What is the minimum number of amino acids that would have been needed to encode complex protein folds similar to those found in nature today? Here we show that a small beta-sheet protein, the SH3 domain, can be largely encoded by a five letter amino acid alphabet but not by a three letter alphabet. Furthermore, despite the dramatic changes in sequence, the folding rates of the reduced alphabet proteins are very close to that of the naturally occurring SH3 domain. This finding suggests that despite the vast size of the search space, the rapid folding of biological sequences to their native states is not the result of extensive evolutionary optimization. Instead, the results support the idea that the interactions which stabilize the native state induce a funnel shape to the free energy landscape sufficient to guide the folding polypeptide chain to the proper structure.

Phage display selection of ligand residues important for Src homology 3 domain binding specificity.

Rickles RJ, Botfield MC, Zhou XM, Henry PA, Brugge JS, Zoller MJ
Proc Natl Acad Sci U S A. 1995; 92: 10909-13

The Src homology 3 (SH3) domain is a 50-aa modular unit present in many cellular proteins involved in intracellular signal transduction. It functions to direct protein-protein interactions through the recognition of proline-rich motifs on associated proteins. SH3 domains are important regulatory elements that have been demonstrated to specify distinct regulatory pathways important for cell growth, migration, differentiation, and responses to the external milieu. By the use of synthetic peptides, ligands have been shown to consist of a minimum core sequence and to bind to SH3 domains in one of two pseudosymmetrical orientations, class I and class II. The class I sites have the consensus sequence ZP(L/P)PP psi P whereas the class II consensus is PP psi PPZ (where psi is a hydrophobic residue and Z is a SH3 domain-specific residue). We previously showed by M13 phage display that the Src, Fyn, Lyn, and phosphatidylinositol 3-kinase (PI3K) SH3 domains preferred the same class I-type core binding sequence, RPLPP psi P. These results failed to explain the specificity for cellular proteins displayed by SH3 domains in cells. In the current study, class I and class II core ligand sequences were displayed on the surface of bacteriophage M13 with five random residues placed either N- or C-terminal of core ligand residues. These libraries were screened for binding to the Src, Fyn, Lyn, Yes, and PI3K SH3 domains. By this approach, additional ligand residue preferences were identified that can increase the affinity of SH3 peptide ligands at least 20-fold compared with core peptides. The amino acids selected in the flanking sequences were similar for Src, Fyn, and Yes SH3 domains; however, Lyn and PI3K SH3 domains showed distinct binding specificities. These results indicate that residues that flank the core binding sequences shared by many SH3 domains are important determinants of SH3 binding affinity and selectivity.

Association of p62, a multifunctional SH2- and SH3-domain-binding protein, with src family tyrosine kinases, Grb2, and phospholipase C gamma-1.

Richard S, Yu D, Blumer KJ, Hausladen D, Olszowy MW, Connelly PA, Shaw AS
Mol Cell Biol. 1995; 15: 186-97

src family tyrosine kinases contain two noncatalytic domains termed src homology 3 (SH3) and SH2 domains. Although several other signal transduction molecules also contain tandemly occurring SH3 and SH2 domains, the function of these closely spaced domains is not well understood. To identify the role of the SH3 domains of src family tyrosine kinases, we sought to identify proteins that interacted with this domain. By using the yeast two-hybrid system, we identified p62, a tyrosine-phosphorylated protein that associates with p21ras GTPase-activating protein, as a src family kinase SH3-domain-binding protein. Reconstitution of complexes containing p62 and the src family kinase p59fyn in HeLa cells demonstrated that complex formation resulted in tyrosine phosphorylation of p62 and was mediated by both the SH3 and SH2 domains of p59fyn. The phosphorylation of p62 by p59fyn required an intact SH3 domain, demonstrating that one function of the src family kinase SH3 domains is to bind and present certain substrates to the kinase. As p62 contains at least five SH3-domain-binding motifs and multiple tyrosine phosphorylation sites, p62 may interact with other signalling molecules via SH3 and SH2 domain interactions. Here we show that the SH3 and/or SH2 domains of the signalling proteins Grb2 and phospholipase C gamma-1 can interact with p62 both in vitro and in vivo. Thus, we propose that one function of the tandemly occurring SH3 and SH2 domains of src family kinases is to bind p62, a multifunctional SH3 and SH2 domain adapter protein, linking src family kinases to downstream effector and regulatory molecules.

Solid phase synthesis of a biased mini tetrapeptoid-library for the discovery of monodentate ITAM mimics as ZAP-70 inhibitors.

Revesz L, Bonne F, Manning U, Zuber JF
Bioorg Med Chem Lett. 1998; 8: 405-8

The biased library was composed of a novel phosphotyrosine mimic fixed in the P1 position of a tetrapeptoid and combined with three lipophilic N-substituents at the remaining positions giving a total of 27 single compounds. Screening for ZAP-70 antagonism identified 8 as a novel selective monodentate ZAP-70 antagonist and lead in the search for new immunosuppressive drugs.

pH-Dependent self-association of the Src homology 2 (SH2) domain of the Src homologous and collagen-like (SHC) protein.

Rety S, Futterer K, Grucza RA, Munoz CM, Frazier WA, Waksman G
Protein Sci. 1996; 5: 405-13

The Src homologous and collagen-like (SHC) protein plays an essential role in signal transduction pathways in that it participates in the chain of events that leads to the activation of the protein Ras. The crystal structure of the SH2 domain of SHC has been determined using the method of multiple isomorphous replacement at a resolution of 2.5 A. The SH2 domain of SHC is similar in fold to other SH2 domains. The peptide-binding surfaces resemble that of the SH2 domain of Src in that a deep pocket is formed where the third amino acid C-terminal to the phosphotyrosine can insert. A novel feature of this structure is the observation of a disulfide bond and an extensive dimer interface between two symmetry-related molecules. Solution studies under reducing conditions using analytical centrifugation and PAGE suggest that the SH2 domain of SHC dimerizes in a pH-dependent manner where low pH conditions (approximately 4.5) are conducive to dimer formation. Dimerization of SHC may have important biological implications in that it may promote the assembly of large heteromultimeric signaling complexes.

Myristylation and palmitylation of Src family members: the fats of the matter.

Resh MD
Cell. 1994; 76: 411-3

Abl protein-tyrosine kinase selects the Crk adapter as a substrate using SH3-binding sites.

Ren R, Ye ZS, Baltimore D
Genes Dev. 1994; 8: 783-95

To understand the normal and oncogenic functions of the protein-tyrosine kinase Abl, the yeast two-hybrid system has been used for identifying proteins that interact with it. One interacting protein is Crk-I, an SH3/SH2-containing adapter protein that was originally identified as the oncogenic element in the avian sarcoma virus CT10. Direct interaction between the Crk-I SH3 and Abl at novel, approximately 10 amino acid sites just carboxy-terminal to the Abl kinase domain occurs in vitro and in mammalian cells. There is a nearby site specific for binding another adapter, Nck, and these sites also bind Grb-2. When bound to Abl, Crk-I was phosphorylated on tyrosine. Thus, the SH3-binding sites on Abl serve as substrate recognition sites for the relatively nonspecific kinase of Abl. In Crk-I-transformed cells, Crk-I associates with endogenous c-Abl and is phosphorylated on tyrosine. The association of Crk and Abl suggests that Abl could play a role in v-Crk and Crk-I transformation and that normal Abl function may be partly mediated through bound adapter molecules.

Requirement of phosphatidylinositol 3-kinase in focal adhesion kinase-promoted cell migration.

Reiske HR, Kao SC, Cary LA, Guan JL, Lai JF, Chen HC
J Biol Chem. 1999; 274: 12361-6

We have previously shown that overexpression of focal adhesion kinase (FAK) in Chinese hamster ovary (CHO) cells promoted their migration on fibronectin. This effect was dependent on the phosphorylation of FAK at Tyr-397. This residue was known to serve as a binding site for both Src and phosphatidylinositol 3-kinase (PI3K), implying that either one or both are required for FAK to promote cell migration. In this study, we have examined the role of PI3K in FAK-promoted cell migration. We have demonstrated that the PI3K inhibitors, wortmannin and LY294002, were able to inhibit FAK-promoted migration in a dose-dependent manner. Furthermore, a FAK mutant capable of binding Src but not PI3K was generated by a substitution of Asp residue 395 with Ala. When overexpressed in CHO cells, this differential binding mutant failed to promote cell migration although its association with Src was retained. Together, these results strongly suggest that PI3K binding is required for FAK to promote cell migration and that the binding of Src and p130(Cas) to FAK may not be sufficient for this event.

Sequence homology between Wingless/Wnt-1 and a lipid-binding domain in secreted phospholipase A2.

Reichsman F, Moore HM, Cumberledge S
Curr Biol. 1999; 9: 3535-3535

Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein.

Reedquist KA, Fukazawa T, Druker B, Panchamoorthy G, Shoelson SE, Band H
Proc Natl Acad Sci U S A. 1994; 91: 4135-9

Tyrosine phosphorylation of cellular proteins is the earliest identifiable event following T-cell antigen receptor (TCR) stimulation and is essential for activating downstream signaling machinery. Two Src-family protein-tyrosine kinases, the TCR-associated p59fyn (Fyn) and the CD4/8-associated p56lck (Lck), have emerged as the likely mediators of early tyrosine phosphorylation in T cells. Here, we show direct binding of a 120-kDa TCR-induced phosphotyrosyl polypeptide, p120, to glutathione S-transferase fusion proteins of the Src homology 3 (SH3) domains of Fyn, Lck, and p60src (Src) but not other proteins. While binding of p120 to Fyn SH2 domain was phosphotyrosine-dependent as expected, its binding to the SH3 domain was independent of tyrosine phosphorylation, as shown by lack of competition with a phosphotyrosyl competitor peptide. In contrast, an SH3-specific proline-rich peptide completely abolished p120 binding to SH3. p120 was tyrosine-phosphorylated within 10 sec following stimulation of Jurkat cells with anti-CD3 monoclonal antibody, with maximal phosphorylation at 30 sec. Importantly, p120 was found associated with Fyn and Lck proteins in unstimulated Jurkat cells and served as an in vitro substrate for these kinases. These results provide evidence for a role of the SH3 domains of Fyn and Lck in the recruitment of early tyrosine-phosphorylation substrates to the TCR-associated tyrosine kinases.

The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis.

Rath A, Davidson AR
Protein Sci. 2000; 9: 2457-69

We have characterized the thermodynamic stability of the SH3 domain from the Saccharomyces cerevisiae Abp1p protein and found it to be relatively low compared to most other SH3 domains, with a Tm of 60 degrees C and a deltaGu of 3.08 kcal/mol. Analysis of a large alignment of SH3 domains led to the identification of atypical residues at eight positions in the wild-type Abp1p SH3 domain sequence that were subsequently replaced by the residue seen most frequently at that position in the alignment. Three of the eight mutants constructed in this way displayed increases in Tm ranging from 8 to 15 degrees C with concomitant increases in deltaGu of up to 1.4 kcal/mol. The effects of these substitutions on folding thermodynamics and kinetics were entirely additive, and a mutant containing all three was dramatically stabilized with a Tm greater than 90 degrees C and a deltaGu more than double that of the wild-type domain. The folding rate of this hyperstable mutant was 10-fold faster than wild-type, while its unfolding rate was fivefold slower. All of the stabilized mutants were still able to bind a target peptide with wild-type affinity. We have analyzed the stabilizing amino acid substitutions isolated in this study and several other similar sequence alignment based studies. In approximately 25% of cases, increased stability can be explained by enhanced propensity of the substituted residue for the local backbone conformation at the mutagenized site.

The proline-rich region of Vav binds to Grb2 and Grb3-3.

Ramos-Morales F, Romero F, Schweighoffer F, Bismuth G, Camonis J, Tortolero M, Fischer S
Oncogene. 1995; 11: 1665-9

Vav has structural features found in signaling proteins and is expressed only in hematopoietic cells. The recent development of mice Vav -/- has confirmed a major role of Vav in early blood cell development. We previously showed that Vav constitutively interacts with glutathione-S-transferase-Grb2. Coimmunoprecipitation experiments supported the idea of a complex formed by Vav-Grb2 in vivo. This complex is of potential interest in signaling of hematopoietic cells. In this work we localize the domains of Vav and Grb2 involved in this interaction. By the use of an in vivo genetic approach (the double hybrid system) and through in vitro experiments (glutathione-S-transferase fusion proteins) we furnish evidence that the interaction between Vav and Grb2 involves the C-SH3 domain of Grb2 and the proline-rich region located in the N-SH3 of Vav. Furthermore this was confirmed by the use of both Vav and Sos derived proline-rich peptides which blocked the binding. In addition we show that Vav also interacts with Grb3-3, a naturally occurring Grb2 isoform wich lacks functional SH2 domain.

Phosphatidylinositol (3,4,5)P3 interacts with SH2 domains and modulates PI 3-kinase association with tyrosine-phosphorylated proteins.

Rameh LE, Chen CS, Cantley LC
Cell. 1995; 83: 821-30

Src homology 2 (SH2) domains on the regulatory subunit of phosphoinositide 3-kinase (PI 3-kinase) mediate its binding to specific tyrosine-phosphorylated proteins in stimulated cells. Using a pharmacological and genetic approach, we show that the amount of PI 3-kinase associated with tyrosine-phosphorylated proteins inversely correlates with the amount of PI 3-kinase lipid products present in the cell. An explanation for this observation is provided by our finding that phosphatidylinositol (3,4,5)trisphosphate (Ptdlns [3,4,5]P3) binds directly and selectively to the SH2 domains of the 85 kDa subunit of PI 3-kinase and thereby blocks binding to tyrosine-phosphorylated proteins. The SH2 domain of pp60C-STC also specifically bound Ptdlns (3,4,5)P3, and the binding was competed by a phosphopeptide specific for the Src SH2 domain. These results indicate that production of Ptdlns (3,4,5)P3 at the membrane disrupts the binding of PI 3-kinase to phosphoproteins. This lipid may also recruit other SH2-containing proteins to the membrane to initiate downstream signaling.

A synthetic peptidic substrate of minimal size and semioptimal sequence for the protein tyrosine kinase pp60c-src.

Ramdas L, Obeyesekere NU, McMurray JS, Budde RJ
Arch Biochem Biophys. 1996; 326: 73-8

We used a novel approach to determine the minimal size and semioptimal sequence of a peptide to serve as an inhibitor and/or substrate for the protein tyrosine kinase pp60c-src. The preferred amino acids surrounding tyrosine were determined by a systematic study in which we increased the length of a series of linear peptides starting from the tripeptide EYG. Using an iterative cycle, the size of the peptide was increased one residue at a time, first at the amino terminus and then at the carboxy terminus. A series of six analogs were synthesized at each position and assayed as inhibitors and substrates. The amino acids G, A, L, F, E, and K were used to semioptimize each position. The tripeptide EYG was not a substrate nor an efficient inhibitor. With increasing size of the peptide, the Ki decreased from 10.0 to 0.10 mM. The smallest peptide to serve as a substrate was a hexapeptide. The best overall peptide obtained from this method, EFEYAFF, had a Ki value of 0.13 mM with Km and Vmax values of 0.21 mM and 680 nmol/min/mg, respectively. Our best peptide was found to have higher substrate specificity than all other commerically available peptidic substrates for pp60c-src.

Structural basis for specificity of Grb2-SH2 revealed by a novel ligand binding mode.

Rahuel J, Gay B, Erdmann D, Strauss A, Garcia-Echeverria C, Furet P, Caravatti G, Fretz H, Schoepfer J, Grutter MG
Nat Struct Biol. 1996; 3: 586-9

Structural basis for the high affinity of amino-aromatic SH2 phosphopeptide ligands.

Rahuel J, Garcia-Echeverria C, Furet P, Strauss A, Caravatti G, Fretz H, Schoepfer J, Gay B
J Mol Biol. 1998; 279: 1013-22

An anthranyl moiety placed at the N terminus of a phosphotyrosine peptide potentiates the inhibitory effect of this small peptide on the binding of the Grb2 SH2 domain to the EGF receptor. Using molecular modeling procedures based on the Lck SH2 domain structure, this observation was rationalized in terms of a suitably favorable pi-pi stacking interaction between the anthranyl moiety and the arginine alphaA2 (ArgalphaA2) residue side-chain of Grb2 SH2. The crystal structure of the Grb2 SH2 domain in complex with the inhibitor 2-Abz-EpYINQ-NH2 (IC50 26 nM) has been solved in two different crystal forms at 2.1 and 1.8 A resolution. This structure confirms the modeling based on the Lck SH2 domain. The ArgalphaA2 residue is conserved in most SH2 domains. Thus, as expected, the anthranyl group also confers high affinity to small peptide ligands of other SH2 domains such as Lck-, PLC-gamma-amino-terminal and p85 amino-terminal SH2 domains as demonstrated by structure affinity relationships (SAR) data. These potent peptides with an amino-terminal surrogate group and the structure of Grb2 SH2 domain in complex with one such peptide represent good starting points for the design and optimization of new inhibitors of many SH2 domains.

Purification of recombinant pp60v-src protein tyrosine kinase and phosphorylation of peptides with different secondary structure preference.

Radziejewski C, Miller WT, Mobashery S, Goldberg AR, Kaiser ET
Biochemistry. 1989; 28: 9047-52

The expression of the transforming gene product of Rous sarcoma virus (pp60v-src) in Saccharomyces cerevisiae has recently been reported (Kornbluth et al., 1987; Brugge et al., 1987). To carry out biochemical and structural studies of this enzyme, a facile purification was developed. The purification was accomplished in four chromatographic steps: Q-Sepharose, Affi-Gel Blue, phosphoagarose, and hydroxylapatite chromatography. The tyrosine kinase was isolated in milligram quantities as two highly active proteolytic fragments (52 and 54 kDa). Three model tyrosine kinase substrates with propensities to adopt helical or omega-loop conformations were synthesized and characterized. The peptides were based on the sites of phosphorylation of pp60v-src, lipocortin I, and lipocortin II. Circular dichroism spectroscopy was used to study the conformation of the helix-forming peptides in 50 mM Tris and in 50% trifluoroethanol/Tris. Peptide 1, which was designed to form an amphiphilic alpha-helix, displayed 24.2% helicity in buffer and 40.2% helicity in 50% TFE/buffer. Similar experiments for peptide 3, the other helix former, showed a lower helicity (8.1% helical and 26.0% helical in buffer and in 50% TFE/buffer, respectively). All three peptides were shown to be substrates for the recombinant tyrosine kinase. Kinetic measurements using high-voltage paper electrophoresis indicated that the helix-forming peptides exhibited low KM values (approximately 450 microM) for the purified src gene product, consistent with the notion that elements of secondary structure may be important in substrate recognition by tyrosine kinases.

High-resolution structure of the diphtheria toxin repressor complexed with cobalt and manganese reveals an SH3-like third domain and suggests a possible role of phosphate as co-corepressor.

Qiu X, Pohl E, Holmes RK, Hol WG
Biochemistry. 1996; 35: 12292-302

The crystal structure of diphtheria toxin repressor (DtxR) in complex with the corepressor Co2+ has been determined at 2.0 A resolution and in complex with Mn2+ at 2.2 A resolution. The structure of the flexible third domain could be determined at this high resolution. It appears to contain five antiparallel strands exhibiting a fold very similar to the SH3 domain. A superposition of 46 equivalent C alpha atoms of DtxR and alpha-spectrin SH3 resulted in an rms deviation of 3.0 A. The sequence identity is only 7%. This third domain of DtxR appears to have no interactions with the DNA binding domain nor with the metal binding domain of the repressor. Yet, flexibility in the region between the second and the third domain allows in principle significant conformational changes such as might occur upon DNA binding. The two metal binding sites in the second domain have been unraveled in considerable detail. Metal binding site 1 was well occupied in both the cobalt and manganese structures and showed a surprising sulfate ion as ligand. The sulfate was proven beyond doubt by the high peak at its position in a selenate versus sulfate difference Fourier. The presence of the intriguing sulfate ion at such a crucial position near the metal corepressor suggests the possibility that under physiological conditions phosphate may act as a "co-corepressor" for this class of metal-regulated DNA binding proteins in Corynebacteria, Mycobacteria, and related organisms. The second metal binding site is significantly different in these two DtxR structures. In the 2.0 A cobalt structure, the site is not occupied by a metal ion. In the 2.2 A manganese structure the site is well occupied, at approximately the same position as observed previously in cadmium DtxR. The ligands are Glu105, His106, the carbonyl oxygen of Cys102, and a water molecule. The reasons for differential occupancy of this site in different structures are intriguing and require further investigations.

Assignment of 1H and 15N resonances of murine Tec SH3 domain.

Pursglove SE, Mulhern TD, Hinds MG, Norton RS, Booker GW
J Biomol NMR. 1998; 12: 461-2

Molecular rulers: an assessment of distance and spatial relationships of Src tyrosine kinase Sh2 and active site regions.

Profit AA, Lee TR, Niu J, Lawrence DS
J Biol Chem. 2001; 276: 9446-51

The three-dimensional structures of the inactive conformations of Hck and Src, members of the Src protein-tyrosine kinase family, have recently been described. In both cases, the catalytic domain lies on the opposite face of the enzyme from the SH2 and SH3 domains. The active conformation of these enzymes has not yet been described. Given the known role of the SH2 and SH3 domains in promoting substrate binding, enzyme activation likely reorients the relative spatial arrangement between the SH2/SH3 domains and the active site region. We describe herein a series of "molecular rulers" and their use in assessing the topological and spatial relationships of the SH2 and active site regions of the Src protein-tyrosine kinase. These synthetic compounds contain sequences that are active site-directed (-Glu-Glu-Ile-Ile-(F(5))Phe-, where (F(5))Phe is pentafluorophenylalanine) and SH2-directed (-Tyr(P)-Glu-Glu-Ile-Glu-), separated by a sequence of variable length. The most potent bivalent compound, acetyl-Glu-Glu-Leu-Leu-(F(5))Phe-(GABA)(3)-Tyr(P)-Glu-Glu-Ile-Glu-amide (where GABA is gamma-aminobutyric acid), displays a >120-fold enhancement in inhibitory potency relative to the simple monovalent active site-directed species, acetyl-Glu-Glu-Leu-Leu-(F(5))Phe-amide. The short linker length (3 GABA residues) between the active site- and SH2-directed peptide fragments suggests that the corresponding domains on the Src kinase can assume a nearly contiguous spatial arrangement in the active form of the enzyme.

Computational binding studies of human pp60c-src SH2 domain with a series of nonpeptide, phosphophenyl-containing ligands.

Price DJ, Jorgensen WL
Bioorg Med Chem Lett. 2000; 10: 2067-70

Monte Carlo/free energy perturbation (MC/FEP) simulations were performed on a series of nonpeptide ligands of the human pp60c-src SH2 domain in order to calculate relative free energies of binding for each compound and to understand the structural requirements for high affinity binding. The amido compound, exhibiting the highest experimental affinity, takes advantage of an interaction with a previously unobserved structural water.

Regulation of protein tyrosine phosphatase 1C: opposing effects of the two src homology 2 domains.

Pregel MJ, Shen SH, Storer AC
Protein Eng. 1995; 8: 1309-16

The regulatory roles of the two src homology 2 (SH2) domains of protein tyrosine phosphatase 1C were investigated by comparing recombinant full-length PTP1C with mutants in which either the N-terminal SH2 (N-SH2) domain (PTP1C delta NSH2), the C-terminal SH2 (C-SH2) domain (PTP1C delta CSH2) or both SH2 domains were deleted (PTP1C delta NSH2 delta CSH2). This revealed that the SH2 domains have opposing and independent effects on activity: strong inhibition by N-SH2 (42-fold) and weak activation by C-SH2 (2.1-fold). C-SH2 caused activation across a wide pH range while N-SH2 inhibited most at neutral and high pH through a shift of the basic limb of the pH profile of kcat/Km, apparently via perturbation of an active-site pKa value. A phosphotyrosyl peptide derived from the erythropoietin receptor caused an approximately 30-fold activation of PTP1C and PTP1C delta CSH2 but had no effect on PTP1C delta NSH2 or PTP1C delta NSH2 delta CSH2, indicating that binding of this peptide to N-SH2 abolished its inhibition. Since C-SH2 separates N-SH2 from the catalytic domain in full-length PTP1C and activation is observed for PTP1C delta CSH2, it appears that the inhibitory effect of N-SH2 is independent of the position in the sequence and that intermolecular interactions may also be possible.

Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.

Prasad KV, Janssen O, Kapeller R, Raab M, Cantley LC, Rudd CE
Proc Natl Acad Sci U S A. 1993; 90: 7366-70

The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex.

Structural basis for the activity of pp60(c-src) protein tyrosine kinase inhibitors.

Prabhu NV, Siddiqui SA, McMurray JS, Pettitt BM
Biopolymers. 2001; 59: 167-79

Conformational searches on three closely related pp60(c-src) protein tyrosine kinase inhibitors of varying potencies were performed to determine a structural basis for their activity. The first was a linear peptide (PDNEYAFFQf), the second its 10-membered cyclic analogue, and the third a cyclic analogue with a para carboxyphenylalanine in place of one the F residues. A common backbone conformation with an antiparallel beta-sheet-like geometry capped by similar beta-turns was found for all three peptides, which may be a binding conformation and gives a candidate pharmacophore for further testing. The interaction between some polar side chains and between some of the aromatic rings may be important for maintaining the correct conformation. The differences in potencies of these inhibitors may be attributed to certain thermodynamic and chemical reasons.

Development of highly selective SH3 binding peptides for Crk and CRKL which disrupt Crk-complexes with DOCK180, SoS and C3G.

Posern G, Zheng J, Knudsen BS, Kardinal C, Muller KB, Voss J, Shishido T, Cowburn D, Cheng G, Wang B, Kruh GD, Burrell SK, Jacobson CA, Lenz DM, Zamborelli TJ, Adermann K, Hanafusa H, Feller SM
Oncogene. 1998; 16: 1903-12

Many Src Homology 3 (SH3) domains function as molecular adhesives in intracellular signal transduction. Based on previous ultrastructural studies, short motifs which bind to the first SH3 domains of the adapters Crk and CRKL were selectively mutagenised to generate Crk/CRKL SH3-binding peptides of very high affinity and selectivity. Affinities were increased up to 20-fold compared to the best wildtype sequences, while the selectivity against a similar SH3 domain [Grb2SH3(N)] was not only retained, but sometimes increased. Blot techniques with GST-fusion peptides and in solution precipitation assays with biotinylated high affinity Crk binding peptides (HACBPs) were subsequently used to analyse the binding of these sequences to a large panel of SH3 domain-containing fusion proteins. Only those proteins which contained the CrkSH3(1) or CRKLSH3(1) domains bound efficiently to the HACBPs. A GST-HACBP fusion protein precipitated Crk and CRKL proteins out of 35S-labelled and unlabelled cell lysates. Very little binding of other cellular proteins to HACBP was detectable, indicative of a great preference for Crk and CRKL when compared to the wide variety of other endogenous cellular proteins. Moreover, HACBP disrupted in vitro preexisting Crk-complexes with DOCK180 and the exchange factors SoS and C3G, which are known targets of Crk adapters, in a concentration dependent manner. HACBP-based molecules should therefore be useful as highly selective inhibitors of intracellular signalling processes involving Crk and CRKL.

Reciprocal regulation of Hck activity by phosphorylation of Tyr(527) and Tyr(416). Effect of introducing a high affinity intramolecular SH2 ligand.

Porter M, Schindler T, Kuriyan J, Miller WT
J Biol Chem. 2000; 275: 2721-6

The Src family tyrosine kinase Hck possesses two phosphorylation sites, Tyr(527) and Tyr(416), that affect the catalytic activity in opposite ways. When phosphorylated, Tyr(527) and residues C-terminal to it are involved in an inhibitory intramolecular interaction with the SH2 domain. However, this sequence does not conform to the sequence of the high affinity SH2 ligand, pYEEI. We mutated this sequence to YEEI and show that this mutant form of Hck cannot be activated by exogenous SH2 ligands. The SH3 domain of Hck is also involved in an inhibitory interaction with the catalytic domain. The SH3 ligand Nef binds to and activates YEEI-Hck mutant in a similar manner to wild-type Hck, indicating that disrupting the SH3 interaction overrides the strengthened SH2 interaction. The other phosphorylation site, Tyr(416), is the autophosphorylation site in the activation loop. Phosphorylation of Tyr(416) is required for Hck activation. We mutated this residue to alanine and characterized its catalytic activity. The Y416A mutant shows a higher K(m) value for peptide and a lower V(max) than autophosphorylated wild-type Hck. We also present evidence for cross-talk between the activation loop and the intramolecular binding of the SH2 and SH3 domains.

Hydrophobic D-amino acids in the design of peptide ligands for the pp60src SH2 domain.

Plummer MS, Lunney EA, Para KS, Prasad JV, Shahripour A, Singh J, Stankovic CJ, Humblet C, Fergus JH, Marks JS, Sawyer TK
Drug Des Discov. 1996; 13: 75-81

Src homology-2 (SH2) domains, containing approximately 100 amino acid residues, are noncatalytic motifs involved with intracellular signal transduction. These domains can be found on nonreceptor kinases, phosphatases, and in regulatory adapter proteins among others. SH2 domains bind proteins containing phosphotyrosine (pTyr) residues in a sequence specific manner. Our efforts have focused on designing peptide mimetic ligands for the SH2 domain of the nonreceptor tyrosine kinase pp60src. We employed the cocrystal structure of the 11mer Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-IIe-Tyr-Leu IC50 = 800 nM as a starting point for our design efforts. These efforts have resulted in the discovery of tripeptide ligands containing D-amino acids that are only 2-fold less potent than the 11mer.

Design, synthesis, and cocrystal structure of a nonpeptide Src SH2 domain ligand.

Plummer MS, Holland DR, Shahripour A, Lunney EA, Fergus JH, Marks JS, McConnell P, Mueller WT, Sawyer TK
J Med Chem. 1997; 40: 3719-25

The specific association of an SH2 domain with a phosphotyrosine (pTyr)-containing sequence of another protein precipitates a cascade of intracellular molecular interactions (signals) which effect a wide range of intracellular processes. The nonreceptor tyrosine kinase Src, which has been associated with breast cancer and osteoporosis, contains an SH2 domain. Inhibition of Src SH2-phosphoprotein interactions by small molecules will aid biological proof-of-concept studies which may lead to the development of novel therapeutic agents. Structure-based design efforts have focused on reducing the size and charge of Src SH2 ligands while increasing their ability to penetrate cells and reach the intracellular Src SH2 domain target. In this report we describe the synthesis, binding affinity, and Src SH2 cocrystal structure of a small, novel, nonpeptide, urea-containing SH2 domain ligand.

Activation of phosphatidylinositol-3' kinase by Src-family kinase SH3 binding to the p85 subunit.

Pleiman CM, Hertz WM, Cambier JC
Science. 1994; 263: 1609-12

Engagement of antigen receptor complexes induces rapid activation of Src-family kinases and association with phosphatidylinositol-3' kinase (PI-3 kinase). Here it was found that the Src homology 3 (SH3) domain of Lyn and Fyn bound to a proline-rich region (residues 84 to 99) within the 85-kilodalton subunit (p85) of PI-3 kinase. The binding of SH3 to the purified kinase led to a five- to sevenfold increase in the specific activity of PI-3 kinase. Ligand-induced receptor stimulation activated PI-3 kinase, and this activation was blocked by a peptide containing residues 84 to 99 of p85. These data demonstrate a mechanism for PI-3 kinase activation and show that binding of SH3 domains to proline-rich target sequences can regulate enzymatic activity.

The folding kinetics and thermodynamics of the Fyn-SH3 domain.

Plaxco KW, Guijarro JI, Morton CJ, Pitkeathly M, Campbell ID, Dobson CM
Biochemistry. 1998; 37: 2529-37

The equilibrium unfolding and the kinetic folding and unfolding of the 67 residue Fyn-SH3 domain have been investigated. Equilibrium unfolding experiments indicate that, despite the lack of both disulfide bonds and prosthetic groups, Fyn-SH3 is relatively stable with a free energy of folding of -6.0 +/- 0.6 kcal mol-1 at 20 degrees C. Kinetic experiments indicate that the domain refolds in a rapid two-state manner without significant population of intermediates (k = 94.3 s-1 in H2O at 20 degrees C). Despite the presence of two proline residues, the refolding of the domain is monophasic, and no significant proline isomerization-like refolding phase is observed. This can be attributed to an extremely low level of the incorrect (cis) isomer of the structurally important Pro134 residue in the protein denatured in 8 M guanidine hydrochloride. Analysis of the temperature and guanidine hydrochloride dependence of the folding rate suggests that the folding transition state of this protein is relatively well organized. A comparison with the refolding kinetics and thermodynamics of other homologous SH3 domains indicates that these exhibit an equivalent degree of transition state organization. This potentially arises from conservation of key features of the transition state conformation despite sometimes relatively low overall sequence identity. Such a comparison further suggests that relative thermodynamic stability is an important factor in determining the relative folding rates of natural proteins with a common fold, but that specific details of the amino acid sequence can also play a significant role in individual cases.

Characterization of the proline-rich region of mouse MAPKAP kinase 2: influence on catalytic properties and binding to the c-abl SH3 domain in vitro.

Plath K, Engel K, Schwedersky G, Gaestel M
Biochem Biophys Res Commun. 1994; 203: 1188-94

The primary structure of mouse MAP kinase-activated protein (MAPKAP) kinase 2 contains a proline-rich N-terminal region which might function as a src-homology 3 (SH3) domain-binding motif in vivo. To demonstrate the ability of this region to bind SH3 domains, we analyzed the interaction of the SH3 domain of the protein tyrosine kinase c-abl with MAPKAP kinase 2. It is demonstrated, that the proline-rich region specifically binds c-abl-SH3 domain in vitro. Furthermore, it is shown, that deletion of this proline-rich region does not significantly influence the substrate binding properties of the enzyme when analyzed with the substrate small heat shock protein Hsp25. The data suggest that the proline-rich region of MAPKAP kinase 2 could interact with proteins containing SH3-domains also in vivo regulating its cellular localization and/or modulating its enzymatic properties.

Crystal structure of the abl-SH3 domain complexed with a designed high-affinity peptide ligand: implications for SH3-ligand interactions.

Pisabarro MT, Serrano L, Wilmanns M
J Mol Biol. 1998; 281: 513-21

The Abl-SH3 domain is implicated in negative regulation of the Abl kinase by mediating protein-protein interactions. High-affinity SH3 ligands could compete for these interactions and specifically activate the Abl kinase, providing control and a better understanding of the molecular interactions that underlie diseases where SH3 domains are involved. The p41 peptide (APSYSPPPPP) is a member of a group of peptide ligands designed to bind specifically the Abl-SH3 domain. It binds to Abl-SH3 with a Kd of 1.5 microM, whereas its affinity for the Fyn-SH3 domain is 273 microM. We have determined the crystal structure of the Abl-SH3 domain in complex with the high-affinity peptide ligand p41 at 1.6 A resolution. In the crystal structure, this peptide adopts a polyproline type II helix conformation through residue 5 to 10, and it binds in type I orientation to the Abl-SH3 domain. The tyrosine side-chain in position 4 of the peptide is hydrogen bonded to two residues in the RT-loop of the Abl-SH3 domain. The tight fit of this side-chain into the RT-loop pocket is enhanced by conformational adjustment of the main chain at position 5. The SH3 ligand peptides can be divided into two distinct parts. The N-terminal part binds to the SH3 domain in the region formed by the valley between the nSrc and RT-loops. It determines the specificity for different SH3 domains. The C-terminal part adopts a polyproline type II helix conformation. This binds to a well-conserved hydrophobic surface of the SH3 domain. Analysis of two "half"-peptides, corresponding to these ligand parts, shows that both are essential components for strong binding to the SH3 domains. The crystal structure of the Abl-SH3:p41 complex explains the high affinity and specificity of the p41 peptide towards the Abl-SH3 domain, and reveals principles that will be exploited for future design of small, high-affinity ligands to interfere efficiently with the in vivo regulation of Abl kinase activity.

Rational design of specific high-affinity peptide ligands for the Abl-SH3 domain.

Pisabarro MT, Serrano L
Biochemistry. 1996; 35: 10634-40

SH3 domains bind proline-rich peptides with affinities in the order of 0.2-50 microM. In general, these domains are quite promiscuous, and the same peptide can bind to several different SH3 domains with similar affinities (i.e., 3BP1 peptide to Abl- and Fyn-SH3). This poor affinity and specificity make it difficult to elucidate their role in vivo as well as the use of peptides to specifically bind to a single domain. Here, we report that by using existing biocomputing tools, as well as simple physicochemical reasoning, it is possible to design mutations in the 3BP1 peptide (Met4-Tyr, Pro5-Ser, and Leu8-Pro), so that the affinity for Abl-SH3 increases 20-fold (p40 peptide: APTYSPPPPP; Kd = 0.4 microM), while that for the closely related domain, Fyn-SH3, decreases 10-fold. Both the RT and n-Src loops are responsible for regulating the specificity for Pro-rich ligands and more specifically residues Ser15, Thr19, and Glu38 in Abl-SH3. The first six positions in the 3BP1 peptide are important for determining the specificity for SH3 domains, while the remaining four seem to be more important for the affinity. Moreover, by choosing rationally the substituents, it is possible to replace some of the Pro residues postulated to be essential for the interaction with SH3 domains and still have a significant affinity. This indicates that the sequence repertoire that could interact with a specific SH3 domain could be larger than previously thought.

Molecular modeling of the interaction of polyproline-based peptides with the Abl-SH3 domain: rational modification of the interaction.

Pisabarro MT, Ortiz AR, Viguera AR, Gago F, Serrano L
Protein Eng. 1994; 7: 1455-62

A molecular model of the interaction of polyproline-rich peptides with the Abl-SH3 domain is proposed, based on docking calculations with the DOCK program coupled with molecular dynamics simulations. Two distinct binding modes of the peptide to the same aromatic-rich region (Tyr10, Phe12, Trp39, Trp50, Tyr55) of the domain were obtained. It is proposed that these two models could represent different binding modes of proline-rich peptides to Src homology region 3 domains. Several peptide mutants were designed to determine whether the two orientations were possible. Analysis of the Kd values and fluorescence emission of these peptides indicate that one of the orientations is more plausible and that residues at position 4 of the peptide interact with the RT loop, being important in modulating the peptide affinity for the Abl-SH3 domain.

Homology modeling of the Abl-SH3 domain.

Pisabarro MT, Ortiz AR, Serrano L, Wade RC
Proteins. 1994; 20: 203-15

A tertiary structure model of the Abl-SH3 domain is predicted by using homology modeling techniques coupled to molecular dynamics simulations. Two template proteins were used, Fyn-SH3 and Spc-SH3. The refined model was extensively checked for errors using criteria based on stereochemistry, packing, solvation free-energy, accessible surface areas, and contact analyses. The different checking methods do not totally agree, as each one evaluates a different characteristic of protein structures. Several zones of the protein are more susceptible to incorporating errors. These include residues 13, 15, 35, 39, 45, 46, 50, and 60. An interesting finding is that the measurement of the C alpha chirality correlated well with the rest of the criteria, suggesting that this parameter might be a good indicator of correct local conformation. Deviations of more than 4 degrees may be indicative of poor local structure.

Solution studies of the SH2 domain from the fyn tyrosine kinase: secondary structure, backbone dynamics and protein association.

Pintar A, Hensmann M, Jumel K, Pitkeathly M, Harding SE, Campbell ID
Eur Biophys J. 1996; 24: 371-80

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155-270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of 1H-15N-correlated 2D HSQC, 15N-edited 3D TOCSY-HMQC, and 15N-edited 3D NOESY-HMQC spectra. By analysis of the alpha-proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain. To investigate the internal dynamics of the protein backbone, T1 and T2 relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.

SH3GLB, a new endophilin-related protein family featuring an SH3 domain.

Pierrat B, Simonen M, Cueto M, Mestan J, Ferrigno P, Heim J
Genomics. 2001; 71: 222-34

A new cDNA encoding a protein of 362 amino acids designated SH3GLB1, for SH3 domain GRB2-like endophilin B1, was identified in a yeast two-hybrid screen devoted to the identification of new partners interacting with the apoptosis inducer Bax. SH3GLB1 shows strong similarities to the SH3 domain-containing proteins of the endophilin family and presumably represents the human homologue of the potential Caenorhabditis elegans SH3 containing-protein identified by systematic translation of the C. elegans genome (GenBank Accession No. U46675). Reversing prey to bait in the yeast screen, a second protein, SH3GLB2, of 395 amino acids showing 65% identity to SH3GLB1 was identified as an interacting partner of SH3GLB1. The discovery of SH3GLB1 itself in the screening with SH3GLB1 as a bait and further mapping experiments demonstrated that a core coiled-coil-type region is required for the formation of SH3GLB homo- and/or heterodimers, whereas the SH3 domain is not involved in these interactions. Interestingly, the similarities with the endophilin proteins cover the entire sequence of the SH3GLB family, suggesting a common fold and presumably a common mode of action. Furthermore, SH3GLB members colocalize to the cytoplasmic compartment of the cell together with Bax and are excluded from the nucleus. SH3GLB1 and SH3GLB2 do not significantly influence the onset and time course of Bax-mediated apoptosis in HeLa or 293T cells. Copyright 2001 Academic Press.

The synthesis and use of pp60src-related peptides and phosphopeptides as substrates for enzymatic phosphorylation studies.

Perich JW, Meggio F, Valerio RM, Johns RB, Pinna LA, Reynolds EC
Bioorg Med Chem. 1993; 1: 381-8

A series of peptides and phosphopeptides corresponding to the auto-phosphorylation site of pp60src, -Asn-Glu-Tyr416-Thr-Ala-, were prepared by either Boc/solution or Fmoc/solid phase peptide synthesis and used as substrates to study their enzymatic phosphorylation by various casein kinases. The Tyr(P)-containing peptide, Asn-Glu-Tyr(P)-Thr-Ala, was prepared by the use of Fmoc-Tyr(PO3Bzl2)-OH in Fmoc/solid phase peptide synthesis followed by acidolytic treatment of the peptide-resin with 5% anisole/CF3CO2H. Both Asn-Glu-Tyr-Thr-Ala and Asn-Glu-Ser(P)-Thr-Ala were prepared by the Boc/solution phase peptide synthesis and employed hydrogenolytic deprotection of the protected peptides. Enzymatic phosphorylation studies established that (A) the Tyr residue acted as an unusual positive determinant for directing phosphorylation to the Thr-residue, (B) the rate of Thr-phosphorylation was markedly facilitated by a change from the Tyr-residue to the Tyr(P)-residue, and (C) a Ser(P)-residue was as effective as the Tyr(P)-residue in facilitating Thr-phosphorylation. A subsequent structure-function study using Asn-Glu-Phe-Thr-Ala, Asn-Glu-Tyr(Me)-Thr-Ala (prepared by Fmoc/solid phase peptide synthesis) and Asn-Glu-Cha-Thr-Ala (prepared by hydrogenation of Asn-Glu-Tyr-Thr-Ala) established that the rate of Thr-phosphorylation was influenced by the extent of hydrophobic-hydrophobic interactions by the aralkyl side-chain group (either aromatic or aliphatic) of the 416-residue with casein kinase-2; the rate of Thr-phosphorylation being decreased by the introduction of methyl or hydroxyl groups at the 4-position of the aromatic group (i.e. Tyr(Me) and Tyr respectively) but enhanced by the introduction of the hydrophilic phosphate group (i.e. as Tyr(P)).

Enhanced phosphorylation of Src family kinase substrates containing SH2 domain binding sites.

Pellicena P, Stowell KR, Miller WT
J Biol Chem. 1998; 273: 15325-8

Src family protein-tyrosine kinases possess several modular domains important for regulation of catalytic activity and interaction with potential substrates. Here, we explore interactions between the SH2 domain of Hck, a Src family kinase, and substrates containing SH2 domain-binding sites. We have synthesized a series of peptide substrates containing a high affinity SH2 domain binding site, (phospho)Tyr-Glu-Glu-Ile. We show that the presence of this sequence in a peptide results in a dramatic increase in the phosphorylation rate of a second tyrosine located at the N terminus. Enhanced phosphorylation is not a consequence of stimulation of enzymatic activity by C-terminal tail displacement but is imparted instead by a 10-fold reduction in the Km of the phosphotyrosine-containing peptide when compared with a control. The isolated catalytic domain of the non-receptor tyrosine kinase Abl does not show a preference for the pYEEI motif-containing peptide; however, the preference is restored when the SH2 domain of Src is introduced into Abl. Furthermore, enhanced phosphorylation is dependent on the distance between SH2 domain-binding site and phosphorylatable tyrosine, with the minimum distance requirement being seven amino acids. Reversing the orientation of the pYEEI motif with respect to the substrate sequence decreases phosphorylation by down-regulated Hck, but both orientations are utilized equally well by activated Hck. We discuss the possible implications of these results for processive phosphorylation of substrates in vivo by Src family kinases.

CD43-specific activation of T cells induces association of CD43 to Fyn kinase.

Pedraza-Alva G, Merida LB, Burakoff SJ, Rosenstein Y
J Biol Chem. 1996; 271: 27564-8

CD43, the most abundant membrane protein of T lymphocytes, is able to initiate signal transduction pathways that lead to Ca2+ mobilization and interleukin-2 production, yet the molecular events involved in CD43's signal transduction pathway are poorly understood. In the present report we show that activation of both purified T lymphocytes and Jurkat cells, through CD43 cross-linking with the anti-CD43 L10 monoclonal antibody, induced CD43 association to Fyn kinase. This association is mediated by the Src homology 3 (SH3) domain of Fyn, since a glutathione S-transferase-Fyn SH3 fusion protein was able to precipitate CD43 from lysates of CD43-activated T cells. A synthetic peptide containing the SH3 binding sites of p85, located within the amino acid sequence 300ERQPAPALPPKPPKP314, was able to inhibit binding of CD43 to Fyn as well as to the glutathione S-transferase-Fyn SH3 fusion protein. We also provide evidence that upon CD43 cross-linking, Fyn is tyrosine-phosphorylated in a time-dependent manner. Our results suggest that CD43 cross-linking on the T cell surface induces the interaction between CD43 and Fyn, presumably through the Fyn SH3 domain and a putative SH3 binding site in CD43, leading to Fyn tyrosine phosphorylation and signal propagation.

The phosphopeptide-binding specificity of Src family SH2 domains.

Payne G, Stolz LA, Pei D, Band H, Shoelson SE, Walsh CT
Chem Biol. 1994; 1: 99-105

BACKGROUND: Src homology 2 (SH2) domains mediate protein/protein interactions by binding phosphotyrosyl proteins with high specificity. The protein Lck, a Src-like lymphocyte-specific tyrosine kinase which is important in signals involved in T-cell development, contains one such domain. The crystal structure of a complex of the Lck SH2 domain with a high-affinity ligand, pY324, is known. This ligand has the sequence EPQpYEEIPIYL. RESULTS: We designed and synthesized a series of phosphopeptides with single amino-acid changes in the four residues C-terminal to the phosphotyrosine (pTyr) in pY324. Surprisingly, the Glu one residue C-terminal to the phosphotyrosine (at position pY + 1) is sensitive to substitution, whereas the Ile at position pY + 3 is much less sensitive, accommodating a Glu with only modest loss of binding affinity. Replacement of the Glu and Pro on either side of the Ile had little effect, as predicted. Truncated phosphopeptides that end at position pY + 5 and have only an acetyl group N-terminal to the pTyr bound with only slightly lower affinity than pY324. In addition, naturally occurring phosphopeptide sequences that span a 1,000-fold range in binding affinity for the Lck SH2 domain have been identified. CONCLUSIONS: The Lck SH2 domain is highly selective for phosphotyrosyl-peptide binding; its specificity is dictated by the first and third residues C-terminal to the pTyr. The unexpected effects of some amino-acid substitutions indicate that the interactions seen between SH2 domains and ligand in the crystal structure may not be identical to those that occur in solution.

Kinetics of p56lck and p60src Src homology 2 domain binding to tyrosine-phosphorylated peptides determined by a competition assay or surface plasmon resonance.

Payne G, Shoelson SE, Gish GD, Pawson T, Walsh CT
Proc Natl Acad Sci U S A. 1993; 90: 4902-6

Src homology 2 (SH2) domains are phosphotyrosine-binding modules found within various signal-transducing proteins. We have determined by 125I competition assay and surface plasmon resonance that the SH2 domains of Src and Lck bind to a variety of phosphopeptides with similar affinity and specificity. Both bound with highest affinity [Kd(app) approximately 3.7 nM; ka = 2.4 x 10(5) M-1 x s-1; kd = 1.2 x 10(-3) s-1] a phosphopeptide having a Tyr(P)-Glu-Glu-Ile motif found in the hamster polyomavirus middle-sized tumor antigen. Intermediate affinity (5- to 40-fold lower) was observed with phosphopeptides corresponding to the regulatory domains of Src and Lck, containing Tyr527 and Tyr505, respectively. Lowest affinity (80- to 300-fold lower) was observed with phosphopeptides corresponding to phosphorylated tyrosines of GTPase-activating protein, insulin receptor substrate 1, and SH2 domain-containing protein-tyrosine-phosphatase 1.

Protein modules and signalling networks.

Pawson T
Nature. 1995; 373: 573-80

Communication between cells assumes particular importance in multicellular organisms. The growth, migration and differentiation of cells in the embryo, and their organization into specific tissues, depend on signals transmitted from one cell to another. In the adult, cell signalling orchestrates normal cellular behaviour and responses to wounding and infection. The consequences of breakdowns in this signalling underlie cancer, diabetes and disorders of the immune and cardiovascular systems. Conserved protein domains that act as key regulatory participants in many of these different signalling pathways are highlighted.

New impressions of Src and Hck.

Pawson T
Nature. 1997; 385: 582-3

SH3 domain of Bruton's tyrosine kinase can bind to proline-rich peptides of TH domain of the kinase and p120cbl.

Patel HV, Tzeng SR, Liao CY, Chen SH, Cheng JW
Proteins. 1997; 29: 545-52

X-linked agammaglobulinemia (XLA), an inherited disease, is caused by mutations in the Bruton's tyrosine kinase (BTK). The absence of functional BTK leads to failure of B-cell differentiation; this incapacitates antibody production in XLA patients, who suffer from recurrent, sometimes lethal, bacterial infections. BTK plays an important role in B-cell development; it interacts with several proteins in the context of signal transduction. Point mutation in the BTK gene that leads to deletion of C-terminal 14 aa residues of BTK SH3 domain was found in a patient family. To understand the role of BTK, we studied binding of BTK SH3 domain (aa 216-273, 58 residues) and truncated SH3 domain (216-259, 44 residues) with proline-rich peptides; the first peptide constitutes the SH3 domain of BTK, while the latter peptide lacks 14 amino acid residues of the C terminal. Proline-rich peptides selected from TH domain of BTK and p120cbl were studied. It is known that BTK TH domain binds to SH3 domains of various proteins. We found that BTK SH3 domain binds to peptides of BTK TH domain. This suggests that BTK SH3 and TH domains may associate in inter- or intramolecular fashion, which raises the possibility that the kinase may be regulating its own activity by restricting the availability of both its ligand-binding modules. We also found that truncated SH3 domain binds to BTK TH domain peptide less avidly than does normal SH3 domain. Also, we show that the SH3 and truncated SH3 domains bind to peptide of p120cbl, but the latter domain binds weakly. It is likely that the truncated SH3 domain fails to present to the ligand the crucial residues in the correct context, hence the weaker binding. These results delineate the importance of C-terminal in binding of SH3 domains and indicate also that improper folding and the altered binding behavior of mutant BTK SH3 domain likely leads to XLA.

Structural and dynamic characterization of the phosphotyrosine binding region of a Src homology 2 domain--phosphopeptide complex by NMR relaxation, proton exchange, and chemical shift approaches.

Pascal SM, Yamazaki T, Singer AU, Kay LE, Forman-Kay JD
Biochemistry. 1995; 34: 11353-62

Arginine side chains are often involved in protein--protein and protein--nucleic acid interactions. Due to a number of factors, resonance assignment and detection of NOEs involving the arginine side chains via standard NMR techniques can be difficult. We present here an approach to characterization of the interaction between a phosphopeptide (pY1021) and four arginine residues that line the phosphotyrosine-binding pocket of the C-terminal SH2 domain of phospholipase C-gamma 1 (PLCC SH2). Previously published [Pascal, S. M., et al. (1994) Cell 77, 461] NOE data provide a partial description of this interaction, including contacts between the aliphatic region of Arg 59 and the phosphotyrosine (pTyr) aromatic ring. Further characterization has now been accomplished by using 15N and 13C NMR relaxation studies of arginine N episilon and C zeta spins, respectively, and proton exchange rates of arginine H episilon nuclei. Differences between the chemical shifts of the arginine guanidino groups of the free SH2 domain in imidazole and phosphate buffers or in complex with pY1021 have provided insight into specific interactions with the phosphate and the aromatic ring of the pTyr. The resulting data are consistent with the most stable hydrogen bonds to phosphate donated by the Arg 39 epsilon-NH and the two Arg 37 eta-NH2 groups and with pTyr aromatic ring interactions involving the Arg 39 and possibly the Arg 18 guanidino groups.

Engineering temperature-sensitive SH3 domains.

Parrini MC, Mayer BJ
Chem Biol. 1999; 6: 679-87

BACKGROUND: The ability to control specific protein-protein interactions conditionally in vivo would be extremely helpful for analyzing protein-protein interaction networks. SH3 (Src homology 3) modular protein binding domains are found in many signaling proteins and they play a crucial role in signal transduction by binding to proline-rich sequences. RESULTS: Random in vitro mutagenesis coupled with yeast two-hybrid screening was used to identify mutations in the second SH3 domain of Nck that render interaction with its ligand temperature sensitive. Four of the mutants were functionally temperature sensitive in mammalian cells, where temperature sensitivity was correlated with a pronounced instability of the mutant domains at the nonpermissive temperature. Two of the mutations affect conserved residues in the hydrophobic core (Val133 and Val160), suggesting a general strategy for engineering temperature-sensitive SH3-containing proteins. Indeed mutagenesis of the corresponding positions in another SH3 domain, that of Crk-1, rendered the full-length Crk-1 protein temperature sensitive for function and stability in mammalian cells. CONCLUSIONS: Construction of temperature-sensitive SH3 domains is a novel approach to regulating the function of SH3 domains in vivo. Such mutants will be valuable in dissecting SH3-mediated signaling pathways. Furthermore, the methodology described here to isolate temperature-sensitive domains should be widely applicable to any domain involved in protein-protein interactions.

Regulation of Btk function by a major autophosphorylation site within the SH3 domain.

Park H, Wahl MI, Afar DE, Turck CW, Rawlings DJ, Tam C, Scharenberg AM, Kinet JP, Witte ON
Immunity. 1996; 4: 515-25

Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development. Overexpression of Btk with a Src family kinase increases tyrosine phosphorylation and catalytic activity of Btk. This occurs by transphosphorylation at Y551 in the Btk catalytic domain and the enhancement of Btk autophosphorylation at a second site. A gain-of-function mutant called Btk* containing E41 to K change within the pleckstrin homology domain induces fibroblast transformation. Btk* enhances the transphosphorylation of Y551 by endogenous Src family tyrosine kinases and autophosphorylation at the second site. We mapped the major Btk autophosphorylation site to Y223 within the SH3 domain. Mutation of Y223 to F blocks Btk autophosphorylation and dramatically potentiates the transforming activity of Btk* in fibroblasts. The location of Y223 in a potential ligand-binding pocket suggests that autophosphorylation regulates SH3-mediated signaling by Btk.

Son of sevenless binds to the SH3 domain of src-type tyrosine kinase.

Park C, Choi Y, Yun Y
Mol Cells. 1998; 8: 518-23

To identify molecules which bind to the SH3 domains of p56lck, we screened a mouse T-cell lymphoma cDNA library using the yeast two-hybrid system. As a result, we obtained several positive clones including the Son of Sevenless gene which encodes a mammalian homolog of Drosophila Ras GDP/GTP exchange factor. In a subsequent analysis with the yeast two-hybrid system, Sos associated only with the constitutively active form of p56lck (F505) but not with wild type p56lck (Y505), indicating the requirement for an active conformation of p56lck for binding to Sos. Subsequently, we have demonstrated in vitro that the SH3 domain of p56lck as well as the proline-rich sequences of Sos are responsible for this association. In addition, the proline-rich domain of Sos also bound to the SH3 domains of other src-type tyrosine kinases, src and fyn, but not to that of PLC-gamma. More importantly, the p56lck SH3-Sos interaction was enhanced by serum stimulation, suggesting the possibility that the direct interaction between p56lck SH3 and Sos may contribute to the regulation of the Ras pathway.

Characterization of a novel Src-like adapter protein that associates with the Eck receptor tyrosine kinase.

Pandey A, Duan H, Dixit VM
J Biol Chem. 1995; 270: 19201-4

The Eph family of receptor protein tyrosine kinases (RPTKs) is the largest family of RPTKs. The signal transduction pathways initiated by this family have only recently begun to be explored. Using a yeast two-hybrid screen to identify molecules that interact with the cytoplasmic domain of Eck, it was previously shown that activated Eck RPTK bound to and stimulated phosphatidylinositol 3-kinase (Pandey, A., Lazar, D.F., Saltiel, A. R., and Dixit, V.M. (1994) J. Biol. Chem. 269, 30154-30157). Also isolated from this same screen was a novel protein containing SH3 and SH2 adapter modules that had striking homology to those found in the Src family of non-receptor tyrosine kinases. However, unlike other Src family members, it lacked a catalytic tyrosine kinase domain. Hence, this protein was designated SLAP for Src-like adapter protein. Using glutathione S-transferase fusion Proteins, it was demonstrated that SLAP bound to activated Eck receptor tyrosine kinase. Therefore, SLAP is a novel candidate downstream signaling intermediate and the first member of the Src family that resembles an adapter molecule.

Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn.

Panchamoorthy G, Fukazawa T, Stolz L, Payne G, Reedquist K, Shoelson S, Zhou S, Cantley L, Walsh C, Band H
Mol Cell Biol. 1994; 14: 6372-85

The Src family protein tyrosine kinases participate in signalling through cell surface receptors that lack intrinsic tyrosine kinase domains. All nine members of this family possess adjacent Src homology (SH2 and SH3) domains, both of which are essential for repression of the enzymatic activity. The repression is mediated by binding between the SH2 domain and a C-terminal phosphotyrosine, and the SH3 domain is required for this interaction. However, the biochemical basis of functional SH2-SH3 interaction is unclear. Here, we demonstrate that when the SH2 and SH3 domains of p59fyn (Fyn) were present as adjacent domains in a single protein, binding of phosphotyrosyl peptides and proteins to the SH2 domain was enhanced, whereas binding of a subset of cellular polypeptide ligands to the SH3 domain was decreased. An interdomain communication was further revealed by occupancy with domain-specific peptide ligands: occupancy of the SH3 domain with a proline-rich peptide enhanced phosphotyrosine binding to the linked SH2 domain, and occupancy of the SH2 domain with phosphotyrosyl peptides enhanced binding of certain SH3-specific cellular polypeptides. Second, we demonstrate a direct binding between purified SH2 and SH3 domains of Fyn and Lck Src family kinases. Heterologous binding between SH2 and SH3 domains of closely related members of the Src family, namely, Fyn, Lck, and Src, was also observed. In contrast, Grb2, Crk, Abl, p85 phosphatidylinositol 3-kinase, and GTPase-activating protein SH2 domains showed lower or no binding to Fyn or Lck SH3 domains. SH2-SH3 binding did not require an intact phosphotyrosine binding pocket on the SH2 domain; however, perturbations of the SH2 domain induced by specific high-affinity phosphotyrosyl peptide binding abrogated binding of the SH3 domain. SH3-SH2 binding was observed in the presence of proline-rich peptides or when a point mutation (W119K) was introduced in the putative ligand-binding pouch of the Fyn SH3 domain, although these treatments completely abolished the binding to p85 phosphatidylinositol 3-kinase and other SH3-specific polypeptides. These biochemical SH2-SH3 interactions suggest novel mechanisms of regulating the enzymatic activity of Src kinases and their interactions with other proteins.

Potent dipeptide inhibitors of the pp60c-src SH2 domain.

Pacofsky GJ, Lackey K, Alligood KJ, Berman J, Charifson PS, Crosby RM, Dorsey GF Jr, Feldman PL, Gilmer TM, Hummel CW, Jordan SR, Mohr C, Shewchuk LM, Sternbach DD, Rodriguez M
J Med Chem. 1998; 41: 1894-908

The design, synthesis, and evaluation of dipeptide analogues as ligands for the pp60c-src SH2 domain are described. The critical binding interactions between Ac-Tyr-Glu-N(n-C5H11)2 (2) and the protein are established and form the basis for our structure-based drug design efforts. The effects of changes in both the C-terminal (11-27) and N-terminal (51-69) portions of the dipeptide are explored. Analogues with reduced overall charge (92-95) are also investigated. We demonstrate the feasibility of pairing structurally diverse subunits in a modest dipeptide framework with the goal of increasing the druglike attributes without sacrificing binding affinity.

Crystal structure of the amphiphysin-2 SH3 domain and its role in the prevention of dynamin ring formation.

Owen DJ, Wigge P, Vallis Y, Moore JD, Evans PR, McMahon HT
EMBO J. 1998; 17: 5273-85

The amphiphysins are brain-enriched proteins, implicated in clathrin-mediated endocytosis, that interact with dynamin through their SH3 domains. To elucidate the nature of this interaction, we have solved the crystal structure of the amphiphysin-2 (Amph2) SH3 domain to 2.2 A. The structure possesses several notable features, including an extensive patch of negative electrostatic potential covering a large portion of its dynamin binding site. This patch accounts for the specific requirement of amphiphysin for two arginines in the proline-rich binding motif to which it binds on dynamin. We demonstrate that the interaction of dynamin with amphiphysin SH3 domains, unlike that with SH3 domains of Grb2 or spectrin, prevents dynamin self-assembly into rings. Deletion of a unique insert in the n-Src loop of Amph2 SH3, a loop adjacent to the dynamin binding site, significantly reduces this effect. Conversely, replacing the n-Src loop of the N-terminal SH3 domain of Grb2 with that of Amph2 causes it to favour dynamin ring disassembly. Transferrin uptake assays show that shortening the n-Src loop of Amph2 SH3 reduces the ability of this domain to inhibit endocytosis in vivo. Our data suggest that amphiphysin SH3 domains are important regulators of the multimerization cycle of dynamin in endocytosis.

Secondary structure of Src homology 2 domain of c-Abl by heteronuclear NMR spectroscopy in solution.

Overduin M, Mayer B, Rios CB, Baltimore D, Cowburn D
Proc Natl Acad Sci U S A. 1992; 89: 11673-7

The Src homology 2 (SH2) domain is a recognition motif thought to mediate the association of the cytoplasmic proteins involved in signal transduction by binding to phosphotyrosyl-containing sequences in proteins. Assignments of nearly all 1H and 15N resonances of the SH2 domain from the c-Abl protein-tyrosine kinase have been obtained from homonuclear and heteronuclear NMR experiments. The secondary structure has been elucidated from the pattern of nuclear Overhauser effects, from vicinal coupling constants, and from observation of slowly exchanging amino hydrogens. The secondary structure contains two alpha-helices and eight beta-strands, six of which are arranged in two contiguous, antiparallel beta-sheets. Residues believed to be involved in phosphotyrosyl ligand binding are on a face of one beta-sheet. The alignment of homologous sequences on the basis of secondary structure suggests a conserved global fold in a family of SH2 domains.

Multiple src-related kinase genes, srk1-4, in the fresh water sponge Spongilla lacustris.

Ottilie S, Raulf F, Barnekow A, Hannig G, Schartl M
Oncogene. 1992; 7: 1625-30

In one of the simplest metazoan organisms, the sponge Spongilla lacustris, at least four different src-related kinase genes (srk1-4) are expressed, all of which show a high degree of similarity to the c-src genes of vertebrates. Whereas srk2 and srk3 are clearly unrelated at the nucleic acid level, srk1 and srk4 share identical sequences in the 5' parts of their cDNAs. The cloning of several primer extension clones and genomic polymerase chain reaction experiments confirmed the hypothesis of an alternative splicing of tandemly arranged carboxy-terminal parts of srk1 and srk4. The genomic sequence encoding both proteins was found to be interrupted at the splice point by an intron which is located in the same position as one of the introns in the chicken src gene, which is the only gene conserved in invertebrates and vertebrates. All four srk genes are expressed in adult sponges as mRNA transcripts of about 2.2 kb. Tyrosine kinase activity of a src-related kinase could be detected in adult sponges but not in their resting form (gemmulae), and may reflect the activity of the srk protein products. Spongilla lacustris is the simplest organism from which a protein tyrosine kinase gene has been isolated. The presence of at least four such genes in the evolutionary ancient and primitive phylum Porifera suggests that tyrosine kinase genes arose concomitantly with or shortly after the appearance of multicellular organisms and that their activity may be involved in aggregation and cell-cell recognition.

[Recent progress in the studies of phosphatidylinositol 3-kinase]

Otsu M
Seikagaku. 1993; 65: 1299-316

SH3 ligands in the dopamine D3 receptor.

Oldenhof J, Ray A, Vickery R, Van Tol HH
Cell Signal. 2001; 13: 411-6

It has recently been observed that G protein-coupled receptors (GPCRs) can interact with SH3 domains through polyproline motifs. These interactions appear to be involved in receptor internalization and MAPK signalling. Here we report that the third cytoplasmic loop of the dopamine D3 receptor can interact in vitro with the adaptor protein Grb2. While the amino- and carboxy-terminal SH3 domains of Grb2 separately did not interact with the D3 receptor loop, the interaction is at least partially maintained with a Grb2 mutant for the amino-terminal SH3 domain, but disrupted for a Grb2 mutant with a nonfunctional carboxy-terminal SH3 domain. The data indicate the need of structural integrity of the entire Grb2 protein for the interaction and dominant role of the carboxy-terminal SH3 domain in the interaction. Disruption of the PXXP motifs in the D3 receptor did not affect the interaction with Grb2. These results indicate that GPCRs may contain SH3 ligands that do not contain the postulated minimal consensus sequence PXXP.

Grb2 forms an inducible protein complex with CD28 through a Src homology 3 domain-proline interaction.

Okkenhaug K, Rottapel R
J Biol Chem. 1998; 273: 21194-202

CD28 provides a costimulatory signal that results in optimal activation of T cells. The signal transduction pathways necessary for CD28-mediated costimulation are presently unknown. Engagement of CD28 leads to its tyrosine phosphorylation and subsequent binding to Src homology 2 (SH2)-containing proteins including the p85 subunit of phosphatidylinositol 3'-kinase (PI3K); however, the contribution of PI3K to CD28-dependent costimulation remains controversial. Here we show that CD28 is capable of binding the Src homology 3 (SH3) domains of several proteins, including Grb2. The interaction between Grb2 and CD28 is mediated by the binding of Grb2-SH3 domains to the C-terminal diproline motif present in the cytoplasmic domain of CD28. While the affinity of the C-terminal SH3 domain of Grb2 for CD28 is greater than that of the N-terminal SH3 domain, optimal binding requires both SH3 domains. Ligation of CD28, but not tyrosine-phosphorylation, is required for the SH3-mediated binding of Grb2 to CD28. We propose a model whereby the association of Grb2 with CD28 occurs via an inducible SH3-mediated interaction and leads to the recruitment of tyrosine-phosphorylated proteins such as p52(shc) bound to the SH2 domain of Grb2. The inducible interaction of Grb2 to the C-terminal region of CD28 may form the basis for PI3K-independent signaling through CD28.

Differential binding of pp60c-src and pp60v-src to cytoskeleton is mediated by SH2 and catalytic domains.

Okamura H, Resh MD
Oncogene. 1994; 9: 2293-303

The transforming protein of Rous sarcoma virus, pp60v-src, and its normal cellular homolog, pp60c-src, differ not only in oncogenic potential but also in their subcellular localization and cytoskeletal binding ability. pp60v-src has been shown to stably associate with a detergent-insoluble cytoskeletal matrix, whereas pp60c-src does not. We have generated a series of precise deletion and truncations of the Src homology domains within pp60v-src and pp60c-src, based on the crystal and solution structures of these regions, to determine not only the region responsible for cytoskeletal association but also the mechanism accounting for the differential binding observed. Here we show that the SH2 domain, but not the SH3 domain, mediates cytoskeletal association of pp60v-src through a phosphotyrosine-dependent interaction. The ability to interact with the cytoskeletal matrix is regulated by the catalytic (SH1) domain. Truncation of the pp60v-src catalytic domain results in lower binding while removal of the catalytic domain of pp60c-src results in the acquisition of cytoskeletal binding similar to that of the analogous v-src construct. These results indicate that the SH2 and catalytic domains function coordinately to regulate the cytoskeletal association of pp60v-src and pp60c-src.

Role of the basic, proline-rich region of dynamin in Src homology 3 domain binding and endocytosis.

Okamoto PM, Herskovits JS, Vallee RB
J Biol Chem. 1997; 272: 11629-35

The GTPase dynamin has been implicated in the regulation of the scission of coated and noncoated pits during the early stages of endocytosis. Various macromolecules including microtubules, acidic phospholipids, and Src homology 3 (SH3) domains have been shown to interact with the basic, proline-rich region of dynamin and act as effectors of its GTPase activity. The interaction of dynamin with SH3 domain-containing proteins is of particular interest since SH3 domains are known to mediate protein-protein interactions in signal transducing complexes. In this study, we have systematically defined three distinct SH3 binding regions within the dynamin proline-rich C terminus. These binding regions conform to either the Class I or II SH3 binding consensus sequence, and their location coincides with a region previously shown to be important in the colocalization of dynamin with clathrin-coated pits. Two of these SH3 binding regions are well conserved among four dynamin isoforms, and we show that the overall binding pattern for SH3 domains is comparable among the isoforms. We also demonstrate that neither transferrin nor platelet-derived growth factor receptor uptake is restored upon removal of the basic, proline-rich region in a dominant negative dynamin GTP binding mutant. Together with earlier evidence from our laboratory, these findings suggest that SH3 domains may serve to target dynamin to coated pits and are not the direct targets of dominant inhibitory mutants of dynamin.

Confocal fluorescence microscopy for antibodies against a highly conserved sequence in SH2 domains.

Okabe T, Teshima R, Furuno T, Torigoe C, Sawada J, Nakanishi M
Biochem Biophys Res Commun. 1996; 223: 245-9

We have prepared monoclonal antibodies for a highly conserved sequence (GTFLVRESETTK) in SH2 domains. Mouse IgG1s (12E and 32D) prepared against a peptide-conjugated keyhole lympet hemocyanin specifically bound the antigenic peptide but not the carrier protein. Western blot analysis showed that one IgG1 (12E) recognized mainly 62 kDa proteins (possibly src-family tyrosine kinases) from triton X-100 extracts of RBL-2H3 cells and that another (32D) recognized mainly 32 and 110 kDa proteins. Confocal fluorescence microscopy showed that the SH2 domains had a diffuse cytoplasmic distribution and were not present in the nucleus. Following antigen stimulation, a markedly different cellular distribution was observed in the cells stained with 12E and 32D IgGs. 12E IgGs strongly stained the plasma membranes while 32D IgGs stained small granules in the cytoplasm. As 12E IgGs bound 62 kDa proteins on Western blotting, the results suggest that tyrosine kinases cluster along the plasma membranes and/or than conformational changes occur in the domains after antigen stimulation.

Dot far-western blot analysis of relative binding affinities of the Src homology 3 domains of Efs and its related proteins.

Ohba T, Ishino M, Aoto H, Sasaki T
Anal Biochem. 1998; 262: 185-92

The Src homology 3 (SH3) domains are a modular structure of about 60 amino acid residues found in many proteins important in signal transduction. Each SH3 domain has a binding specificity to sequences containing a PXXP motif in ligand proteins. We found that a focal adhesion kinase (FAK)-related protein, cell adhesion kinase beta (CAKbeta), was bound in vitro by the SH3 domain of embryonal Fyn-associated substrate (Efs), a docking protein structurally related to p130Cas (Cas) and HEF1. Here, we employed a dot far-Western blotting technique to evaluate the affinity and specificity of the binding by the SH3 domains of Efs and its related proteins. The SH3 domains and their ligands were prepared as glutathione S-transferase fusion proteins, and one of the binding components was immobilized on membranes while the other was labeled with 32P to use as a probe. The amount of the bound probe was determined by autoradiography using an imaging plate and a bioimaging analyzer. A competitive binding assay showed that Efs, compared with Cas and HEF1, had a SH3 domain with a lower relative affinity to CAKbeta and FAK and with a preference to interact with FAK rather than CAKbeta. Our assay based on dot far-Western blotting is a simple and sensitive method to evaluate fine differences in the binding affinity of SH3-mediated interactions.

Xenopus laevis TRK-fused gene (TFG) is an SH3 domain binding protein highly expressed in the cement gland.

Ohan N, Sabourin D, Booth RA, Liu XJ
Mol Reprod Dev. 2000; 56: 336-44

TRK-fused gene (TFG) was originally identified in humans as the N-terminus of an oncogenic fusion protein TRK-T3, associated with papillary thyroid carcinoma. An amino-terminal coiled coil domain of TFG is responsible for mediating oligomerization of the TRK-T3 oncoprotein, resulting in constitutive activation of the TRK protein tyrosine kinase and oncogenesis. We have cloned the Xenopus laevis homologue of TFG and demonstrated that xTFG was highly expressed in the cement gland of tailbud embryos. Overexpression of xTFG2-136 (including the coiled coil domain) in early embryos, via mRNA microinjection as well as transgenic expression using the recently described restriction enzyme mediated integration (REMI) transgenesis, did not alter embryonic development or development of a functional cement gland, despite clear evidence that xTFG2-136 strongly interacted with endogenous xTFG. Finally, we have identified a potential SH3 binding motif in xTFG (and in TFG) and have demonstrated that xTFG selectively interacted with SH3 domains of Src, PLCgamma, and the p85 phosphatidylinositol 3-kinase subunit.

Identification and characterization of two novel SH2 domain-containing proteins from a yeast two hybrid screen with the ABL tyrosine kinase.

Oda T, Kujovich J, Reis M, Newman B, Druker BJ
Oncogene. 1997; 15: 1255-62

To further our understanding of the molecular mechanism of Bcr-Abl mediated transformation, a yeast two hybrid screen was used to identify proteins binding to the Abl tyrosine kinase. Two partial cDNAs encoding novel SH2 domain-containing proteins were cloned and designated Shd and She. Both have homology to Shb, a previously reported SH2 domain-containing protein. Northern blot analysis showed that She is expressed in heart, lung, brain, and skeletal muscle, while expression of Shd is restricted to the brain. The deduced amino acid sequence of the full length mouse Shd cDNA contains an amino-terminal proline-rich region, and a carboxyterminal SH2 domain. A bacterially expressed Shd domain bound multiple tyrosine-phosphorylated proteins with relative molecular weights of 200, 170, 130, 100, 90, 78, 72 and 32 kDa from K562 cell lysates. Shd contains five YXXP motifs, a substrate sequence preferred by Abl tyrosine kinases. Shd was tyrosine phosphorylated in COS-7 cells co-transfected with Shd and c-Abl or Bcr-Abl. These results suggest that Shd may be a physiological substrate of c-Abl and may function as an adapter protein in the central nervous system.

Identification of Trk binding sites for SHC and phosphatidylinositol 3'-kinase and formation of a multimeric signaling complex.

Obermeier A, Lammers R, Wiesmuller KH, Jung G, Schlessinger J, Ullrich A
J Biol Chem. 1993; 268: 22963-6

Phosphotyrosine-containing synthetic peptides were used to identify the binding sites for cellular polypeptides involved in nerve growth factor receptor/Trk-mediated signal transduction. In vitro association of SHC and the p85 subunit of phosphatidylinositol 3'-kinase with the Trk tyrosine kinase was prevented only by phosphorylated Y-490- and Y-751-containing peptides, respectively. In spite of the close proximity of the p85 binding site to that of phospholipase C gamma (Y-785), both target proteins are able to interact with the same receptor molecule simultaneously.

Mouse alpha1-Syntrophin Binding to Grb2: Further Evidence of a Role for Syntrophin in Cell Signaling.

Oak SA, Russo K, Petrucci TC, Jarrett HW
Biochemistry. 2001; 40: 11270-8

Syntrophins have been proposed to serve as adapter proteins. Syntrophins are found in the dystrophin glycoprotein complex (DGC); defects in the constituents of this complex are linked to various muscular dystrophies. Blot overlay experiments demonstrate that alpha-dystroglycan, beta-dystroglycan, and syntrophins all bind Grb2, the growth factor receptor bound adapter protein. Mouse alpha1-syntrophin sequences were produced as chimeric fusion proteins in bacteria and found to also bind Grb2 in a Ca(2+)-independent manner. This binding was localized to the proline rich sequences adjacent to and overlapping with the N-terminal pleckstrin homology domain (PH1). Grb2 bound syntrophin with an apparent K(D) of 563 +/- 15 nM. Grb2-C-SH3 domain bound syntrophin with slightly higher affinity than Grb2-N-SH3 domain. Crk-L, an SH2/SH3 protein of similar domain structure but different specificity, does not bind these syntrophin sequences.

Crystal structure of the PI 3-kinase p85 amino-terminal SH2 domain and its phosphopeptide complexes.

Nolte RT, Eck MJ, Schlessinger J, Shoelson SE, Harrison SC
Nat Struct Biol. 1996; 3: 364-74

Crystal structures of the amino-terminal SH2 domain of the p85alpha subunit of phosphatidylinositol (PI) 3-kinase, alone and in complex with phosphopeptides bearing pTyr-Met/Val-Xaa-Met motifs, show that phosphopeptides bind in the two-pronged manner seen in high-affinity Lck and Src SH2 complexes, with conserved interactions between the domain and the peptide segment from phosphotyrosine to Met+3. Peptide binding requires the rearrangement of a tyrosyl side chain in the BG loop to create the hydrophobic Met+3 binding pocket. The structures suggest a mechanism for the biological specificity exhibited by PI 3-kinase in its interactions with phosphoprotein partners.

Nonphosphorylatable tyrosine surrogates. Implications for protein kinase inhibitor design.

Niu J, Lawrence DS
J Biol Chem. 1997; 272: 1493-9

Tyrosine-specific protein kinases are known to utilize short synthetic tyrosine-containing peptides as substrates and, as a consequence, a number of inhibitory peptides have been prepared by replacing the tyrosine moiety in these peptides with a nonphosphorylatable phenylalanine residue. Unfortunately, the inhibitory efficacy of these phenylalanine-based peptides is often disappointing. These results demonstrate the need for nonphosphorylatable tyrosine surrogates that enhance enzyme affinity. As a consequence, we prepared nearly two dozen different phenethylamine derivatives, attached them to the C terminus of an active site-directed peptide (Glu-Glu-Leu-Leu), and examined their effectiveness as inhibitors of pp60(c-)src. Three derivatives exhibit enhanced inhibitory activity (relative to phenethylamine), including para-substituted sulfonamide and guanidino analogs as well as a pentafluoro-containing species. The para-sulfonamide derivative was selected for further study and was found to function as a competitive inhibitor versus variable peptide substrate and as a noncompetitive inhibitor versus variable ATP. In short, the enhanced inhibitory activity of the sulfonamide derivative is not due to the association of this moiety with the ATP binding site. Furthermore, peptides containing the para-guanidino and pentafluoro derivatives of phenylalanine were prepared. These species also display enhanced inhibitory activity toward pp60(c-)src relative to the corresponding phenylalanine-based peptide.

Tight-binding inhibitory sequences against pp60(c-src) identified using a random 15-amino-acid peptide library.

Nishi T, Budde RJ, McMurray JS, Obeyesekere NU, Safdar N, Levin VA, Saya H
FEBS Lett. 1996; 399: 237-40

A bacteriophage peptide library containing a random 15-amino-acid insert was screened for identification of peptide sequence(s) that bind pp60(c-src). Sequencing the random insert from more than 100 virions indicated that more than 60% of the phage virions that bound to this enzyme contained a GXXG sequence motif in which X was frequently a hydrophobic residue. The GXXG sequence was often repeated as GXXGXXG. Two nonameric peptides were synthesized to determine whether or not the peptide inhibits pp60(c-src) tyrosine kinase activity and the importance of the glycine residues within this sequence. The peptide containing glycine had a Ki of 24 microM, whereas replacing the glycines with proline increased the Ki value to 3.1 mM.

Novel recognition mode between Vav and Grb2 SH3 domains.

Nishida M, Nagata K, Hachimori Y, Horiuchi M, Ogura K, Mandiyan V, Schlessinger J, Inagaki F
EMBO J. 2001; 20: 2995-3007

Vav is a guanine nucleotide exchange factor for the Rho/Rac family that is expressed exclusively in hematopoietic cells. Growth factor receptor-bound protein 2 (Grb2) has been proposed to play important roles in the membrane localization and activation of Vav through dimerization of its C-terminal Src-homology 3 (SH3) domain (GrbS) and the N-terminal SH3 domain of Vav (VavS). The crystal structure of VavS complexed with GrbS has been solved. VavS is distinct from other SH3 domain proteins in that its binding site for proline-rich peptides is blocked by its own RT loop. One of the ends of the VavS beta-barrel forms a concave hydrophobic surface. The GrbS components make a contiguous complementary interface with the VavS surface. The binding site of GrbS for VavS partially overlaps with the canonical binding site for proline-rich peptides, but is definitely different. Mutations at the interface caused a decrease in the binding affinity of VavS for GrbS by 4- to 40-fold. The structure reveals how GrbS discriminates VavS specifically from other signaling molecules without binding to the proline-rich motif.

Improving SH3 domain ligand selectivity using a non-natural scaffold.

Nguyen JT, Porter M, Amoui M, Miller WT, Zuckermann RN, Lim WA
Chem Biol. 2000; 7: 463-73

BACKGROUND: Src homology 3 (SH3) domains bind sequences bearing the consensus motif PxxP (where P is proline and x is any amino acid), wherein domain specificity is mediated largely by sequences flanking the PxxP core. This specificity is limited, however, as most SH3 domains show high ligand cross-reactivity. We have recently shown that diverse N-substituted residues (peptoids) can replace the prolines in the PxxP motif, yielding a new source of ligand specificity. RESULTS: We have tested the effects of combining multiple peptoid substitutions with specific flanking sequences on ligand affinity and specificity. We show that by varying these different elements, a ligand can be selectively tuned to target a single SH3 domain in a test set. In addition, we show that by making multiple peptoid substitutions, high-affinity ligands can be generated that completely lack the canonical PxxP motif. The resulting ligands can potently disrupt natural SH3-mediated interactions. CONCLUSIONS: Peptide-peptoid hybrid scaffolds yield SH3 ligands with markedly improved domain selectivity, overcoming one of the principal challenges in designing inhibitors against these domains. These compounds represent important leads in the search for orthogonal inhibitors of SH3 domains, and can serve as tools for the dissection of complex signaling pathways.

The nonreceptor protein-tyrosine kinase CSK complexes directly with the GTPase-activating protein-associated p62 protein in cells expressing v-Src or activated c-Src.

Neet K, Hunter T
Mol Cell Biol. 1995; 15: 4908-20

CSK is a predominantly cytosolic protein-tyrosine kinase (PTK) that negatively regulates Src family PTKs by phosphorylation of a conserved tyrosine near their C termini. Little is known about how CSK itself is regulated. On the basis of immunofluorescence studies, a model has been proposed that when c-Src is activated, it is redistributed to podosomes, in which substrates become phosphorylated, creating binding sites for CSK. CSK is recruited to these sites of c-Src activation via its SH2 and SH3 domains and is then in a position to downregulate c-Src activity (B. W. Howell and J. A. Cooper, Mol. Cell. Biol. 14:5402-5411, 1994). To identify phosphotyrosine (P.Tyr)-containing proteins that may mediate translocation of CSK due to c-Src activation, we have examined the whole spectrum of P.Tyr-containing proteins that associate with CSK in v-Src NIH 3T3 cells by anti-P.Tyr immunoblotting. Nine P.Tyr-containing proteins coimmunoprecipitated with CSK from v-Src NIH 3T3 cells. One of these, an approximately 62-kDa protein, also associated with CSK in NIH 3T3 cells treated with vanadate prior to lysis and in NIH 3T3 cells expressing an activated c-Src mutant. This 62-kDa protein was shown to be identical to the GTPase-activating protein (GAP)-associated p62 (GAP-A.p62) protein. The interaction between CSK and GAP-A.p62 could be reconstituted in vitro with glutathione S-transferase fusion proteins containing full-length CSK or the CSK SH2 domain. Furthermore, our data show that CSK interacts directly with GAP.A-p62 and that the complex between the two proteins is localized in subcellular membrane or cytoskeletal fractions. Our results suggest that GAP-A.p62 may function as a docking protein and may mediate translocation of proteins, including GAP and CSK, to membrane or cytoskeletal regions upon c-Src activation.

Coevolution of the domains of cytoplasmic tyrosine kinases.

Nars M, Vihinen M
Mol Biol Evol. 2001; 18: 312-21

Many signaling molecules are multidomain proteins that have other domains in addition to the catalytic kinase domain. Protein tyrosine kinases almost without exception contain Src homology 2 (SH2) and/or SH3 domains that can interact with other signaling proteins. Here, we studied evolution of the tyrosine kinases containing SH2 and/or SH3 and kinase domains. The three domains seem to have duplicated together, since the phylogenetic analysis using parsimony gave almost identical evolutionary trees for the separate domains and the multidomain complexes. The congruence analysis of the sequences for the separate domains also suggested that the domains have coevolved. There are several reasons for the domains to appear in a cluster. Kinases are regulated in many ways, and the presence of SH2 and SH3 domains at proper positions is crucial. Because all three domains can recognize different parts of ligands and substrates, their evolution has been interconnected. The reasons for the clustering and coevolution of the three domains in protein tyrosine kinases (PTKs) are discussed.

Crystal structure of the tandem phosphatase domains of RPTP LAR.

Nam HJ, Poy F, Krueger NX, Saito H, Frederick CA
Cell. 1999; 97: 449-57

Most receptor-like protein tyrosine phosphatases (RPTPs) contain two conserved phosphatase domains (D1 and D2) in their intracellular region. The carboxy-terminal D2 domain has little or no catalytic activity. The crystal structure of the tandem D1 and D2 domains of the human RPTP LAR revealed that the tertiary structures of the LAR D1 and D2 domains are very similar to each other, with the exception of conformational differences at two amino acid positions in the D2 domain. Site-directed mutational changes at these positions (Leu-1644-to-Tyr and Glu-1779-to-Asp) conferred a robust PTPase activity to the D2 domain. The catalytic sites of both domains are accessible, in contrast to the dimeric blocked orientation model previously suggested. The relative orientation of the LAR D1 and D2 domains, constrained by a short linker, is stabilized by extensive interdomain interactions, suggesting that this orientation might be favored in solution.

Intramolecular interactions of the regulatory domains of the Bcr-Abl kinase reveal a novel control mechanism.

Nam HJ, Haser WG, Roberts TM, Frederick CA
Structure. 1996; 4: 1105-14

BACKGROUND. The Abl nonreceptor tyrosine kinase is implicated in a range of cellular processes and its transforming variants are involved in human leukemias. The N-terminal regulatory region of the Abl protein contains Src homology domains SH2 and SH3 which have been shown to be important for the regulation of its activity in vivo. These domains are often found together in the same protein and biochemical data suggest that the functions of one domain can be influenced by the other. RESULTS. We have determined the crystal structure of the Abl regulatory region containing the SH3 and SH2 domains. In general, the individual domains are very similar to those of previously solved structures, although the Abl SH2 domain contains a loop which is extended so that one side of the resulting phosphotyrosine-binding pocket is open. In our structure the protein exists as a monomer with no intermolecular contacts to which a biological function may be attributed. However, there is a significant intramolecular contact between a loop of the SH3 domain and the extended loop of the SH2 domain. This contact surface includes the SH2 loop segment that is responsible for binding the phosphate moiety of phosphotyrosine-containing proteins and is therefore critical for orienting peptide interactions. CONCLUSIONS. The crystal structure of the composite Abl SH3-SH2 domain provides the first indication of how SH2 and SH3 domains communicate with each other within the same molecule and why the presence of one directly influences the activity of the other. This is the first clear evidence that these two domains are in contact with each other. The results suggest that this direct interaction between the two domains may affect the ligand binding properties of the SH2 domain, thus providing an explanation for biochemical and functional data concerning the Bcr-Abl kinase.

Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src.

Nada S, Okada M, MacAuley A, Cooper JA, Nakagawa H
Nature. 1991; 351: 69-72

The protein-tyrosine kinase activity of the proto-oncogene product p60c-src is negatively regulated by the phosphorylation of a tyrosine residue close to the C terminus, tyrosine 527. The phosphorylation might be catalysed by a so-far-unidentified tyrosine kinase, distinct from p60c-src. Recently we purified a protein-tyrosine kinase that specifically phosphorylates tyrosine 527 of p60c-src from neonatal rat brain. We have now confirmed the specificity of this enzyme by using a mutant p60c-src that has a phenylalanine instead of tyrosine 527, and cloned a complementary DNA that encodes the enzyme. The enzyme is similar to kinases of the src family in that it has two conserved regions, Src-homology regions 2 and 3, upstream of a tyrosine kinase domain. The amino-acid identity of each region is no more than 47%, however, and the enzyme lacks phosphorylation sites corresponding to tyrosines 416 and 527 of p60c-src and has no myristylation signal. These results suggest that this protein-tyrosine kinase, which might negatively regulate p60c-src, represents a new type of tyrosine kinase.

Structure and function of the SH3 domain.

Musacchio A, Wilmanns M, Saraste M
Prog Biophys Mol Biol. 1994; 61: 283-97

SH3--an abundant protein domain in search of a function.

Musacchio A, Gibson T, Lehto VP, Saraste M
FEBS Lett. 1992; 307: 55-61

Src-homology 3 is a small protein domain of about 60 amino acid residues. It is probably made of beta-sheets. SH3 is present in a large number of eukaryotic proteins which are involved in signal transduction, cell polarization and membrane-cytoskeleton interactions. Here we review its occurrence and discuss possible functions of this domain.

Molecular astrology: the case of the Myb DNA binding domain.

Murthy K
Protein Eng. 1993; 6: 129-31

Suppression of c-Src activity by C-terminal Src kinase involves the c-Src SH2 and SH3 domains: analysis with Saccharomyces cerevisiae.

Murphy SM, Bergman M, Morgan DO
Mol Cell Biol. 1993; 13: 5290-300

The kinase activity of c-Src is normally repressed in vertebrate cells by extensive phosphorylation of Y-527. C-terminal Src kinase (CSK) is a candidate for the enzyme that catalyzes this phosphorylation. We have used budding yeast to study the regulation of c-Src activity by CSK in intact cells. Expression of c-Src in Saccharomyces cerevisiae, which lacks endogenous c-Src and Y-527 kinases, induces a kinase-dependent growth inhibition. Coexpression of CSK in these cells results in phosphorylation of c-Src on Y-527 and suppression of the c-Src phenotype. CSK does not fully suppress the activity of c-Src mutants lacking portions of the SH2 or SH3 domains, even though these mutant proteins are phosphorylated on Y-527 by CSK both in vivo and in vitro. These results suggest that both the SH2 and SH3 domains of c-Src are required for the suppression of c-Src activity by Y-527 phosphorylation.

A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction.

Muller AJ, Pendergast AM, Havlik MH, Puil L, Pawson T, Witte ON
Mol Cell Biol. 1992; 12: 5087-93

SH2 (src homology region 2) domains are implicated in protein-protein interactions involved in signal transduction pathways. Isolated SH2 domains bind proteins that are tyrosine phosphorylated. A novel, phosphotyrosine-independent binding interaction between BCR, the Philadelphia chromosome breakpoint cluster region gene product, and the SH2 domain of its translocation partner c-ABL has recently been reported. We have examined the ability of additional SH2 domains to bind phosphotyrosine-free BCR and compared this with their ability to bind tyrosine-phosphorylated c-ABL 1b. Of 11 individual SH2 domains examined, 8 exhibited relatively high affinity for c-ABL 1b, whereas only 4 exhibited relatively high affinity for BCR. Binding of tyrosine-phosphorylated c-ABL 1b by the relatively high-affinity ABL and ARG SH2 domains was quantitatively analyzed, and equilibrium dissociation constants for both interactions were estimated to be in the range of 5 x 10(-7) M. The ABL SH2 domain exhibited relatively high affinity for phosphotyrosine-free BCR as well; however, this interaction appears to be about two orders of magnitude weaker than binding of tyrosine-phosphorylated c-ABL 1b. The ARG SH2 domain exhibited relatively weak affinity for BCR and was determined to bind about 10-fold less strongly than the ABL SH2 domain. The ABL and ARG SH2 domains differ by only 10 of 91 amino acids, and the substitution of ABL-specific amino acids into either the amino- or carboxy-terminal half of the ARG SH2 domain was found to increase its affinity for BCR. We discuss these results in terms of a model which has been proposed for peptide binding by class I histocompatibility glycoproteins.

The SH2 domain from the tyrosine kinase Fyn in complex with a phosphotyrosyl peptide reveals insights into domain stability and binding specificity.

Mulhern TD, Shaw GL, Morton CJ, Day AJ, Campbell ID
Structure. 1997; 5: 1313-23

BACKGROUND: SH2 domains are found in a variety of signal transduction proteins; they bind phosphotyrosine-containing sequences, allowing them to both recognize target molecules and regulate intramolecular kinase activity. Fyn is a member of the Src family of tyrosine kinases that are involved in signal transduction by association with a number of membrane receptors. The kinase activity of these signalling proteins is modulated by switching the binding mode of their SH2 and SH3 domains from intramolecular to intermolecular. The molecular basis of the signalling roles observed for different Src family members is still not well understood; although structures have been determined for the SH2 domains of other Src family molecules, this is the first structure of the Fyn SH2 domain. RESULTS: The structure of the Fyn SH2 domain in complex with a phosphotyrosyl peptide (EPQpYEEIPIYL) was determined by high resolution NMR spectroscopy. The overall structure of the complex is analogous to that of other SH2-peptide complexes. Noteworthy aspects of the structure are: the BG loop, which contacts the bound peptide, contains a type-I' turn; a capping-box-like interaction is present at the N-terminal end of helix alpha A; cis-trans isomerization of the Val beta G1-Pro beta G2 peptide bond causes conformational heterogeneity of residues near the N and C termini of the domain. CONCLUSIONS: Comparison of the Fyn SH2 domain structure with other structures of SH2 domains highlights several interesting features. Conservation of helix capping interactions among various SH2 domains is suggestive of a role in protein stabilisation. The presence of a type-I' turn in the BG loop, which is dependent on the presence of a glycine residue at position BG3, is indicative of a binding pocket, characteristic of the Src family, SykC and Abl, rather than a binding groove found in PLC-gamma 1C, p85 alpha N and Shc, for example.

Solution structure and peptide binding of the SH3 domain from human Fyn.

Morton CJ, Pugh DJ, Brown EL, Kahmann JD, Renzoni DA, Campbell ID
Structure. 1996; 4: 705-14

BACKGROUND: The Src family of tyrosine kinases is involved in the propagation of intracellular signals from many transmembrane receptors. Each member of the family contains two domains that regulate interactions with other molecules, one of which is the Src homology 3 (SH3) domain. Although structures have previously been determined for SH3 domains, and ideas about peptide-binding modes have been proposed, their physiological role is still unclear. RESULTS: We have determined the solution structure of the SH3 domain from the Src family tyrosine kinase Fyn in two forms: unbound and complexed with a peptide corresponding to a putative ligand sequence from phosphatidylinositol 3' kinase. Fyn SH3 shows the typical SH3 topology of two perpendicular three-stranded beta sheets and a single turn of 3(10) helix. The interaction of SH3 with three potential ligand peptides was investigated, demonstrating that they all bind to the same site on the molecule. A previous model for ligand binding to SH3 domains predicts binding in one of two orientations (class I or II), each characterized by a consensus sequence. The ligand with the closest match to the class I consensus sequence bound with highest affinity and in the predicted orientation. CONCLUSIONS: The Fyn SH3 domain has a well-defined structure in solution. The relative binding affinities of the three ligand peptides and their orientation within the Fyn SH3 complex were consistent with recently proposed models for the binding of 'consensus' polyproline sequences. Although the affinities of consensus and non-consensus peptides are different, the degree of difference is not very large, suggesting that SH3 domains bind to polyproline peptides in a promiscuous manner.

The SH3 domain of Bruton's tyrosine kinase displays altered ligand binding properties when auto-phosphorylated in vitro.

Morrogh LM, Hinshelwood S, Costello P, Cory GO, Kinnon C
Eur J Immunol. 1999; 29: 2269-79

Btk is a member of the Tec family of protein tyrosine kinases expressed in B cells. It is stimulated following cross-linking of the B cell receptor which leads to the autophosphorylation of a specific residue in the SH3 domain, Y223. Previous work using Btk-derived fusion proteins has shown that the Btk SH3 domain binds to c-Cbl and Wiskott-Aldrich syndrome protein (WASP) in vitro. We show here that when the Btk SH3 domain fusion protein is autophosphorylated, its ability to take part in protein interactions is altered as compared to the non-phosphorylated fusion protein. Although the phosphorylated Btk SH3 domain still binds c-Cbl, it no longer binds WASP and instead acquires a high affinity for kinase-active Syk. The region of Syk responsible for this interaction is contained within its C terminus, suggesting a novel mechanism by which these proteins may interact.

Src kinase associates with a member of a distinct subfamily of protein-tyrosine phosphatases containing an ezrin-like domain.

Moller NP, Moller KB, Lammers R, Kharitonenkov A, Sures I, Ullrich A
Proc Natl Acad Sci U S A. 1994; 91: 7477-81

A 6.2-kb full-length clone encoding a distinct protein-tyrosine phosphatase (PTP; EC 3.1.3.48), PTPD1, was isolated from a human skeletal muscle cDNA library. The cDNA encodes a protein of 1174 amino acids with N-terminal sequence homology to the ezrin-band 4.1-merlin-radixin protein family, which also includes the two PTPs H1 and MEG1. The PTP domain is positioned in the extreme C-terminal part of PTPD1, and there is an intervening sequence of about 580 residues without any apparent homology to known proteins separating the ezrin-like and the PTP domains. Thus, PTPD1 and the closely related, partially characterized, PTPD2 belong to the same family as PTPH1 and PTPMEG1, but because of distinct features constitute a different PTP subfamily. Northern blot analyses indicate that PTPD1 and PTPD2 are expressed in a variety of tissues. In transient coexpression experiments PTPD1 was found to be efficiently phosphorylated by and associated with the src kinase pp60src.

Activation of the Src-family tyrosine kinase Hck by SH3 domain displacement.

Moarefi I, LaFevre-Bernt M, Sicheri F, Huse M, Lee CH, Kuriyan J, Miller WT
Nature. 1997; 385: 650-3

The protein Hck is a member of the Src family of non-receptor tyrosine kinases which is preferentially expressed in haematopoietic cells of the myeloid and B-lymphoid lineages. Src kinases are inhibited by tyrosine-phosphorylation at a carboxy-terminal site. The SH2 domains of these enzymes play an essential role in this regulation by binding to the tyrosine-phosphorylated tail. The crystal structure of the downregulated form of Hck has been determined and reveals that the SH2 domain regulates enzymatic activity indirectly; intramolecular interactions between the SH3 and catalytic domains appear to stabilize an inactive form of the kinase. Here we compare the roles of the SH2 and SH3 domains in modulating the activity of Hck in an investigation of the C-terminally phosphorylated form of the enzyme. We show that addition of the HIV-1 Nef protein, which is a high-affinity ligand for the Hck SH3 domain, to either the downregulated or activated form of Hck causes a large increase in Hck catalytic activity. The intact SH3-binding motif in Nef is crucial for Hck activation. Our results indicate that binding of the Hck SH3 domain by Nef causes a more marked activation of the enzyme than does binding of the SH2 domain, suggesting a new mechanism for regulation of the activity of tyrosine kinases.

Critical amino acid substitutions in the Src SH3 domain that convert c-Src to be oncogenic.

Miyazaki K, Senga T, Matsuda S, Tanaka M, Machida K, Takenouchi Y, Nimura Y, Hamaguchi M
Biochem Biophys Res Commun. 1999; 263: 759-64

The Src homology 3 (SH3) domain, originally identified in v-Crk, plays an important role in signal transduction. The comparative study with c-src has revealed that v-src oncogene of Schmidt-Ruppin strain of Rous sarcoma virus has three point mutations in its SH3 domain and one in the upstream of SH3. To assess the role of these mutations, each of the single mutations was introduced into c-Src by oligonucleotide-directed mutagenesis and its effect on cell transformation was examined. While variant Src proteins that carry each one of single mutations could not transform cells, double mutation at positions 95 and 117 converted c-Src to be oncogenic and active in kinase. An additional mutation at position 124 together with one at 95 and 117 further activated Src kinase. By use of GST-fusion forms of v-Src SH3 and c-Src SH3, we found that these mutations in SH3 suppressed the binding of SH3 with c-Src protein, possibly with a linker region, while v-SrcSH3 retained the ability to bind a subset of cellular protein to the level similar to those of c-SrcSH3. Taken together, our results suggest that point mutations accumulated in SH3 region can activate, in concert, Src kinase by relaxing the interaction between SH3 and the linker region and subsequently convert Src to be oncogenic.

Isolation and characterization of a novel human gene (NESH) which encodes a putative signaling molecule similar to e3B1 protein.

Miyazaki K, Matsuda S, Ichigotani Y, Takenouchi Y, Hayashi K, Fukuda Y, Nimura Y, Hamaguchi M
Biochim Biophys Acta. 2000; 1493: 237-41

Using a conventional cloning technique, a novel full-length cDNA was isolated and sequenced from a human placental cDNA library. This cDNA consists of 2129 bp and has a predicted open reading frame encoding 366 amino acids. It possesses a Src homology 3 (SH3) motif, proline-rich region, serine-rich region and no catalytic domain, suggesting that it seems to be a signaling protein most similar to e3B1, an eps8 SH3 binding protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 17q21.3 near the marker D17S1795.

The crystal structures of the SH2 domain of p56lck complexed with two phosphonopeptides suggest a gated peptide binding site.

Mikol V, Baumann G, Keller TH, Manning U, Zurini MG
J Mol Biol. 1995; 246: 344-55

Src homology-2 (SH2) domains are protein modules found within a wide variety of cytoplasmic signalling molecules that bind with high affinity to phosphotyrosyl-containing protein sequences. In order to develop SH2 inhibitors that contain phosphotyrosyl analogues resistant to cellular phosphatases, we have solved the crystal structures of the SH2 domain of p56lck in separate complexes with two high-affinity p-(phosphonomethyl)phenylalanine-containing peptides. The structures have been determined at 2.3 A and 2.25 A, and refined to crystallographic R-factors of 19.2% and 18.5%, respectively. The conformation of the SH2 domain of p56lck is essentially similar to that observed in Src and Lck complexed with a phosphotyrosine-containing peptide except in some loops and especially in the loop that connects the second and third beta-strands. This loop, which was involved in hydrogen-bond interactions with the phosphotyrosine moiety, has moved away in the phosphonopeptide complexes as a rigid body by about 7 A on two hinges leaving the tyrosine phosphate mimetic moiety accessible to the solvent. Some intramolecular hydrogen bonds with other residues of the third and fourth beta-strands stabilize an open conformation of the lid, suggesting a flap mechanism for peptide binding.

Mutational analysis of the STAT6 SH2 domain.

Mikita T, Daniel C, Wu P, Schindler U
J Biol Chem. 1998; 273: 17634-42

The SH2 domain of the STAT family of transcription factors is essential for STAT binding to phosphorylated cytoplasmic domains of activated cytokine receptors. Furthermore, the same domain mediates dimerization of activated STAT monomers, a prerequisite for DNA binding by this family of proteins. To identify amino acid residues within the STAT protein that mediate these various interactions, we have carried out an extensive mutational analysis of the Stat6 SH2 domain. Recombinant proteins carrying C-terminal deletions or double alanine substitutions were expressed in mammalian and insect cells and assayed for DNA binding, transcription activation, tyrosine phosphorylation, and the ability to interact with a tyrosine-phosphorylated peptide derived from the interleukin-4 receptor signaling chain. From these studies, we have identified amino acids that are required for both DNA binding and interleukin-4 receptor interaction, as well as residues that when mutated impair only one of the two functions. Our results suggest that the structural homology between the SH2 domain of Stat6 and that of the distantly related Src protein may be higher than predicted on the basis of primary amino acid sequence comparisons. However, the two types of SH2 domains may differ at their C-terminal ends.

Association of Ash/Grb-2 with dynamin through the Src homology 3 domain.

Miki H, Miura K, Matuoka K, Nakata T, Hirokawa N, Orita S, Kaibuchi K, Takai Y, Takenawa T
J Biol Chem. 1994; 269: 5489-92

Ash/Grb-2 is an adaptor protein composed only of Src homology (SH) 2 and SH3 domains that is considered to be essential for Ras activation. To clarify the downstream of Ash signaling, we investigated Ash-bound proteins. Ash-glutathione S-transferase (GST) fusion proteins were used to affinity-purify proteins bound to Ash. We found 180-, 150-, 100-, and 70-kDa proteins bound to GST-Ash, among which the 100 kDa protein was found to be dynamin by amino acid sequencing and Western blot with anti-dynamin antibody. Next, the in vitro and in vivo associations between Ash and dynamin were examined using PC12 cells. Dynamin in PC12 cell lysates bound to GST-Ash independent of NGF treatment. Also, Ash and dynamin co-precipitated when cell lysates of PC12 were immunoprecipitated with anti-Ash antibody or anti-dynamin antibody. Using various GST-Ash constructs, we studied the importance of the individual domains in binding and found that the SH3 domain is necessary for binding. This binding was inhibited by a synthetic peptide (GPPQVPSRPNRC, amino acids 827-838 in dynamin). These data show that Ash SH3 domains bind to the proline-rich region of dynamin. Considering the function of dynamin in membrane trafficking, Ash may regulate endocytosis in addition to Ras activation.

Binding of A/T-rich DNA by three high mobility group-like domains in c-Abl tyrosine kinase.

Miao YJ, Wang JY
J Biol Chem. 1996; 271: 22823-30

The c-Abl tyrosine kinase has been shown previously to bind DNA. Using polymerase chain reaction-based binding site-selection methods, no consensus high affinity binding site for c-Abl was found. Instead, oligonucleotides with runs of A/T sequences were isolated, and purified c-Abl was shown to bind A/T-containing oligonucleotides better than those without A/T sequences. DNA binding of c-Abl was dependent on three high mobility group 1-like boxes (HLBs), which bound cooperatively to the A/T-rich oligonucleotides. To distinguish binding to A/T sequences per se from binding to nonspecific DNA with a bend at the A/T-rich region, two oligonucleotides were compared for binding to c-Abl. Both oligonucleotides contained A/T sequences. In one, the A/T motif was part of an 80-mer duplex DNA. In another, the A/T motif was in the duplex arm of an 80-mer "bubble DNA" containing an internal unpaired 20-mer region to provide a flexible hinge. Interestingly, the HLBs of c-Abl bound better to the oligonucleotide containing the bubble, suggesting a higher affinity for bent DNA rather than A/T sequences per se. Taken together, these observations define a new class of DNA binding domains, the HLBs, which do not bind DNA with a high degree of sequence specificity, but may selectively bind to bent DNA or to sequences that are easier to distort.

Structure-based design and solid-phase parallel synthesis of phosphorylated nonpeptides to explore hydrophobic binding at the Src SH2 domain.

Metcalf CA 3rd, Eyermann CJ, Bohacek RS, Haraldson CA, Varkhedkar VM, Lynch BA, Bartlett C, Violette SM, Sawyer TK
J Comb Chem. 2000; 2: 305-13

Using a novel, solid-phase parallel synthetic route and a computational docking program, a series of phosphorylated nonpeptides were generated to determine their structure-activity relationships (SAR) for binding at the SH2 domain of pp60src (Src). A functionalized benzoic acid intermediate was attached to solid support via Rink amide linkage, which upon acid cleavage generated the desired benzamide template-based nonpeptides in a facile manner. Compounds were synthesized using a combination of solid- and solution-phase techniques. Purification using reversed-phase, semipreparative HPLC allowed for quantitative SAR studies. Specifically, this work focused on functional group modifications, in a parallel fashion, designed to explore hydrophobic binding at the pY+3 pocket of the Src SH2 domain.

FAP52, a novel, SH3 domain-containing focal adhesion protein.

Merilainen J, Lehto VP, Wasenius VM
J Biol Chem. 1997; 272: 23278-84

Src-homology 3 (SH3) domain is a 60-70-amino acid motif present in a large variety of signal transduction and cytoskeletal proteins. We used reverse transcriptase-polymerase chain reaction with degenerate and specific primers and chicken brain mRNA to clone a cDNA that codes for a novel SH3 domain-containing protein. The sequence predicts a 448-amino acid polypeptide with a molecular mass of 51, 971 daltons. In the amino terminus, it shows a very high propensity for alpha-helicity, suggesting coiled-coil and possibly a higher order oligomeric arrangement. In the carboxyl terminus, there is a unique SH3 sequence. In Northern blotting, a major 3.7-kilobase and a minor 7.2-kilobase transcript was detected in most chicken tissues. In immunofluorescence microscopy and immunoelectron microscopy on cultured chicken fibroblasts, the protein was localized to focal adhesions in which it showed a distinct codistribution with the focal adhesion proteins vinculin, talin, and paxillin. Phosphoamino acid analysis showed that in cultured chicken heart fibroblasts, the protein contains phosphoserine, but no phosphothreonine or phosphotyrosine, and that the phosphorylation is not dependent on fibronectin. We propose this protein the name FAP52, for Focal Adhesion Protein of 52 kDa, and suggest that it forms part of the multimolecular complex constituting focal adhesion sites.

Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase.

Meng W, Sawasdikosol S, Burakoff SJ, Eck MJ
Nature. 1999; 398: 84-90

Cbl is an adaptor protein that functions as a negative regulator of many signalling pathways that start from receptors at the cell surface. The evolutionarily conserved amino-terminal region of Cbl (Cbl-N) binds to phosphorylated tyrosine residues and has cell-transforming activity. Point mutations in Cbl that disrupt its recognition of phosphotyrosine also interfere with its negative regulatory function and, in the case of v-cbl, with its oncogenic potential. In T cells, Cbl-N binds to the tyrosine-phosphorylated inhibitory site of the protein tyrosine kinase ZAP-70. Here we describe the crystal structure of Cbl-N, both alone and in complex with a phosphopeptide that represents its binding site in ZAP-70. The structures show that Cbl-N is composed of three interacting domains: a four-helix bundle (4H), an EF-hand calcium-binding domain, and a divergent SH2 domain that was not recognizable from the amino-acid sequence of the protein. The calcium-bound EF hand wedges between the 4H and SH2 domains and roughly determines their relative orientation. In the ligand-occupied structure, the 4H domain packs against the SH2 domain and completes its phosphotyrosine-recognition pocket. Disruption of this binding to ZAP-70 as a result of structure-based mutations in the 4H, EF-hand and SH2 domains confirms that the three domains together form an integrated phosphoprotein-recognition module.

Cyclic peptides as probes of the substrate binding site of the cytosolic tyrosine kinase, pp60c-src.

McMurray JS, Budde RJ, Ke S, Obeyesekere NU, Wang W, Ramdas L, Lewis CA
Arch Biochem Biophys. 1998; 355: 124-30

A series of 48 cyclic peptides based on the amino acid sequence surrounding the autophosphorylation site of pp60(c-src) was synthesized and each was tested as both a substrate and an inhibitor of this protein tyrosine kinase. Starting with cyclo(Asp1-Asn2-Gln3-Tyr4-Ala5-Ala6-Arg7-Gln8-d- Phe9-Pro10) a six-amino-acid survey was performed at positions 1 through 8 to determine which positions were critical for affinity and phosphorylation and which amino acids produced the greatest activity. Our survey found that Arg7 was detrimental for binding and phosphorylation and that aromatic residues were preferred at this position. Further increases in affinity were obtained with hydrophobic residues at position 6 with the optimum for both affinity and phosphorylation being Phe. Changes on the "amino-terminal" side of Tyr4 resulted in reduced Vmax values, illustrating the requirement for acidic residues in peptidic tyrosine kinase substrates. The result of the survey was cyclo(Asp1-Asn2-Gln3-Tyr4-Ala5-Phe6-Phe7-Gln8-d-Phe 9-Pro10). The change of residues 6 and 7 resulted in a 42-fold increase in affinity and no increase in Vmax. As a substrate, this peptide displayed Michaelis-Menten kinetics at saturating ATP conditions. As an inhibitor, mixed inhibition was observed. A linear version of this peptide was 13-fold less potent an inhibitor than the cyclic peptide.

Mutation analysis of the mouse myosin VIIA deafness gene.

Mburu P, Liu XZ, Walsh J, Saw D Jr, Cope MJ, Gibson F, Kendrick-Jones J, Steel KP, Brown SD
Genes Funct. 1997; 1: 191-203

The shaker-1 (Myo7a) mouse deafness locus is encoded by an unconventional myosin gene: myosin VIIA [Gibson, Walsh, Mburu, Varela, Brown, Antonio, Biesel, Steel and Brown (1995) Nature (London) 374, 62-64]. The myosin VIIA gene is expressed in hair cells in the cochlea, where it is thought to function in the development of the critical neuroepithelium where auditory transduction takes place. In order to understand better the function of myosin VIIA, we have determined the complete sequence of the mouse myosin VIIA cDNA and employed the wild-type sequence for mutational analysis of a number of shaker-1 alleles. Analysis of the mouse myosin VIIA tail sequence demonstrates a large internal repeat with regions of similarity to myosins IV, X and XII as well as members of the band 4.1 family. In addition, the myosin VIIA repeats are similar along their entire length to a tail domain from a plant kinesin. The mouse myosin VIIA tail also contains a putative Src homology 3 (SH3) domain. Along with three previously reported shaker-1 mutations, mutations for seven shaker-1 alleles in total have now been identified. The mutational changes have been analysed in terms of their predicted effect on both myosin motor head and tail domain function and the predictions related to the known phenotypes of the shaker-1 alleles. Five of the mutations lie in the motor head, and analysis of their likely effect on myosin head structure correlates well with the known severity of the shaker-1 alleles. Of the two mutations in the tail, one is a missense mutation within the kinesin and myosin IV, X and XII homology domains that substitutes a conserved amino acid and leads to a severe deafness phenotype. This and other data suggest that myosin VIIA may have properties of a myosin-motor-kinesin-tail hybrid and be involved in membrane turnover within the actin-rich environment of the apical hair cell surface.

The noncatalytic src homology region 2 segment of abl tyrosine kinase binds to tyrosine-phosphorylated cellular proteins with high affinity.

Mayer BJ, Jackson PK, Baltimore D
Proc Natl Acad Sci U S A. 1991; 88: 627-31

Several proteins implicated in the regulation of cell proliferation contain a common noncatalytic domain, src homology region 2 (SH2). We have used the bacterially expressed SH2 domain of abl protein-tyrosine kinase to evaluate the ability of this domain to bind to cellular proteins. ablSH2 specifically bound to a number of tyrosine-phosphorylated proteins from cells transformed by tyrosine kinase oncogenes in a filter-binding assay and to a subset of those proteins in solution. The SH2 probe bound almost exclusively to tyrosine-phosphorylated proteins, and binding was eliminated by dephosphorylation of cell proteins. Free phosphotyrosine could partially disrupt SH2 binding, suggesting that phosphotyrosine is directly involved in the binding interaction. These results demonstrate that an SH2 domain is sufficient to confer direct, high-affinity phosphotyrosine-dependent binding to proteins and suggest a general role for SH2 domains in cellular signaling pathways.

Functions of SH2 and SH3 domains.

Mayer BJ, Gupta R
Curr Top Microbiol Immunol. 1998; 228: 1-22

SH3 domains. Minding your p's and q's.

Mayer BJ, Eck MJ
Curr Biol. 1995; 5: 364-7

SH3 domains mediate many important protein-protein interactions. The molecular basis of the binding of these domains to their ligands has been revealed, making it possible to identify SH3-binding sites in new proteins.

Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase.

Mayer BJ, Baltimore D
Mol Cell Biol. 1994; 14: 2883-94

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.

SH3 domains: complexity in moderation.

Mayer BJ
J Cell Sci. 2001; 114: 1253-63

The SH3 domain is perhaps the best-characterized member of the growing family of protein-interaction modules. By binding with moderate affinity and selectivity to proline-rich ligands, these domains play critical roles in a wide variety of biological processes ranging from regulation of enzymes by intramolecular interactions, increasing the local concentration or altering the subcellular localization of components of signaling pathways, and mediating the assembly of large multiprotein complexes. SH3 domains and their binding sites have cropped up in many hundreds of proteins in species from yeast to man, which suggests that they provide the cell with an especially handy and adaptable means of bringing proteins together. The wealth of genetic, biochemical and structural information available provides an intimate and detailed portrait of the domain, serving as a framework for understanding other modular protein-interaction domains. Processes regulated by SH3 domains also raise important questions about the nature of specificity and the overall logic governing networks of protein interactions.

Identification of the binding site for Gqalpha on its effector Bruton's tyrosine kinase.

Ma YC, Huang XY
Proc Natl Acad Sci U S A. 1998; 95: 12197-201

Heterotrimeric G proteins and tyrosine kinases are two major cellular signal transducers. Although G proteins are known to activate tyrosine kinases, the activation mechanism is not clear. Here, we demonstrate that G protein Gqalpha binds directly to the nonreceptor Bruton's tyrosine kinase (Btk) to a region composed of a Tec-homology (TH) domain and a sarcoma virus tyrosine kinase (Src)-homology 3 (SH3) domain both in vitro and in vivo. Only active GTP-bound Gqalpha, not inactive GDP-bound Gqalpha, can bind to Btk. Mutations of Btk that disrupt its ability to bind Gqalpha also eliminate Btk stimulation by Gqalpha, suggesting that this interaction is important for Btk activation. Remarkably, the structure of this TH (including a proline-rich sequence) -SH3 fragment of the Btk family of tyrosine kinases shows an intramolecular interaction. Furthermore, the crystal structure of the Src family of tyrosine kinases reveals that the intramolecular interaction of SH3 and its ligand is the major determining factor keeping the kinase inactive. Thus, we propose an activation model that entails binding of Gqalpha to the TH-SH3 region, thereby disrupting the TH-SH3 intramolecular interaction and activating Btk.

Mutagenesis of a buried polar interaction in an SH3 domain: sequence conservation provides the best prediction of stability effects.

Maxwell KL, Davidson AR
Biochemistry. 1998; 37: 16172-82

The SH3 domain from the Fyn tyrosine kinase possesses a buried hydrogen bond between the side chains of a glutamate (Glu24) and a serine (Ser41) residue. Multiple amino acid substitutions were made at these positions to determine the role of this interaction in the stability and conformational specificity of the domain and to assess the relationship between the thermodynamic stability of mutants and sequence conservation seen in the SH3 domain family. Analysis of single and double alanine mutations indicated that the Glu24-Ser41 interaction contributes 0.50 kcal/mol to the stability of the domain. However, disruption of the Glu24-Ser41 interaction did not impair peptide binding function, suggesting that the interaction is not critical for conformational specificity. The stability of the domain was not increased by the replacement of these residues with different combinations of hydrophobic residues or with potential salt bridge forming residues. Despite their similar structural roles in the Fyn SH3 domain, the Ser41 position was considerably more tolerant to substitution than was the Glu24 position. An alignment of >350 different SH3 domains has been completed in our laboratory. A statistically significant correlation was found between the conservation data for the Glu24 and Ser41 positions and the thermodynamic stabilities of the mutants constructed at these positions. Surprisingly, our analysis of sequence alignment data provided a more accurate prediction of the stability of mutants than did examination of the three-dimensional structure of the domain.

Cloning of ASH, a ubiquitous protein composed of one Src homology region (SH) 2 and two SH3 domains, from human and rat cDNA libraries.

Matuoka K, Shibata M, Yamakawa A, Takenawa T
Proc Natl Acad Sci U S A. 1992; 89: 9015-9

The protein ASH (for abundant Src homology), composed of one Src homology region (SH) 2 and two SH3 domains, was cloned by screening human and rat cDNA libraries with an oligonucleotide probe directed to a consensus sequence of the SH2 domains. The rat-derived ASH peptide was comprised of 217 amino acids with a molecular mass of 25-28 kDa and was found to be ubiquitous in rat tissues. A human cDNA clone was also found to code for part of the same protein, suggesting that ASH is common to human and rat. The amino acid sequence of ASH was strikingly similar to Sem-5, the product of a nematode cell-signaling gene, and ASH is most probably a mammalian homologue of Sem-5. ASH bound in vitro to phosphotyrosine-containing proteins, including activated epidermal growth factor receptor, the ASH SH2 domain being responsible for the binding. Induced expression of an antisense ASH cDNA led to a reduction in cell growth. Considering these observations and the structural homology to Sem-5, ASH is likely to function as a ubiquitous signal transducer, possibly resembling Sem-5, which communicates between a receptor protein tyrosine kinase and a Ras protein.

[The Src homology regions 2 and 3, mysterious associates with protein tyrosine kinases]

Matuoka K
Seikagaku. 1992; 64: 1268-72

Identification and characterization of a novel SH3-domain binding protein, Sab, which preferentially associates with Bruton's tyrosine kinase (BtK).

Matsushita M, Yamadori T, Kato S, Takemoto Y, Inazawa J, Baba Y, Hashimoto S, Sekine S, Arai S, Kunikata T, Kurimoto M, Kishimoto T, Tsukada S
Biochem Biophys Res Commun. 1998; 245: 337-43

Protein interaction cloning method was used to identify a novel molecule, Sab, which binds to the SH3 domain of Bruton's tyrosine kinase (Btk), the deficient cytoplasmic tyrosine kinase in human X-linked agammaglobulinemia and murine X-linked immunodeficiency. Immunoprecipitation using the anti-Sab antibody identified the protein product of the gene as a 70 kDa molecule. While Sab does not have a proline-rich sequence, it was shown to bind to Btk through the commonly conserved structure among SH3 domains. Remarkably, Sab exhibited a high preference for binding to Btk rather than to other cytoplasmic tyrosine kinases, which suggests a unique role of Sab in the Btk signal transduction pathway.

Interaction between the amino-terminal SH3 domain of CRK and its natural target proteins.

Matsuda M, Ota S, Tanimura R, Nakamura H, Matuoka K, Takenawa T, Nagashima K, Kurata T
J Biol Chem. 1996; 271: 14468-72

CRK is a human homolog of chichen v-Crk, which is an adaptor protein. The SH2 domain of CRK binds to several tyrosine-phosphorylated proteins, including the epidermal growth factor receptor, p130(Cas), Shc, and paxillin. The SH3 domain, in turn, binds to cytosolic proteins of 135-145, 160, 180, and 220 kDa. We screened expression libraries by Far Western blotting, using CRK SH3 as a probe, and identified partial cDNA sequences of four distinct proteins, including C3G, DOCK180, EPS15, and clone ST12. The consensus sequence of the CRK SH3 binding sites as deduced from their amino acid sequences was Pro+3-Pro+2-X+1-Leu0-Pro-1-X-2-Lys-3. The interaction of the CRK SH3 domain with the DOCK180 peptide was examined with an optical biosensor, based on the principles of surface plasmon resonance. A low dissociation constant of the order of 10(-7) resulted from a high association rate constant (kassoc = 3 x 10(4)) and low dissociation rate constant (kdiss = 3 x 10(-3)). All CRK-binding proteins except clone ST12 also bound to another adaptor protein, Grb2. Mutational analysis revealed that glycine at position +1 of ST12 inhibited the binding to Grb2 while retaining the high affinity binding to CRK SH3. The result suggests that the amino acid at position +1 also contributes to the high affinity binding of the peptides to the SH3 domain of Grb2, but not to that of CRK.

RN-tre identifies a family of tre-related proteins displaying a novel potential protein binding domain.

Matoskova B, Wong WT, Seki N, Nagase T, Nomura N, Robbins KC, Di Fiore PP
Oncogene. 1996; 12: 2563-71

Eps8 is a recently identified SH3-containing substrate for tyrosine kinase receptors. To understand the role of eps8 in receptor-mediated signaling, we cloned cDNAs encoding proteins that bind to its SH3 domain. One of these cDNAs predicts the synthesis of an 828 amino acid protein with homology to the N-terminal region of the tre oncogene. We designated this protein RN-tre for Related to the N-terminus of tre. RN-tre is ubiquitously expressed and maps to 10p13, a region known to be involved in translocations in various leukemias. In addition, a 10p13 monosomy syndrome, characterized by developmental alterations, has been reported. The regional homology between RN-tre and tre, which is limited to their N-terminal portion, prompted us to investigate the origin of the tre oncogene transcriptional unit. We were able to show that tre is the fusion product of a 5' genetic element, homologous to RN-tre and a 3' element, encoding a de-ubiquinating enzyme. Moreover, we identified, within the N-terminus of RN-tre and tre, a domain (named TrH, for Tre Homology), which is conserved within several proteins from yeast to mammals and has protein-binding properties in vitro.

RN-tre specifically binds to the SH3 domain of eps8 with high affinity and confers growth advantage to NIH3T3 upon carboxy-terminal truncation.

Matoskova B, Wong WT, Nomura N, Robbins KC, Di Fiore PP
Oncogene. 1996; 12: 2679-88

We isolated a cDNA encoding a protein, RN-tre, which shows homology to the N-terminus of the tre oncogene product and has SH3-binding ability as well as an evolutionarily conserved domain, termed TrH, with protein-binding ability in vitro. In the present study, we identify the product of the RN-tre gene as a 97-100 kDa protein. We demonstrate stable association in vivo and in vitro between RN-tre and eps8, mediated by the SH3 domain of the latter. In vitro, RN-tre displayed remarkable preference for binding to the eps8-SH3, as compared to eight other SH3s. The Kd for the in vitro interaction between RN-tre and eps8-SH3 was between 10(-8) and 10(-7) M. A role for RN-tre in cell proliferation was suggested by the finding that a C-terminal truncated mutant was able to confer proliferative advantage and reduced serum-requirement to NIH3T3 fibroblasts. Finally, comparison of the structure and biological activities of RN-tre and of the tre oncogene product, provided insight into the mechanism of oncogenic activation of tre.

Crystal structures of the transposon Tn5-carried bleomycin resistance determinant uncomplexed and complexed with bleomycin.

Maruyama M, Kumagai T, Matoba Y, Hayashida M, Fujii T, Hata Y, Sugiyama M
J Biol Chem. 2001; 276: 9992-9

The transposon Tn5 carries a gene designated ble that confers resistance to bleomycin (Bm). In this study, we determined the x-ray crystal structures of the ble gene product, designated BLMT, uncomplexed and complexed with Bm at 1.7 and 2.5 A resolution, respectively. The structure of BLMT is a dimer with two Bm-binding pockets composed of two large concavities and two long grooves. This crystal structure of BLMT complexed with Bm gives a precise mode for binding of the antibiotic to BLMT. The conformational change of BLMT generated by binding to Bm occurs at a beta-turn composed of the residues from Gln(97) to Thr(102). Crystallographic analysis of Bm bound to BLMT shows that two thiazolium rings of the bithiazole moiety are in the trans conformation. The axial ligand, which binds a metal ion, seems to be the primary amine in the beta-aminoalanine moiety. This report, which is the first with regard to the x-ray crystal structure of Bm, shows that the bithiazole moiety of Bm is far from the metal-binding domain. That is, Bm complexed with BLMT takes a more extended form than the drug complexed with DNA.

The hunting of the Src.

Martin GS
Nat Rev Mol Cell Biol. 2001; 2: 467-75

The non-receptor tyrosine kinase Src is important for many aspects of cell physiology. The viral src gene was the first retroviral oncogene to be identified, and its cellular counterpart was the first proto-oncogene to be discovered in the vertebrate genome. Src has been important, not only as an object of study in itself, but also as an entry point into the molecular genetics of cancer.

The folding transition state between SH3 domains is conformationally restricted and evolutionarily conserved.

Martinez JC, Serrano L
Nat Struct Biol. 1999; 6: 1010-6

The protein engineering analysis of the alpha-spectrin SH3 domain at three different stability conditions (pH 7.0, 3.5 and 2.5) reveals a folding transition state structured around the distal loop beta-hairpin and the 310-helix. This region is impervious to overall changes in protein stability, suggesting a transition state ensemble with little conformational variability. Comparison with the Src SH3 domain (36% sequence homology) indicates that the transition state in this protein family may be conserved. Discrepancies at some positions can be rationalized in terms of the different interactions made by the different side chains in both domains. Bronsted plot analysis confirms the straight phidouble dagger-U results and shows two folding subdomains for this small protein. These results, together with previous data on circular permutants of the alpha-spectrin SH3 domain, indicate that polypeptide topology and chain connectivity play a major role in the folding reaction of this protein family.

SH2 domain specificity and activity modified by a single residue.

Marengere LE, Songyang Z, Gish GD, Schaller MD, Parsons JT, Stern MJ, Cantley LC, Pawson T
Nature. 1994; 369: 502-5

Many intracellular targets of protein-tyrosine kinases possess Src homology 2 (SH2) domains that directly recognize phosphotyrosine-containing sites on autophosphorylated growth factor receptors and cytoplasmic proteins, and thereby mediate the activation of biochemical signalling pathways. SH2 domains possess relatively well conserved residues that form the phosphotyrosine-binding pocket, and more variable residues that are implicated in determining binding specificity by recognition of the three amino acids carboxy-terminal to phosphotyrosine (the +1 to +3 positions). One such residue, occupying the EF1 position of the +3-binding pocket, is a Thr in the SH2 domain of the Src tyrosine kinase, but is predicted to be a Trp in the SH2 domain of the Sem-5/drk/Grb2 adaptor protein. Here we report that changing this residue in the Src SH2 domain from Thr to Trp switches its selectivity to resemble that of the Sem-5/drk/Grb2 SH2 domain. Furthermore, this mutant Src SH2 domain effectively substitutes for the SH2 domain of the Sem-5 protein in activation of the Ras pathway in vivo. These results identify a residue that can modify SH2 selectivity, and indicate that the biological activity of an SH2 domain correlates with its binding specificity.

Activation of NFAT-dependent gene expression by Nef: conservation among divergent Nef alleles, dependence on SH3 binding and membrane association, and cooperation with protein kinase C-theta.

Manninen A, Huotari P, Hiipakka M, Renkema GH, Saksela K
J Virol. 2001; 75: 3034-7

Here we show that the potential to regulate NFAT is a conserved property of different Nef alleles and that Nef residues involved in membrane targeting and SH3 binding are critical for this function. Cotransfection of an activated protein kinase C-theta (PKC-theta) with Nef implicated PKC-theta as a possible physiological cofactor of Nef in promoting NFAT-dependent gene expression and T-cell activation.

SH3-Domain binding function of HIV-1 Nef is required for association with a PAK-related kinase.

Manninen A, Hiipakka M, Vihinen M, Lu W, Mayer BJ, Saksela K
Virology. 1998; 250: 273-82

HIV-1 Nef has previously been shown to bind to Src homology-3 (SH3) domains of a subset of Src family tyrosine kinases. In addition, Nef has been reported to coprecipitate with a serine/threonine kinase activity termed NAK (for Nef-associated kinase). The identity of NAK remains uncertain, but it has been suggested to represent a novel member of the p21-activated kinase (PAK) family. We report here that NAK autophosphorylation is increased not only by an activated form of the p21-family GTPase cdc42 but also by a plasma membrane-targeted fragment of the adapter protein Nck, thus providing further evidence that NAK is related to PAKs. A detailed structure-based mutational analysis of Nef revealed that all amino acid changes that inhibited the Nef/Hck-SH3 interaction, as measured by surface-plasmon resonance, also abolished coprecipitation of NAK. As PAK family proteins do not contain SH3 domains, these observations are best explained by a protein complex in which Nef, NAK, and an SH3-protein all contact each other. In addition, a number of conserved amino acids in Nef that are not involved in SH3 binding were also found to be crucial for association with NAK. Molecular modeling suggests that these residues are involved in formation of an adjacent binding surface for NAK or another critical component of the NAK/Nef complex.

Slap negatively regulates Src mitogenic function but does not revert Src-induced cell morphology changes.

Manes G, Bello P, Roche S
Mol Cell Biol. 2000; 20: 3396-406

Src-like adapter protein (Slap) is a recently identified protein that negatively regulates mitogenesis in murine fibroblasts (S. Roche, G. Alonso, A. Kazlausakas, V. M. Dixit, S. A. Courtneidge, and A. Pandey, Curr. Biol. 8:975-978, 1998) and comprises an SH3 and SH2 domain with striking identity to the corresponding Src domains. In light of this, we sought to investigate whether Slap could be an antagonist of all Src functions. Like Src, Slap was found to be myristylated in vivo and largely colocalized with Src when coexpressed in Cos7 cells. Microinjection of a Slap-expressing construct into quiescent NIH 3T3 cells inhibited platelet-derived growth factor (PDGF)-induced DNA synthesis, and the inhibition was rescued by the transcription factor c-Myc but not by c-Jun/c-Fos expression. Fyn (or Src) overexpression overrides the G(1)/S block induced by both SrcK- and a Slap mutant with a deletion of its C terminus (SlapDeltaC), but not the block induced by Slap or SlapDeltaSH3, implying that the C terminus is a noncompetitive inhibitor of Src mitogenic function. Furthermore, a chimeric adapter comprising SrcDeltaK fused to the Slap C terminus (Src/SlapC) also inhibited Src function during the PDGF response in a noncompetitive manner, as Src coexpression could not rescue PDGF signaling. Slap, however, did not reverse deregulated Src-induced cell transformation, as it was unable to inhibit depolymerization of actin stress fibers while still being able to inhibit SrcY527F-induced DNA synthesis. This was attributed to a distinct Slap SH3 binding specificity, since the chimeric Slap/SrcSH3 molecule, in which the Slap SH3 was replaced by the Src SH3 sequence, substantially restored stress fiber formation. Indeed, three amino acids important for ligand binding in Src SH3 were replaced in the Slap SH3 sequence; Slap SH3 did not bind to the Src SH3 partners p85alpha, Shc, and Sam68 in vitro, and the chimeric tyrosine kinase Slap/SrcK, composed of SlapDeltaC fused to the SH2 linker kinase sequence of Src, was not regulated in vivo. Furthermore, the Src SH3 domain is required for signaling during mitogenesis and since Slap/SrcK behaved as a dominant negative in the PDGF mitogenic response when microinjected into quiescent fibroblasts. We conclude that Slap is a negative regulator of Src during mitogenesis involving both the SH2 and the C terminus domains in a noncompetitive manner, but it does not regulate all Src function due to specific SH3 binding substrates.

Some NMR experiments and a structure determination employing a [15N,2H] enriched protein.

Mal TK, Matthews SJ, Kovacs H, Campbell ID, Boyd J
J Biomol NMR. 1998; 12: 259-76

We present the results of studies of an aqueous sample of a highly [15N,2H] enriched protein, the SH3 domain from Fyn. Measurements of 1H relaxation and interactions between H2O solvent and exchangeable protons are given, as well as a method for increasing the effective longitudinal relaxation of solvent exchangeable proton resonances. The long-range isotope shifts are measured, for 1H and 15N, which arise due to perdeuteration. Simulations, which employed a 7 or 8 spin relaxation matrix analysis, were compared to the experimental data from a time series of 2D NOESY datasets for some resonances. The agreement between experiment and simulation suggest that, with this 1H dilute sample, relatively long mixing times (up to 1.2 s) can be used to detect specific dipolar interactions between amide protons up to about 7A apart. A set of 155 inter-amide NOEs and 7 side chain NOEs were thus identified in a series of 3D HSQC-NOESY-HSQC experiments. These data, alone and in combination with previously collected restraints, were used to calculate sets of structures using X-PLOR. These results are compared to the available X-ray and NMR structures of the Fyn SH3 domain.

Nitrotyrosine mimics phosphotyrosine binding to the SH2 domain of the src family tyrosine kinase lyn.

Mallozzi C, Di Stasi AM, Minetti M
FEBS Lett. 2001; 503: 189-95

The nitration of tyrosine residues in protein occurs through the action of reactive oxygen and nitrogen species and is considered a marker of oxidative stress under pathological conditions. The most active nitrating species so far identified is peroxynitrite, the product of the reaction between nitric oxide and superoxide anion. Previously, we have reported that in erythrocytes peroxynitrite irreversibly upregulates lyn, a tyrosine kinase of the src family. In this study we investigated the possible role of tyrosine nitration in the mechanism of lyn activation. We found that tyrosine containing peptides modelled either on the C-terminal tail of src kinases or corresponding to the first 15 amino acids of human erythrocyte band 3 were able to activate lyn when the tyrosine was substituted with 3-nitrotyrosine. The activity of nitrated peptides was shared with phosphorylated but not with unphosphorylated, chlorinated or scrambled peptides. Recombinant lyn src homology 2 (SH2) domain blocked the capacity of the band 3-derived nitrotyrosine peptide to activate lyn and we demonstrated that this peptide specifically binds the SH2 domain of lyn. We propose that nitropeptides may activate src kinases through the displacement of the phosphotyrosine in the tail from its binding site in the SH2 domain. These observations suggest a new mechanism of peroxynitrite-mediated signalling that may be correlated with the upregulation of tyrosine phosphorylation observed in several pathological conditions.

Identification of amino acid residues required for a specific interaction between Src-tyrosine kinase and proline-rich region of phosphatidylinositol-3' kinase.

Mak P, He Z, Kurosaki T
FEBS Lett. 1996; 397: 183-5

The binding of ligand to B-cell antigen receptors (BCR) leads to the activation of receptor-associated Src-family kinases and phosphatidylinositol-3' kinase (PI-3 kinase). Although it has been demonstrated that SH3 domains of several Src-family kinases interact with PI-3 kinase by binding to a proline-rich region of PI-3 kinase in vitro, there is no direct evidence to support their interaction in vivo. Thus, we utilized the yeast two-hybrid assay to reconstitute this protein-protein interaction. This genetic screen clearly indicates that the interaction between SH3 domain of Fyn and the proline-rich region (residues: 80-104) of PI-3 kinase is highly specific. Mutational analysis revealed that amino acid residues Asp92, Tyr93, Arg96 and Thr97 of the SH3 domain of Fyn are essential for interacting with the proline-rich peptide of PI-3 kinase.

Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

Mahajan S, Fargnoli J, Burkhardt AL, Kut SA, Saouaf SJ, Bolen JB
Mol Cell Biol. 1995; 15: 5304-11

Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation.

The carboxy-terminal sequence of p56lck can regulate p60c-src.

MacAuley A, Cooper JA
Mol Cell Biol. 1988; 8: 3560-4

A chimera containing the coding region for residues 1 to 516 of p60c-src and residues 495 to 509 (the carboxy terminus) of p56lck was constructed and expressed in mouse fibroblasts. The chimeric protein appeared to be phosphorylated and regulated in the same fashion as p60c-src.

Structural differences between repressed and derepressed forms of p60c-src.

MacAuley A, Cooper JA
Mol Cell Biol. 1989; 9: 2648-56

The kinase activity of p60c-src is derepressed by removal of phosphate from Tyr-527, mutation of this residue to Phe, or binding of a carboxy-terminal antibody. We have compared the structures of repressed and active p60c-src, using proteases. All forms of p60c-src are susceptible to proteolysis at the boundary between the amino-terminal region and the kinase domain, but there are several sites elsewhere that are more sensitive to trypsin digestion in repressed than in derepressed forms of p60c-src. The carboxy-terminal tail (containing Tyr-527) is more sensitive to digestion by pronase E and thermolysin when Tyr-527 is not phosphorylated. The kinase domain fragment released with trypsin has kinase activity. Relative to intact p60c-src, the kinase domain fragment shows altered substrate specificity, diminished regulation by the phosphorylated carboxy terminus, and novel phosphorylation sites. The results identify parts of p60c-src that change conformation upon kinase activation and suggest functions for the amino-terminal region.

A fluorescence polarization based Src-SH2 binding assay.

Lynch BA, Loiacono KA, Tiong CL, Adams SE, MacNeil IA
Anal Biochem. 1997; 247: 77-82

The tyrosine kinase pp60c.src has been implicated as being a potential therapeutic target in several human diseases including cancer and osteoporosis. An important region within this kinase is the SH2 domain (Src homology 2) which binds to phosphorylated tyrosine residues contained within specific peptide sequences. Homologous domains are found in a variety of cytoplasmic proteins and have been shown to be essential for controlling many important signaling pathways. Developing specific inhibitors of SH2 interactions would therefore be extremely useful for modulating a variety of signaling pathways and potentially be useful for the treatment of human disease. Current methodology for the development of organic molecules as drug leads requires the ability to test thousands of individual compounds or natural product extracts in biochemical assays. Such tests must be reproducible, simple, and versatile. This paper describes an assay based on fluorescence polarization for measuring the binding of compounds to the Src-SH2 domain. The assay is insensitive to changes in fluorescence intensity working even in solutions with moderate optical density and functions in the presence of up to 20% dimethyl sulfoxide. These features make it especially useful for high-throughput screening of both natural and synthetic compound libraries.

A protein kinase assay system for both acidic and basic peptides.

Lou Q, Wu J, Lam KS
Anal Biochem. 1996; 235: 107-9

Potent pseudosubstrate-based peptide inhibitors for p60(c-src) protein tyrosine kinase.

Lou Q, Leftwich ME, McKay RT, Salmon SE, Rychetsky L, Lam KS
Cancer Res. 1997; 57: 1877-81

We recently reported the identification of GIYWHHY as an efficient and specific substrate for p60(c-src) protein tyrosine kinase (PTK) by screening a secondary random peptide library (Q. Lou et al., Bioorg. Med. Chem., 4: 677-682, 1996). Based on the primary structure of GIYWHHY, we designed and synthesized several pseudosubstrate-based peptide inhibitors. Some of these peptide inhibitors are highly potent and specific with IC50 in the low micromolar range. Because both YIYGSFK and GIYWHHY are efficient and specific substrates for p60(c-src) PTK, chimeric branched peptides based on these two sequences were synthesized. These branched peptides inhibit p60(c-src) PTK with high potency, indicating that the enzyme-active site of p60(c-src) PTK can accommodate more than a linear motif. This may explain why seemingly several peptides with very different linear structures can all be phosphorylated by this enzyme.

Identification of GIYWHHY as a novel peptide substrate for human p60c-src protein tyrosine kinase.

Lou Q, Leftwich ME, Lam KS
Bioorg Med Chem. 1996; 4: 677-82

We have recently determined that -Ile-Tyr- were the two critical residues as a peptide substrate for p60c-src protein tyrosine kinase (Lou, Q. et al., Lett. Peptide Sci., 1995, 2, 289). Here, we report on the design and synthesis of a secondary 'one-bead, one-compound' combinatorial peptide library based on this dipeptide motif (XIYXXXX, where X = all 19 eukaryotic amino acids except for cysteine). This secondary library was screened for its ability to be phosphorylated by p60c-src PTK using [gamma 32P]ATP as a tracer. Five of the strongest [32P]-labeled peptide-beads were identified and microsequenced: GIYWHHY, KIYDDYE, EIYEENG, EIYEEYE, and YIYEEED. A solid-phase phosphorylation assay was used to evaluate the structure-activity relationship of GIYWHHY. It was determined that Ile2, Tyr3, His5, and His6 were crucial for its activity as a substrate.

Solution structure of the DNA binding domain of HIV-1 integrase.

Lodi PJ, Ernst JA, Kuszewski J, Hickman AB, Engelman A, Craigie R, Clore GM, Gronenborn AM
Biochemistry. 1995; 34: 9826-33

The solution structure of the DNA binding domain of HIV-1 integrase (residues 220-270) has been determined by multidimensional NMR spectroscopy. The protein is a dimer in solution, and each subunit is composed of a five-stranded beta-barrel with a topology very similar to that of the SH3 domain. The dimer is formed by a stacked beta-interface comprising strands 2, 3, and 4, with the two triple-stranded antiparallel beta-sheets, one from each subunit, oriented antiparallel to each other. One surface of the dimer, bounded by the loop between strands beta 1 and beta 2, forms a saddle-shaped groove with dimensions of approximately 24 x 23 x 12 A in cross section. Lys264, which has been shown from mutational data to be involved in DNA binding, protrudes from this surface, implicating the saddle-shaped groove as the potential DNA binding site.

Two isoforms of murine hck, generated by utilization of alternative translational initiation codons, exhibit different patterns of subcellular localization.

Lock P, Ralph S, Stanley E, Boulet I, Ramsay R, Dunn AR
Mol Cell Biol. 1991; 11: 4363-70

Mammalian hck, a member of the src family of tyrosine kinases, is expressed predominantly in cells of the myeloid and B-lymphoid lineages. Using mutational analysis, we have investigated the molecular basis of two immunoreactive forms of murine hck of 56 and 59 kDa found in numerous hemopoietic cell types. Our results indicate that translation of murine p59hck initiates from a CTG codon located 21 codons 5' of an ATG that is utilized to generate p56hck. We provide evidence that two human hck isoforms are generated by the same mechanism. Subcellular fractionation studies reveal that while p59hck and p56hck are associated with membranes of various murine B-lymphoid and myeloid cell lines, p59hck alone is also located in the cytosol. In contrast to membrane-associated p59hck, which is metabolically labeled with [3H]myristic acid and exhibits amphiphilic properties in Triton X-114 detergent, cytosolic p59hck is hydrophilic, suggesting that it is not acylated. Possible mechanisms are proposed to account for these observations.

Biochemistry of the Src protein-tyrosine kinase: regulation by SH2 and SH3 domains.

Liu X, Pawson T
Recent Prog Horm Res. 1994; 49: 149-60

pp60c-Srs (c-Src) is the prototype for a family of cytoplasmic protein-tyrosine kinases involved in the control of signal transduction. In addition to the enzymatic kinase domain, c-Src has several noncatalytic domains which regulate Src tyrosine kinase activity in both a positive and a negative fashion. Phosphorylation of c-Src at Tyr527 in the noncatalytic C-terminal tail is a key mechanism for repression of c-Src tyrosine kinase activity. This inhibitory phosphorylation is apparently catalyzed by another cytoplasmic tyrosine kinase (Csk). Recent evidence suggests that the c-Src SH2 domain participates in this phosphorylation-dependent repression of kinase activity through an intramolecular association with the phosphotyrosine-containing C-terminus. The SH3 domain of c-Src also negatively regulates c-Src tyrosin kinase activity, although the mechanism is as yet unknown. However, in the background of constitutively active transforming Src variants, such as a c-Src mutant with an amino acid substitution eliminating Tyr527 (527F c-Src) or the retroviral oncogene v-src product pp60v-src (v-Src), both the SH2 and SH3 domains contribute positively to the enzymatic and biological activities of the Src tyrosine kinase through interactions with Src substrates and/or cellular regulators.

The v-Src SH3 domain binds phosphatidylinositol 3'-kinase.

Liu X, Marengere LE, Koch CA, Pawson T
Mol Cell Biol. 1993; 13: 5225-32

Fibroblasts transformed by v-src or by related oncogenes encoding activated tyrosine kinases contain elevated levels of polyphosphoinositides with phosphate at the D-3 position of the inositol ring, as a result of the activation of phosphatidylinositol (PI) 3'-kinase. v-src-transformed cells also contain increased levels of PI 3'-kinase activity immunoprecipitable with anti-phosphotyrosine antibodies; furthermore, PI 3'-kinase can be detected in association with the v-Src tyrosine kinase. To identify regions of v-Src that can interact with PI 3'-kinase, the v-Src SH2 and SH3 domains were expressed in bacteria and incubated with lysates of normal chicken embryo fibroblasts. In vitro, the v-Src SH3 domain, but not the SH2 domain, bound PI 3'-kinase in lysates of uninfected chicken embryo fibroblasts. Substitutions of two highly conserved SH3 residues implicated in ligand binding abolished the ability of the v-Src SH3 domain to associate with PI 3'-kinase. Furthermore, the v-Src SH3 domain bound in vitro to the amino-terminal region of the p85 alpha subunit of PI 3'-kinase. These results suggest that the v-Src SH3 domain may mediate an interaction between the v-Src tyrosine kinase and PI 3'-kinase, by direct binding to p85.

Regulation of c-Src tyrosine kinase activity by the Src SH2 domain.

Liu X, Brodeur SR, Gish G, Songyang Z, Cantley LC, Laudano AP, Pawson T
Oncogene. 1993; 8: 1119-26

The protein-tyrosine kinase activity of pp60c-src (c-Src) is inhibited by phosphorylation of tyr527, within the c-Src c-terminal tail. Genetic and biochemical data have suggested that this negative regulation requires an intact Src homology 2 (SH2) domain. Since SH2 domains recognize phosphotyrosine, it is possible that these two non-catalytic domains associate, and thereby repress c-Src kinase activity. Consistent with this model, an isolated Src SH2 domain expressed in bacteria as a GST fusion protein bound in vitro to a synthetic phosphotyrosine-containing peptide modeled on the C-terminal 13 residues of the c-Src tail. Binding was absolutely dependent on phosphorylation of tyr527 in the tail peptide, and was modified by both the length and sequence of the peptide. Competition experiments indicated only a moderate binding affinity between the Src SH2 domain and the phosphorylated tail. A distinct phosphotyrosine-containing peptide previously identified as binding the Src SH2 domain with high affinity stimulated c-Src tyrosine kinase activity in vitro, possibly by competing with the endogenous tail phosphorylation site for binding to the SH2 domain. Indeed, this activation was competitively inhibited by purified bacterial Src SH2 domain. These data provide direct evidence that the c-Src tail has an intrinsic affinity for the Src SH2 domain, and suggest that such an interaction in the intact molecule contributes to maintaining c-Src in an inactive form.

Binding of SH2 containing proteins to the insulin receptor: a new way for modulating insulin signalling.

Liu F, Roth RA
Mol Cell Biochem. 1998; 182: 73-8

Prior studies have established a role in insulin action for the tyrosine phosphorylation of substrates and their subsequent complexing with SH2 containing proteins. More recently, SH2 proteins have been identified which can tightly bind to the tyrosine phosphorylated insulin receptor. The major protein identified so far (called Grb-IR or Grb10) of this type appears to be present in at least 3 isoforms, varying in the presence of a pleckstrin homology domain and in the sequence of its amino terminus. The binding of this protein to the insulin receptor appears to inhibit signalling by the receptor. The present review will discuss the current knowledge of the structure and function of this protein.

Prediction of the structure of human Janus kinase 2 (JAK2) comprising the two carboxy-terminal domains reveals a mechanism for autoregulation.

Lindauer K, Loerting T, Liedl KR, Kroemer RT
Protein Eng. 2001; 14: 27-37

The structure of human Janus kinase 2 (JAK2) comprising the two C-terminal domains (JH1 and JH2) was predicted by application of homology modelling techniques. JH1 and JH2 represent the tyrosine kinase and tyrosine kinase-like domains, respectively, and are crucial for function and regulation of the protein. A comparison between the structures of the two domains is made and structural differences are highlighted. Prediction of the relative orientation of JH1 and JH2 was aided by a newly developed method for the detection of correlated amino acid mutations. Analysis of the interactions between the two domains led to a model for the regulatory effect of JH2 on JH1. The predictions are consistent with available experimental data on JAK2 or related proteins and provide an explanation for inhibition of JH1 tyrosine kinase activity by the adjacent JH2 domain.

Critical residues in an SH3 domain from Sem-5 suggest a mechanism for proline-rich peptide recognition.

Lim WA, Richards FM
Nat Struct Biol. 1994; 1: 221-5

Src homology 3 (SH3) domains bind specific proline-rich peptide motifs. To identify interactions involved in peptide recognition, we have mutated residues on the putative binding surface of an SH3 domain from the Caenorhabditis elegans protein Sem-5. Among the most critical positions are three adjacent aromatic residues, which appear to participate in highly stereospecific packing interactions with the ligand. The co-planar arrangement of two of these residues closely matches the periodicity of a poly-proline II (PPII) helix. Thus, a model for recognition has the peptide adopting a PPII helix, with the pyrrolidine rings on one helical face interlocking with the aromatic SH3 residues.

Reading between the lines: SH3 recognition of an intact protein.

Lim WA
Structure. 1996; 4: 657-9

The structure of Nef-SH3 domain complex reveals how an SH3 domain can more effectively 'read' its linear proline-rich recognition element when it is presented within the context of a folded protein.

Regulated association of microtubule-associated protein 2 (MAP2) with Src and Grb2: evidence for MAP2 as a scaffolding protein.

Lim RW, Halpain S
J Biol Chem. 2000; 275: 20578-87

Microtubule-associated protein 2 (MAP2) and tau, which is involved in Alzheimer's disease, are major cytoskeletal proteins in neurons. These proteins are involved in microtubule assembly and stability. To further characterize MAP2, we took a strategy of identifying potential MAP2 binding partners. The low molecular weight MAP2c protein has 11 PXXP motifs that are conserved across species, and these PXXP motifs could be potential ligands for Src homology 3 (SH3) domains. We tested for MAP2 interaction with SH3 domain-containing proteins. All neuronal MAP2 isoforms bound specifically to the SH3 domains of c-Src and Grb2 in an in vitro glutathione S-transferase-SH3 pull-down assay. Interactions between endogenous proteins were confirmed by co-immunoprecipitation using brain lysate. All three proteins were also found co-expressed in neuronal cell bodies and dendrites. Surprisingly, the SH3 domain-binding site was mapped to the microtubule-binding domain that contains no PXXP motif. Src bound primarily the soluble, non-microtubule-associated MAP2c in vitro. This specific MAP2/SH3 domain interaction was inhibited by phosphorylation of MAP2c by the mitogen-activated protein kinase extracellular signal-regulated kinase 2 but not by protein kinase A. This phosphorylation-regulated association of MAP2 with proteins of intracellular signal transduction pathways suggests a possible link between cellular signaling and neuronal cytoskeleton, with MAP2 perhaps acting as a molecular scaffold upon which cytoskeleton-modifying proteins assemble and dissociate in response to neuronal activity.

SPIN90 (SH3 protein interacting with Nck, 90 kDa), an adaptor protein that is developmentally regulated during cardiac myocyte differentiation.

Lim CS, Park ES, Kim DJ, Song YH, Eom SH, Chun JS, Kim JH, Kim JK, Park D, Song WK
J Biol Chem. 2001; 276: 12871-8

In the yeast two-hybrid screening, we have isolated a cDNA clone from a human heart library using Nck Src homology 3 (SH3) domains as bait. The full-length cDNA, which encoded 722 amino acids, was identified as a VIP54-related gene containing an SH3 domain, proline-rich motifs, a serine/threonine-rich region, and a long C-terminal hydrophobic region. We refer to this protein as SPIN90 (SH3 Protein Interacting with Nck, 90 kDa). The amino acid sequence of the SH3 domain has the highest homology with those of Fyn, Yes, and c-Src. SPIN90 was broadly expressed in human tissues; in particular, it was highly expressed in heart, brain, and skeletal muscle, and its expression was developmentally regulated during cardiac myocyte differentiation. SPIN90 is able to bind to the first and third SH3 domains of Nck, in vitro, and is colocalized with Nck at sarcomere Z-discs within cardiac myocytes. Moreover, treatment with antisera raised against SPIN90 disrupted sarcomere structure, suggesting that this protein may play an important role in the maintenance of sarcomere structure and/or in the assembly of myofibrils into sarcomeres.

Kinetics, thermodynamics and evolution of non-native interactions in a protein folding nucleus.

Li L, Mirny LA, Shakhnovich EI
Nat Struct Biol. 2000; 7: 336-42

A lattice model with side chains was used to investigate protein folding with computer simulations. In this model, we rigorously demonstrate the existence of a specific folding nucleus. This nucleus contains specific interactions not present in the native state that, when weakened, slow folding but do not change protein stability. Such a decoupling of folding kinetics from thermodynamics has been observed experimentally for real proteins. From our results, we conclude that specific non-native interactions in the transition state would give rise to straight phi-values that are negative or larger than unity. Furthermore, we demonstrate that residue Ile 34 in src SH3, which has been shown to be kinetically, but not thermodynamically, important, is universally conserved in proteins with the SH3 fold. This is a clear example of evolution optimizing the folding rate of a protein independent of its stability and function.

An evolutionary trace method defines binding surfaces common to protein families.

Lichtarge O, Bourne HR, Cohen FE
J Mol Biol. 1996; 257: 342-58

X-ray or NMR structures of proteins are often derived without their ligands, and even when the structure of a full complex is available, the area of contact that is functionally and energetically significant may be a specialized subset of the geometric interface deduced from the spatial proximity between ligands. Thus, even after a structure is solved, it remains a major theoretical and experimental goal to localize protein functional interfaces and understand the role of their constituent residues. The evolutionary trace method is a systematic, transparent and novel predictive technique that identifies active sites and functional interfaces in proteins with known structure. It is based on the extraction of functionally important residues from sequence conservation patterns in homologous proteins, and on their mapping onto the protein surface to generate clusters identifying functional interfaces. The SH2 and SH3 modular signaling domains and the DNA binding domain of the nuclear hormone receptors provide tests for the accuracy and validity of our method. In each case, the evolutionary trace delineates the functional epitope and identifies residues critical to binding specificity. Based on mutational evolutionary analysis and on the structural homology of protein families, this simple and versatile approach should help focus site-directed mutagenesis studies of structure-function relationships in macromolecules, as well as studies of specificity in molecular recognition. More generally, it provides an evolutionary perspective for judging the functional or structural role of each residue in protein structure.

Crystal structure of P13K SH3 domain at 20 angstroms resolution.

Liang J, Chen JK, Schreiber ST, Clardy J
J Mol Biol. 1996; 257: 632-43

The P13K SH3 domain, residues 1 to 85 of the P1-3 kinase p85 subunit, has been characterized by X-ray diffraction. Crystals belonging to space group P4(3)2(1)2 diffract to 2.0 angstroms resolution and the structure was phased by single isomorphous replacement and anomalous scattering (SIRAS). As expected, the domain is a compact beta barrel with an over-all confirmation very similar to the independently determined NMR structures. The X-ray structure illuminates a discrepancy between the two NMR structures on the conformation of the loop region unique to P13K SH3. Furthermore, the ligand binding pockets of P13K SH3 domain are occupied by amino acid residues from symmetry-related P13K SH3 molecules: the C-terminal residues I(82) SPP of one and R18 of another. The interaction modes clearly resemble those observed for the P13K SH3 domain complexed with the synthetic peptide RLP1, a class 1 ligand, although there are significant differences. The solid-state interactions suggest a model of protein-protein aggregation that could be mediated by SH3 domains.

The C-terminal SH3 domain of the adapter protein Grb2 binds with high affinity to sequences in Gab1 and SLP-76 which lack the SH3-typical P-x-x-P core motif.

Lewitzky M, Kardinal C, Gehring NH, Schmidt EK, Konkol B, Eulitz M, Birchmeier W, Schaeper U, Feller SM
Oncogene. 2001; 20: 1052-62

The adapter Grb2 is an important mediator of normal cell proliferation and oncogenic signal transduction events. It consists of a central SH2 domain flanked by two SH3 domains. While the binding specificities of the Grb2 SH2 and N-terminal SH3 domain [Grb2 SH3(N)] have been studied in detail, binding properties of the Grb2 SH3(C) domain remained poorly defined. Gab1, a receptor tyrosine kinase substrate which associates with Grb2 and the c-Met receptor, was previously shown to bind Grb2 via a region which lacks a Grb2 SH3(N)-typical motif (P-x-x-P-x-R). Precipitation experiments with the domains of Grb2 show now that Gab1 can bind stably to the Grb2 SH3(C) domain. For further analyses, Gab1 mutants were generated by PCR to test in vivo residues thought to be crucial for Grb2 SH3(C) binding. The Grb2 SH3(C) binding region of Gab1 has significant homology to a region of the adapter protein SLP-76. Peptides corresponding to epitopes SLP-76, Gab1, SoS and other proteins with related sequences, as well as mutant peptides were synthesized and analysed by tryptophan-fluorescence spectrometry and by in vitro competition experiments. These experiments define a 13 amino acid sequence with the unusual consensus motif P-x-x-x-R-x-x-K-P as required for a stable binding to the SH3(C) domain of Grb2. Additional analyses point to a distinct binding specificity of the Grb2-homologous adapter protein Mona (Gads), indicating that the proteins of the Grb2 adapter family may have partially overlapping, yet distinct protein binding properties.

The extraordinary active site substrate specificity of pp60c-src. A multiple specificity protein kinase.

Lee TR, Niu J, Lawrence DS
J Biol Chem. 1995; 270: 5375-80

We report the first active site substrate specificity analysis of a tyrosine-specific protein kinase, namely pp60c-src. Like the cAMP-dependent protein kinase and protein kinase C, pp60c-src will phosphorylate an assortment of achiral residues attached to active site-directed peptides. Furthermore, pp60c-src phosphorylates both aromatic and aliphatic alcohols. However, the substrate specificity of pp60c-src is much broader than that of the two previously examined serine/threonine-specific protein kinases. We have previously shown that both the cAMP-dependent protein kinase and protein kinase C will utilize a wide array of non-amino acid residues as substrates, as long as the distance between the hydroxyl moiety and the adjacent peptide backbone is comparable with that present in serine and threonine (Kwon, Y.-G., Mendelow, M., and Lawrence, D. S. (1994) J. Biol. Chem. 269, 4839-4844). In marked contrast, pp60c-src does not discriminate against substrates on the basis of chain length, catalyzing the phosphorylation of residues that contain anywhere from 2-12 carbons between the alcohol functional group and the adjacent peptide bond. In addition, pp60c-src phosphorylates L-serine in an active site-directed peptide. The possible structural basis for the multiple specificity of pp60c-src is discussed. Finally, the active site specificity of pp60c-src is not just limited to L-amino acid residues, but also extends into the realm of D-amino acids as well.

A single amino acid in the SH3 domain of Hck determines its high affinity and specificity in binding to HIV-1 Nef protein.

Lee CH, Leung B, Lemmon MA, Zheng J, Cowburn D, Kuriyan J, Saksela K
EMBO J. 1995; 14: 5006-15

We have examined the differential binding of Hck and Fyn to HIV-1 Nef to elucidate the structural basis of SH3 binding affinity and specificity. Full-length Nef bound to Hck SH3 with the highest affinity reported for an SH3-mediated interaction (KD 250 nM). In contrast to Hck, affinity of the highly homologous Fyn SH3 for Nef was too weak (KD > 20 microM) to be accurately determined. We show that this distinct specificity lies in a variable loop, the 'RT loop', positioned close to conserved SH3 residues implicated in the binding of proline-rich (PxxP) motifs. A mutant Fyn SH3 with a single amino acid substitution (R96I) in its RT loop had an affinity (KD 380 nM) for Nef comparable with that of Hck SH3. Based on additional mutagenesis studies we propose that the selective recognition of Nef by Hck SH3 is determined by hydrophobic interactions involving an isoleucine residue in its RT loop. Although Nef contains a PxxP motif which is necessary for the interaction with Hck SH3, high affinity binding was only observed for intact Nef protein. The binding of a peptide containing the Nef PxxP motif showed > 300-fold weaker affinity for Hck SH3 than full-length Nef.

Genetic mapping of eight SH3 domain genes on seven mouse chromosomes.

Lee CG, Morse HC 3rd, Kay BK, Kozak CA
Mamm Genome. 1999; 10: 402-4

A unique insert in the linker domain of Syk is necessary for its function in immunoreceptor signalling.

Latour S, Zhang J, Siraganian RP, Veillette A
EMBO J. 1998; 17: 2584-95

Accumulating data indicate that the 'linker' region of Syk, which lies between its tandem Src homology 2 (SH2) domains and kinase region, provides a critical function for the biological activity of Syk. This importance has been ascribed to the presence of tyrosine phosphorylation sites capable of mediating the recruitment of cellular effectors. We and others previously identified an alternatively spliced variant of Syk, termed SykB, which lacks a 23 amino acid sequence in the linker domain. As this 'linker insert' is also not present in the closely related enzyme Zap-70, it seems plausible that Syk possesses this unique sequence for functional reasons. To understand its role better, we have compared the abilities of Syk and SykB to participate in immunoreceptor-triggered signal transduction. The results of our experiments revealed that, unlike Syk, SykB was inefficient at coupling stimulation of FcepsilonRI on basophils or the antigen receptor on T cells to the early and late events of cellular activation. Further studies showed that the functional defect in SykB was not caused by the absence of crucial tyrosine phosphorylation sites, or by a reduced intrinsic kinase activity. Rather, it correlated with the reduced ability of SykB to bind phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) in vitro and in vivo. In combination, these results demonstrated that the unique insert in the linker domain of Syk is crucial for its capacity to participate in immunoreceptor signalling. Furthermore, they provided evidence that the linker region can regulate the ability of Syk to bind ITAMs, thus identifying a novel function for this domain.

The identification of conserved interactions within the SH3 domain by alignment of sequences and structures.

Larson SM, Davidson AR
Protein Sci. 2000; 9: 2170-80

The SH3 domain, comprised of approximately 60 residues, is found within a wide variety of proteins, and is a mediator of protein-protein interactions. Due to the large number of SH3 domain sequences and structures in the databases, this domain provides one of the best available systems for the examination of sequence and structural conservation within a protein family. In this study, a large and diverse alignment of SH3 domain sequences was constructed, and the pattern of conservation within this alignment was compared to conserved structural features, as deduced from analysis of eighteen different SH3 domain structures. Seventeen SH3 domain structures solved in the presence of bound peptide were also examined to identify positions that are consistently most important in mediating the peptide-binding function of this domain. Although residues at the two most conserved positions in the alignment are directly involved in peptide binding, residues at most other conserved positions play structural roles, such as stabilizing turns or comprising the hydrophobic core. Surprisingly, several highly conserved side-chain to main-chain hydrogen bonds were observed in the functionally crucial RT-Src loop between residues with little direct involvement in peptide binding. These hydrogen bonds may be important for maintaining this region in the precise conformation necessary for specific peptide recognition. In addition, a previously unrecognized yet highly conserved beta-bulge was identified in the second beta-strand of the domain, which appears to provide a necessary kink in this strand, allowing it to hydrogen bond to both sheets comprising the fold.

Abl tyrosine protein kinase.

Laneuville P
Semin Immunol. 1995; 7: 255-66

Mammalian c-Abl belongs to an evolutionary conserved family of non-receptor tyrosine kinases. It is distributed both in the cytoplasm in association with F-actin, and in the nucleus where it binds chromatin. The normal function of c-Abl is poorly understood. Nevertheless, there has been rapid progress in the characterization of the structural features, signal transduction pathways, substrates and ligands involved in the action of c-Abl and Abl-derived oncogenes. These developments suggest that several mechanisms co-operate to allow regulation of normal cell growth by c-Abl and induction of leukemias by Bcr-abl.

Structure of the protein tyrosine kinase domain of C-terminal Src kinase (CSK) in complex with staurosporine.

Lamers MB, Antson AA, Hubbard RE, Scott RK, Williams DH
J Mol Biol. 1999; 285: 713-25

The crystal structure of the kinase domain of C-terminal Src kinase (CSK) has been determined by molecular replacement, co-complexed with the protein kinase inhibitor staurosporine (crystals belong to the space group P21212 with a=44.5 A, b=120.6 A, c=48.3 A). The final model of CSK has been refined to an R-factor of 19.9 % (Rfree=28.7 %) at 2.4 A resolution. The structure consists of a small, N-terminal lobe made up mostly of a beta-sheet, and a larger C-terminal lobe made up mostly of alpha-helices. The structure reveals atomic details of interactions with staurosporine, which binds in a deep cleft between the lobes. The polypeptide chain fold of CSK is most similar to c-Src, Hck and fibroblast growth factor receptor 1 kinase (FGFR1K) and most dissimilar to insulin receptor kinase (IRK).Interactions between the N and C-terminal lobe are mediated by the bound staurosporine molecule and by hydrogen bonds. In addition, there are several water molecules forming lobe-bridging hydrogen bonds, which may be important for maintaining the catalytic integrity of the kinase. Furthermore, the conserved Lys328 and Glu267 residues utilise water in the formation of a molecular pivot which is essential in allowing relative movement of the N and C-terminal lobes. An analysis of the residues around the ATP-binding site reveals structural differences with other protein tyrosine kinases. Most notable of these are different orientations of the conserved residues Asp332 and Phe333, suggesting that inhibitor binding proceeds via an induced fit.These structural observations have implications for understanding protein tyrosine kinase catalytic mechanisms and for the design of ATP-mimicking inhibitors of protein kinases.

Intramolecular regulatory interactions in the Src family kinase Hck probed by mutagenesis of a conserved tryptophan residue.

LaFevre-Bernt M, Sicheri F, Pico A, Porter M, Kuriyan J, Miller WT
J Biol Chem. 1998; 273: 32129-34

Intramolecular interactions between the Src homology domains (SH2 and SH3) and the catalytic domains of Src family kinases result in repression of catalytic activity. The crystal structure of the Src family kinase Hck, with its regulatory domains intact, has been solved. It predicts that a conserved residue, Trp260, at the end of the linker between the SH2 and the catalytic domains plays an important role in regulation by the SH3 and SH2 domains. We have mutated this residue and compared the activities of C-terminally phosphorylated wild type Hck and W260A Hck. The W260A mutant has a higher specific activity than wild type Hck. The W260A mutant requires autophosphorylation at Tyr416 for full activity, but it is not activated by ligand binding to the SH3 or SH2 domains. This mutation also changes the accessibility of the SH2 and SH3 domains to their cognate peptide ligands. Our results indicate that Trp260 plays a critical role in the coupling of the regulatory domains to the catalytic domain, as well as in positioning the ligand binding surfaces.

Measurement of the binding of tyrosyl phosphopeptides to SH2 domains: a reappraisal.

Ladbury JE, Lemmon MA, Zhou M, Green J, Botfield MC, Schlessinger J
Proc Natl Acad Sci U S A. 1995; 92: 3199-203

Src homology 2 (SH2) domain-mediated interactions with phosphotyrosine residues are critical in many intracellular signal transduction pathways. Attempts to understand the determinants of specificity and selectivity of these interactions have prompted many binding studies that have used several techniques. Some discrepancies, in both the absolute and relative values of the dissociation constants for particular interactions, are apparent. To establish the correct dissociation constants and to understand the origin of these differences, we have analyzed three previously determined interactions using the techniques of surface plasmon resonance and isothermal titration calorimetry. We find that the binding of SH2 domains to phosphopeptides is weaker than generally presumed. A phosphopeptide based on the hamster polyoma middle tumor antigen interacts with the SH2 domain from Src with an equilibrium dissociation constant (Kd) of 600 nM; a phosphopeptide based on one binding site from the platelet-derived growth factor receptor binds to the N-terminal SH2 domain of the 1-phosphatidylinositol 3-kinase p85 subunit with a Kd of 300 nM; and a phosphopeptide based on the C terminus of Lck binds to the SH2 domain of Lck with a Kd of 4 microM. In addition, we demonstrate that avidity effects that result from the dimerization of glutathione S-transferase fusion proteins with SH2 domains could be responsible for overestimates of affinities for these interactions previously studied by surface plasmon resonance.

Alternative modes of tyrosyl phosphopeptide binding to a Src family SH2 domain: implications for regulation of tyrosine kinase activity.

Ladbury JE, Hensmann M, Panayotou G, Campbell ID
Biochemistry. 1996; 35: 11062-9

Src homology 2 (SH2) domains interact with proteins containing phosphorylated tyrosine residues and as such play a key role in mediating tyrosine kinase signal transduction. Determination of how these interactions maintain specificity is central to understanding the mechanism of this intracellular signal processing. In the Src family tyrosine kinases specificity is enhanced by a form of regulation based on binding of a phosphotyrosine, pY, and its proximal amino acid sequence from the C-terminus to the SH2 domain of the same protein (autoregulation) or to a similar protein (homodimeric regulation). Activation of the protein is accomplished by removal of this regulatory interaction by competition from a "specific" interacting ligand. We adopt the SH2 domain from a member of the Src family, Fyn (whose predominant physiological role is in initiation of signals from the T-cell receptor complex), to explore the differences in structural, thermodynamic, and kinetic determinants of regulatory and specific interactions using tyrosyl phosphopeptides based on the C-terminus and on a putative physiological interacting species from the hamster middle-sized tumor antigen. The specific peptide interacts with micromolar affinity via embedding the pY and an isoleucine residue (in the pY + 3 position) in two deep pockets. This leads to a large favorable enthalpic contribution to free energy. The regulatory peptide interacts in the pY pocket which forms a pivot for the rest of the molecule which is dynamic. These structural data for the regulatory peptide are supported by the observation of a more favorable entropic term and a complex mode of binding revealed by kinetic analysis.

Molecular recognition properties of the C-terminal Sh3 domain of the Cbl associated protein, Cap.

Kurakin A, Hoffman NG, Kay BK
J Pept Res. 1998; 52: 331-7

A phage-displayed combinatorial peptide library was used to define the specificity of one of the three Src homology 3 (SH3) domains in a novel cytoskeletal protein, named CAP, for Cbl Associated Protein. The C-terminal SH3 domain was used to affinity select peptides with the consensus, PXPPXRXSSL, from a library of X6PXXPX6 peptides. Peptide sequences resembling this consensus were identified in two signal transduction proteins, c-Cbl and son-on-sevenless (Sos), previously shown to interact with the C-terminal SH3 domain of CAP. Genetic fusion of 16 and 14 amino acid segments of c-Cbl and Sos, respectively, to bacterial alkaline phosphatase confirmed that these segments were potential ligand sites for the C-terminal SH3 domain of CAP. Alanine-scanning mutagenesis of the c-Cbl peptide ligand confirmed that most of the residues, which were conserved among the peptide ligands selected from the combinatorial peptide library, contributed to binding to the C-terminal SH3 domain of CAP.

Native protein sequences are close to optimal for their structures.

Kuhlman B, Baker D
Proc Natl Acad Sci U S A. 2000; 97: 10383-8

How large is the volume of sequence space that is compatible with a given protein structure? Starting from random sequences, low free energy sequences were generated for 108 protein backbone structures by using a Monte Carlo optimization procedure and a free energy function based primarily on Lennard-Jones packing interactions and the Lazaridis-Karplus implicit solvation model. Remarkably, in the designed sequences 51% of the core residues and 27% of all residues were identical to the amino acids in the corresponding positions in the native sequences. The lowest free energy sequences obtained for ensembles of native-like backbone structures were also similar to the native sequence. Furthermore, both the individual residue frequencies and the covariances between pairs of positions observed in the very large SH3 domain family were recapitulated in core sequences designed for SH3 domain structures. Taken together, these results suggest that the volume of sequence space optimal for a protein structure is surprisingly restricted to a region around the native sequence.

Characterization of promoter region and genomic structure of the murine and human genes encoding Src like adapter protein.

Kratchmarova I, Sosinowski T, Weiss A, Witter K, Vincenz C, Pandey A
Gene. 2001; 262: 267-73

Src-like adapter protein (SLAP) was identified as a signaling molecule in a yeast two-hybrid system using the cytoplasmic domain of EphA2, a receptor protein tyrosine kinase (Pandey et al., 1995. Characterization of a novel Src-like adapter protein that associates with the Eck receptor tyrosine kinase. J. Biol. Chem. 270, 19201-19204). It is very similar to members of the Src family of cytoplasmic tyrosine kinases in that it contains very homologous SH3 and SH2 domains (Abram and Courtneidge, 2000. Src family tyrosine kinases and growth factor signaling. Exp. Cell. Res. 254, 1-13.). However, instead of a kinase domain at the C-terminus, it contains a unique C-terminal region. In order to exclude the possibility that an alternative form exists, we have isolated genomic clones containing the murine Slap gene as well as the human SLA gene. The coding regions of murine Slap and human SLA genes contain seven exons and six introns. Absence of any kinase domain in the genomic region confirm its designation as an adapter protein. Additionally, we have cloned and sequenced approximately 2.6 kb of the region 5' to the initiator methionine of the murine Slap gene. When subcloned upstream of a luciferase gene, this fragment increased the transcriptional activity about 6-fold in a human Jurkat T cell line and approximately 52-fold in a murine T cell line indicating that this region contains promoter elements that dictate SLAP expression. We have also cloned the promoter region of the human SLA gene. Since SLAP is transcriptionally regulated by retinoic acid and by activation of B cells, the cloning of its promoter region will permit a detailed analysis of the elements required for its transcriptional regulation.

1H and 15N assignments and secondary structure of the PI3K SH3 domain.

Koyama S, Yu H, Dalgarno DC, Shin TB, Zydowsky LD, Schreiber SL
FEBS Lett. 1993; 324: 93-8

The sequential 1H and 15N assignments of the SH3 domain of human phosphatidyl inositol 3'-kinase (PI3K) were determined by a combination of homonuclear and heteronuclear NMR experiments. With the exception of several protons belonging to lysine and proline residues, all proton and proton-bearing amide nitrogen resonances were assigned. Based on the sequential nuclear Overhauser effects (NOEs), 3JNH-C alpha H coupling constants and locations of slowly exchanging amide protons, we determined that the secondary structures of the protein consists of six beta-strands, two beta-turns and four short helices. Additional long range NOEs indicate that these beta-strands form two antiparallel beta-sheets. The topology of secondary structural elements of the PI3K SH3 domain is similar to those of the SH3 domains from c-Src and alpha-spectrin, suggesting that the SH3 family has a common tertiary structural motif.

Essential motions and energetic contributions of individual residues in a peptide bound to an SH3 domain.

Kolafa J, Perram JW, Bywater RP
Biophys J. 2000; 79: 646-55

We have studied protein-ligand interactions by molecular dynamics simulations using software designed to exploit parallel computing architectures. The trajectories were analyzed to extract the essential motions and to estimate the individual contributions of fragments of the ligand to overall binding enthalpy. Two forms of the bound ligand are compared, one with the termini blocked by covalent derivatization, and one in the underivatized, zwitterionic form. The ends of the peptide tend to bind more loosely in the capped form. We can observe significant motions in the bound ligand and distinguish between motions of the peptide backbone and of the side chains. This could be useful in designing ligands, which fit optimally to the binding protein. We show that it is possible to determine the different contributions of each residue in a peptide to the enthalpy of binding. Proline is a major net contributor to binding enthalpy, in keeping with the known propensity for this family of proteins to bind proline-rich peptides.

Structural requirements for the efficient regulation of the Src protein tyrosine kinase by Csk.

Koegl M, Courtneidge SA, Superti-Furga G
Oncogene. 1995; 11: 2317-29

Protein tyrosine kinases of the Src family are negatively regulated by phosphorylation in the C-terminal tail of the molecule. A different protein tyrosine kinase, Csk, is largely responsible for this regulation. The phosphorylated tail of c-Src engages with the SH2 domain in a conformation that is associated with low kinase activity and which involves stabilization by the SH3 domain. Inducible expression of c-Src in fission yeast is lethal unless Csk is coexpressed. Using this assay we present evidence that Src regulation by C-terminal phosphorylation does not require the myristylation signal or the unique domain at the N-terminus of the Src protein. Mutagenesis of the SH3 and SH2 domains of Csk show that neither are necessary in yeast or in vitro for efficient regulation of Src. Mutation of Tyr416 of Src, a site of autophosphorylation common to most protein tyrosine kinases, abolished the ability of Src to arrest growth of phosphorylate endogenous proteins. Tyr416 had the same effect on a shorter form of Src consisting of the kinase domain only, indicating that the mutation affects a property intrinsic to the catalytic domain. The residual activity of full-length Src mutated at Tyr416 is efficiently repressed by Csk action, suggesting that regulation by C-terminal phosphorylation does not act by preventing phosphorylation at Tyr416.

Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins.

Knudsen BS, Zheng J, Feller SM, Mayer JP, Burrell SK, Cowburn D, Hanafusa H
EMBO J. 1995; 14: 2191-8

The specificity of SH3 domain complex formation plays an important role in determining signal transduction events. We have previously identified a highly specific interaction between the first CrkSH3 domain [CrkSH3(1)] and proline-rich sequences in the guanine nucleotide exchange factor C3G. A 10 amino acid peptide derived from the first proline-rich sequence (P3P4P5A6L7P8P9K10K11R12) bound with a Kd of 1.89 +/- 0.06 microM and fully retained the high affinity and unique selectivity for the CrkSH3(1) domain. Mutational analysis showed that P5, P8, L7 and K10 are critical for high affinity binding. A conservative mutation, K10R, significantly decreased the affinity for the CrkSH3(1) domain while increasing the affinity for Grb2. Comparative binding studies with the K10R and K10A mutant peptides to c-Crk and v-Crk further suggested that K10 binds via a charge-dependent and a charge-independent interaction to the RT loop of the CrkSH3(1) domain. Besides determining important structural features necessary for high affinity and specificity binding to the CrkSH3(1) domain, our results also demonstrate that a conservative mutation in a single amino acid can significantly alter the specificity of an SH3 binding peptide.

Four proline-rich sequences of the guanine-nucleotide exchange factor C3G bind with unique specificity to the first Src homology 3 domain of Crk.

Knudsen BS, Feller SM, Hanafusa H
J Biol Chem. 1994; 269: 32781-7

The widely expressed cellular Crk protein has the domain structure SH2-SH3-SH3. We have previously demonstrated that the more N-terminal SH3 domain of Crk (CrkSH3(N)) specifically binds several cytoplasmic proteins. A cDNA encoding one of these proteins was isolated and found to have two different splice forms. The sequence is virtually identical to C3G, a guanine-nucleotide exchange factor. The center region of the 145-155-kDa protein contains four similar proline-rich sequences which are capable of binding individually to the SH3(N) domains of c-Crk and v-Crk. Comparison of these sequences in C3G to proline-rich sequences in other Crk-binding proteins suggests that positively charged amino acids following the prolines play an important role in the binding to the CrkSH3(N) domain. The endogenous C3G could be coprecipitated with Crk from cell lysates of cells expressing high levels of c-Crk or v-Crk, suggesting high binding affinity and a possible interaction in vivo. Unlike many other SH3-binding proteins which interact with multiple SH3 domains, C3G from cell lysates binds preferentially to the CrkSH3(N) domain. This unique binding specificity supports the idea that C3G plays an important role in Crk signaling pathways.

Molecular cloning and analysis of cDNA encoding the murine c-yes tyrosine protein kinase.

Klages S, Adam D, Eiseman E, Fargnoli J, Dymecki SM, Desiderio SV, Bolen JB
Oncogene. 1993; 8: 713-9

The cellular yes (c-yes) gene is a member of the class of proto-oncogenes that encode non-receptor tyrosine protein kinases. In this report we describe the isolation of cDNAs that encode the murine c-yes gene product and analysis of the nucleotide sequence of the murine c-yes cDNA clones. The reading frame encodes a protein of 541 amino acids with a calculated molecular mass of 60.63 kDa that is reactive with anti-Yes antisera and possesses protein kinase activity.

Ctk: a protein-tyrosine kinase related to Csk that defines an enzyme family.

Klages S, Adam D, Class K, Fargnoli J, Bolen JB, Penhallow RC
Proc Natl Acad Sci U S A. 1994; 91: 2597-601

We used the polymerase chain reaction with degenerate oligonucleotide primers to search for Csk-related kinases. A cDNA coding for a Csk-like protein-tyrosine kinase was cloned from mouse brain and was designated ctk, for csk-type protein-tyrosine kinase. The 1.9-kb ctk mRNA was found to be expressed predominantly in brain and capable of encoding a 52-kDa protein-tyrosine kinase. The amino acid sequence of Ctk was found to possess 53% identity with mouse Csk, shared all the predicted structural features of Csk, and was capable of phosphorylating the carboxyl-terminal conserved tyrosine of Src family members. Our results thereby indicate that ctk represents a gene that defines a family of structurally and functionally related Csk-like protein-tyrosine kinases.

Molecular cloning of p125Nap1, a protein that associates with an SH3 domain of Nck.

Kitamura T, Kitamura Y, Yonezawa K, Totty NF, Gout I, Hara K, Waterfield MD, Sakaue M, Ogawa W, Kasuga M
Biochem Biophys Res Commun. 1996; 219: 509-14

Binding proteins to the Src homology 3 (SH3) domains of Nck were screened by the use of glutathione S-transferase fusion proteins. Two proteins of 140 and 125 kDa were detected, both of which associated preferentially with the first SH3 domain of Nck. The 125-kDa protein, designated as Nap1 for Nck-associated protein 1, was purified and the corresponding rat cDNA was isolated. The predicted amino acid sequence revealed that p125Nap1 does not contain any known functional motif but shows sequence homology to Hem family gene. Using specific antibodies, p125Nap1 was shown to associate with Nck both in vitro and in intact cells. Further characterization of p125Nap1 may clarify the protein-protein interaction in the downstream signaling of Nck.

Effect of pH and salt bridges on structural assembly: molecular structures of the monomer and intertwined dimer of the Eps8 SH3 domain.

Kishan KV, Newcomer ME, Rhodes TH, Guilliot SD
Protein Sci. 2001; 10: 1046-55

The SH3 domain of Eps8 was previously found to form an intertwined, domain-swapped dimer. We report here a monomeric structure of the EPS8 SH3 domain obtained from crystals grown at low pH, as well as an improved domain-swapped dimer structure at 1.8 A resolution. In the domain-swapped dimer the asymmetric unit contains two "hybrid-monomers." In the low pH form there are two independently folded SH3 molecules per asymmetric unit. The formation of intermolecular salt bridges is thought to be the reason for the formation of the dimer. On the basis of the monomer SH3 structure, it is argued that Eps8 SH3 should, in principle, bind to peptides containing a PxxP motif. Recently it was reported that Eps8 SH3 binds to a peptide with a PxxDY motif. Because the "SH3 fold" is conserved, alternate binding sites may be possible for the PxxDY motif to bind. The strand exchange or domain swap occurs at the n-src loops because the n-src loops are flexible. The thermal b-factors also indicate the flexible nature of n-src loops and a possible handle for domain swap initiation. Despite the loop swapping, the typical SH3 fold in both forms is conserved structurally. The interface of the acidic form of SH3 is stabilized by a tetragonal network of water molecules above hydrophobic residues. The intertwined dimer interface is stabilized by hydrophobic and aromatic stacking interactions in the core and by hydrophilic interactions on the surface.

Direct binding of p130(Cas) to the guanine nucleotide exchange factor C3G.

Kirsch KH, Georgescu MM, Hanafusa H
J Biol Chem. 1998; 273: 25673-9

p130(Cas) (Cas; crk-associated substrate) belongs to a new family of docking molecules. It contains one Src homology (SH) 3 domain in its amino-terminal region followed by a region containing binding motifs for SH2 and SH3 domains. To gain further insight into Cas signaling we used the SH3 domain of Cas in a two-hybrid screen to search a human placenta library for binding partners. The screen confirmed a previous finding of its binding to the focal adhesion kinase (FAK) but also identified C3G, a guanine nucleotide exchange factor. We found direct interaction between Cas and C3G in vitro and in vivo. A series of analysis with C3G deletion mutants revealed a proline-rich Cas-binding site (Ala0-Pro1-Pro2-Lys3-Pro4-Pro5-Leu6-Pro7) located NH2-terminal to the previously characterized Crk binding motifs in C3G. Mutagenesis studies showed that Pro1, Lys3, and Pro4 within the ligand-binding site are critical for high affinity interaction. These results, combined with sequence alignments of proline-rich binding elements from proteins known for Cas binding, define the consensus sequence XXPXKPX which is recognized by the CasSH3 domain. Cas shows structural characteristics of a docking molecule and may serve to bring C3G to specific compartments within the cell.

Vinexin: a novel vinculin-binding protein with multiple SH3 domains enhances actin cytoskeletal organization.

Kioka N, Sakata S, Kawauchi T, Amachi T, Akiyama SK, Okazaki K, Yaen C, Yamada KM, Aota S
J Cell Biol. 1999; 144: 59-69

Using the yeast two-hybrid system and an in vitro binding assay, we have identified a novel protein termed vinexin as a vinculin-binding protein. By Northern blotting, we identified two types of vinexin mRNA that were 3 and 2 kb in length. Screening for full-length cDNA clones and sequencing indicated that the two mRNA encode 82- and 37-kD polypeptides termed vinexin alpha and beta, respectively. Both forms of vinexin share a common carboxyl-terminal sequence containing three SH3 domains. The larger vinexin alpha contains an additional amino-terminal sequence. The interaction between vinexin and vinculin was mediated by two SH3 domains of vinexin and the proline-rich region of vinculin. When expressed, vinexin alpha and beta localized to focal adhesions in NIH 3T3 fibroblasts, and to cell-cell junctions in epithelial LLC-PK1 cells. Furthermore, expression of vinexin increased focal adhesion size. Vinexin alpha also promoted upregulation of actin stress fiber formation. In addition, cell lines stably expressing vinexin beta showed enhanced cell spreading on fibronectin. These data identify vinexin as a novel focal adhesion and cell- cell adhesion protein that binds via SH3 domains to the hinge region of vinculin, which can enhance actin cytoskeletal organization and cell spreading.

Post-translational modification of the N-terminal His tag interferes with the crystallization of the wild-type and mutant SH3 domains from chicken src tyrosine kinase.

Kim KM, Yi EC, Baker D, Zhang KY
Acta Crystallogr D Biol Crystallogr. 2001; 57: 759-62

Structural studies of the wild type and mutants of the src SH3 domain were initiated to elucidate the correlation of the native-state topology with protein thermostability and folding kinetics. An extra mass of 178 Da arising from the post-translational modification at the N-terminal His tag was observed. The spontaneous alpha-N-6 gluconoylation at the amino group of the His-tagged SH3 domain contributed to the observed extra mass. The partial modification of the N-terminal His-tag produced heterogeneity, both in size and in charge, in the Escherichia coli expressed SH3 domain. The removal of the His tag from the SH3 domain was essential for the crystallization of both wild-type and mutant src SH3. Both the wild type and the W43I mutant were crystallized by hanging-drop vapor diffusion and are in the hexagonal space group P6(5)22 with one molecule in the asymmetric unit. Data sets were collected to 1.8 and 1.95 A resolution for the the wild type and the W43I mutant, respectively.

Structural basis for specificity switching of the Src SH2 domain.

Kimber MS, Nachman J, Cunningham AM, Gish GD, Pawson T, Pai EF
Mol Cell. 2000; 5: 1043-9

The Src SH2 domain binds pYEEI-containing phosphopeptides in an extended conformation with a hydrophobic pocket, which includes ThrEF1, binding Ile(pY +3). Mutating ThrEF1 to tryptophan switches specificity to an Asn(pY +2) requirement, yielding a biological mimic of the Grb2 SH2 domain. Here we show that the Src ThrEF1Trp SH2 domain mutant binds pYVNV phosphopeptides in a beta turn conformation, which, despite differing conformations of the interacting tryptophan, closely resembles the native Grb2/pYVNV cognate peptide binding mode. The ThrEF1Trp substitution therefore switches specificity by physically occluding the pTyr +3 binding pocket and by providing additional interaction surface area for Asn(pY +2). This demonstrates structurally how novel SH2 domain specificities may rapidly evolve through single amino acid substitutions and suggests how new signaling pathways may develop.

Molecular cloning of a new mouse gene tagL containing a lysozyme-like domain.

Kibardin AV, Mirkina II, Korneeva EA, Gnuchev NV, Georgiev GP, Kiselev SL
Dokl Biochem. 2000; 372: 103-5

Identification of a mouse orthologue of the human ras-GAP-SH3-domain binding protein and structural confirmation that these proteins contain an RNA recognition motif.

Kennedy D, Wood SA, Ramsdale T, Tam PP, Steiner KA, Mattick JS
Biomed Pept Proteins Nucleic Acids. 1996; 2: 93-9

Human ras-GTPase-activating protein (GAP120) SH3-domain-binding protein (G3BP) has recently been identified on the basis of its specific binding to the GAP120 SH3 binding domain. Here we report the identification of a mouse G3BP cDNA and the confirmation by three dimensional modelling of an RNA recognition motif (RRM) in the encoded protein. Mouse G3BP also contains an RGG domain, an acid-rich amino acid domain, and several SH3 domain-binding consensus sequences, indicating that mammalian G3BPs represent a new family of signal transduction proteins which connect tyrosine kinase-linked receptors to cellular RNA metabolism.

Signalling by the p60c-src family of protein-tyrosine kinases.

Kefalas P, Brown TR, Brickell PM
Int J Biochem Cell Biol. 1995; 27: 551-63

The c-src gene family has nine known members (blk, c-fgr, fyn, hck, lck, lyn, c-src, c-yes and yrk), each encoding a cytoplasmic protein-tyrosine kinase (PTK) believed to be involved in signal transduction. The c-src PTKs contain three domains (SH1, SH2 and SH3) that are found in many other signalling proteins. The SH1 domain has PTK activity, whilst the SH2 and SH3 domains are involved in mediating protein-protein interactions by binding to phosphotyrosine-containing and proline-rich motifs, respectively. The expression patterns of the c-src PTKs suggest that they function in a broad range of biological situations, in many cases regulating the behaviour of terminally-differentiated, post-mitotic cell types. Targeted disruption of members of the c-src family in transgenic mice has confirmed important roles for p56lck and p59fym(T) in T-lymphocyte maturation and activation, but has also revealed unexpected roles for p60c-src in bone maintenance and for p59fym(B) in learning and memory. There is increasingly detailed information about the biochemical nature of the signalling pathways in which the c-src PTKs operate and about the other signalling proteins with which they interact. The c-src PTKs can associate with activated receptor PTKs, including the receptors for platelet-derived growth factor and epidermal growth factor, by means of SH2-phosphotyrosine binding. The c-src PTKs also associated with transmembrane proteins that lack PTK activity, frequently by means of interactions involving their unique amino-terminal sequences.

The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.

Kay BK, Williamson MP, Sudol M
FASEB J. 2000; 14: 231-41

Acommon focus among molecular and cellular biologists is the identification of proteins that interact with each other. Yeast two-hybrid, cDNA expression library screening, and coimmunoprecipitation experiments are powerful methods for identifying novel proteins that bind to one's favorite protein for the purpose of learning more regarding its cellular function. These same techniques, coupled with truncation and mutagenesis experiments, have been used to define the region of interaction between pairs of proteins. One conclusion from this work is that many interactions occur over short regions, often less than 10 amino acids in length within one protein. For example, mapping studies and 3-dimensional analyses of antigen-antibody interactions have revealed that epitopes are typically 4-7 residues long (1). Other examples include protein-interaction modules, such as Src homology (SH) 2 and 3 domains, phosphotyrosine binding domains (PTB), postsynaptic density/disc-large/ZO1 (PDZ) domains, WW domains, Eps15 homology (EH) domains, and 14-3-3 proteins that typically recognize linear regions of 3-9 amino acids. Each of these domains has been the subject of recent reviews published elsewhere (2 3 4 5 6 7). Among the primary structures of many ligands for protein-protein interactions, the amino acid proline is critical. In particular, SH3, WW, and several new protein-interaction domains prefer ligand sequences that are proline-rich. In addition, even though ligands for EH domains and 14-3-3 domains are not proline-rich, they do include a single proline residue. This review highlights the analysis of those protein-protein interactions that involve proline residues, the biochemistry of proline, and current drug discovery efforts based on proline peptidomimetics.-Kay, B. K., Williamson, M. P., Sudol, M. The importance of being proline: the interaction of proline-rich motifs in signaling proteins with their cognate domains.

A novel phosphotyrosine mimetic 4'-carboxymethyloxy-3'-phosphonophenylalanine (cpp): exploitation in the design of nonpeptide inhibitors of pp60(Src) SH2 domain.

Kawahata N, Yang MG, Luke GP, Shakespeare WC, Sundaramoorthi R, Wang Y, Johnson D, Merry T, Violette S, Guan W, Bartlett C, Smith J, Hatada M, Lu X, Dalgarno DC, Eyermann CJ, Bohacek RS, Sawyer TK
Bioorg Med Chem Lett. 2001; 11: 2319-23

The novel phosphotyrosine (pTyr) mimetic 4'-carboxymethyloxy-3'-phosphonophenylalanine (Cpp) has been designed and incorporated into a series of nonpeptide inhibitors of the SH2 domain of pp60(c-Src) (Src) tyrosine kinase. A 2.2 A X-ray crystal structure of 1a bound to a mutant form of Lck SH2 domain provides insight regarding the structure-activity relationships and supports the design concept of this new pTyr mimetic.

Nef proteins of distinct HIV-1 or -2 isolates differ in their binding properties for HCK: isolation of a novel Nef binding factor with characteristics of an adaptor protein.

Karn T, Hock B, Holtrich U, Adamski M, Strebhardt K, Rubsamen-Waigmann H
Virology. 1998; 246: 45-52

The Nef gene of the human and simian immunodeficiency viruses HIV and SIV has been implicated in pathogenicity; however, the mechanism by which Nef induces disease is still unknown. An impact on signal transduction in cells has been suggested by the interaction of Nef from an HIV-1 strain and tyrosine kinases like HCK and LCK as well as serine/threonine kinases. We have confirmed the binding of HCK to HIV-1 subtype B Nef and demonstrated an equally strong interaction with a subtype E Nef protein but weaker binding to Nef of HIV-2 subtype A (HIV-2D194). No binding, however, was observed to HIV-2 subtype B Nef (HIV-2D205). Instead, this protein bound to a novel cellular protein, Nefin 1, with characteristics of an adaptor protein and strong expression in all human hematopoietic tissues. Nefin 1 binds through an amino-terminal domain, which is related to SH3 domains. For interaction of Nef with Nefin 1, the PxxP motif and the three-dimensional conformation of the molecule appear necessary. In conclusion, this study demonstrates that Nef proteins of divergent strains of HIV-1 and HIV-2 may use different elements of signal transduction pathways for the induction of pathogenicity in vivo.

Association of the amino-terminal half of c-Src with focal adhesions alters their properties and is regulated by phosphorylation of tyrosine 527.

Kaplan KB, Bibbins KB, Swedlow JR, Arnaud M, Morgan DO, Varmus HE
EMBO J. 1994; 13: 4745-56

We have characterized the mechanism by which the subcellular distribution of c-Src is controlled by the phosphorylation of tyrosine 527. Mutation of this tyrosine dramatically redistributes c-Src from endosomal membranes to focal adhesions. Redistribution to focal adhesions occurs independently of kinase activity and cellular transformation. In cells lacking the regulatory kinase (CSK) that phosphorylates tyrosine 527, c-Src is also found predominantly in focal adhesions, confirming that phosphorylation of tyrosine 527 affects the location of c-Src inside the cell. The first 251 amino acids of c-Src are sufficient to allow association with focal adhesions, indicating that at least one signal for positioning c-Src in focal adhesions resides in the amino-terminal half. Point mutations and deletions in the first 251 amino acids of c-Src reveal that association with focal adhesions requires the myristylation site needed for membrane attachment, as well as the SH3 domain. Expression of the amino-terminal region alters both the structural and biochemical properties of focal adhesions. Focal adhesions containing this non-catalytic portion of c-Src are larger and exhibit increased levels of phosphotyrosine staining. Our results suggest that c-Src may regulate focal adhesions and cellular adhesion by a kinase-independent mechanism.

Identification of two SH3-binding motifs in the regulatory subunit of phosphatidylinositol 3-kinase.

Kapeller R, Prasad KV, Janssen O, Hou W, Schaffhausen BS, Rudd CE, Cantley LC
J Biol Chem. 1994; 269: 1927-33

Src homology 3 (SH3) domains have been recently shown to bind to proline-rich sequences contained in 3BP1, 3BP2, and SOS. In a recent study we demonstrated that phosphatidylinositol 3-kinase (PI 3-kinase) associates with the Fyn SH3 domain. Here we show that p85, the regulatory subunit of PI 3-kinase, binds directly to the SH3 domains of Abl, Lck, Fyn, and p85 itself. An examination of p85 amino acid sequence revealed two proline-rich sequences in its N-terminal region similar to those present in 3BP1, 3BP2, and SOS. To test whether these sequences mediate the association of p85 with SH3 domains two peptides with amino acid composition corresponding to the p85 alpha proline-rich sequences were synthesized and used in competition assays. Both peptides worked equally well in inhibiting the binding of PI 3-kinase activity and p85 alpha to Fyn SH3 domain, whereas a control peptide had no effect. These results indicate that, as in 3BP1 and SOS, the proline-rich sequences in p85 mediate its interaction with SH3 domains. These results also suggest that the SH3 domain of p85 may "self-associate" with the proline-rich motifs of the same subunit as part of the PI 3-kinase regulatory mechanism.

The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110.

Kanner SB, Reynolds AB, Wang HC, Vines RR, Parsons JT
EMBO J. 1991; 10: 1689-98

Transformation of chicken embryo cells with the tyrosine kinase oncogene src results in the tyrosine phosphorylation of numerous cellular proteins. We have recently generated monoclonal antibodies to individual tyrosine phosphorylated cellular src substrates, several of which are directed to the phosphotyrosine-containing proteins p130 and p110. These proteins form stable complexes with activated variants of pp60src. Mutagenesis of the src homology domains (SH2 and SH3) of activated pp60src resulted in src variants with altered association with p130 and p110. Analysis of these variants showed that the SH3 domain was required for association of p110, while the SH2 domain contained residues necessary for the formation of the ternary complex involving p130, p110 and pp60src. Both the tyrosine phosphorylation status and pp60src association of p130 and p110 appeared to correlate, in part, with the extent of cell transformation. Biochemical analysis demonstrated that p130 and p110 were substrates of both serine/threonine and tyrosine kinases. In addition, p130 was redistributed from the nucleus to cellular membranes upon src transformation, whereas p110, which normally colocalized with cytoskeletal elements, was observed in adhesion plaques (podosomes) in src transformed cells. These data indicate that tyrosine phosphorylation of two different phosphoproteins may play a role during src transformation either by directing their interaction with pp60src, by redirecting subcellular distribution or both.

Tyrosine phosphorylation of connexin 43 by v-Src is mediated by SH2 and SH3 domain interactions.

Kanemitsu MY, Loo LW, Simon S, Lau AF, Eckhart W
J Biol Chem. 1997; 272: 22824-31

Reduction of gap junctional communication in v-src transformed cells is accompanied by tyrosine phosphorylation of the gap junction protein, connexin 43 (Cx43). Cx43 is phosphorylated on tyrosine by v-Src. The Src homology 3 (SH3) and Src homology 2 (SH2) domains of v-Src mediate interactions with substrate proteins. SH3 domains interact with proline-rich peptide motifs. SH2 domains associate with short amino acid sequences containing phosphotyrosine. We present evidence that the SH3 and SH2 domains of v-Src bind to proline-rich motifs and a phosphorylated tyrosine residue in the C-terminal tail of Cx43. Cx43 bound to the SH3 domain of v-Src, but not c-Src, in vitro. Tyrosine-phosphorylated Cx43 bound to the SH2 domain of v-Src in vitro. v-Src coprecipitated with Cx43 from v-src-transformed Rat-1 fibroblasts. Mutations in the SH3 and SH2 domains of v-Src, and in the proline-rich region or tyrosine 265 of Cx43, reduced interactions between v-Src and Cx43 in vivo. Tyrosine phosphorylation of Cx43 was dependent on the association of v-Src and Cx43. These results provide further evidence for the direct involvement of v-Src in tyrosine phosphorylation of Cx43 and inhibition of gap junctional communication in v-src-transformed cells.

Computational and functional analysis of the putative SH2 domain in Janus Kinases.

Kampa D, Burnside J
Biochem Biophys Res Commun. 2000; 278: 175-82

Src homology 2 (SH2) domains interact in a highly specific manner with phosphorylated tyrosine residues on other signaling molecules. Protein tyrosine kinases (PTK) frequently contain SH2 domains, which often control signaling specificity. The Janus Kinases (JAKs) are a family of PTKs involved in signal transduction pathways mediated by various cytokines. Initial characterization of JAKs showed no identifiable SH2 domain. However, we have found substantial evidence supporting the existence of an SH2 domain in JAKs through the use of various web-based computational analysis programs. Predictive secondary and tertiary structures recognize an SH2 domain in JAKs. In addition, a three-dimensional homology model was constructed using the SH2 domains of Src tyrosine kinase and Syp tyrosine phosphatase as templates. These results, in conjunction with preliminary binding studies showing interactions with tyrosine phosphorylated proteins in activated splenocytes, suggest a functional role for this domain in JAKs.

Crystal structure of estrogen sulphotransferase.

Kakuta Y, Pedersen LG, Carter CW, Negishi M, Pedersen LC
Nat Struct Biol. 1997; 4: 904-8

The structure of estrogen sulphotransferase has been solved in the presence of inactive cofactor PAP and substrate 17 beta-estradiol. This structure reveals structural similarities between cytosolic sulphotransferases and nucleotide kinases.

Drosophila abelson interacting protein (dAbi) is a positive regulator of abelson tyrosine kinase activity.

Juang JL, Hoffmann FM
Oncogene. 1999; 18: 5138-47

Human and mouse Abelson interacting proteins (Abi) are SH3-domain containing proteins that bind to the proline-rich motifs of the Abelson protein tyrosine kinase. We report a new member of this gene family, a Drosophila Abi (dAbi) that is a substrate for Abl kinase and that co-immunoprecipitates with Abl if the Abi SH3 domain is intact. We have identified a new function for both dAbi and human Abi-2 (hAbi-2). Both proteins activate the kinase activity of Abl as assayed by phosphorylation of the Drosophila Enabled (Ena) protein. Removal of the dAbi SH3 domain eliminates dAbi's activation of Abl kinase activity. dAbi is an unstable protein in cells and is present at low steady state levels but its protein level is increased coincident with phosphorylation by Abl kinase. Expression of the antisense strand of dAbi reduces dAbi protein levels and abolishes activation of Abl kinase activity. Modulation of Abi protein levels may be an important mechanism for regulating the level of Abl kinase activity in the cell.

Modulation of the catalytic activity of the Src family tyrosine kinase Hck by autophosphorylation at a novel site in the unique domain.

Johnson TM, Williamson NA, Scholz G, Jaworowski A, Wettenhall RE, Dunn AR, Cheng HC
J Biol Chem. 2000; 275: 33353-64

Autophosphorylation is a key event in the activation of protein kinases. In this study, we demonstrate that autophosphorylation of the recombinant Src family kinase Hck leads to a 20-fold increase in its specific enzymatic activity. Hck was found to autophosphorylate readily to a stoichiometry of 1.3 mol of phosphate per mol of enzyme, indicating that the kinase autophosphorylated at more than one site. Solid phase sequencing and two-dimensional mapping of the phosphopeptide fragments derived from the autophosphorylated enzyme revealed that the kinase can undergo autophosphorylation at the following two sites: (i) Tyr-388, which is located to the consensus autophosphorylation site commonly found in the activation loop of many protein kinases, and (ii) Tyr-29, which is located in the unique domain of Hck. Hck purified from mouse bone marrow-derived macrophages could also autophosphorylate in vitro at both Tyr-388 and Tyr-29, indicating that naturally occurring Hck can also autophosphorylate at Tyr-29. Furthermore, Hck transiently expressed in human embryonic kidney 293T cells was found to be phosphorylated at Tyr-29 and Tyr-388, proving that Hck can also undergo autophosphorylation at both sites in vivo. The recombinant enzyme carrying the mutation of Tyr-388 to Phe was also able to autophosphorylate at Tyr-29, albeit at a significantly slower rate. A 2-fold increase in the specific enzymatic activity was seen with this mutant despite the stoichiometry of autophosphorylation only approaching 0.2 mol of phosphate per mol of enzyme. This indicates that autophosphorylation of Tyr-29 contributes significantly to the activation of Hck. Regulation of the catalytic activity by phosphorylation of Tyr-29 in the unique domain may represent a new mechanism of regulation of Src family tyrosine kinases.

Common and differential recognition of structural features in synthetic peptides by the catalytic domain and the Src-homology 2 (SH2) domain of pp60c-src.

Johnson TM, Perich JW, Bjorge JD, Fujita DJ, Cheng HC
J Pept Res. 1997; 50: 365-71

The relative efficiencies of the catalytic domain of the src-family kinase pp60c-src in phosphorylating four peptide substrates including (i) src-optimal peptide (AEEEIYGEFEAKKKK), (ii) "-YEEI-peptide" (KKTHQEEEEPQYEEIPIYL), (iii) cdc2(6-20) (KVEKIGEGTYGVVYK), (iv) src-autophosphorylation site peptide (ADFGLARLIEDNEYTARG) and the relative efficiencies of its SH2 domain in binding the phosphorylated forms of these peptide substrates were compared. The results show that the src-optimal peptide, "-YEEI-peptide," cdc2(6-20) peptide were phosphorylated by the catalytic domain with high efficiency and that the phosphorylated form of all three peptides could bind the SH2 domain of the kinase, confirming the hypothesis proposed by Songyang and co-workers that the catalytic domain of pp60c-src phosphorylates sites which are recognized by its own SH2 domain (Songyang et al. (1995) Nature 373, 536-539). The four peptides were phosphorylated by the kinase with relative efficiencies in the order of Src-optimal peptide > "-YEEI-peptide" > cdc2(6-20) >> src-autophosphorylation site peptide. However, the Tyr(P)-Src-optimal peptide and [pY]15cdc2(6-20) bound to the SH2 domain of the kinase with an affinity at least an order of magnitude lower than that of the tight-binding peptide, "-pYEEI-peptide." Thus, our study suggests that the catalytic and SH2 domains of pp60c-src recognize overlapping but not identical determinants in the local structure around the tyrosine phosphorylation site of the substrate peptides.

Biochemistry. Ubiquitination--more than two to tango.

Joazeiro CA, Hunter T
Science. 2000; 289: 2061-2

Chemical and physical characterization of a proline-rich polypeptide from sheep colostrum.

Janusz M, Staroscik K, Zimecki M, Wieczorek Z, Lisowski J
Biochem J. 1981; 199: 9-15

A proline-rich polypeptide isolated from sheep colostrum is described. The molecular weight of the polypeptide determined by gel filtration is 17 200. However, in the presence of guanidinium chloride the molecular weight found is about 6000. The polypeptide contains about 22% of proline, a high proportion of non-polar amino acids, a low percentage of glycine, and no alanine, arginine and cysteine residues. The only N-terminal amino acid found is leucine. C.d. spectra in water and in 50% (v/v) trifluoroethanol suggest the presence of block sequences of proline residues forming helices of polyproline II type. The proline-rich polypeptide is soluble at 4 degrees C but is reversibly precipitated on warming to room temperature. Maximal precipitation is observed at pH 4.6 and at ionic strength above 0.6. The precipitation depends on the concentration of the polypeptide. No effect of other proteins, Ca2+ and Zn2+ ions on the precipitation of the polypeptide was found. The proline-rich polypeptide is not an amphipathic protein. The lack of effect of the polypeptide on proteolytic enzymes ruled out the possibility that it is an inhibitor of proteinases.

Structural determinants of the interaction between the erbB2 receptor and the Src homology 2 domain of Grb7.

Janes PW, Lackmann M, Church WB, Sanderson GM, Sutherland RL, Daly RJ
J Biol Chem. 1997; 272: 8490-7

The Src homology 2 (SH2) domain-containing protein Grb7 and the erbB2 receptor tyrosine kinase are overexpressed in a subset of human breast cancers. They also co-immunoprecipitate from cell lysates and associate directly in vitro. Whereas the Grb7 SH2 domain binds strongly to erbB2, the SH2 domain of Grb14, a protein closely related to Grb7, does not. We have investigated the preferred binding site of Grb7 within the erbB2 intracellular domain and the SH2 domain residues that determine the high affinity of Grb7 compared with Grb14 for this site. Phosphopeptide competition and site-directed mutagenesis revealed that Tyr-1139 of erbB2 is the major binding site for the Grb7 SH2 domain, indicating an overlap in binding specificity between the Grb7 and Grb2 SH2 domains. Substituting individual amino acids in the Grb14 SH2 domain with the corresponding residues from Grb7 demonstrated that a Gln to Leu change at the betaD6 position imparted high affinity erbB2 interaction, paralleled by a marked increase in affinity for the Tyr-1139 phosphopeptide. The reverse switch at the betaD6 position abrogated Grb7 binding to erbB2. This residue therefore represents an important determinant of SH2 domain specificity within the Grb7 family.

Molecular cloning of a cDNA encoding a phosphoprotein, Efs, which contains a Src homology 3 domain and associates with Fyn.

Ishino M, Ohba T, Sasaki H, Sasaki T
Oncogene. 1995; 11: 2331-8

Src homology 2 and 3 (SH2 and SH3) domains mediate protein-protein interactions in intracellular signaling by protein-tyrosine kinases (PTKs). We have isolated cDNA clones from mouse embryo cDNA expression library that encode a new signaling protein which we call Efs (Embryonal Fyn-associated Substrate). The deduced amino acid sequence of 560 residues in length revealed one SH3 domain at its amino-terminal region, two proline-rich motifs with the consensus sequences of binding to Src-family SH3s, and a cluster of YXXP motifs that are possibly tyrosine-phosphorylated to serve as ligands binding to SH2 domains. Structure and alignment of these characteristics sequences are homologous to those of p130Cas, but Efs and p130Cas are different proteins. Expression of the Efs gene was higher in placenta, embryo and brain than in other adult tissues. Transfection of COS-7 cells with a plasmid encoding an epitope-tagged Efs resulted in the expression of a 83 kDa protein. The epitope-tagged Efs was hyperphosphorylated when cotransfected with a vector expressing Fyn. In an in vitro kinase assay with the PCC4 cell lysate, Efs became phosphorylated on tyrosine residues and coprecipitated with p59fyn and p62yes; the result suggests that Efs is a physiological substrate of these PTKs.

Identification of a novel nuclear localization signal in Sam68.

Ishidate T, Yoshihara S, Kawasaki Y, Roy BC, Toyoshima K, Akiyama T
FEBS Lett. 1997; 409: 237-41

Sam68, a nuclear RNA binding protein, binds to Src and is phosphorylated at tyrosine residues in an M-phase specific manner. Here we identified a stretch of 24 amino acid residues in the COOH-terminal portion of Sam68 which function as a nuclear localization signal. This signal sequence bears no apparent homology to any other known nuclear localization sequence. However, this sequence was found to contain a motif, PPXXR (P, Pro; R, Arg), which is conserved in various RNA binding proteins including hnRNP proteins. Replacement of Arg in this motif with Ala abolished the nuclear accumulation of a GFP fusion protein, suggesting that this residue is important in translocating the protein to the nucleus.

Regulation of Src family kinases in the developing rat brain: correlation with their regulator kinase, Csk.

Inomata M, Takayama Y, Kiyama H, Nada S, Okada M, Nakagawa H
J Biochem (Tokyo). 1994; 116: 386-92

We have so far suggested that the functions of Src family protein-tyrosine kinases are under the control of their regulator kinase, Csk. To evaluate the role of Csk-mediated regulation in neural tissues, we examined the correlation between the activities of Src family kinases and the expression level of Csk during development of the rat brain. Csk was expressed at high levels in the developing embryonic brain and then rapidly decreased as the brain matured. Consistent with the decrease in the Csk level, the kinase activity of a member of the Src family, Fyn, was greatly enhanced, but that of Src was not correlated inversely with the level of Csk expression. Src exhibited elevated activity in the developing brain, in which a neuronal form of Src (N-Src) is expressed as the dominant form of Src. Although N-Src was readily down-regulated by Csk when coexpressed in yeast, it showed much higher specific activity than c-Src, even in the repressed form. These findings suggest that neural tissues acquire high activities of Src family kinases, which might be important for differentiation and development of the nervous system, through induction of the active form of Src (N-Src) and down-regulation of their suppresser, Csk.

Enhancement of guanine-nucleotide exchange activity of C3G for Rap1 by the expression of Crk, CrkL, and Grb2.

Ichiba T, Kuraishi Y, Sakai O, Nagata S, Groffen J, Kurata T, Hattori S, Matsuda M
J Biol Chem. 1997; 272: 22215-20

Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1 in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.

Insights into autoregulation from the crystal structure of twitchin kinase.

Hu SH, Parker MW, Lei JY, Wilce MC, Benian GM, Kemp BE
Nature. 1994; 369: 581-4

Many protein kinases are self-regulated by an intrasteric mechanism where part of the enzyme's structure directly inhibits the active site. This inhibitory structure is called a pseudosubstrate and specific regulators are required to remove it from the active site to allow substrates access. Removal of the pseudosubstrate sequence from members of the myosin light-chain kinase subfamily, including twitchin kinase, activates them but it is not known whether the pseudosubstrate sequence binds to the active site. Native twitchin is a 753K protein (6,839 residues) located in muscle A-bands of the nematode Caenorhabditis elegans and because of its size has not been easy to study. We have determined the crystal structure, refined to 2.8 A resolution, of a recombinant fragment (residues 5,890 to 6,262) of twitchin kinase that contains the catalytic core and a 60 residue carboxy-terminal tail. The C-terminal tail extends through the active site, wedged between the small and large lobes of the structure and making extensive contacts with the catalytic core which accounts for autoinhibition and provides direct support for the intrasteric mechanism of protein kinase regulation.

Engagement of the osteoclast integrin alpha v beta 3 by osteopontin stimulates phosphatidylinositol 3-hydroxyl kinase activity.

Hruska KA, Rolnick F, Huskey M, Alvarez U, Cheresh D
Ann N Y Acad Sci. 1995; 760: 151-65

Mouse disabled (mDab1): a Src binding protein implicated in neuronal development.

Howell BW, Gertler FB, Cooper JA
EMBO J. 1997; 16: 121-32

Here, we identify a mouse homolog of the Drosophila Disabled (Dab) protein, mDab1, and show it is an adaptor molecule functioning in neural development. We find that mDab1 is expressed in certain neuronal and hematopoietic cell lines, and is localized to the growing nerves of embryonic mice. During mouse embryogenesis, mDab1 is tyrosine phosphorylated when the nervous system is undergoing dramatic expansion. However, when nerve tracts are established, mDab1 lacks detectable phosphotyrosine. Tyrosine-phosphorylated mDab1 associates with the SH2 domains of Src, Fyn and Abl. An interaction between mDab1 and Src is observed when P19 embryonal carcinoma (EC) cells undergo differentiation into neuronal cell types. mDab1 can also form complexes with cellular phosphotyrosyl proteins through a domain that is related to the phosphotyrosine binding (PTB) domains of the Shc family of adaptor proteins. The mDab1 PTB domain binds to phosphotyrosine-containing proteins of 200, 120 and 40 kDa from extracts of embryonic mouse heads. The properties of mDab1 and genetic analysis of Dab in Drosophila suggest that these molecules function in key signal transduction pathways involved in the formation of neural networks.

Csk suppression of Src involves movement of Csk to sites of Src activity.

Howell BW, Cooper JA
Mol Cell Biol. 1994; 14: 5402-11

Csk phosphorylates Src family members at a key regulatory tyrosine in the C-terminal tail and suppresses their activities. It is not known whether Csk activity is regulated. To examine the features of Csk required for Src suppression, we expressed Csk mutants in a cell line with a disrupted csk gene. Expression of wild-type Csk suppressed Src, but Csk with mutations in the SH2, SH3, and catalytic domains did not suppress Src. An SH3 deletion mutant of Csk was fully active against in vitro substrates, but two SH2 domain mutants were essentially inactive. Whereas Src repressed by Csk was predominantly perinuclear, the activated Src in cells lacking Csk was localized to structures resembling podosomes. Activated mutant Src was also in podosomes, even in the presence of Csk. When Src was not active, Csk was diffusely located in the cytosol, but when Src was active, Csk colocalized with activated Src to podosomes. Csk also localizes to podosomes of cells transformed by an activated Src that lacks the major tyrosine autophosphorylation site, suggesting that the relocalization of Csk is not a consequence of the binding of the Csk SH2 domain to phosphorylated Src. A catalytically inactive Csk mutant also localized with Src to podosomes, but SH3 and SH2 domain mutants did not, suggesting that the SH3 and SH2 domains are both necessary to target Csk to places where Src is active. The failure of the catalytically active SH3 mutant of Csk to regulate Src may be due to its inability to colocalize with active Src.

Herpesvirus saimiri protein StpB associates with cellular Src.

Hor S, Ensser A, Reiss C, Ballmer-Hofer K, Biesinger B
J Gen Virol. 2001; 82: 339-44

Subgroup B isolates of Herpesvirus saimiri are less efficient in T lymphocyte transformation when compared with subgroups A or C. Here it is shown that subgroup B strain SMHI encodes a protein, StpB, at a position equivalent to those of the ORFs for the saimiri transforming proteins (Stp) of subgroups A and C. StpB shares little similarity with StpA or StpC, but interacts with the SH2 domain of cellular Src, as does StpA. Thus, factors other than c-Src binding determine the efficiency of primary T cell transformation by Herpesvirus saimiri.

Dynamics of the Hck-SH3 domain: comparison of experiment with multiple molecular dynamics simulations.

Horita DA, Zhang W, Smithgall TE, Gmeiner WH, Byrd RA
Protein Sci. 2000; 9: 95-103

Molecular dynamics calculations provide a method by which the dynamic properties of molecules can be explored over timescales and at a level of detail that cannot be obtained experimentally from NMR or X-ray analyses. Recent work (Philippopoulos M, Mandel AM, Palmer AG III, Lim C, 1997, Proteins 28:481-493) has indicated that the accuracy of these simulations is high, as measured by the correspondence of parameters extracted from these calculations to those determined through experimental means. Here, we investigate the dynamic behavior of the Src homology 3 (SH3) domain of hematopoietic cell kinase (Hck) via 5N backbone relaxation NMR studies and a set of four independent 4 ns solvated molecular dynamics calculations. We also find that molecular dynamics simulations accurately reproduce fast motion dynamics as estimated from generalized order parameter (S2) analysis for regions of the protein that have experimentally well-defined coordinates (i.e., stable secondary structural elements). However, for regions where the coordinates are not well defined, as indicated by high local root-mean-square deviations among NMR-determined structural family members or high B-factors/low electron density in X-ray crystallography determined structures, the parameters calculated from a short to moderate length (less than 5-10 ns) molecular dynamics trajectory are dependent on the particular coordinates chosen as a starting point for the simulation.

Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain.

Holmes TC, Fadool DA, Ren R, Levitan IB
Science. 1996; 274: 2089-91

The human Kv1.5 potassium channel (hKv1.5) contains proline-rich sequences identical to those that bind to Src homology 3 (SH3) domains. Direct association of the Src tyrosine kinase with cloned hKv1.5 and native hKv1.5 in human myocardium was observed. This interaction was mediated by the proline-rich motif of hKv1.5 and the SH3 domain of Src. Furthermore, hKv1.5 was tyrosine phosphorylated, and the channel current was suppressed, in cells coexpressing v-Src. These results provide direct biochemical evidence for a signaling complex composed of a potassium channel and a protein tyrosine kinase.

Crystal structure of the tyrosine phosphatase SHP-2.

Hof P, Pluskey S, Dhe-Paganon S, Eck MJ, Shoelson SE
Cell. 1998; 92: 441-50

The structure of the SHP-2 tyrosine phosphatase, determined at 2.0 angstroms resolution, shows how its catalytic activity is regulated by its two SH2 domains. In the absence of a tyrosine-phosphorylated binding partner, the N-terminal SH2 domain binds the phosphatase domain and directly blocks its active site. This interaction alters the structure of the N-SH2 domain, disrupting its phosphopeptide-binding cleft. Conversely, interaction of the N-SH2 domain with phosphopeptide disrupts its phosphatase recognition surface. Thus, the N-SH2 domain is a conformational switch; it either binds and inhibits the phosphatase, or it binds phosphoproteins and activates the enzyme. Recognition of bisphosphorylated ligands by the tandem SH2 domains is an integral element of this switch; the C-terminal SH2 domain contributes binding energy and specificity, but it does not have a direct role in activation.

Binding properties of SH3 peptide ligands identified from phage-displayed random peptide libraries.

Hoffman NG, Sparks AB, Carter JM, Kay BK
Mol Divers. 1996; 2: 5-12

Combinatorial libraries have yielded high-affinity ligands for SH3 domains of a number of different proteins. We have shown that synthetic peptides containing these SH3 ligand sequences serve as specific probes of SH3 domains. Direct binding of the N-terminal biotinylated peptide ligands was conveniently detected in ELISA, filter-blotting, and dot-blotting experiments with the use of streptavidin-conjugated enzymes. In some cases, detection of peptide-SH3 interactions required that the biotinylated peptides first were preconjugated with streptavidin to form a multivalent complex. Interestingly, these nominally tetravalent SH3 peptide ligands cross-react to varying degrees with different SH3 domains. We have used such complexes to screen lambda cDNA expression libraries and have isolated clones that encode both known and novel SH3-domain-containing proteins. Based on the success of this methodology, we propose a general strategy by which ligands of a modular domain-containing protein can be isolated from random peptide libraries and used to screen cDNA expression libraries systematically for novel modular domain-containing proteins.

Looking for protein family members.

Hoess RH
Nat Biotechnol. 1996; 14: 705-6

Crystal structure of the C-terminal SH2 domain of the p85alpha regulatory subunit of phosphoinositide 3-kinase: an SH2 domain mimicking its own substrate.

Hoedemaeker FJ, Siegal G, Roe SM, Driscoll PC, Abrahams JP
J Mol Biol. 1999; 292: 763-70

The binding properties of Src homology-2 (SH2) domains to phosphotyrosine (pY)-containing peptides have been studied in recent years with the elucidation of a large number of crystal and solution structures. Taken together, these structures suggest a general mode of binding of pY-containing peptides, explain the specificities of different SH2 domains, and may be used to design inhibitors of pY binding by SH2 domain-containing proteins. We now report the crystal structure to 1.8 A resolution of the C-terminal SH2 domain (C-SH2) of the P85alpha regulatory subunit of phosphoinositide 3-kinase (PI3 K). Surprisingly, the carboxylate group of Asp2 from a neighbouring molecule occupies the phosphotyrosine binding site and interacts with Arg18 (alphaA2) and Arg36 (betaB5), in a similar manner to the phosphotyrosine-protein interactions seen in structures of other SH2 domains complexed with pY peptides. It is the first example of a non-phosphate-containing, non-aromatic mimetic of phosphotyrosine binding to SH2 domains, and this could have implications for the design of substrate analogues and inhibitors. Overall, the crystal structure closely resembles the solution structure, but a number of loops which demonstrate mobility in solution are well defined by the crystal packing. C-SH2 has adopted a binding conformation reminiscent of the ligand bound N-terminal SH2 domain of PI3K, apparently induced by the substrate mimicking of a neighbouring molecule in the crystal.

Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein.

Hodge DR, Dunn KJ, Pei GK, Chakrabarty MK, Heidecker G, Lautenberger JA, Samuel KP
J Biol Chem. 1998; 273: 15727-33

Nef is a membrane-associated cytoplasmic phosphoprotein that is well conserved among the different human (HIV-1 and HIV-2) and simian immunodeficiency viruses and has important roles in down-regulating the CD4 receptor and modulating T-cell signaling pathways. The ability to modulate T-cell signaling pathways suggests that Nef may physically interact with T-cell signaling proteins. In order to identify Nef binding proteins and map their site(s) of interaction, we targeted a highly conserved acidic sequence at the carboxyl-terminal region of Nef sharing striking similarity with an acidic sequence at the c-Raf1-binding site within the Ras effector region. Here, we used deletion and site-specific mutagenesis to generate mutant Nef proteins fused to bacterial glutathione S-transferase in in vitro precipitation assays and immunoblot analysis to map the specific interaction between the HIV-1LAI Nef and c-Raf1 to a conserved acidic sequence motif containing the core sequence Asp-Asp-X-X-X-Glu (position 174-179). Significantly, we demonstrate that substitution of the nonpolar glycine residue for either or both of the conserved negatively charged aspartic acid residues at positions 174 and 175 in the full-length recombinant Nef protein background completely abrogated binding of c-Raf1 in vitro. In addition, lysates from a permanent CEM T-cell line constitutively expressing the native HIV-1 Nef protein was used to coimmunoprecipitate a stable Nef-c-Raf1 complex, suggesting that molecular interactions between Nef and c-Raf1, an important downstream transducer of cell signaling through the c-Raf1-MAP kinase pathway, occur in vivo. This interaction may account for the Nef-induced perturbations of T-cell signaling and activation pathways in vitro and in vivo.

SH3 domain-dependent interaction of the proto-oncogene product Vav with the focal contact protein zyxin.

Hobert O, Schilling JW, Beckerle MC, Ullrich A, Jallal B
Oncogene. 1996; 12: 1577-81

Scr homology 3 (SH3) domain-mediated protein-protein interactions have been implicated in the localization of proteins to specific sites within the cell. We present evidence that the product of the vav proto-oncogene, p95vav, interacts specifically with the focal adhesion protein zyxin both in vitro and in yeast two hybrid system. Solution binding and two-hybrid system experiments demonstrate that association of Vav with the LIM domain protein zyxin is mediated by the C-terminal SH3 domain of the Vav and involves the proline-rich N-terminus of zyxin. The interaction appears to be selective, since no binding of the proline-rich N-terminus of zyxin with other SH3 domain-containing proteins such as GRB-2, phospholipase C gamma, GTPase-activating protein, or p85 was detected.

Novel signaling pathway suggested by SH3 domain-mediated p95vav/heterogeneous ribonucleoprotein K interaction.

Hobert O, Jallal B, Schlessinger J, Ullrich A
J Biol Chem. 1994; 269: 20225-8

The role of the protooncogene product p95vav in signal transduction was investigated by characterizing its interactions with proteins that may represent components of a novel signaling pathway. We demonstrate here stable association of p95vav with the heterogeneous ribonucleoprotein K (hnRNP-K), a protein that not only was found to be part of hnRNP particles but has also been implicated in transcriptional regulation of the c-myc gene. Through the PLPPPPPPRG sequence, hnRNP-K specifically interacts with the SH3 domain of p95vav and thus represents a novel SH3 binding protein that may be capable of translating cell surface receptor signals through p95vav activation into regulatory events on the level of gene expression.

Regulation of the human c-fes protein tyrosine kinase (p93c-fes) by its src homology 2 domain and major autophosphorylation site (Tyr-713).

Hjermstad SJ, Peters KL, Briggs SD, Glazer RI, Smithgall TE
Oncogene. 1993; 8: 2283-92

The c-fes proto-oncogene product is expressed predominantly in hematopoietic cells of the myeloid lineage and has been implicated in the regulation of myeloid differentiation. The c-fes locus encodes a 93-kDa protein tyrosine kinase (p93c-fes) that possesses several structural features characteristic of the cytoplasmic class of protein tyrosine kinases, including a consensus sequence for autophosphorylation surrounding Tyr-713 and a src homology 2 (SH2) domain. To assess the effect of each of these potential regulatory sites on p93c-fes protein tyrosine kinase activity, we specifically deleted the c-fes SH2 domain using the polymerase chain reaction and replaced Tyr-713 with phenylalanine by oligonucleotide-directed mutagenesis (Y713F mutant). The resulting mutants were expressed in Escherichia coli and assayed for changes in protein tyrosine kinase activity using an immune complex kinase assay. Both mutations produced a marked decrease in the rate and extent of autophosphorylation and phosphorylation of the model substrate, enolase. To test whether the c-fes SH2 domain could interact with the autophosphorylated kinase domain, the SH2 domain was expressed as a fusion protein with glutathione S-transferase and immobilized on glutathione-agarose. The recombinant c-fes SH2 domain precipitated p93c-fes as readily as a monoclonal antibody. Binding of the SH2 domain to p93c-fes was completely dependent upon autophosphorylation, as a kinase-defective mutant of p93c-fes was not precipitated by the SH2 domain. High-affinity binding was also observed with recombinant SH2 domains from v-src and v-fps, raising the possibility of protein-protein interactions between various members of the cytoplasmic PTK family. These results indicate that the c-fes SH2 domain and consensus autophosphorylation site (Tyr-713) play major roles in the positive regulation of p93c-fes tyrosine kinase activity, possibly through intramolecular interaction.

Cloning and sequencing of human gp330, a Ca(2+)-binding receptor with potential intracellular signaling properties.

Hjalm G, Murray E, Crumley G, Harazim W, Lundgren S, Onyango I, Ek B, Larsson M, Juhlin C, Hellman P, Davis H, Akerstrom G, Rask L, Morse B
Eur J Biochem. 1996; 239: 132-7

We present here the complete primary structure of human gp330, the human variant of the principal kidney autoantigen causing Heymann membranous glomerulonephritis in rats. The deduced 4655 amino acid residues give a calculated molecular mass of 519636 Da for the mature protein and consists of a probable 25-amino-acid N-terminal signal peptide sequence, an extracellular region of 4398 amino acids, a single transmembrane-spanning domain of 23 amino acids, and an intracellular C-terminal region of 209 amino acid residues. Three types of cysteine-rich repeats characteristic of the low density lipoprotein receptor (LDLR) superfamily are present in human gp330. In the extracellular region, there are a total of 36 LDLR ligand-binding repeats, comprising four distinct domains, 16 growth factor repeats separated by eight YWTD spacer regions, and one epidermal growth factor-like repeat. No consensus cleavage sequence for the processing endoprotease furin is detected in human gp330. The intracellular tail contains not only two copies of the F(X)NPXY coated-pit mediated internalization signal characteristic of LDLR superfamily members, but also intriguing and potentially functional motifs including several Src-homology 3 recognition motifs, one Src-homology 2 recognition motif for the p85 regulatory subunit of phosphatidylinositol 3-kinase, and additional sites for protein kinase C, casein kinase II and cAMP-/cGMP-dependent protein kinase. There is approximately 77% amino acid identity between human and rat gp330 with minor differences between the extracellular and intracellular regions. Recently gp330 has been implicated in Ca2+ regulation in the parathyroid, the placenta, and the renal tubule, but its overall physiological and pathological role still remains uncertain.

Determination of the solution structure of the SH3 domain of human p56 Lck tyrosine kinase.

Hiroaki H, Klaus W, Senn H
J Biomol NMR. 1996; 8: 105-22

The solution structure of the SH3 domain of human p56 Lck tyrosine kinase (Lck-SH3) has been determined by multidimensional heteronuclear NMR spectroscopy. The structure was calculated from a total of 935 experimental restraints comprising 785 distance restraints derived from 1017 assigned NOE cross peaks and 150 dihedral angel restraints derived from 160 vicinal coupling constants. A novel combination of the constant-time 1H-13C NMR correlation experiment recorded with various delays of the constant-time refocusing delays and a fractionally 13C-labelled sample was exploited for the stereospecific assignment of prochiral methyl groups. Additionally, 28 restraints of 14 identified hydrogen bonds were included. A family of 25 conformers was selected to characterize the solution structure. The average root-mean-square deviations of the backbone atoms (N, C alpha, C', O) among the 25 conformers is 0.42 A for residues 7 to 63. The N- and C-terminal residues, 1 to 6 and 64 to 81, are disordered, while the well-converged residues 7 to 63 correspond to the conserved sequences of other SH3 domains. The topology of the SH3 structure comprises five anti-parallel beta-strands arranged to form two perpendicular beta-sheets, which are concave and twisted in the middle part. The overall secondary structure and the backbone conformation of the core beta-strands are almost identical to the X-ray structure of the fragment containing the SH2-SH3 domains of p56 Lck [Eck et al. (1994) Nature, 368, 764-769]. The X-ray structure of the SH3 domain in the tandem SH2-SH3 fragment is spatially included within the ensemble of the 25 NMR conformers, except for the segment of residues 14 to 18, which makes intermolecular contacts with an adjacent SH2 molecule and the phosphopeptide ligand in the crystal lattice. Local structural differences from other known SH3 domains are also observed, the most prominent of which is the absence in Lck-SH3 of the two additional short beta-strands in the regions Ser15 to Glu17 and Gly25 to Glu27 flanking the so-called "RT-Src' loop. This loop (residues Glu17 to Leu24), together with the "n-Src' loop (residues Gln37 to Ser46) confines the ligand interaction site which is formed by a shallow patch of hydrophobic amino acids (His14, Tyr16, Trp41, Phe54 and Phe59). Both loops are flexible and belong to the most mobile regions of the protein, which is assessed by the heteronuclear 15N, 1H-NOE values characterizing the degree of internal backbone motions. The aromatic residues of the ligand binding site are arranged such that they form three pockets for interactions with the polyproline ligand.

SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef.

Hiipakka M, Poikonen K, Saksela K
J Mol Biol. 1999; 293: 1097-106

The avid binding of HIV-1 Nef to the Src homology-3 (SH3) domain of Hck (KD 250 nM) has been shown to involve an interaction between the RT-loop of Hck-SH3 and residues in Nef outside of its prototypic polyproline type II (PPII) helix-containing SH3-ligand region. Such distinctive interactions are thought to provide specificity and affinity for other SH3/ligand protein complexes as well. Here, we have constructed and successfully displayed on the surface of M13 bacteriophage particles a complex library of SH3 domains, which are derived from Hck but carry a random hexapeptide substitution in their RT-loops (termed RRT-SH3). Using this strategy we have identified individual RRT-SH3 domains that can bind to Nef up to 40-fold more avidly than Hck-SH3. Some of these high-affinity RRT-SH3 domains resembled Hck-SH3 in that they bound much less well to a Nef variant containing an engineered F90R mutation that interferes with docking of the native Hck RT-loop. In addition, we could also select RRT-SH3 domains with an opposite specificity, which were dependent on the Arg90 residue for strong binding, and bound 100-fold less well to unmodified Nef. These results demonstrate the utility of phage-display in engineering of signaling protein interaction domains, and emphasize the importance of the RT-loop in SH3 ligand selection, thus suggesting a general strategy for creating SH3 domains with desired binding properties.

Inhibition of cellular functions of HIV-1 Nef by artificial SH3 domains.

Hiipakka M, Huotari P, Manninen A, Renkema GH, Saksela K
Virology. 2001; 286: 152-9

SH3 domains regulate many normal and pathological cellular processes by guiding specific protein interactions. Studies on binding of HIV-1 Nef to the SH3 domain of the Hck tyrosine kinase have indicated an important role for the SH3 RT-loop region in ligand binding. Here we have tested the potential of artificial Hck-derived SH3 domains carrying tailored RT-loops providing high affinity for Nef as intracellular inhibitors of Nef. These artificial SH3 domains efficiently associated with Nef in cells and thereby potently inhibited SH3-dependent Nef functions, such as association with p21-activated kinase-2 and induction of the transcription factor NFAT. On the other hand, biochemical and functional data indicated that the Nef-targeted SH3 domains were not prone to compete with normal SH3-mediated processes. Thus, RT-loop-modified SH3 domains represent a novel approach for selectively interfering with cellular signaling events, which could be exploited in research as well as in therapeutic applications. Copyright 2001 Academic Press.

Using CLUSTAL for multiple sequence alignments.

Higgins DG, Thompson JD, Gibson TJ
Methods Enzymol. 1996; 266: 383-402

We have tested CLUSTAL W in a wide variety of situations, and it is capable of handling some very difficult protein alignment problems. If the data set consists of enough closely related sequences so that the first alignments are accurate, then CLUSTAL W will usually find an alignment that is very close to ideal. Problems can still occur if the data set includes sequences of greatly different lengths or if some sequences include long regions that are impossible to align with the rest of the data set. Trying to balance the need for long insertions and deletions in some alignments with the need to avoid them in others is still a problem. The default values for our parameters were tested empirically using test cases of sets of globular proteins where some information as to the correct alignment was available. The parameter values may not be very appropriate with nonglobular proteins. We have argued that using one weight matrix and two gap penalties is too simplistic to be of general use in the most difficult cases. We have replaced these parameters with a large number of new parameters designed primarily to help encourage gaps in loop regions. Although these new parameters are largely heuristic in nature, they perform surprisingly well and are simple to implement. The underlying speed of the progressive alignment approach is not adversely affected. The disadvantage is that the parameter space is now huge; the number of possible combinations of parameters is more than can easily be examined by hand. We justify this by asking the user to treat CLUSTAL W as a data exploration tool rather than as a definitive analysis method. It is not sensible to automatically derive multiple alignments and to trust particular algorithms as being capable of always getting the correct answer. One must examine the alignments closely, especially in conjunction with the underlying phylogenetic tree (or estimate of it) and try varying some of the parameters. Outliers (sequences that have no close relatives) should be aligned carefully, as should fragments of sequences. The program will automatically delay the alignment of any sequences that are less than 40% identical to any others until all other sequences are aligned, but this can be set from a menu by the user. It may be useful to build up an alignment of closely related sequences first and to then add in the more distant relatives one at a time or in batches, using the profile alignments and weighting scheme described earlier and perhaps using a variety of parameter settings. We give one example using SH2 domains. SH2 domains are widespread in eukaryotic signalling proteins where they function in the recognition of phosphotyrosine-containing peptides. In the chapter by Bork and Gibson ([11], this volume), Blast and pattern/profile searches were used to extract the set of known SH2 domains and to search for new members. (Profiles used in database searches are conceptually very similar to the profiles used in CLUSTAL W: see the chapters [11] and [13] for profile search methods.) The profile searches detected SH2 domains in the JAK family of protein tyrosine kinases, which were thought not to contain SH2 domains. Although the JAK family SH2 domains are rather divergent, they have the necessary core structural residues as well as the critical positively charged residue that binds phosphotyrosine, leaving no doubt that they are bona fide SH2 domains. The five new JAK family SH2 domains were added sequentially to the existing alignment of 65 SH2 domains using the CLUSTAL W profile alignment option. Figure 6 shows part of the resulting alignment. Despite their divergent sequences, the new SH2 domains have been aligned nearly perfectly with the old set. No insertions were placed in the original SH2 domains. In this example, the profile alignment procedure has produced better results than a one-step full alignment of all 70 SH2 domains, and in considerably less time. (ABSTRACT TRUNCATED)

Microtubules and Src homology 3 domains stimulate the dynamin GTPase via its C-terminal domain.

Herskovits JS, Shpetner HS, Burgess CC, Vallee RB
Proc Natl Acad Sci U S A. 1993; 90: 11468-72

Dynamin is a 100-kDa GTPase that plays a critical role in the initial stages of endocytosis. Dynamin binds to microtubules, which potently stimulate its GTPase activity. Binding to Src homology 3 (SH3) domains of proteins involved in signal transduction has also recently been reported. In the present study, the protein was digested with a variety of proteases to define its functional domains. Limited digestion with papain split the protein into an approximately 7- to 9-kDa microtubule-binding fragment and a 90-kDa nonbinding fragment. Immunoblotting with an antibody to the C-terminal 20 amino acids of rat dynamin showed the small fragment to derive from the C-terminal end of the polypeptide. Microtubule-activated GTPase activity, but not basal GTPase activity, was abolished by papain digestion, identifying the basic, proline-rich C-terminal region of dynamin as an important regulatory site. Bacterially expressed growth factor receptor-bound protein 2 (GRB2) and the SH3 domain of c-Src were also found to stimulate GTPase activity, although to a lesser extent than microtubules. Stimulation of GTPase activity by the recombinant proteins was similarly abolished by papain digestion. These results identify the basic, proline-rich C-terminal region of dynamin as the binding site for both microtubules and SH3 domains and demonstrate an allosteric interaction between this region of the molecule and the N-terminal GTPase domain.

Phosphopeptide binding to the N-terminal SH2 domain of the p85 alpha subunit of PI 3'-kinase: a heteronuclear NMR study.

Hensmann M, Booker GW, Panayotou G, Boyd J, Linacre J, Waterfield M, Campbell ID
Protein Sci. 1994; 3: 1020-30

The N-terminal src-homology 2 domain of the p85 alpha subunit of phosphatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosine-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated beta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2-N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D-(15N-1H) heteronuclear single-quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2-N domain, these changes indicate a peptide-binding surface on the protein. Line shape analysis of 1D profiles of individual (15N-1H)-HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the (15N-1H) heteronuclear NOE for the backbone amide of each residue was determined for the SH2-N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85 alpha SH2-N domain is suggested by this study.

The tyrosine kinase Hck is an inhibitor of HIV-1 replication counteracted by the viral vif protein.

Hassaine G, Courcoul M, Bessou G, Barthalay Y, Picard C, Olive D, Collette Y, Vigne R, Decroly E
J Biol Chem. 2001; 276: 16885-93

The virus infectivity factor (Vif) protein facilitates the replication of human immunodeficiency virus type 1 (HIV-1) in primary lymphocytes and macrophages. Its action is strongly dependent on the cellular environment, and it has been proposed that the Vif protein counteracts cellular activities that would otherwise limit HIV-1 replication. Using a glutathione S-transferase pull-down assay, we identified that Vif binds specifically to the Src homology 3 domain of Hck, a tyrosine kinase from the Src family. The interaction between Vif and the full-length Hck was further assessed by co-precipitation assays in vitro and in human cells. The Vif protein repressed the kinase activity of Hck and was not itself a substrate for Hck phosphorylation. Within one single replication cycle of HIV-1, Hck was able to inhibit the production and the infectivity of vif-deleted virus but not that of wild-type virus. Accordingly, HIV-1 vif- replication was delayed in Jurkat T cell clones stably expressing Hck. Our data demonstrate that Hck controls negatively HIV-1 replication and that this inhibition is suppressed by the expression of Vif. Hck, which is present in monocyte-macrophage cells, represents the first identified cellular inhibitor of HIV-1 replication overcome by Vif.

The crystal structure of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase reveals distinct domain homologies.

Hasemann CA, Istvan ES, Uyeda K, Deisenhofer J
Structure. 1996; 4: 1017-29

BACKGROUND. Glucose homeostasis is maintained by the processes of glycolysis and gluconeogenesis. The importance of these pathways is demonstrated by the severe and life threatening effects observed in various forms of diabetes. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate, a potent regulator of glycolysis. Thus this bifunctional enzyme plays an indirect yet key role in the regulation of glucose metabolism. RESULTS. We have determined the 2.0 A crystal structure of the rat testis isozyme of this bifunctional enzyme. The enzyme is a homodimer of 55 kDa subunits arranged in a head-to-head fashion, with each monomer consisting of independent kinase and phosphatase domains. The location of ATPgammaS and inorganic phosphate in the kinase and phosphatase domains, respectively, allow us to locate and describe the active sites of both domains. CONCLUSIONS. The kinase domain is clearly related to the superfamily of mononucleotide binding proteins, with a particularly close relationship to the adenylate kinases and the nucleotide-binding portion of the G proteins. This is in disagreement with the broad speculation that this domain would resemble phosphofructokinase. The phosphatase domain is structurally related to a family of proteins which includes the cofactor independent phosphoglycerate mutases and acid phosphatases.

Intermolecular interactions between the SH3 domain and the proline-rich TH region of Bruton's tyrosine kinase.

Hansson H, Okoh MP, Smith CI, Vihinen M, Hard T
FEBS Lett. 2001; 489: 67-70

The SH3 domain of Bruton's tyrosine kinase (Btk) is preceded by the Tec homology (TH) region containing proline-rich sequences. We have studied a protein fragment containing both the Btk SH3 domain and the proline-rich sequences of the TH region (PRR-SH3). Intermolecular NMR cross-relaxation measurements, gel permeation chromatography profiles, titrations with proline-rich peptides, and (15)N NMR relaxation measurements are all consistent with a monomer-dimer equilibrium with a dissociation constant on the order of 60 microM. The intermolecular interactions do, at least in part, involve proline-rich sequences in the TH region. This behavior of Btk PRR-SH3 may have implications for the functional action of Btk.

Solution structure of the SH3 domain from Bruton's tyrosine kinase.

Hansson H, Mattsson PT, Allard P, Haapaniemi P, Vihinen M, Smith CI, Hard T
Biochemistry. 1998; 37: 2912-24

X-linked agammaglobulinemia (XLA) is a heritable immunodeficiency caused by mutations in the gene coding for Bruton's tyrosine kinase (Btk). Btk belongs to the Tec family of tyrosine kinases. Each member of the family contains five regions and mutations causing XLA have been isolated in all five regions. We have determined the solution structure of the Src homology 3 (SH3) domain of Btk using two- and three-dimensional nuclear magnetic resonance (NMR) spectroscopy on natural abundance and 15N-labeled protein material. The structure determination is complemented by investigation of backbone dynamics based on 15N NMR relaxation. The Btk SH3 forms a well-defined structure and shows the typical SH3 topology of two short antiparallel beta-sheets packed almost perpendicular to each other in a sandwich-like fold. The N- and C-termini are more flexible as are peptide fragments in the RT and n-Src loops. The studied Btk SH3 fragment adopts two slowly interconverting conformations with a relative concentration ratio of 7:1. The overall fold of the minor form is similar to that of the major form, as judged on the basis of observed NOE connectivities and small chemical shift differences. A tryptophan (W251) ring flip is the favored mechanism for interconversion, although other possibilities cannot be excluded. The side chain of Y223, which becomes autophosphorylated upon activation of Btk, is exposed within the potential SH3 ligand binding site. Finally, we compare the present Btk SH3 structure with other SH3 structures.

Interaction of peptides derived from the Fas ligand with the Fyn-SH3 domain.

Hane M, Lowin B, Peitsch M, Becker K, Tschopp J
FEBS Lett. 1995; 373: 265-8

Interaction of the widely expressed Fas with its membrane-bound ligand (FasL) leads to rapid cell death via apoptosis. To avoid pathological tissue damage, the activity of FasL requires tight regulation. Here, we report that the Src homology 3 (SH3) domain of Fyn binds to the proline-rich cytoplasmic region of FasL. Binding of the SH3 domain occurs between amino acid residues 44-71 which contains several potential SH3 interaction sites. This binding is specific, as SH3 domains of Lck, Grb2 and ras-GAP bind only weakly or not at all. We suggest that FasL activity may be modulated by SH3 domains of the src-like Fyn kinase.

A potential catalytic site revealed by the 1.7-A crystal structure of the amino-terminal signalling domain of Sonic hedgehog.

Hall TM, Porter JA, Beachy PA, Leahy DJ
Nature. 1995; 378: 212-6

Within the past few years, members of the hedgehog (hh) family of secreted signalling proteins have emerged as the primary signals generated by certain embryonic patterning centres. In vertebrate embryos, for example, sonic hedgehog expression in the notochord appears to be responsible for the local and long-range induction of ventral cell types within the neural tube and somites (reviewed in refs 1, 2). Protein products encoded by hh family members are synthesized as precursors that undergo autoprocessing to generate an amino-terminal domain that appears to be responsible for both local and long-range signalling activities, and a carboxy-terminal domain that contains the autoprocessing activity. As part of an effort to understand how hh family members participate in cell-to-cell signalling, we have determined and report here the crystal structure at 1.7 A of the amino-terminal domain of murine Sonic hedgehog (Shh-N). The structure revealed a tetrahedrally coordinated zinc ion that appears to be structurally analogous to the zinc coordination sites of zinc hydrolases, such as thermolysin and carboxypeptidase A. This previously unsuspected catalytic site represents a distinct activity from the autoprocessing activity that resides in the carboxy-terminal domain.

The crystal structure of the N-terminal SH3 domain of Grb2.

Guruprasad L, Dhanaraj V, Timm D, Blundell TL, Gout I, Waterfield MD
J Mol Biol. 1995; 248: 856-66

The 3-D structure of the N-terminal SH3 domain of the regulatory protein Grb2 has been determined by X-ray analysis at 2.8 A resolution and refined to a crystallographic R factor of 21.5%. The structure, which is very similar to those of other SH3 domains, consists of two orthogonal, antiparallel up-down beta-sheets, with three variable loops and a 3(10) helix. Docking of the proline-rich peptide, 3BP1 on Grb2-N SH3, shows that the polyproline type II helix can bind the SH3 domain forming conserved hydrogen bonds between the main-chain carbonyl oxygens of Met4 and Pro7 of the proline-rich peptide and the reoriented side-chains of Trp36 and Asn51, respectively, and a hydrogen bond between the main-chain carbonyl of Leu8 of the proline rich peptide with the side-chain OH of Tyr52 of the Grb2-N SH3. The peptide side-chain binding occurs on the surface of SH3 domain at three major sites involving the side-chains of the residues in the hydrophobic patch (Tyr7, Phe9, Trp36, Phe47, Pro49 and Tyr52) and the RT-Src and n-Src loops of the SH3 domain. The proline-rich peptides could bind the Grb2-N SH3 in either orientation and maintain the key hydrogen bonds because of the pseudo-symmetry of the polyproline type II helix. However, for the mSos1 peptide a salt bridge can be formed between the arginine of the proline-rich peptide and the protein at Asp15, Glu16 and Glu31 only in one direction; this orientation seems to be strongly preferred. The conservatively varied RGD sequence motif (sometimes KGE or KGD) in SH3 domains might be involved in interactions at the cell membrane.

The SH3 domain of Bruton's tyrosine kinase interacts with Vav, Sam68 and EWS.

Guinamard R, Fougereau M, Seckinger P
Scand J Immunol. 1997; 45: 587-95

Bruton tyrosine kinase (BTK) is a cytoplasmic protein tyrosine kinase which controls crucial steps of differentiation of B lymphocytes. Mutations affecting either the PH, SH3, SH2 or kinase domain of BTK all give rise to X linked agammaglobulinaemia (XLA) in humans. In this study, the authors report that the BTK-SH3 domain binds to a set of proteins expressed in pro-B, pre-B and B cell lines. Three of them were characterized as Vav, Sam68 and EWS. The authors show that a Pro-->Leu substitution in a region of the SH3 domain, which is deleted in an XLA patient, is sufficient to abolish BTK-SH3 binding potential. The authors also report that several of the BTK-SH3 binding proteins, including Sam68, EWS and Vav, are tyrosine phosphorylated in conditions that also promote BTK kinase activity. For EWS and Sam68 this tyrosine phosphorylation was cell cycle dependent.

The SH3-fold family: experimental evidence and prediction of variations in the folding pathways.

Guerois R, Serrano L
J Mol Biol. 2000; 304: 967-82

To investigate the relationships between protein topology, amino acid sequence and folding mechanisms, the folding transition state of the Sso7d protein has been characterised both experimentally and theoretically. Although Sso7d protein has a similar topology to that of the SH3 domains, the structure of its transition state is different from that of alpha-spectrin and src SH3 domains previously studied. The folding algorithm, Fold-X, including an energy function with specific sequence features, accounts for these differences and reproduces with a good agreement the set of experimental phi(double dagger-U) values obtained for the three proteins. Our analysis shows that taking into account sequence features underlying protein topology is critical for an accurate prediction of the folding process.

The integrity of the SH3 binding motif of AFAP-110 is required to facilitate tyrosine phosphorylation by, and stable complex formation with, Src.

Guappone AC, Flynn DC
Mol Cell Biochem. 1997; 175: 243-52

The actin filament-associated protein AFAP-110 forms a stable complex with activated variants of Src in chick embryo fibroblast cells. Stable complex formation requires the integrity of the Src SH2 and SH3 domains. In addition, AFAP-110 encodes two adjacent SH3 binding motifs and six candidate SH2 binding motifs. These data indicate that both SH2 and SH3 domains may work cooperatively to facilitate Src/AFAP-110 stable complex formation. As a test for this hypothesis, we sought to understand whether one or both SH3 binding motifs in AFAP-110 modulate interactions with the Src SH3 domain and if this interaction was required to present AFAP-110 for tyrosine phosphorylation by, and stable complex formation with, Src. A proline to alanine site-directed mutation in the amino terminal SH3 binding motif (SH3bm I) was sufficient to abrogate absorption of AFAP-110 with GST-SH3STC. Co-expression of activated Src (pp60(527F)) with AFAP-110 in Cos-1 cells permit tyrosine phosphorylation of AFAP-110 and stable complex formation with pp60(527F). However, co-expression of the SH3 null-binding mutant (AFAP71A) with pp60(527F) revealed a 2.7 fold decrease in steady-state levels of tyrosine phosphorylation, compared to AFAP-110. Although a lower but detectable level of AFAP71A was phosphorylated on tyrosine, AFAP71A could not be detected in stable complex with pp60(527F), unlike AFAP-110. These data indicate that SH3 interactions facilitate presentation of AFAP-110 for tyrosine phosphorylation and are also required for stable complex formation with pp60(527F).

Role of native topology investigated by multiple unfolding simulations of four SH3 domains.

Gsponer J, Caflisch A
J Mol Biol. 2001; 309: 285-98

The relative importance of amino acid sequence and native topology in the unfolding process of two SH3 domains and two circular permutants was investigated by 120 molecular dynamics runs at 375 K for a total simulation time of 0.72 micros. The alpha-spectrin (aSH3) and src SH3 (sSH3) domains, which have the same topology and a sequence identity of only 34%, show similar unfolding pathways. The disappearance of the three-stranded antiparallel beta-sheet is the last unfolding event, in agreement with a large repertoire of kinetic data derived from point mutations as well as glycine insertions and disulfide crosslinks. Two alternative routes of beta-sheet unfolding have emerged from the analysis of the trajectories. One is statistically preferred in aSH3 (n-src loop breaks before distal hair-pin) and the inverse in sSH3. An elongation of the beta2-beta3 hairpin was observed during the unfolding of sSH3 at 375 K and in 300 K simulations started from the putative transition state of sSH3 in accord with unusual kinetic data for point mutations at the n-src loop. The change of connectivity in the permutants influenced the sequence of unfolding events mainly at the permutation site. Regions where the connectivity remained unaffected showed the same chronology of contact disappearance. Taken together with previous folding simulations of two designed three-stranded antiparallel beta-sheet peptides, these results indicate that, at least for small beta-sheet proteins, the folding mechanism is primarily defined by the native state topology, whilst specific interactions determine the statistically predominant folding route.

Refined solution structure and backbone dynamics of HIV-1 Nef.

Grzesiek S, Bax A, Hu JS, Kaufman J, Palmer I, Stahl SJ, Tjandra N, Wingfield PT
Protein Sci. 1997; 6: 1248-63

The tendency of HIV-1 Nef to form aggregates in solution, particularly at pH values below 8, together with its large fraction of highly mobile residues seriously complicated determination of its three-dimensional structure, both for heteronuclear solution NMR (Grzesiek et al., 1996a, Nat Struct Biol 3:340-345) and for X-ray crystallography (Lee et al., 1996, Cell 85:931-942). Methods used to determine the Nef structure by NMR at pH 8 and 0.6 mM concentration are presented, together with a detailed description of Nef's secondary and tertiary structure. The described techniques have general applicability for the NMR structure determination of proteins that are aggregating and/or have limited stability at low pH values. Extensive chemical shift assignments are reported for backbone and side chain 1H, 13C, and 15N resonances of the HIV-1 Nef deletion mutants NEF delta 2-39, NEF delta 2-39, delta 159-173, and of NEF delta 2-39, delta 159-173 in complex with the SH3 domain of the Hck tyrosine protein kinase. Besides a type II polyproline helix, Nef's structure consists of three alpha-helices, a 3(10) helix, and a five-stranded anti-parallel beta-sheet. The analysis of 15N relaxation parameters of the backbone amide sites reveals that all the secondary structure elements are non-mobile on the picosecond to nanosecond and on the millisecond time scale. A large number of slowly exchanging amide protons provides evidence for the stability of the Nef core even on the time scale of hours. Significant internal motions on the ps to ns time scale are detected for residues 60 to 71 and for residues 149 to 180, which form solvent-exposed loops. The residues of the HIV-1 protease cleavage site (W57/L58) do not exhibit large amplitude motions on the sub-nanosecond time scale, and their side chains insert themselves into a hydrophobic crevice formed between the C-terminus of helix 1 and the N-terminus of helix 2. A refined structure has been determined based on additional constraints for side-chain and backbone dihedral angles derived from a large number of three-bond J-coupling and ROE data.

The solution structure of HIV-1 Nef reveals an unexpected fold and permits delineation of the binding surface for the SH3 domain of Hck tyrosine protein kinase.

Grzesiek S, Bax A, Clore GM, Gronenborn AM, Hu JS, Kaufman J, Palmer I, Stahl SJ, Wingfield PT
Nat Struct Biol. 1996; 3: 340-5

The solution structure of HIV-1 Nef has been solved by multidimensional heteronuclear NMR spectroscopy. The construct employed to circumvent problems associated with aggregation was a double-deletion mutant (delta2-39, delta159-173) in which conformationally disordered regions of the protein at the N terminus and in a long solvent-exposed flexible loop were removed, without affecting the properties or structural integrity of the remainder of the protein. Despite the absence of any sequence similarity, the overall fold of Nef is reminiscent of that of the family of winged helix-turn-helix DNA binding proteins. The binding surface of Nef for the SH3 domain of Hck tyrosine protein kinase has been mapped and reveals a non-contiguous (in terms of amino-acid sequence) interaction surface. This unique feature may suggest possible avenues for drug design aimed at inhibiting the interaction between Nef and SH3 domains.

SH2 domains: from structure to energetics, a dual approach to the study of structure-function relationships.

Grucza RA, Bradshaw JM, Futterer K, Waksman G
Med Res Rev. 1999; 19: 273-93

Mutagenic analysis of Vav reveals that an intact SH3 domain is required for transformation.

Groysman M, Nagano M, Shaanan B, Katzav S
Oncogene. 1998; 17: 1597-606

The vav proto-oncogene encodes a protein with multiple modulae domains that enable it to function as a mediator, linking tyrosine signaling to downstream events in hematopoietic cells. Circumstantial evidence suggests that protein-protein interactions exerted by two of these domains, the Src homology 2 (SH2) and the Src homology 3 (SH3), play an important role in the regulation of Vav activity. To study the relevance of the SH3 domain for the function of vav as a transforming gene, we have created several mutations in the SH3 domain located at its carboxy region. Substitution of the non-conserved aspartic acid 797 (to asparagine, D797N) retained the transforming potential of the vav oncogene, whereas substitutions of five highly conserved amino-acids: alanine 789 (to asparagine, A789N), leucine 801 (to arginine, L801R), tryptophan 821 (to arginine, W821R), glycine 830 (to valine, G830V) and valine 837 (to glutamic acid, V837E) greatly reduced its transforming potential. The mutant proteins resemble Vav in many biochemical properties; however, while the transforming mutant protein (D797N) associates with several unidentified proteins in a manner similar to that of Vav, the non-transforming mutant Vav proteins react very poorly with these proteins. Among the known Vav-interacting proteins, hnRNP-K associates with all mutant proteins except A789N and V837E whereas binding of Zyxin to any of the mutant proteins is not affected. Taken together, our results clearly demonstrate that the SH3 domain has a positive effect on vav activity and is needed for vav transformation. The vavSH3C associating protein(s) that are crucial for its activity as a transforming gene have probably not yet been identified.

Evidence for a functional association between phosphatidylinositol 3-kinase and c-src in the spreading response of osteoclasts to colony-stimulating factor-1.

Grey A, Chen Y, Paliwal I, Carlberg K, Insogna K
Endocrinology. 2000; 141: 2129-38

Osteoclasts are bone-resorbing cells whose normal function depends in part upon their ability to migrate over the bone surface to initiate new sites of bone resorption. The growth factor/cytokine, colony-stimulating factor-1 (CSF-1), potently stimulates osteoclast motility, in a c-src-dependent fashion. The intracellular signaling molecules that participate with c-src in CSF-1-induced remodeling of the osteoclast cytoskeleton have not been identified. Here we demonstrate, using the inhibitors wortmannin and LY294002, that activation of phosphatidylinositol 3-kinase (PI3-K) is required for CSF-1-induced spreading in osteoclasts. After CSF-1 treatment of osteoclast-like cells, PI3-K activity associated with the CSF-1 receptor c-fms, is increased, and the 85-kDa regulatory subunit of PI3-K and c-src coimmunoprecipitate. CSF-1 induces redistribution of PI3-K to the periphery of the cell. The association between p85 and c-src is due in part to a direct interaction between the proline-rich sequences of p85 and the SH3 domain of c-src. In vitro, the c-src SH3 domain stimulates PI3-K activity. Taken together, the current data suggest that c-src, via its SH3 domain, may participate in CSF-1-induced activation of PI3-K and that PI3-K and c-src are in the signaling pathway that subserves CSF-1-induced cytoskeletal changes in osteoclasts.

Sequence requirements for association of protein-tyrosine phosphatase PEP with the Src homology 3 domain of inhibitory tyrosine protein kinase p50(csk).

Gregorieff A, Cloutier JF, Veillette A
J Biol Chem. 1998; 273: 13217-22

Previously, we reported that the inhibitory tyrosine protein kinase p50(csk) is physically associated with the protein-tyrosine phosphatase PEP in hematopoietic cells. This interaction was shown to involve the Src homology 3 (SH3) region of Csk and a proline-rich sequence of PEP termed P1 (SRRTDDEIPPPLPERTPESFIVVEE). In this report, we have attempted to understand the structural basis for the highly specific association of these two molecules in vivo. Our studies revealed that the proline-rich core of the P1 region of PEP (PPPLPERT) was necessary but not sufficient for binding to p50(csk). Additional sequences located carboxyl to this motif were also needed for binding to the Csk SH3 domain in vitro and in vivo. Further analyses revealed that two aliphatic residues (isoleucine 625 and valine 626; PESFIVVEE) were especially important for this effect. In addition to clarifying the molecular basis for the selective ability of PEP to associate with Csk, these results constitute further evidence that sequences outside proline-rich cores dictate the specificity of SH3 domain-mediated interactions in vivo.

HIV-1 Nef: a critical factor in viral-induced pathogenesis.

Greenway AL, Holloway G, McPhee DA
Adv Pharmacol. 2000; 48: 299-343

Simian immunodeficiency virus and human immunodeficiency virus type 1 nef proteins show distinct patterns and mechanisms of Src kinase activation.

Greenway AL, Dutartre H, Allen K, McPhee DA, Olive D, Collette Y
J Virol. 1999; 73: 6152-8

The nef gene from human and simian immunodeficiency viruses (HIV and SIV) regulates cell function and viral replication, possibly through binding of the nef product to cellular proteins, including Src family tyrosine kinases. We show here that the Nef protein encoded by SIVmac239 interacts with and also activates the human Src kinases Lck and Hck. This is in direct contrast to the inhibitory effect of HIV type 1 (HIV-1) Nef on Lck catalytic activity. Unexpectedly, however, the interaction of SIV Nef with human Lck or Hck is not mediated via its consensus proline motif, which is known to mediate HIV-1 Nef binding to Src homology 3 (SH3) domains, and various experimental analyses failed to show significant interaction of SIV Nef with the SH3 domain of either kinase. Instead, SIV Nef can bind Lck and Hck SH2 domains, and its N-terminal 50 amino acid residues are sufficient for Src kinase binding and activation. Our results provide evidence for multiple mechanisms by which Nef binds to and regulates Src kinases.

Human immunodeficiency virus type 1 Nef binds directly to Lck and mitogen-activated protein kinase, inhibiting kinase activity.

Greenway A, Azad A, Mills J, McPhee D
J Virol. 1996; 70: 6701-8

It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis.

Important role of hydrogen bonds in the structurally polarized transition state for folding of the src SH3 domain.

Grantcharova VP, Riddle DS, Santiago JV, Baker D
Nat Struct Biol. 1998; 5: 714-20

Experimental and theoretical studies on the folding of small proteins such as the chymotrypsin inhibitor 2 (CI-2) and the P22 Arc repressor suggest that the folding transition state is an expanded version of the native state with most interactions partially formed. Here we report that this picture does not hold generally: a hydrogen bond network involving two beta-turns and an adjacent hydrophobic cluster appear to be formed in the folding transition state of the src SH3 domain, while the remainder of the polypeptide chain is largely unstructured. Comparison with data on other small proteins suggests that this structural polarization is a consequence of the topology of the SH3 domain fold. The non-uniform distribution of structure in the folding transition state provides a challenging test for computational models of the folding process.

Folding dynamics of the src SH3 domain.

Grantcharova VP, Baker D
Biochemistry. 1997; 36: 15685-92

The thermodynamics and kinetics of folding of the chicken src SH3 domain were characterized using equilibrium and stopped-flow fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) hydrogen exchange experiments. As found for other SH3 domains, guanidinium chloride (GdmCl) denaturation melts followed by both fluorescence and circular dichroism were nearly superimposable, indicating the concerted formation of secondary and tertiary structure. Kinetic studies confirmed the two-state character of the folding reaction. Except for a very slow refolding phase due to proline isomerization, both folding and unfolding traces fit well to single exponentials over a wide range of GdmCl concentrations, and no burst phase in amplitude was observed during the dead time of the stopped-flow instrument. The entropy, enthalpy, and heat capacity changes upon unfolding were determined by global fitting of temperature melts at varying GdmCl concentrations (0.4-3.7 M). Estimates of the free energy of unfolding, DeltaGUH2O, from guanidine denaturation, thermal denaturation, and kinetic experiments were in good agreement. To complement these data on the global characteristics of src SH3 folding, individual hydrogen-deuterium (HD) exchange rates were measured for approximately half of the backbone amides in 0 and 0.7 M GdmCl. The calculated free energies of the opening reaction leading to exchange (DeltaGHD) indicated that unfolding is highly cooperative--slowly exchanging protons were distributed throughout the core of the protein. The slowly exchanging protons exhibited DeltaGHD values higher than the global DeltaGUH2O by approximately 1 kcal/mol, suggesting that the denatured state might be somewhat compact under native conditions. Comparison of the src SH3 with homologous SH3 domains as well as with other small well-characterized beta-sheet proteins provides insights into the determinants of folding kinetics and protein stability.

Sequences surrounding the Src-homology 3 domain of phospholipase Cgamma-1 increase the domain's association with Cbl.

Graham LJ, Stoica BA, Shapiro M, DeBell KE, Rellahan B, Laborda J, Bonvini E
Biochem Biophys Res Commun. 1998; 249: 537-41

SH3 domains are protein modules that interact with proline-rich polypeptide fragments. Cbl is an adapter-like protein known to interact with several SH3 domains, including the PLCgamma1 SH3 domain and the Grb2 amino terminal SH3 domain. Here we explore whether sequences surrounding the PLCgamma1 SH3 domain core sequence (aa.796-851) can affect the binding to Cbl, a target used as a prototypical ligand. Consistent with previous reports, our results demonstrated a weak binding of Cbl to GST fusion proteins that strictly encompass the structural core of the PLCgamma1 SH3 domain but a high-avidity binding to the Grb2 amino-terminal SH3 domain. Inclusion of amino acids immediately flanking the PLCgamma1 SH3 core domain, however, substantially increased binding of Cbl to a level comparable to that of the Grb2 amino-terminal SH3 domain. The interaction of this extended PLCgamma1 SH3 domain fusion protein with Cbl was shown to depend entirely upon the interaction of the domain with a proline-rich motif in Cbl, ruling out the possibility that amino acids adjacent to the core SH3 domain of PLCgamma1 provide independent Cbl binding. These data suggest that sequences surrounding the SH3 domain of PLCgamma1 may contribute to or stabilize the association of the domain with the target protein, thus increasing its binding efficiency.

The GTPase dynamin binds to and is activated by a subset of SH3 domains.

Gout I, Dhand R, Hiles ID, Fry MJ, Panayotou G, Das P, Truong O, Totty NF, Hsuan J, Booker GW, et al.
Cell. 1993; 75: 25-36

Src homology 3 (SH3) domains have been implicated in mediating protein-protein interactions in receptor signaling processes; however, the precise role of this domain remains unclear. In this report, affinity purification techniques were used to identify the GTPase dynamin as an SH3 domain-binding protein. Selective binding to a subset of 15 different recombinant SH3 domains occurs through proline-rich sequence motifs similar to those that mediate the interaction of the SH3 domains of Grb2 and Abl proteins to the guanine nucleotide exchange protein, Sos, and to the 3BP1 protein, respectively. Dynamin GTPase activity is stimulated by several of the bound SH3 domains, suggesting that the function of the SH3 module is not restricted to protein-protein interactions but may also include the interactive regulation of GTP-binding proteins.

Interaction of SH3 domain of Hck tyrosine kinase with cellular proteins containing proline-rich regions: evidence for modulation by unique domain.

Gouri BS, Swarup G
Indian J Biochem Biophys. 1997; 34: 29-39

The Hck tyrosine kinase, a member of Src family, is predominantly expressed in myeloid cells. In this report we have analyzed interaction of cellular proteins with Src homology 3 (SH3) domain of Hck. For this purpose we used various GST-Hck fusion proteins comprising a part of unique region, complete unique region and/or complete SH3 domain of Hck, and glutathione S-transferase (GST). When these fusion proteins (or GST), immobilized on glutathione-agarose beads were incubated with [35S] methionine labelled cell extracts, multiple proteins which interact specifically with SH3 domain of Hck were detected by SDS-PAGE followed by autoradiography. The Hck interacting proteins could also be detected by a tandem blot binding assay in which the blot was incubated with purified fusion protein (or GST) and then the interacting proteins were identified by using antibody against GST. When a part of or complete unique domain was present along with SH3 domain, the interaction of some specific proteins was reduced several fold. These results raise the possibility of unique domain altering the properties of SH3 domain, thus modulating or restricting the interaction of SH3 domain with specific cellular proteins. This modulatory effect of unique domain was localized to 28 amino acids upstream of SH3 domain. SH3 interacting proteins were associated with serine/threonine and tyrosine kinase activities towards exogenous substrates. Most of the SH3 binding proteins were soluble in Triton X-100. Differentiation of promyelocytic leukemia cell line HL-60 into macrophage like cells resulted in appearance of novel SH3 binding proteins. Hck was detected in the eluate of WGA-Sepharose column, suggesting that it interacts with WGA binding glycoprotein (s). A rat spleen cDNA library was screened for the SH3 binding proteins by protein interaction cloning. Sequence analysis of the clones showed the presence of proline rich regions containing PPXP motifs.

NMR structure of the N-terminal SH3 domain of GRB2 and its complex with a proline-rich peptide from Sos.

Goudreau N, Cornille F, Duchesne M, Parker F, Tocque B, Garbay C, Roques BP
Nat Struct Biol. 1994; 1: 898-907

GRB2 is a small adaptor protein of 217 amino acids comprising one SH2 domain surrounded by two SH3 domains. GRB2 couples receptor tyrosine kinase activation to Ras signalling by interacting, through its SH3 domains, to the carboxy-terminal proline-rich regions of the guanine nucleotide exchange factor Sos. Here we report the synthesis and solution structure of the amino-terminal SH3 domain of GRB2 and of its more stable Ser 32 mutant. 1H NMR analysis of the complex between the Ser-32-GRB2-N-SH3 domain and the proline-rich peptide VPPPVPPRRR, derived from h-Sos, shows that relative to the SH3 peptide complexes described for PI3K, Fyn and Abl, the proline-rich peptide in this complex binds in the opposite orientation.

The solution structure of Abl SH3, and its relationship to SH2 in the SH(32) construct.

Gosser YQ, Zheng J, Overduin M, Mayer BJ, Cowburn D
Structure. 1995; 3: 1075-86

BACKGROUND: The Src homology domains, SH3 and SH2, of Abl protein tyrosine kinase regulate enzymatic activity in vivo. Abl SH3 suppresses kinase activity, whereas Abl SH2 is required for the transforming activity of the activated form of Abl. We expect that the solution structures of Abl SH3, Abl SH2 and Abl SH(32) (a dual domain comprising SH3 and SH2 subdomains) will contribute to a structural basis for understanding the mechanism of the Abl 'regulatory apparatus'. RESULTS: We present the solution structure of the free Abl SH3 domain and a structural characterization of the Abl regulatory apparatus, the SH(32) dual domain. The solution structure of Abl SH3 was determined using multidimensional double resonance NMR spectroscopy. It consists of two antiparallel beta sheets packed orthogonally, an arrangement first shown in spectrin SH3. Compared with the crystal structure of the Abl SH3 complexed with a natural ligand, there is no significant difference in overall folding pattern. The structure of the Abl SH(32) dual domain was characterized by NMR spectroscopy using the 1H and 15N resonance assignment of Abl SH3 and Abl SH2. On the basis of the high degree of similarity in chemical shifts and hydrogen/deuterium exchange pattern for the individual domains of SH3 and SH2 compared with those of the SH(32) dual domain, a structural model of the Abl SH(32) regulatory apparatus is suggested. This model is in good agreement with the ligand-binding characteristics of Abl SH3, SH2 and SH(32). The binding constants for isolated SH3 and SH2 domains when binding to natural ligands, measured by intrinsic fluorescence quenching, do not differ significantly from the constants of these domains within SH(32). CONCLUSION: The solution structures of free Abl SH3 and Abl SH2, and the structural model of Abl SH(32), provide information about the overall topology of these modular domains. The structural model of Abl SH(32), a monomer, consists of the SH3 and SH2 domains connected by a flexible linker. Sites of ligand binding for the two subdomains are independent.

Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2.

Gorina S, Pavletich NP
Science. 1996; 274: 1001-5

Mutations in the p53 tumor suppressor are among the most frequently observed genetic alterations in human cancer and map to the 200-amino acid core domain of the protein. The core domain contains the sequence-specific DNA binding activity and the in vitro 53BP2 protein binding activity of p53. The crystal structure of the p53 core domain bound to the 53BP2 protein, which contains an SH3 (Src homology 3) domain and four ankyrin repeats, revealed that (i) the SH3 domain binds the L3 loop of p53 in a manner distinct from that of previously characterized SH3-polyproline peptide complexes, and (ii) an ankyrin repeat, which forms an L-shaped structure consisting of a beta hairpin and two alpha helices, binds the L2 loop of p53. The structure of the complex shows that the 53BP2 binding site on the p53 core domain consists of evolutionarily conserved regions that are frequently mutated in cancer and that it overlaps the site of DNA binding. The six most frequently observed p53 mutations disrupt 53BP2 binding in vitro. The structure provides evidence that the 53BP2-p53 complex forms in vivo and may have a critical role in the p53 pathway of tumor suppression.

The role of the linker between the SH2 domain and catalytic domain in the regulation and function of Src.

Gonfloni S, Williams JC, Hattula K, Weijland A, Wierenga RK, Superti-Furga G
EMBO J. 1997; 16: 7261-71

The crystal structures of the regulated Src and Hck tyrosine kinases show intramolecular interactions between the phosphorylated tail and the SH2 domain as well as between the SH3 domain, the SH2-catalytic domain linker (SH2-CD linker) and the catalytic domain. The relative contribution of these interactions to regulation of activity is poorly understood. Mutational analysis of Src and Lck revealed that interaction of the SH2-CD linker with the SH3 domain is crucial for regulation. Moreover, three sites of interaction of the linker with the catalytic domain, one at the beginning (Trp260) and two at the back of the small lobe, opposite the catalytic cleft (beta2/beta3 loop; alphaC/beta4 loop), impinge on Src activity. Other activating mutations map to the front of the catalytic domain in the loop preceding the alphaC-helix (beta3/alphaC loop). SH2-CD linker mutants are deregulated in mammalian cells but transform fibroblasts weakly, suggesting that the linker may bind cellular components. Interpretation of our results on the basis of the crystal structure of Src favours a model in which the correctly positioned SH2-CD linker exerts an inhibitory function on catalysis of Src family members by facilitating displacement of the alphaC-helix. This study may provide a template for the generation of deregulated versions of other protein kinases.

Crosstalk between the catalytic and regulatory domains allows bidirectional regulation of Src.

Gonfloni S, Weijland A, Kretzschmar J, Superti-Furga G
Nat Struct Biol. 2000; 7: 281-6

The catalytic activity of Src family tyrosine kinases is inhibited by intramolecular interactions between the regulatory SH3 and SH2 domains and the catalytic domain. In the inactive state, the critical alphaC-helix in the catalytic domain is positioned such that the formation of the Glu 310-Lys 295 salt bridge is precluded, Tyr 416 in the activation loop is unphosphorylated, and the SH2 and SH3 domains are unavailable for interactions with other proteins. We found that phosphorylation of the activation loop or mutation of the loop preceding the alphaC-helix activates Src and increases the accessibility of the SH3 domain for ligands. Interaction of the alphaC-helix with the activation loop is a central component of this regulatory system. Our data suggest a bidirectional regulation mechanism in which the regulatory domains inhibit Src activity, and Src activity controls the availability of the regulatory domains. By this mechanism, Src family kinases can be activated by proteins phosphorylating or changing the conformation of the catalytic domain. Once active, Src family kinases become less prone to regulation, implying a positive feedback loop on their activity.

Leucine 255 of Src couples intramolecular interactions to inhibition of catalysis.

Gonfloni S, Frischknecht F, Way M, Superti-Furga G
Nat Struct Biol. 1999; 6: 760-4

The activity of the c-Src tyrosine kinase is regulated through intramolecular interactions between the catalytic and SH2/SH3 domains. However, the exact mechanism by which this occurs remains obscure. In the crystal structure of c-Src, the peptide that links the SH2 and catalytic domain (SH2-CD linker) is sandwiched between the latter and the SH3 domain. A residue in the linker, Leu 255, inserts its side chain into a deep hydrophobic pocket present on the surface of the catalytic domain. To investigate the possible regulatory role of this prominent interaction, we mutated Leu 255 to different hydrophobic residues. We found that the length and 'bulkiness' of the side chain had a profound influence on c-Src regulation. Src-L255V was highly active but showed reduced SH3 accessibility in vitro as well as an altered localization in vivo when compared to other deregulated forms of Src. Our analyses lead us to suggest that the Leu 255-pocket interaction is a critical component of the intramolecular inhibition mechanism of Src family kinases.

Amino-terminal regions of polyomavirus middle T antigen are required for interactions with protein phosphatase 2A.

Glenn GM, Eckhart W
J Virol. 1995; 69: 3729-36

Polyomavirus middle T antigen (MT) is the major transforming protein of the virus. It functions through interactions with a number of cellular proteins involved in cell proliferation. MT forms complexes with protein phosphatase 2A (PP2A), pp60c-src, phosphatidylinositol 3-kinase, and Shc. We introduced both deletion and point mutations into three regions of MT and examined their ability to associate with PP2A and pp60c-src. The first 25 amino acid residues of MT are required for association with PP2A and pp60c-src. Amino acids 105 to 111, comprising the sequence Cys-Arg-Met-Pro-Leu-Thr-Cys, is also required for complex formation between MT and PP2A. However, the sequence Asp-Lys-Gly-Gly (amino acids 44 to 47), also found in the B subunit of PP2A, is dispensable for complex formation between MT and PP2A. We find a strict correlation between the ability of MT to associate with PP2A and the ability of MT to associate with pp60c-src. One mutant, L5E, associates with a phosphatase other than PP2A, pp60c-src, and phosphatidylinositol 3-kinase in a manner similar to that of wild-type MT yet is reduced in its transforming ability on NIH 3T3 cells.

Biochemical analysis of SH2 domain-mediated protein interactions.

Gish G, Larose L, Shen R, Pawson T
Methods Enzymol. 1995; 254: 503-23

Modelling of insulin receptor tyrosine kinase in its active form: a case study for validation of theoretical methods.

Ginalski K, Wojciechowski M, Lesyng B
Acta Biochim Pol. 1999; 46: 601-7

An active form of an insulin receptor tyrosine kinase (IRK) catalytic core was modelled based on its experimentally known inactive form and the active form of a serine/threonine kinase, protein kinase A (PKA). This theoretical model was compared with the crystallographic structure of the active form of IRK reported later. The structures are very similar, which shows that all the most important features and interactions have been taken into account in the modelling procedure. The elaborated procedure can be applied to other tyrosine kinases. This would allow designing of a wide class of tyrosine kinase inhibitors, very important potential anti-cancer and/or anti-viral drugs.

Peptide inhibitors of src SH3-SH2-phosphoprotein interactions.

Gilmer T, Rodriguez M, Jordan S, Crosby R, Alligood K, Green M, Kimery M, Wagner C, Kinder D, Charifson P, et al.
J Biol Chem. 1994; 269: 31711-9

Activated pp60c-src has been implicated in a number of human malignancies including colon carcinoma and breast adenocarcinoma. Association of the src SH2 domain with tyrosine-phosphorylated proteins plays a role in src-mediated signal transduction. Inhibitors of src SH2 domain-phosphoprotein interactions are, thus, of great interest in defining the role(s) of src in signal transduction pathways. To facilitate such studies, an enzyme-linked immunosorbent assay (ELISA) was developed to detect inhibitors of src SH2-phosphoprotein interactions. This assay measures inhibition of binding of a fusion construct (glutathione S-transferase src SH3-SH2) with autophosphorylated epidermal growth factor receptor tyrosine kinase domain. Activities of phosphopeptide segments derived from potential src SH2 cognate phosphoprotein partners were determined, with the focal adhesion kinase-derived segment VSETDDY*AEIIDE yielding the highest inhibitory activity. Structure activity studies starting from acetyl (Ac)-Y*EEIE have identified Ac-Y*Y*Y*IE as the most active compound screened in the ELISA. This compound is at least 20-fold more active than the parent peptide Ac-Y*EEIE. A high resolution (2 A) crystal structure of human src SH2 complexed with Ac-Y*EEIE was obtained and provided a useful framework for understanding the structure-activity relationships. Additionally, Ac-Y*EEIE was able to block interactions between src and its cellular phosphoprotein partners in vanadate-treated cell lysates from MDA-MB-468 breast carcinoma cells. However, it is unable to abrogate proliferation of MDA-MB-468 cells in culture, presumably because of poor cell penetration and/or lability of the phosphate group on tyrosine.

Domain assembly, surface accessibility and sequence conservation in full length HIV-1 Nef.

Geyer M, Peterlin BM
FEBS Lett. 2001; 496: 91-5

The accessory Nef protein from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. Here we present an assembly of the full length structure of HIV-1 Nef, allele NL4-3, based on the previously solved anchor and core domain structures. The center part of the 33 residue encompassing flexible loop at the C-terminus of Nef, involved in Nef internalization and CD4 endocytosis, has been modelled. The degree of sequence conservation in HIV-1 Nef proteins was determined using a total of 186 different strains from five different subtypes. The sequence conservation has been correlated with the accessible surface area and with secondary structure features for individual residues. The high amount of flexible regions in Nef accounts for the large surface and the multiple interaction sites the protein exhibits.

Structure of the anchor-domain of myristoylated and non-myristoylated HIV-1 Nef protein.

Geyer M, Munte CE, Schorr J, Kellner R, Kalbitzer HR
J Mol Biol. 1999; 289: 123-38

Negative factor (Nef) is a regulatory myristoylated protein of human immunodeficiency virus (HIV) that has a two-domain structure consisting of an anchor domain and a core domain separated by a specific cleavage site of the HIV proteases. For structural analysis, the HIV-1 Nef anchor domain (residues 2-57) was synthesized with a myristoylated and non-myristoylated N terminus. The structures of the two peptides were studied by1H NMR spectroscopy and a structural model was obtained by restrained molecular dynamic simulations. The non-myristoylated peptide does not have a unique, compactly folded structure but occurs in a relatively extended conformation. The only rather well-defined canonical secondary structure element is a short two-turn alpha-helix (H2) between Arg35 and Gly41. A tendency for another helical secondary structure element (H1) can be observed for the arginine-rich region (Arg17 to Arg22). Myristoylation of the N-terminal glycine residue leads to stabilization of both helices, H1 and H2. The first helix in the arginine-rich region is stabilized by the myristoylation and now contains residues Pro14 to Arg22. The second helix appears to be better defined and to contain more residues (Ala33 to Gly41) than in the absence of myristoylation. In addition, the hydrophobic N-terminal myristic acid residue interacts closely with the side-chain of Trp5 and thereby forms a loop with Gly2, Gly3 and Lys4 in the kink region. This interaction could possibly be disturbed by phosphorylation of a nearby serine residue, and modifiy the characteristic membrane interactions of the HIV-1 Nef anchor domain.

Structure-function relationships in HIV-1 Nef.

Geyer M, Fackler OT, Peterlin BM
EMBO Rep. 2001; 2: 580-5

The accessory Nef protein of HIV and SIV is essential for viral pathogenesis, yet it is perplexing in its multitude of molecular functions. In this review we analyse the structure-function relationships of motifs recently proposed to play roles in aspects of Nef modification, signalling and trafficking, and thereby to impinge on the ability of the virus to survive in, and to manipulate, its cellular host. Based on the full-length structure assembly of HIV Nef, we correlate surface accessibility with secondary structure elements and sequence conservation. Motifs involved in Nef-mediated CD4 and MHC I downregulation are located in flexible regions of Nef, suggesting that the formation of the transient trafficking complexes involved in these processes depends on the recognition of primary sequences. In contrast, the interaction sites for signalling molecules that contain SH3 domains or the p21-activated kinases are associated with the well folded core domain, suggesting the recognition of highly structured protein surfaces.

The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site.

Gervais FG, Chow LM, Lee JM, Branton PE, Veillette A
Mol Cell Biol. 1993; 13: 7112-21

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.

Drosophila photoreceptor axon guidance and targeting requires the dreadlocks SH2/SH3 adapter protein.

Garrity PA, Rao Y, Salecker I, McGlade J, Pawson T, Zipursky SL
Cell. 1996; 85: 639-50

SUMMARY: Mutations in the Drosophila gene dreadlocks (dock) disrupt photoreceptor cell (R cell) axon guidance and targeting. Genetic mosaic analysis and cell-type-specific expression of dock transgenes demonstrate dock is required in R cells for proper innervation. Dock protein contains one SH2 and three SH3 domains, implicating it in tyrosine kinase signaling, and is highly related to the human proto-oncogene Nck. Dock expression is detected in R cell growth cones in the target region. We propose Dock transmits signals in the growth cone in response to guidance and targeting cues. These findings provide an important step for dissection of signaling pathways regulating growth cone motility.

Phosphopeptide occupancy and photoaffinity cross-linking of the v-Src SH2 domain attenuates tyrosine kinase activity.

Garcia P, Shoelson SE, Drew JS, Miller WT
J Biol Chem. 1994; 269: 30574-9

Phosphorylation of c-Src at carboxyl-terminal Tyr-527 suppresses tyrosine kinase activity and transforming potential, presumably by facilitating the intramolecular interaction of the C terminus of Src with its SH2 domain. In addition, it has been shown previously that occupancy of the c-Src SH2 domain with a phosphopeptide stimulates c-Src kinase catalytic activity. We have performed analogous studies with v-Src, the transforming protein from Rous sarcoma virus, which has extensive homology with c-Src. v-Src lacks an autoregulatory phosphorylation site, and its kinase domain is constitutively active. Phosphopeptides corresponding to the sequences surrounding c-Src Tyr-527 and a Tyr-Glu-Glu-Ile motif from the hamster polyoma virus middle T antigen inhibit tyrosine kinase activity of baculovirus-expressed v-Src 2- and 4-fold, respectively. To determine the mechanism of this regulation, the Tyr-527 phosphopeptide was substituted with the photoactive amino acid p-benzoylphenylalanine at the adjacent positions (N- and C-terminal) to phosphotyrosine. These peptides photoinactivate the v-Src tyrosine kinase 5-fold in a time- and concentration-dependent manner. Furthermore, the peptides cross-link an isolated Src SH2 domain with similar rates and specificity. These data indicate that occupancy of the v-Src SH2 domain induces a conformational change that is transmitted to the kinase domain and attenuates tyrosine kinase activity.

bca: an activation-related B-cell gene.

Gangi-Peterson L, Peterson SN, Shapiro LH, Golding A, Caricchio R, Cohen DI, Margulies DH, Cohen PL
Mol Immunol. 1998; 35: 55-63

We have identified a novel activation related B-cell gene (bca) through differential hybridization screening of a murine B cell cDNA library. The deduced amino acid sequence predicted a protein of 482 amino acids with strong sequence similarity to the SH2 and SH3 domains present within the non-catalytic regions of several protein tyrosine kinases. Northern analysis of RNA from several murine B-cell lines revealed a transcript of 1.8 kb, which was not detected in T-cell and non-lymphoid cell lines. bca was transcribed at low levels in resting spleen cells from a variety of normal mouse strains and was strongly expressed in kidney RNA. bca expression was markedly increased in RNA prepared from mitogen activated B cells, and in freshly isolated spleen and lymph node cells of MRL/lpr and NZB autoimmune strains. The unique sequence of bca, which bears no obvious similarity to any specific class of proteins containing SH2 and SH3 domains, suggests that this gene encodes a novel protein potentially involved in B-cell signal transduction.

Structural basis for Syk tyrosine kinase ubiquity in signal transduction pathways revealed by the crystal structure of its regulatory SH2 domains bound to a dually phosphorylated ITAM peptide.

Futterer K, Wong J, Grucza RA, Chan AC, Waksman G
J Mol Biol. 1998; 281: 523-37

The Syk family of kinases, consisting of ZAP-70 and Syk, play essential roles in a variety of immune and non-immune cells. This family of kinases is characterized by the presence of two adjacent SH2 domains which mediate their localization to the membrane through receptor encoded tyrosine phosphorylated motifs. While these two kinases share many structural and functional features, the more ubiquitous nature of Syk has suggested that this kinase may accommodate a greater variety of motifs to mediate its function. We present the crystal structure of the tandem SH2 domain of Syk complexed with a dually phosphorylated ITAM peptide. The structure was solved by multiple isomorphous replacement at 3.0 A resolution. The asymmetric unit comprises six copies of the liganded protein, revealing a surprising flexibility in the relative orientation of the two SH2 domains. The C-terminal phosphotyrosine-binding site is very different from the equivalent region of ZAP-70, suggesting that in contrast to ZAP-70, the two SH2 domains of Syk can function as independent units. The conformational flexibility and structural independence of the SH2 modules of Syk likely provides the molecular basis for the more ubiquitous involvement of Syk in a variety of signal transduction pathways.

Identification of the major sites of autophosphorylation of the murine protein-tyrosine kinase Syk.

Furlong MT, Mahrenholz AM, Kim KH, Ashendel CL, Harrison ML, Geahlen RL
Biochim Biophys Acta. 1997; 1355: 177-90

The protein tyrosine kinase p72syk (Syk) is expressed in a variety of hematopoietic cell types, including B cells, thymocytes, mast cells and others. Both the activity and phosphotyrosine content of this enzyme increase in these cells in response to engagement of the appropriate cell surface receptors. Herein, we describe the cloning of murine Syk and its expression in Sf9 cells as a catalytically active protein. Full-length Syk and a catalytically active 42.5 kDa carboxyl terminal fragment were also expressed as glutathione S-transferase fusion proteins. Comparative reverse phase HPLC and 40% alkaline gel analysis of tryptic digests of phosphorylated Syk demonstrated that all of the major sites of autophosphorylation were also present in GST-Syk and all but one were contained in the 42.5 kDa fragment. The sites of autophosphorylation were identified using a combination of Edman sequencing and mass spectrometric analysis. Ten sites were identified. One site is located in the amino terminal half of the molecule between the two tandem Src homology 2 (SH2) domains. Five sites are located in the hinge region located between the carboxyl terminal SH2 domain and the kinase domain. Two sites lie in the kinase domain within the catalytic loop and two near the extreme carboxyl terminus. Sequences of phosphorylation sites located within the hinge region predict that Syk serves as a docking site for other SH2 domain-containing proteins. Consistent with this prediction, autophosphorylated Syk efficiently binds the carboxyl terminal SH2 domain of phospholipase C-gamma 1.

Evidence for autoinhibitory regulation of the c-src gene product. A possible interaction between the src homology 2 domain and autophosphorylation site.

Fukami Y, Sato K, Ikeda K, Kamisango K, Koizumi K, Matsuno T
J Biol Chem. 1993; 268: 1132-40

In the previous study (Sato, K., Miki, S., Tachibana, H., Hayashi, F., Akiyama, T., and Fukami, Y. (1990) Biochem. Biophys. Res. Commun. 171, 1152-1159), we found a synthetic peptide, termed peptide A, that inhibited the kinase activity of p60v-src. The peptide A sequence corresponds to residues 137 to 157 of p60v-src which are included in the amino-terminal portion of the src homology 2 domain. In this study, we attempted to specify the inhibitory sequence in this domain and to identify its target site. The most potent peptide A derivative was one that corresponds to residues 140 through 157. The target site of peptide A was assumed to reside in the autophosphorylation site of p60v-src, since synthetic peptides containing the sequence Phe424-Pro-Ile-Lys-Trp428 which is present downstream of the autophosphorylated Tyr416 partially counteracted the inhibitory effect of peptide A. An antibody was prepared against one of such target peptides, termed pepY. Cross-linking experiments showed that 125I-labeled peptide A could bind to p60v-src blotted on a membrane, and the binding was blocked by the anti-pepY antibody but not by other anti-p60v-src antibodies. Conversely, immunoblotting of p60v-src with anti-pepY antibody was blocked by the cross-linking of peptide A to p60v-src. To our surprise, anti-pepY antibody did not affect the p60v-src activity. Furthermore, p60c-src was activated 2- to 6-fold by this antibody. These results suggest that the pepY region in the catalytic domain of p60v-src or of p60c-src is not essential for the catalytic activity but rather is involved in the negative regulation of the kinase activity of p60c-src.

Structure and function of phosphatidylinositol 3-kinase: a potential second messenger system involved in growth control.

Fry MJ, Waterfield MD
Philos Trans R Soc Lond B Biol Sci. 1993; 340: 337-44

Ligand stimulation of growth factor receptors with intrinsic protein-tyrosine kinase activity initiates the assembly of multienzyme signalling complexes. This is mediated by binding of proteins with src homology 2 (SH2) domains to receptor autophosphorylation sites. Among the proteins involved in complex formation is phosphatidylinositol (PI) 3-kinase, a heterodimeric enzyme composed of 85 kDa and 110 kDa subunits, which binds to receptor (and non-receptor) phosphotyrosine residues through the two SH2 domains in the p85 subunit. p85 acts as an adaptor protein and possibly a regulator of the p110 catalytic subunit that phosphorylates phosphoinositides at the D-3 position of the inositol ring. p85 subunit is composed of several distinct functional domains: one SH3 and two SH2 domains, a p110 binding site and a region with homology to BCR. Expression of these domains in E. coli as GST-fusion proteins has allowed definition by nuclear magnetic resonance (NMR) of three-dimensional structures for the SH2 and SH3 domains. The relationship of structure to function for these domains is discussed. The p110 catalytic domain has a region of homology with vps34p of Saccharomyces cerevisiae, a protein involved in protein sorting to the yeast vacuole. Possible clues to the function of PI 3-kinase derived from this and other observations are presented.

A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

Freeman NL, Lila T, Mintzer KA, Chen Z, Pahk AJ, Ren R, Drubin DG, Field J
Mol Cell Biol. 1996; 16: 548-56

Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.

HIV-1 Nef protein: purification, crystallizations, and preliminary X-ray diffraction studies.

Franken P, Arold S, Padilla A, Bodeus M, Hoh F, Strub MP, Boyer M, Jullien M, Benarous R, Dumas C
Protein Sci. 1997; 6: 2681-3

Human immunodeficiency virus Nef protein accelerates virulent progression of AIDS by its interaction with specific cellular proteins involved in cellular activation and signal transduction. Here we report the purification and crystallization of the conserved core of HIV-1LAI Nef protein in the unliganded form and in complex with the wild-type SH3 domain of the P59fyn protein-tyrosine kinase. One-dimensional NMR experiments show that full-length protein and truncated fragment corresponding to the product of HIV-1 protease cleavage have a well-folded compact tertiary structure. The ligand-free HIV-1 Nefcore protein forms cubic crystals belonging to space group P23 with unit cell dimensions of a = b = c = 86.4 A. The Nef-Fyn SH3 cocrystals belong to the space group P6(1)22 or its enantiomorph, P6(5)22, with unit cell dimensions of a = b = 108.2 A and c = 223.7 A. Both crystal forms diffract to a resolution limit of 3.0 A resolution using synchrotron radiation, and are thus suitable for X-ray structure determination.

The HIV Nef protein alters Ca(2+) signaling in myelomonocytic cells through SH3-mediated protein-protein interactions.

Foti M, Cartier L, Piguet V, Lew DP, Carpentier JL, Trono D, Krause KH
J Biol Chem. 1999; 274: 34765-72

Human immunodeficiency virus Nef plays an important role in AIDS pathogenesis. In addition to the well known down-regulation of cell surface receptors (CD4, MHCI), Nef is able to alter cellular signaling. Of particular interest for this study is the ability of Nef to bind with a very high affinity to SH3 domains of myelomonocyte-specific protein-tyrosine kinases of the Src family (Src-like PTK). We have therefore investigated Ca(2+) signaling in HL60 cells retrovirally transduced with wild type Nef or with a Nef mutant deficient in the SH3-interacting proline-rich motif (Nef((PXXP)4(-))). In differentiated HL60 cells, Nef markedly altered cellular Ca(2+) signaling; the amount of intracellularly stored Ca(2+) was increased, and as a consequence, store-operated Ca(2+)-influx was decreased. This effect was not observed in undifferentiated HL60 cells or in CEM T-lymphocytes and correlated with the differentiation-induced up-regulation of Src-like PTK. The Nef effect on Ca(2+) signaling depended entirely on the integrity of its PXXP motif. The Src-like PTK p56/59(hck) co-immunoprecipitated with both Nef and with the inositol 1,4,5-trisphosphate receptor, providing a possible mechanistic link between the viral protein and intracellular Ca(2+) stores of the host cell. Collectively, our results demonstrate that the human immunodeficiency virus 1 Nef protein manipulates intracellular Ca(2+) stores through SH3-mediated interactions in myelomonocytic cells.

Identification of regions of the Wiskott-Aldrich syndrome protein responsible for association with selected Src homology 3 domains.

Finan PM, Soames CJ, Wilson L, Nelson DL, Stewart DM, Truong O, Hsuan JJ, Kellie S
J Biol Chem. 1996; 271: 26291-5

Src homology 3 (SH3) domains have been shown to mediate selected interactions between signaling molecules and are essential for the activation of a number of receptor-driven pathways. The Wiskott-Aldrich syndrome protein was identified as a protein that associated selectively with the SH3 domains derived from c-Src, p85alpha, phospholipase Cgamma1, and c-Fgr. Significantly reduced association was detected to the N-terminal SH3 domain and the tandem SH3 domains of p47(phox), and no binding was detected to the SH3 domain of n-Src, the C-terminal SH3 domain of p47(phox), or either of the SH3 domains of p67(phox). Three peptides corresponding to potential Wiskott-Aldrich syndrome protein SH3 domain binding motifs were found to inhibit its association with c-Src, Fgr, and phospholipase Cgamma1 SH3 domains, but not the p85alpha SH3 domain. These peptides have the sequences MRRQEPLPPPPPPSRG, TGRSGPLPPPPPGA, and KGRSGPLPPVPLGI and show homology with other SH3 domain binding motifs. It is possible that the intracellular association of Wiskott-Aldrich syndrome protein with other signaling proteins is mediated by its SH3 domain-binding regions, and this may play a role in its putative function as a regulatory molecule in immune cells.

A thermodynamic analysis of a family of small globular proteins: SH3 domains.

Filimonov VV, Azuaga AI, Viguera AR, Serrano L, Mateo PL
Biophys Chem. 1999; 77: 195-208

The stability and folding thermodynamics of two SH3-domains, belonging to Fyn and Abl proteins, have been studied by scanning calorimetry and urea-induced unfolding. They undergo an essentially two-state unfolding with parameters similar to those of the previously studied alpha-spectrin SH3 domain. The correlations between the thermodynamic parameters (heat capacity increment, delta Cp,U, the proportionality factor, m, and the Gibbs energy, delta Gw298) of unfolding and some integral structural parameters, such as polar and non-polar areas exposed upon domain denaturation, have been analyzed. The experimental data on delta Cp,U and the m-factor of the linear extrapolation model (LEM) obey the simple empirical correlations deduced elsewhere. The Gibbs energies calculated from the DSC data were compared with those found by fitting urea-unfolding curves to the LEM and the denaturant-binding model (DBM). The delta Gw298 values found with DBM correlate better with the DSC data, while those obtained with LEM are systematically smaller. The systematic difference between the parameters calculated with LEM and DBM are explained by an inherent imperfection of the LEM.

Molecular basis for the binding of SH3 ligands with non-peptide elements identified by combinatorial synthesis.

Feng S, Kapoor TM, Shirai F, Combs AP, Schreiber SL
Chem Biol. 1996; 3: 661-70

BACKGROUND: Protein-structure-based combinatorial chemistry has recently been used to discover several ligands containing non-peptide binding elements to the Src SH3 domain. The encoded library used has the form Cap-M1-M2-M3-PLPPLP, in which the Cap and Mi's are composed of a diverse set of organic monomers. The PLPPLP portion provided a structural bias directing the non-peptide fragment Cap-M1-M2-M3 to the SH3 specificity pocket. Fifteen ligands were selected from > 1.1 million distinct compounds. The structural basis for selection was unknown. RESULTS: The solution structures of the Src SH3 domain complexed with two ligands containing non-peptide elements selected from the library were determined by multidimensional NMR spectroscopy. The non-peptide moieties of the ligands interact with the specificity pocket of Src SH3 domain differently from peptides complexed with SH3 domains. Structural information about the ligands was used to design various homologs, whose affinities for the SH3 domain were measured. The results provide a structural basis for understanding the selection of a few optimal ligands from a large library. CONCLUSIONS: The cycle of protein-structure-based combinatorial chemistry followed by structure determination of the few highest affinity ligands provides a powerful new tool for the field of molecular recognition.

SH2 and SH3 domains as molecular adhesives: the interactions of Crk and Abl.

Feller SM, Ren R, Hanafusa H, Baltimore D
Trends Biochem Sci. 1994; 19: 453-8

The Src homology domains SH2 and SH3 are modular components present in many signal transduction proteins. They allow rapid formation of stable protein complexes and may also regulate protein function through intramolecular binding events. SH2 domains recognize phosphotyrosyl residues in a specific sequence context, while SH3 domains recognize a PxxP motif and additional residues that mediate binding specificity.

Crystal structure of the iron-dependent regulator from Mycobacterium tuberculosis at 2.0-A resolution reveals the Src homology domain 3-like fold and metal binding function of the third domain.

Feese MD, Ingason BP, Goranson-Siekierke J, Holmes RK, Hol WG
J Biol Chem. 2001; 276: 5959-66

Iron-dependent regulators are primary transcriptional regulators of virulence factors and iron scavenging systems that are important for infection by several bacterial pathogens. Here we present the 2.0-A crystal structure of the wild type iron-dependent regulator from Mycobacterium tuberculosis in its fully active holorepressor conformation. Clear, unbiased electron density for the Src homology domain 3-like third domain, which is often invisible in structures of iron-dependent regulators, was revealed by density modification and averaging. This domain is one of the rare examples of Src homology domain 3-like folds in bacterial proteins, and, in addition, displays a metal binding function by contributing two ligands, one Glu and one Gln, to the pentacoordinated cobalt atom at metal site 1. Both metal sites are fully occupied, and tightly bound water molecules at metal site 1 ("Water 1") and metal site 2 ("Water 2") are identified unambiguously. The main chain carbonyl of Leu4 makes an indirect interaction with the cobalt atom at metal site 2 via Water 2, and the adjacent residue, Val5, forms a rare gamma turn. Residues 1-3 are well ordered and make numerous interactions. These ordered solvent molecules and the conformation and interactions of the N-terminal pentapeptide thus might be important in metal-dependent activation.

Src structure crystallizes 20 years of oncogene research.

Featherstone C
Science. 1997; 275: 1066-1066

Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation.

Farrow NA, Muhandiram R, Singer AU, Pascal SM, Kay CM, Gish G, Shoelson SE, Pawson T, Forman-Kay JD, Kay LE
Biochemistry. 1994; 33: 5984-6003

The backbone dynamics of the C-terminal SH2 domain of phospholipase C gamma 1 have been investigated. Two forms of the domain were studied, one in complex with a high-affinity binding peptide derived from the platelet-derived growth factor receptor and the other in the absence of this peptide. 2-D 1H-15N NMR methods, employing pulsed field gradients, were used to determine steady-state 1H-15N NOE values and T1 and T2 15N relaxation times. Backbone dynamics were characterized by the overall correlation time (tau m), order parameters (S2), effective correlation times for internal motions (tau e), and, if required, terms to account for motions on a microsecond-to-millisecond-time scale. An extended two-time-scale formalism was used for residues having relaxation data and that could not be fit adequately using a single-time-scale formalism. The overall correlation times of the uncomplexed and complexed forms of SH2 were found to be 9.2 and 6.5 ns, respectively, suggesting that the uncomplexed form is in a monomer-dimer equilibrium. This was subsequently confirmed by hydrodynamic measurements. Analysis of order parameters reveals that residues in the so-called phosphotyrosine-binding loop exhibited higher than average disorder in both forms of SH2. Although localized differences in order parameters were observed between the uncomplexed and complexed forms of SH2, overall, higher order parameters were not found in the peptide-bound form, indicating that on average, picosecond-time-scale disorder is not reduced upon binding peptide. The relaxation data of the SH2-phosphopeptide complex were fit with fewer exchange terms than the uncomplexed form. This may reflect the monomer-dimer equilibrium that exists in the uncomplexed form or may indicate that the complexed form has lower conformational flexibility on a microsecond-to-millisecond-time scale.

Association of the protein tyrosine phosphatase PTP1C with the protein tyrosine kinase c-Src in human platelets.

Falet H, Ramos-Morales F, Bachelot C, Fischer S, Rendu F
FEBS Lett. 1996; 383: 165-9

Protein tyrosine phosphatase 1C (PTP1C), highly expressed in hematopoietic cells, is a soluble protein tyrosine phosphatase containing two Src homology 2 (SH2) domains at the N-terminus and two putative sites of tyrosine phosphorylation at the C-terminus. This paper reports that PTP1C and c-Src could be coimmunoprecipitated during thrombin-induced platelet activation. Moreover, association between the two signalling proteins occurred only after PTP1C had been tyrosine phosphorylated. In in vitro experiments, PTP1C bound to the SH2 domain of c-Src, suggesting that association between tyrosine phosphorylated PTP1C and c-Src was mediated by the SH2 domain of c-Src. Finally, in resting platelets, PTP1C was mainly found in the Nonidet P-40 soluble fraction whereas following thrombin-induced activation, around 17% of PTP1C was associated with the insoluble fraction.

Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions.

Erpel T, Superti-Furga G, Courtneidge SA
EMBO J. 1995; 14: 963-75

The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its C-terminal tail. The repressed state is achieved through intramolecular interactions involving the phosphorylated tail, the Src homology 2 (SH2) domain and the SH3 domain. Both the SH2 and SH3 domains have also been shown to mediate the intermolecular interaction of Src with several proteins. To test which amino acids of the Src SH3 domain are important for these interactions, and whether the intra- and intermolecular associations involve the same residues, we carried out a detailed mutational analysis of the presumptive interaction surface. All mutations of conserved hydrophobic residues had an effect on both inter- and intramolecular interactions of the Src SH3 domain, although not all amino acids were equally important. Chimeric molecules in which the Src SH3 domain was replaced with those of spectrin or Lck showed derepressed kinase activity, whereas a chimera containing the Fyn SH3 domain was fully regulated. Since spectrin and Lck SH3 domains share the conserved hydrophobic residues characteristic of SH3 domains, other amino acids must be important for specificity. Mutational analysis of non- or semi-conserved residues in the RT and n-Src loops showed that some of these were also involved in inter- and intramolecular interactions. Stable transfection of selected SH3 domain mutants into NIH-3T3 cells showed that despite elevated levels of phosphotyrosine, the cells were morphologically normal, indicating that the SH3 domain was required for efficient transformation of NIH-3T3 cells by Src.

Comparison of SH3 and SH2 domain dynamics when expressed alone or in an SH(3+2) construct: the role of protein dynamics in functional regulation.

Engen JR, Smithgall TE, Gmeiner WH, Smith DL
J Mol Biol. 1999; 287: 645-56

Protein dynamics play an important role in protein function and regulation of enzymatic activity. To determine how additional interactions with surrounding structure affects local protein dynamics, we have used hydrogen exchange and mass spectrometry to investigate the SH2 and SH3 domains of the protein tyrosine kinase Hck. Exchange rates of isolated Hck SH3 and SH2 domains were compared with rates for the same domains when part of a larger SH(3+2) construct. Increased deuterium incorporation was observed for the SH3 domain in the joint construct, particularly near the SH2 interface and the short sequence that connects SH3 to SH2, implying greater flexibility of SH3 when it is part of SH(3+2). Slow cooperative unfolding of the SH3 domain occurred at the same rate in isolated SH3 as in the SH(3+2) construct, suggesting a functional significance for this unfolding. The SH2 domain displayed relatively smaller changes in flexibility when part of the SH(3+2) construct. These results suggest that the domains influence each other. Further, our results imply a link between functional regulation and structural dynamics of SH3 and SH2 domains.

Identification and localization of slow, natural, cooperative unfolding in the hematopoietic cell kinase SH3 domain by amide hydrogen exchange and mass spectrometry.

Engen JR, Smithgall TE, Gmeiner WH, Smith DL
Biochemistry. 1997; 36: 14384-91

Protein unfolding on a fast time scale (milliseconds-minutes) has been widely reported, but slower unfolding events (10 min-hours) have received less attention. Amide hydrogen exchange (HX) and mass spectrometry (MS) were used to investigate the unfolding dynamics of the hematopoietic cell kinase (Hck) SH3 domain. Analysis of mass spectra after deuterium exchange into intact Hck SH3 indicates a cooperative unfolding event involving 24-61% of the domain and occurring with a half-life of approximately 20 min under physiological conditions. To identify the unfolding region, SH3 was incubated in D2O and proteolytically fragmented into peptides that were analyzed by mass spectrometry. Correlation of HX rates and isotope patterns reveals cooperative unfolding in several regions, including the C-terminal half of the RT-loop and a beta-sheet flanking the binding site. Binding of a prolyl-rich segment from the HIV Nef protein slows unfolding by a factor of 3. Further analysis yields a KD of 25 microM for the Nef peptide. These results demonstrate that an inherent flexibility in the SH3 domain may assist interconversion of the closed, intramolecularly ligated state and the open, active state of Src family kinases. Furthermore, this type of previously undetectable, slow unfolding process may provide the basis for new mechanisms in which kinetics of local unfolding combines with thermodynamics to regulate enzymatic activity. The combination of hydrogen exchange and mass spectrometry appears to be the only general method capable of examining these slow unfolding processes.

Investigating protein structure and dynamics by hydrogen exchange MS.

Engen JR, Smith DL
Anal Chem. 2001; 73: 256265-256265

Hydrogen exchange shows peptide binding stabilizes motions in Hck SH2.

Engen JR, Gmeiner WH, Smithgall TE, Smith DL
Biochemistry. 1999; 38: 8926-35

Src-homology-2 domains are small, 100 amino acid protein modules that are present in a number of signal transduction proteins. Previous NMR studies of SH2 domain dynamics indicate that peptide binding decreases protein motions in the pico- to nanosecond, and perhaps slower, time range. We suggest that amide hydrogen exchange and mass spectrometry may be useful for detecting changes in protein dynamics because hydrogen exchange rates are relatively insensitive to the time domains of the dynamics. In the present study, hydrogen exchange and mass spectrometry were used to probe hematopoietic cell kinase SH2 that was either free or bound to a 12-residue high-affinity peptide. Hydrogen exchange rates were determined by exposing free and bound SH2 to D(2)O, fragmenting the SH2 with pepsin, and determining the deuterium level in the peptic fragments. Binding generally decreased hydrogen exchange along much of the SH2 backbone, indicating a widespread reduction in dynamics. Alterations in the exchange of the most rapidly exchanging amide hydrogens, which was detected following acid quench and analysis by mass spectrometry, were used to locate differences in low-amplitude motion when SH2 was bound to the peptide. In addition, the results indicate that hydrogen exchange from the folded form of SH2 is an important process along the entire SH2 backbone.

A biosensor approach to probe the structure and function of the p85 alpha subunit of the phosphatidylinositol 3-kinase complex.

End P, Gout I, Fry MJ, Panayotou G, Dhand R, Yonezawa K, Kasuga M, Waterfield MD
J Biol Chem. 1993; 268: 10066-75

Phosphatidylinositol 3-kinase, which generates putative novel second messenger phospholipids, is a heterodimer composed of regulatory adaptor 85-kDa and catalytic 110-kDa subunits. The p85 alpha subunit contains a NH2-terminal src homology (SH) 3 domain, a region with homology to the product of the breakpoint cluster region (bcr) gene, and a COOH-terminal portion of the molecule which contains two SH2 domains, separated by a spacer region. In this study a panel of monoclonal antibodies (mAb) was raised against recombinant bovine p85 alpha to probe its multidomain structure in relation to function. These mAbs were characterized using a BIAcore biosensor instrument. Epitopes for nine mAbs were mapped in relation to the domain structure of p85 alpha using recombinant protein fragments expressed in bacteria. These mAbs were then used to map the sites on p85 alpha which are involved in growth factor receptor binding. Two interesting classes of functional mAbs were identified. First, mAb U14, whose epitope lies within the NH2-terminal SH2 domain of p85 alpha, blocked the interaction of p85 alpha with activated protein-tyrosine kinase receptors. Second, real-time binding experiments using phospholipid-containing vesicles showed that p85 alpha by itself could specifically bind certain phospholipids. Two mAbs (U9 and U15) with epitopes located in the inter-SH2 spacer region blocked the binding of lipids to this site. The relevance of these observations to understanding the relationship of structure to function of p85 and the phosphatidylinositol 3-kinase are discussed.

Ankyrin(g) ETS domains to DNA.

Ely KR, Kodandapani R
Nat Struct Biol. 1998; 5: 255-9

The DNA-binding domain of HIV-1 integrase has an SH3-like fold.

Eijkelenboom AP, Lutzke RA, Boelens R, Plasterk RH, Kaptein R, Hard K
Nat Struct Biol. 1995; 2: 807-10

We have determined the solution structure of the DNA-binding domain of HIV-1 integrase by nuclear magnetic resonance spectroscopy. In solution, this carboxyterminal region of integrase forms a homodimer, consisting of two structures that closely resemble Src-homology 3 (SH3) domains. Lys 264, previously identified by mutagenesis studies to be important for DNA binding of the integrase, as well as several adjacent basic amino acids are solvent exposed. The identification of an SH3-like domain in integrase provides a new potential target for drug design.

Identification and characterization of a new human gene encoding a small protein with high homology to the proline-rich region of the SH3BGR gene.

Egeo A, Mazzocco M, Arrigo P, Vidal-Taboada JM, Oliva R, Pirola B, Giglio S, Rasore-Quartino A, Scartezzini P
Biochem Biophys Res Commun. 1998; 247: 302-6

As part of an effort to identify genes potentially involved in the Down Syndrome pathogenesis, in this paper we report the identification and characterization of a new human gene (named SH3BGRL), which shows a high homology to the SH3BGR gene, previously mapped to the Down Syndrome region of chromosome 21. The SH3BGRL gene encodes for a small protein of 114 amino acids, sharing 60% identity and 84% conservation on the amino acid level with the middle, proline-rich region of the SH3BGR gene and containing a similar SH3 (Scr homology 3) binding motif. The SH3BGRL and the proline-rich region of SH3BGR proteins appear to be highly conserved, sharing 95 and 98% identity, respectively, with the mouse homologues. A 1.9 kb transcript of the SH3BGRL gene has been found in all the tissues examined, in contrast with the expression pattern of the SH3BGR gene which is transcribed only in heart and skeletal muscle. The SH3BGR gene and its homologue, SH3BGRL, could be members of a new family of genes containing a highly conserved proline-rich functional domain. The SH3BGRL gene has been mapped by fluorescent in situ hybridization to Chromosome Xq13.3.

Spatial constraints on the recognition of phosphoproteins by the tandem SH2 domains of the phosphatase SH-PTP2.

Eck MJ, Pluskey S, Trub T, Harrison SC, Shoelson SE
Nature. 1996; 379: 277-80

The domain organization of many signalling proteins facilitates a segregation of binding, catalytic and regulatory functions. The mammalian SH2 domain protein tyrosine phosphatases (PTPs) contain tandem SH2 domains and a single carboxy-terminal catalytic domain. SH-PTP1 (PTP1C, HCP) and SH-PTP2 (Syp, PTP2C, PTP1D) function downstream from tyrosine kinase-linked insulin, growth factor, cytokine and antigen receptors. As well as directing subcellular localization by binding to receptors and their substrates, the two SH2 domains of these PTPs function together to regulate catalysis. Here we report the structure of the tandem SH2 domains of SH-PTP2 in complex with monophosphopeptides. A fixed relative orientation of the two domains, stabilized by a disulphide bond and a small hydrophobic patch within the interface, separates the peptide binding sites by approximately 40 A. The defined orientation of the SH2 domains in the structure, and data showing that peptide orientation and spacing between binding sites is critical for enzymatic activation, suggest that spatial constraints are important in this multidomain protein-protein interaction.

A novel transmembrane semaphorin can bind c-src.

Eckhardt F, Behar O, Calautti E, Yonezawa K, Nishimoto I, Fishman MC
Mol Cell Neurosci. 1997; 9: 409-19

The semaphorins/collapsins constitute a family of genes unified by the presence of a "semaphorin domain" which has been conserved through metazoan evolution. The semaphorin family comprises both secreted and transmembrane molecules and is thought to be made up of ligands for as yet unidentified receptors. The functions are not known, with the exception of those of sema III (also referred as sem D and collapsin 1), D-sema I, and D-sema II, which have been shown to be involved in axonal pathfinding. Here report the identification of a mouse semaphorin cDNA, termed Sema VIb. Although Sema VIb contains the extracellular semaphorin domain, it lacks the immunoglobulin domain or thrombospondin repeats which are present in other described vertebrate (but not invertebrate) transmembrane semaphorins. During development Sema VIb mRNA is expressed in subregions of the nervous system and is particularly prominent in muscle. In adulthood, Sema VIb mRNA is expressed ubiquitously. The cytoplasmic domain of Sema VIb contains several proline-rich potential SH3 domain binding sites. Using an in vitro binding assay, we show that Sema VIb binds specifically the SH3 domain of the protooncogene c-src. In transfected COS cells Sema VIb coimmunoprecipitates with c-src. These results, along with our evidence that Sema VIb can form dimers, suggests that the semaphorin family not only serves as ligands but may include members, especially those which are transmembrane, which serve as receptors, triggering intracellular signaling via an src-related cascade.

The human immunodeficiency virus type 1 Nef protein binds the Src-related tyrosine kinase Lck SH2 domain through a novel phosphotyrosine independent mechanism.

Dutartre H, Harris M, Olive D, Collette Y
Virology. 1998; 247: 200-11

Primate lentiviruses encode for an unique nef gene with an essential function in both viral replication and pathogenicity in the host. The molecular basis for this function remains however poorly defined. Several Nef-binding cellular proteins are thought to be instrumental in its function. Indeed, Nef contains a proline-rich motif implicated in the binding to the Src-like tyrosine kinase Hck and also to a Ser/Thr kinase of molecular weight 62 kDa. The disruption of this motif affects the binding to both these kinases as well as viral replication. Whereas Hck is expressed in the myeloid lineage and hence may account for the nef function in infected monocytes, we and others have reported previously that Nef also interacts with the T-lymphocyte Src-kinase Lck, leading to specific cell signaling impairment. This interaction occurs through the binding of Nef to both Lck SH2 and SH3 domains. Both the proline motif and phosphorylation of Nef on tyrosine residue were proposed to account for these interactions. Here, we investigate the mechanism of Lck SH2 binding by HIV-1 Nef. Using recombinant fusion proteins to precipitate lysates, we show that although SH2 binding is dependent on phosphorylation events, it occurs in a tyrosine independent manner because it requires neither tyrosine residues in Nef nor the phosphotyrosine binding pocket from the Lck SH2 domain, hence suggesting a role for a phosphoserine or a phosphothreonine residue. Further, we show that Hck SH2 does not interact with Nef, indicating that Hck SH3 binding is sufficient for Nef binding, whereas Lck SH2 cooperate together with SH3 to allow Nef binding to a level similar to Hck SH3. Together, our results establish different mechanisms for Hck and Lck binding by HIV-1 Nef protein, and identify a novel mechanism for Src-like tyrosine kinase targeting by a viral protein.

The murine AIDS virus Gag precursor protein binds to the SH3 domain of c-Abl.

Dupraz P, Rebai N, Klein SJ, Beaulieu N, Jolicoeur P
J Virol. 1997; 71: 2615-20

The Pr60gag protein of the murine AIDS (MAIDS) defective virus promotes the proliferation of the infected target B cells and is responsible for inducing a severe immunodeficiency disease. Using the yeast two-hybrid system, we identified the SH3 domain of c-Abl as interacting with the proline-rich p12 domain of Pr60gag. The two proteins were shown to associate in vitro and in vivo in MAIDS virus-infected B cells. Overexpression of Pr60(gag) in these cells led to a detectable increase of the levels of c-Abl protein and to its translocation at the membrane. These results suggest that this viral protein serves as a docking site for signaling molecules and that c-Abl may be involved in the proliferation of infected B cells.

Cloning, chromosomal localization, and characterization of cDNA from a novel gene, SH3BP4, expressed by human corneal fibroblasts.

Dunlevy JR, Berryhill BL, Vergnes JP, SundarRaj N, Hassell JR
Genomics. 1999; 62: 519-24

The cornea contains, as a major element, a transparent stroma produced and maintained by keratocytes (fibroblasts). Through molecular biology studies using cultured human corneal fibroblasts, a cDNA that was shown to be novel was isolated and sequenced. This novel gene product, named SH3-domain binding protein 4 (SH3BP4), contains a 5.6-kb message that is present in normal human corneal fibroblasts and all tissues examined, with higher levels in pancreas, placenta, heart, and kidney. SH3BP4 was localized by FISH analysis to human chromosome 2q37.1-q37.2 near the telomere. The deduced sequence for SH3BP4 was found to contain a 963-amino-acid open reading frame that has homology to a 479-amino-acid protein in GenBank called EH-binding protein. Although the entire sequence of EH-binding protein aligns with SH3BP4, the alignment is not complete or contiguous. Therefore, SH3BP4 has an additional 73 amino acids at the N-terminus and an additional 411 amino acids near the C-terminus that are not present in EH-binding protein. Consensus sequence domains identified in SH3BP4 include a SH3 domain, three N-P-F motifs, a P-X-X-P motif noted for binding to SH3 domains, a bipartite nuclear targeting signal, and a tyrosine phosphorylation site. SH3BP4 homologies and consensus sequence sites indicate that it may be involved in a newly identified cascade of proteins involved in endocytosis, intracellular sorting, and the cell cycle.

Polyomavirus middle-T antigen associates with the kinase domain of Src-related tyrosine kinases.

Dunant NM, Senften M, Ballmer-Hofer K
J Virol. 1996; 70: 1323-30

Middle-T antigen of mouse polyomavirus, an oncogenic DNA virus, associates with and activates the cellular tyrosine kinases c-Src, c-Yes, and Fyn. This interaction is essential for polyomavirus-mediated transformation of cells in culture and tumor formation in animals. To determine the domain of c-Src directing association with middle-T, mutant c-Src proteins lacking the amino-terminal unique domain and the myristylation signal, the SH2 domain, the SH3 domain, or all three of these domains were coexpressed with middle-T in NIH 3T3 cells. All mutants were found to associate with middle-T, demonstrating that the kinase domain of c-Src, including the carboxy-terminal regulatory tail, is sufficient for association with middle-T. Moreover, we found that Hck, another member of the Src kinase family, does not bind middle-T, while chimeric kinases consisting of the amino-terminal domains of c-Src fused to the kinase domain of Hck or the amino-terminal domains of Hck fused to the kinase domain of c-Src associated with middle-T. Hck mutated at its carboxy-terminal regulatory residue, tyrosine 501, was also found to associate with middle-T. These results suggest that in Hck, the postulated intramolecular interaction between the carboxy-terminal regulatory tyrosine and the SH2 domain prevents association with middle-T. This intramolecular interaction apparently also limits the ability of c-Src to associate with middle-T, since removal of the SH2 or SH3 domain increases the efficiency with which middle-T binds c-Src.

The glycine-rich loop of adenylate kinase forms a giant anion hole.

Dreusicke D, Schulz GE
FEBS Lett. 1986; 208: 301-4

The conformation of the glycine-rich loop of adenylate kinase is described in detail. It forms a giant anion hole for a sulfate ion, which presumably mimicks a nucleotide phosphoryl group. This loop had been called flexible, because at pH values of 6 or below it is displaced in the crystal. In the region of this loop the adenylate kinases are probably homologous to the p21 proteins. Is is known that a mutation in this loop at residue 12 of p21 causes cell transformation and therefore cancer. Other potentially homologous proteins are indicated.

Identification of tricyclic analogs related to ellagic acid as potent/selective tyrosine protein kinase inhibitors.

Dow RL, Chou TT, Bechle BM, Goddard C, Larson ER
J Med Chem. 1994; 37: 2224-31

The plant-derived natural product ellagic acid (1) has recently been identified as a potent, though nonselective, inhibitor of the tyrosine-specific protein kinase pp60src. This report details efforts directed toward the identification of tricyclic structures related to ellagic acid, with enhanced specificity for inhibition of pp60src over other protein kinases. Phenanthridinone and carbazole core structures were selected for investigation, since N-functionalization allows for the synthesis of numerous analogs which can be utilized to probe enzyme-inhibitor interactions. These ring systems were prepared via a general sequence of biaryl bond formation followed by cyclization to form the desired tricyclic ring systems. N-Alkylation, -acylation, or -sulfonylation and deprotection with boron tribromide afford the target tetraphenolic phenanthridinones 5 and carbazoles 9. Several analogs from both of these series have potencies comparable to that of 1 and exhibit substantially enhanced selectivities for inhibition of pp60src relative to protein kinase A (PKA), a serine/threonine protein kinase. Carbazole-based analogs 9j,m,p are submicromolar inhibitors of pp60src, with potency for the target tyrosine kinase comparable to that of ellagic acid (1), however with 2 orders of magnitude greater selectivity versus that for PKA. As seen for ellagic acid, members of the phenanthridinone-based series (e.g., 5a) exhibited inhibition of pp60src in a manner which is partial mixed noncompetitive with respect to ATP, while analogs in the carbazole series (e.g., 9a) inhibit pp60src in an ATP competitive manner.

The GRB2/Sem-5 adaptor protein.

Downward J
FEBS Lett. 1994; 338: 113-7

GRB2/Sem-5 is a 25-kDa adaptor protein which contains a central Src homology type 2 (SH2) domain flanked by two Src homology type 3 (SH3) domains. GRB2/Sem-5 was first identified due to the essential role of the sem-5 gene product in the vulval induction pathway in Caenorhabditis elegans. The SH2 domain of GRB2/Sem-5 binds to a number of tyrosine phosphorylated proteins, most notably the epidermal growth factor receptor, the insulin receptor substrate IRS-1 and another putative adaptor protein, Shc. The SH3 domains bind to Sos, a guanine nucleotide exchange factor for Ras proteins. GRB2/Sem-5 brings together Sos and tyrosine phosphoproteins into a complex and thereby may regulate the nucleotide exchange rate of Ras and hence its activation state.

Complete nucleotide sequence, expression, and chromosomal localisation of human mixed-lineage kinase 2.

Dorow DS, Devereux L, Tu GF, Price G, Nicholl JK, Sutherland GR, Simpson RJ
Eur J Biochem. 1995; 234: 492-500

Protein kinases play pivotal roles in the control of many cellular processes. In a search for protein kinases expressed in human epithelial tumour cells, we discovered two members of a novel protein kinase family [Dorow, D. S., Devereux, L., Dietzsch, E. & de Kretser, T. A. (1993) Eur. J. Biochem. 213, 701-710]. Due to the unique mixture of structural domains within their amino acid sequences, we named the family mixed-lineage kinases (MLK). We initially isolated clones encoding partial cDNAs of MLK1 and 2 from a human colonic cDNA library. The MLK2 cDNA was subsequently used to screen a human brain cDNA library and we have now cloned and sequenced a 3454-bp cDNA encoding the full-length MLK2 protein. The predicted MLK2 polypeptide has 954 amino acids and contains a src homology 3 (SH3) domain, a kinase catalytic domain, a double leucine zipper and basic domain, and a large C-terminal domain. The 22-amino-acid N-terminal region has four glutamic acid residues immediately following the initiator methionine. Beginning at amino acid 23, the 55-amino-acid SH3 domain contains a 5-amino-acid insert in a position corresponding to inserts of 6 and 15 residues in the SH3 domains of n-src and the phosphatidylinositol 3'-kinase. Adjacent to the SH3 domain is a kinase catalytic domain with conserved motifs associated with both serine/threonine and tyrosine specificity. Beginning nine residues C-terminal to the catalytic domain, there are two leucine/isoleucine zippers separated by a 13-amino-acid spacer sequence and followed by a stretch of basic residues. The polybasic sequence contains a motif that is similar to nuclear localisation signals from several proteins. The C-terminal domain is composed of 491 amino acids of which 17% are serine or threonine and 16% are proline. This domain also has a biased ratio of basic to acidic amino acids with a calculated pI of 9.38. When used as a probe to examine mRNA expression in human tissues, a MLK2 cDNA hybridised to a species of 3.8 kb that was expressed at highest levels in RNA from brain and skeletal muscle. The 3454-bp cDNA was also used for fluorescence in situ hybridisation to localise the MLK2 gene to human chromosome 19 q13.2.

Inhibition of SH2 domain/phosphoprotein association by a nonhydrolyzable phosphonopeptide.

Domchek SM, Auger KR, Chatterjee S, Burke TR Jr, Shoelson SE
Biochemistry. 1992; 31: 9865-70

Using the association between the pp60c-src/polyoma virus middle T antigen (mT) complex and phosphatidylinositol 3'-kinase (PI 3-kinase) as a prototype for phosphoprotein-SH2 domain interactions, we tested whether a nonhydrolyzable phosphonopeptide would inhibit association. (Phosphonomethyl)-phenylalanine (Pmp) is a nonnatural analogue of phosphotyrosine in which the > C-O-PO3H2 moiety is replaced by > C-CH2-PO3H2. We synthesized a 13 amino acid phosphonopeptide (mT-Pmp315), a related phosphopeptide (mT-pY315), and an unmodified sequence (mT-Y315), all corresponding to the pp60c-src-phosphorylated site of the mT which is within a YMXM motif common to proteins that bind to and activate PI 3-kinase. Only the phosphonopeptide persistently blocked the in vitro association of the baculovirus-expressed pp60c-src/mT complex with cytosolic PI 3-kinase activity. Sustained inhibition of association by the phosphopeptide required the additional presence of vanadate, a potent protein tyrosine phosphatase (PTPase) inhibitor. The phosphopeptide and L-phosphonopeptide bound tightly (KD approximately 10-20 nM) and specifically to isolated SH2 domains of PI 3-kinase p85, demonstrating that the mechanism of inhibited association is competitive binding to PI 3-kinase SH2 domains. We conclude that the appropriate phosphonopeptide sequence inhibits the interaction between a tyrosine-phosphorylated protein and a cognate SH2 domain-containing protein and is resistant to the actions of PTPases. Proteolytically stable phosphonopeptide derivatives should be useful inhibitors of protein-protein interactions when introduced into cells and may provide a basis for the rational design of a new class of chemotherapeutic agent.

Human genetics. Becoming B cells.

Desiderio S
Nature. 1993; 361: 202-3

Tight association of GRB2 with receptor protein-tyrosine phosphatase alpha is mediated by the SH2 and C-terminal SH3 domains.

den Hertog J, Hunter T
EMBO J. 1996; 15: 3016-27

Receptor protein-tyrosine phosphatase alpha (RPTPalpha), a transmembrane member of the extensive family of protein-tyrosine phosphatases (PTPs), is constitutively phosphorylated on Tyr789, a consensus binding site for the SH2 domain of the SH3-SH2-SH3 adaptor protein GRB2. We have previously shown that GRB2 binds to P.Tyr789 in vivo and in vitro via its SH2 domain. Here, we report that not only the GRB2 SH2 domain, but also the C-terminal SH3 domain is involved in binding to RPTPalpha in vitro and in vivo. Although the N-terminal SH3 domain of GRB2 is essential for binding to the Ras guanine nucleotide exchange factor Son of Sevenless (Sos), an RPTPalpha-GRB2-Sos complex could not be detected. The inclusion of peptides encompassing an hSos1 proline-rich motif in cell lysates resulted in enhanced binding of RPTPalpha to GRB2 in vitro, suggesting that steric hindrance prohibits formation of the RPTPalpha-GRB2-Sos complex. In vitro binding experiments indicated that the binding of GRB2 to Sos/dynamin and RPTPalpha was mutually exclusive. Analysis of in vitro binding kinetics coupled with results from transient co-transfections demonstrated that RPTPalpha is tightly bound to GRB2. The site of interaction of the C-terminal SH3 domain of GRB2 with RPTPalpha was mapped using deletion mutants to an 18-residue region in the N-terminal PTP domain. Arg469, within this region, was identified as one of the residues that is involved in the interaction with the C-terminal SH3 domain of GRB2. RPTPalpha residues 469-486 are localized close to the catalytic site cleft in the structure of the N-terminal PTP-domain, suggesting that interaction with the C-terminal SH3 domain may block access to the catalytic site, thus inhibiting RPTPalpha activity.

The Grb7 family of signalling proteins.

Daly RJ
Cell Signal. 1998; 10: 613-8

The Grb7 family is a rapidly emerging group of Src homology (SH)2 domain-containing signalling proteins that currently contains three members, Grb7, 10 and 14. These proteins possess a conserved multidomain structure, including a central region exhibiting significant homology to the Caenorhabditis elegans protein Mig10. Differences in tissue expression and SH2 binding selectivity suggest that these adaptor proteins function in a tissue-specific manner to link specific receptor tyrosine kinases (RTKs) and other tyrosine phosphorylated proteins to as yet uncharacterised downstream effectors. Interestingly, Grb7 proteins exhibit differential expression amongst a variety of human cancers and cancer cell lines. Consequently, these proteins not only are likely to perform a fundamental signalling role, but may also modulate RTK signalling in cancer cells.

SH3 domains and drug design: ligands, structure, and biological function.

Dalgarno DC, Botfield MC, Rickles RJ
Biopolymers. 1997; 43: 383-400

The ligand binding preferences, structural features, and biological function of SH3 (Src homology 3) domains are discussed. SH3 domains bind "core" Pro-rich peptide ligands (7-9 amino acids in length) in a polyproline II helical conformation in a highly conserved aromatic rich patch on the protein surface (approximately 390 A2). The ligands can interact with the protein in one of two orientations, depending on the position (N- vs C-terminal) of ligand residues binding to the SH3 selectivity pocket. Core SH3 ligands are characterized by relatively weak interactions (KD = 5-100 microM) that show little binding selectivity within SH3 families. Higher affinity, more selective contiguous ligands require additional flanking residues that bind to less conserved portions of the SH3 surface, with corresponding increase in ligand size and complexity. In contrast to peptide ligands, protein ligands of SH3 domains can exploit multiple discontiguous interactions to enhance affinity and selectivity. A protein-SH3 interaction that utilizes unique interactions may permit the design of small high affinity SH3 ligands. At present, the extended nature of the binding site and homologous nature of the core binding region among SH3 domains present key challenges for structure-based drug design.

Abi-2, a novel SH3-containing protein interacts with the c-Abl tyrosine kinase and modulates c-Abl transforming activity.

Dai Z, Pendergast AM
Genes Dev. 1995; 9: 2569-82

A protein has been identified that interacts specifically with both the Src homologous 3 (SH3) domain and carboxy-terminal sequences of the c-Abl tyrosine kinase. The cDNA encoding the Abl interactor protein (Abi-2), was isolated from a human lymphocyte library using the yeast two-hybrid system with the Abl SH3 domain as bait. Abi-2 binds to c-Abl in vitro and in vivo. Abi-2 is a novel protein that contains an SH3 domain and proline-rich sequences critical for binding to c-Abl. A basic region in the amino terminus of Abi-2 is homologous to the DNA-binding sequence of homeo-domain proteins. We show that Abi-2 is a substrate for the c-Abl tyrosine kinase. Expression of an Abi-2 mutant protein that lacks sequences required for binding to the Abl SH3 domain but retains binding to the Abl carboxyl terminus activates the transforming capacity of c-Abl. The properties of Abi-2 are consistent with a dual role as regulator and potential effector of the c-Abl protein and suggest that Abi-2 may function as a tumor suppressor in mammalian cells.

A Sos-derived peptidimer blocks the Ras signaling pathway by binding both Grb2 SH3 domains and displays antiproliferative activity.

Cussac D, Vidal M, Leprince C, Liu WQ, Cornille F, Tiraboschi G, Roques BP, Garbay C
FASEB J. 1999; 13: 31-8

With the aim of interrupting the growth factor-stimulated Ras signaling pathway at the level of the Grb2-Sos interaction, a peptidimer, made of two identical proline-rich sequences from Sos linked by a lysine spacer, was designed using structural data from Grb2 and a proline-rich peptide complexed with its SH3 domains. The peptidimer affinity for Grb2 is 40 nM whereas that of the monomer is 16 microM, supporting the dual recognition of both Grb2 SH3 domains by the dimer. At 50 nM, the peptidimer blocks selectively Grb2-Sos complexation in ER 22 (CCL 39 fibroblasts overexpressing epidermal growth factor receptor) cellular extracts. The peptidimer specifically recognizes Grb2 and does not interact with PI3K or Nck, two SH3 domain-containing adaptors. The peptidimer was modified to enter cells by coupling to a fragment of Antennapedia homeodomain. At 10 microM, the conjugate inhibits the Grb2-Sos interaction (100%) and MAP kinase (ERK1 and ERK2) phosphorylation (60%) without modifying cellular growth of ER 22 cells. At the same concentration, the conjugate also inhibits both MAP kinase activation induced by nerve growth factor or epidermal growth factor in PC12 cells, and differentiation triggered by nerve growth factor. Finally, when tested for its antiproliferative activity, the conjugate was an efficient inhibitor of the colony formation of transformed NIH3T3/HER2 cells grown in soft agar, with an IC50 of around 1 microM. Thus, the designed peptidimers appear to be interesting leads to investigate signaling and intracellular processes and for designing selective inhibitors of tumorigenic Ras-dependent processes.

Analysis of the SH3-binding region of HIV-1 nef: partial functional defects introduced by mutations in the polyproline helix and the hydrophobic pocket.

Craig HM, Pandori MW, Riggs NL, Richman DD, Guatelli JC
Virology. 1999; 262: 55-63

An SH3-binding domain within the Nef protein of primate lentiviruses has been reported to be important to viral replication and infectivity and dispensable for CD4 downregulation, but its precise role remains unclear. This study investigates the effects of mutations in both the polyproline helix and in the hydrophobic pocket that constitute the SH3-binding domain of Nef. The data demonstrate that the well-studied mutation of the central prolines is only partially disruptive to viral infectivity and replication. The central prolines also make a subtle contribution to the efficiency of CD4 downregulation, detectable only using low levels of Nef expression. Mutation of a conserved arginine in the polyproline helix abrogated more completely Nef-mediated enhancement of viral infectivity; this mutation also adversely affected CD4 downregulation at low levels of Nef expression. Only the R77A mutation substantially impaired downregulation of class I MHC. However, mutation of the central prolines and of R77 yielded proteins that were expressed less efficiently than wild-type Nef. The R77A mutant was expressed most poorly, compatible with its defective phenotypes in all assays. Mutations of the hydrophobic pocket were minimally detrimental to both the virologic and the receptor modulatory functions of Nef. Taken together, this analysis suggests that mutations in the SH3-binding domain do not abrogate fully any Nef-associated phenotype in the absence of detrimental effects on protein expression. We suggest that mutations in this domain can introduce incomplete effects caused by subtle impairments to protein expression; these effects may appear selective under certain experimental conditions due to different sensitivities of the assays to the level of Nef expression.

A nuclear SH3 domain-binding protein that colocalizes with mRNA splicing factors and intermediate filament-containing perinuclear networks.

Craggs G, Finan PM, Lawson D, Wingfield J, Perera T, Gadher S, Totty NF, Kellie S
J Biol Chem. 2001; 276: 30552-60

A protein (SNP70) has been isolated that binds to the Src homology domain 3 of p47(phox), p85alpha, and c-src. Cloning and sequencing of the polypeptide revealed it to be a 70-kDa protein that has a number of potential domains, including Src homology 3 binding motifs and several nuclear localization signals. Immunofluorescence using anti-peptide antibodies revealed SNP70 to be primarily concentrated in the nucleus but excluded from nucleoli, in interphase cells. However, it was distributed throughout the cytoplasm in dividing cells. Extraction and subfractionation experiments indicated that SNP70 did not bind directly to DNA but did bind to poly(G)-rich oligonucleotides and was resistant to extraction with non-ionic detergents but was solubilized by treatment with RNase, high salt, or ammonium sulfate. Double-immunofluorescence experiments showed that SNP70 co-localized with two pre-mRNA splicing factors SC35 and U2B" within the nucleus. A population of SNP70 was found outside the nucleus, and double-immunofluorescence and immunoelectron microscopy demonstrated that it associated with vimentin-containing intermediate filaments, particularly those surrounding the nucleus. The data suggest that SNP70 associates with nuclear or perinuclear filaments and may play a role in the regulation of pre-mRNA processing.

Enhanced affinities and specificities of consolidated ligands for the Src homology (SH) 3 and SH2 domains of Abelson protein-tyrosine kinase.

Cowburn D, Zheng J, Xu Q, Barany G
J Biol Chem. 1995; 270: 26738-41

The possible interrelationships between multiple domains of proteins involved in intracellular signal transduction are complex and not easily investigated. We have synthesized a series of bivalent consolidated ligands, which interact simultaneously with the SH2 and SH3 domain of Abelson kinase in a SH(32) dual domain construct, a portion of native Abelson kinase. Affinities were measured by quenching of intrinsic tryptophan fluorescence. Consolidated ligands have enhanced affinity and specificity compared to monovalent equivalents. Affinity is also dependent on the length of the linker joining the two parts, with an optimum distance similar to that expected from structural models of Abl (SH(32). These results suggest that consolidated ligands may be generally useful reagents for probing structural and functional activities of multidomain proteins.

Helical encounter.

Cowburn D
Nat Struct Biol. 1994; 1: 489-91

Much more than the sum of the parts: structures of the dual SH2 domains of ZAP-70 and Syp.

Cowburn D
Chem Biol. 1996; 3: 79-82

Proteins involved in signaling pathways frequently contain one or more SH2 domains. New structural information on proteins that carry two SH2 domains show, surprisingly, that the domains are closely interlinked, so the binding sites are rigidly oriented with respect to each other. Thus, only ligands with the right spacing of the phosphotyrosines will be tightly bound.

Identification of the site in the Syk protein tyrosine kinase that binds the SH2 domain of Lck.

Couture C, Deckert M, Williams S, Russo FO, Altman A, Mustelin T
J Biol Chem. 1996; 271: 24294-9

The Syk protein tyrosine kinase (PTK) is expressed in many hematopoietic cells and is involved in signaling from various receptors for antigen and Fc portions of IgG and IgE. Upon cross-linking of these receptors, Syk is rapidly phosphorylated on tyrosine residues and enzymatically activated. We and others have found that the Lck kinase, a member of the Src family of PTKs, binds through its Src homology (SH) 2 domain to tyrosine phosphorylated Syk and to the related Zap kinase. Here we report that this interaction is direct and identify the two tandem tyrosines at the autophosphorylation site of Syk, Tyr518, and Tyr519, as the binding site for the SH2 domain of Lck. Mutation of either or both tyrosines to phenylalanines abrogated binding, while mutation of a second repetition of the motif at Tyr539 and Tyr540, or of the three tyrosines in the C terminus of Syk, did not. The SH2 domain of Lck bound the autophosphorylation site only when both Tyr518 and Tyr519 were phosphorylated. In intact cells the binding of the SH2 domain of Lck correlated with the ability of Syk to induce tyrosine phosphorylation of cellular proteins.

Protein tyrosine kinases, with emphasis on the Src family.

Courtneidge SA
Semin Cancer Biol. 1994; 5: 239-46

Protein tyrosine phosphorylation is recognized to be an important regulatory event, particularly in those pathways that control cell growth and differentiation. Phosphorylation on tyrosine may either decrease or increase the enzymatic activity of substrate proteins. In addition, tyrosine phosphorylated sequences associate with Src homology 2 (SH2) domains, and thus tyrosine phosphorylation also serves to regulate protein/protein interactions. Many protein tyrosine kinases have been described to date: several are the receptors for peptide growth factors; others are expressed in the cytoplasm and nucleus. The main subject of this review will be the Src family of protein tyrosine kinases, a group of nine enzymes thought to play important roles in several signal transduction pathways.

Ligand-induced strain in hydrogen bonds of the c-Src SH3 domain detected by NMR.

Cordier F, Wang C, Grzesiek S, Nicholson LK
J Mol Biol. 2000; 304: 497-505

Changes in the molecular conformation of proteins can result from a variety of perturbations, and can play crucial roles in the regulation of biological activity. A new solution NMR method has been applied to monitor ligand-induced changes in hydrogen bond geometry in the chicken c-Src SH3 domain. The structural response of this domain to ligand binding has been investigated by measuring trans-hydrogen bond (15)N-(13)C' scalar couplings in the free state and when bound to the high affinity class I ligand RLP2, containing residues RALPPLPRY. A comparison between hydrogen bonds in high resolution X-ray structures of this domain and those observed via (h3)J(NC') couplings in solution shows remarkable agreement. Two backbone-to-side-chain hydrogen bonds are observed in solution, and each appears to play a role in stabilization of loop structure. Reproducible ligand-induced changes in trans-hydrogen bond scalar couplings are observed across the domain that translate into changes in hydrogen bond length ranging between 0.02 to 0.12 A. The observed changes can be rationalized by an induced fit mechanism in which hydrogen bonds across the protein participate in a compensatory response to forces imparted at the protein-ligand interface. Upon ligand binding, mutual intercalation of the two Leu-Pro segments of the ligand between three aromatic side-chains protruding from the SH3 surface wedges apart secondary structural elements within the SH3 domain. This disruption is transmitted in a domino-like effect across the domain through networks of hydrogen bonded peptide planes. The unprecedented resolution obtained demonstrates the ability to characterize subtle structural rearrangements within a protein upon perturbation, and represents a new step in the endeavor to understand how hydrogen bonds contribute to the stabilization and function of biological macromolecules.

Identification of the mitogen-activated protein kinase phosphorylation sites on human Sos1 that regulate interaction with Grb2.

Corbalan-Garcia S, Yang SS, Degenhardt KR, Bar-Sagi D
Mol Cell Biol. 1996; 16: 5674-82

The Son of sevenless proteins (Sos) are guanine nucleotide exchange factors involved in the activation of Ras by cytoplasmic and receptor tyrosine kinases. Growth factor stimulation rapidly induces the phosphorylation of Sos on multiple serine and threonine sites. Previous studies have demonstrated that growth factor-induced Sos phosphorylation occurs at the C-terminal region of the protein and is mediated, in part, by mitogen-activated protein (MAP) kinase. In this report, we describe the identification of five MAP kinase sites (S-1137, S-1167, S-1178, S-1193, and S-1197) on hSos1. We demonstrate that four of these sites, S-1132, S-1167, S-1178, and S-1193, become phosphorylated following growth factor stimulation. The MAP kinase phosphorylation sites are clustered within a region encompassing three proline-rich SH3-binding sites in the C-terminal domain of hSos1. Replacing the MAP kinase phosphorylation sites with alanine residues results in an increase in the binding affinity of Grb2 to hSos1. Interestingly, hSos2 contains only one MAP kinase phosphorylation site and, as demonstrated previously, has an increased affinity toward Grb2 compared with hSos1. These results suggest a role for MAP kinase in the regulation of Grb2-Sos interactions. Since the binding of Grb2 is important for Sos function, the phosphorylation-dependent modulation of Grb2-Sos association may provide a means of controlling Ras activation.

Phospholipases A2 and Wnts are unlikely to share a common ancestor.

Copley RR, Ponting CP, Bork P
Curr Biol. 1999; 9: 7189-7189

The when and how of Src regulation.

Cooper JA, Howell B
Cell. 1993; 73: 1051-4

Physical and functional interaction of Nef with Lck. HIV-1 Nef-induced T-cell signaling defects.

Collette Y, Dutartre H, Benziane A, Ramos-Morales, Benarous R, Harris M, Olive D
J Biol Chem. 1996; 271: 6333-41

The nef gene is unique to the primate lentiviruses and encodes a cytoplasmic membrane-associated protein that affects T-cell signaling and is essential for both maintenance of a high virus load in vivo and for disease progression. Here we investigated the perturbation of cell signaling by Nef in T-cells and found that Nef interacts with the T-cell restricted Lek tyrosine kinase both in vitro and in vivo. The molecular basis for this interaction was analyzed. We show that cell-derived Nef is precipitated in a synergistic manner by the recombinant Src homology 2 (SH2) and SH3 domains from Lck. A functional proline-rich motif and the tyrosine phosphorylation of Nef were evidenced as likely participants in this interaction. The precipitation of Nef by the Lck recombinant proteins was specific, since neither Fyn, Csk, p85 phosphatidylinositol 3-kinase nor phospholipase Cgamma SH2 domains coprecipitated Nef from T-cells. Finally, depressed Lck kinase activity resulted from the presence of Nef, both in vitro and in intact cells, and nef expression resulted in impairment of both proximal and distal Lck-mediated signaling events. These results provide a molecular basis for the Nef-induced T-cell signaling defect and its role in AIDS pathogenesis.

HIV-2 and SIV nef proteins target different Src family SH3 domains than does HIV-1 Nef because of a triple amino acid substitution.

Collette Y, Arold S, Picard C, Janvier K, Benichou S, Benarous R, Olive D, Dumas C
J Biol Chem. 2000; 275: 4171-6

The nef gene is required for optimal viral spread of human and simian immunodeficiency viruses. However, the molecular mechanisms underlying the action of the Nef proteins may not be identical for all viral families. Here we investigate the interaction between the Nef protein of human and simian immunodeficiency viruses and SH3 domains from Src family kinases. Using the yeast two-hybrid system and immunoblotting we show that, in contrast to HIV-1 Nef, SIV and HIV-2 Nef poorly interact with Hck SH3 but bind to Src and Fyn SH3 domains. The molecular basis of these differences in SH3 targeting was revealed by sequence analysis and homology modeling of the putative SH3-Nef structures. Three amino acids (Trp-113, Thr-117, and Gln-118) that localize in a "hydrophobic pocket" implicated in SH3 binding of HIV-1 Nef, are systematically substituted in SIV/HIV-2 alleles (by Tyr, Glu, and Glu, respectively). We demonstrate that site-directed mutagenesis of these residues in SIV(mac239) Nef suffices to restore Hck SH3 binding and co-immunoprecipitation with full-length Hck from transfected cells. Our findings identify fundamental mechanistic differences in targeting of Src family kinases by HIV and SIV Nef. The herein described mechanism of SH3 selection by Nef via a "pocket" proximal to the canonical proline-rich motif may be a common feature for SH3 recognition by their natural ligands.

Evaluation of the catalytic mechanism of recombinant human Csk (C-terminal Src kinase) using nucleotide analogs and viscosity effects.

Cole PA, Burn P, Takacs B, Walsh CT
J Biol Chem. 1994; 269: 30880-7

Tyrosine kinases catalyze phosphoryl transfers from ATP to tyrosine residues in proteins. Despite their growing importance, their kinetic mechanism has remained largely unexplored. In this study, we have investigated the tyrosine kinase reaction catalyzed by purified human recombinant Csk (C-terminal Src kinase). Poly(Glu,Tyr) 4:1 was used as the tyrosine-containing substrate. Both ATP and poly(Glu,Tyr) were shown to be well behaved saturable substrates for recombinant Csk, with Km values that were in reasonable agreement with literature values reported for the non-recombinant enzyme and with kcat about 40 min-1. A sequential kinetic mechanism is suggested by a steady state kinetic analysis. Inhibitor studies with ADP and beta,gamma-imidoadenosine 5'-triphosphate were performed, and these results provided evidence against the possibility that ordered binding of peptide prior to ATP occurs. While a suitable competitive inhibitor of poly(Glu,Tyr) has not yet been identified, other evidence pointed to a rapid equilibrium random mechanism. Csk utilized adenosine 5'-O-(3-thiotriphosphate) in place of ATP. The phosphorothioyl transfer occurred with a kcat about 15-20-fold lower than the ATP reaction but with similar Km values. Deuterium solvent isotope effects on kcat were small for both reactions in a pH-independent range, consistent with the possibility that proton transfer is asymmetric in the reaction transition state. Using viscosity effects, ADP product release was suggested to be partially rate determining for catalysis in the standard ATP reaction. A comparison of the Csk kinetic mechanism with that of protein kinase A is discussed.

Progress in the development of inhibitors of SH2 domains.

Cody WL, Lin Z, Panek RL, Rose DW, Rubin JR
Curr Pharm Des. 2000; 6: 59-98

SH2 domains are discrete structural motifs common to a variety of critical intracellular signaling proteins. Inhibitors of specific SH2 domains have become important therapeutic targets in the treatment and/or prevention of restenosis, cancers (including small cell lung), cardiovascular disease, osteoporosis, apoptosis among others. Considering the social and economic impact of these diseases significant attention has been focused on the development of potent and selective inhibitors of specific SH2 domains. In particular, considerable research has been performed on Src, PI 3-kinase, Grb2 and more recently, Lck. In this review, we will focus on progress in the development of inhibitors for these specific SH2 domains and evaluate potential future targets.

Regulation of the oncogenic activity of the cellular src protein requires the correct spacing between the kinase domain and the C-terminal phosphorylated tyrosine (Tyr-527).

Cobb BS, Payne DM, Reynolds AB, Parsons JT
Mol Cell Biol. 1991; 11: 5832-8

Repression of the tyrosine kinase activity of the cellular src protein (pp60c-src) depends on the phosphorylation of a tyrosine residue (Tyr-527) near the carboxy terminus. Tyr-527 is located 11 residues C terminal from the genetically defined end of the kinase domain (Leu-516) and is therefore in a negative regulatory region. Because the precise sequence of amino acids surrounding Tyr-527 appears to be unimportant for regulation, we hypothesized that the conformational constraints induced by phosphorylated Tyr-527 may require the correct spacing between the kinase domain (Leu-516) and Tyr-527. In this report, we show that deletions at residue 518 of two, four, or seven amino acids or insertions at this residue of two or four amino acids activated the kinase activity and thus the transforming potential of pp60c-src. As is the case for the prototype transforming variant, pp60527F, activation caused by these deletions or insertions was abolished when Tyr-416 (the autophosphorylation site) was changed to phenylalanine. In comparison with wild-type pp60c-src, the src proteins containing the alterations at residue 518 showed a lower phosphorylation state at Tyr-527 regardless of whether residue 416 was a tyrosine or a phenylalanine. Mechanisms dealing with the importance of spacing between the kinase domain and Tyr-527 are discussed.

Regulation of the cellular src protein tyrosine kinase: interactions of the carboxyl terminal sequences residing between the kinase domain and tyrosine-527.

Cobb BS, Parsons JT
Oncogene. 1993; 8: 2897-903

Negative regulation of the cellular Src tyrosine kinase (pp60c-src) is mediated through the phosphorylation of a C-terminal tyrosine residue, Tyr-527. Current models predict that inhibition of c-Src kinase activity results from an interaction of phosphorylated Tyr-527 with the amino terminal SH2 domain. Tyr-527 is located 11 residues C-terminal from the end of the kinase domain. Insertion or deletion of residues within these 11 residues of pp60c-src activates kinase activity and induces morphological transformation. The resultant variant Src proteins also exhibit a reduced level of phosphorylation of Tyr-527. We have used antibodies to phosphotyrosine, susceptibility to tyrosine phosphatases and binding of mutant Src proteins to peptides mimicking the tyrosine phosphorylated C-terminus of pp60c-src to investigate the tyrosine phosphorylated and unphosphorylated forms of such insertion/deletion variants. The reactivity of variant proteins with phosphotyrosine antibodies and the susceptibility of phosphorylated Tyr-527 to tyrosine phosphatases were similar to that of wild type pp60c-src. In addition, the results of binding experiments performed with a C-terminal peptide containing phosphorylated Tyr-527 indicated that only dephosphorylated forms of variant Src proteins bound phospho-peptide. These data suggest that insertion or deletion mutations within the C-terminal region of pp60c-src do not substantially alter the interaction of phosphorylated Tyr-527 with the SH2 domain. Rather, the data are consistent with the hypothesis that the reduction of phosphorylation of Tyr-527 and the accompanying activation of these variants may be due to the action of a tyrosine phosphatase and the inefficient phosphorylation of Tyr-527 by a regulatory kinase.

Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP-rho.

Cicchetti P, Mayer BJ, Thiel G, Baltimore D
Science. 1992; 257: 803-6

A Src homology 3 (SH3) region is a sequence of approximately 50 amino acids found in many nonreceptor tyrosine kinases and other proteins. Deletion of the SH3 region from the protein encoded by the c-abl proto-oncogene activates the protein's transforming capacity, thereby suggesting the participation of the SH3 region in the negative regulation of transformation. A complementary DNA was isolated that encoded a protein, 3BP-1, to which the SH3 region of Abl bound with high specificity and to which SH3 regions from other proteins bound differentially. The sequence of the 3BP-1 protein is similar to that of a COOH-terminal segment of Bcr and to guanosine triphosphatase-activating protein (GAP)-rho, which suggests that it might have GAP activity for Ras-related proteins. The 3BP-1 protein may therefore be a mediator of SH3 function in transformation inhibition and may link tyrosine kinases to Ras-related proteins.

Calculation of ensembles of structures representing the unfolded state of an SH3 domain.

Choy WY, Forman-Kay JD
J Mol Biol. 2001; 308: 1011-32

The N-terminal SH3 domain of drk (drkN SH3 domain) exists in equilibrium between a folded (F(exch)) and an unfolded (U(exch)) form under non-denaturing conditions. In order to further our previous descriptions of the U(exch) state, we have developed a protocol for calculating ensembles of structures, based on experimental spectroscopic data, which broadly represent the unfolded state. A large number of unfolding trajectories were generated, starting from the folded state structure of the protein, in order to provide a reasonable sampling of the conformational space accessible to this sequence. Unfolded state ensembles have been "calculated" using a newly developed program ENSEMBLE, which optimizes the population weights assigned to each structure based on experimental properties of the U(exch) state. Pseudo-energy terms for nuclear Overhauser effects, J-coupling constants, (13)C chemical shifts, translational diffusion coefficients and tryptophan ring burial based on NMR and fluorescence data have been implemented. The population weight assignment procedure was performed for different starting ensembles. Small numbers of structures (<60) dominate the final ensembles compared to the total number in the starting ensembles, suggesting that the drkN SH3 domain U(exch) state can be described by a limited number of lower-energy conformations. The calculated U(exch) state ensembles are much more compact than a "random coil" chain, with significant native-like residual structure observed. In particular, a sizable population of conformers having the n-src loop and distal beta-hairpin structures exist in the calculated U(exch) state ensembles, and Trp36 is involved in a large number of interactions, both native and non-native. Copyright 2001 Academic Press.

The Grb2-mSos1 complex binds phosphopeptides with higher affinity than Grb2.

Chook YM, Gish GD, Kay CM, Pai EF, Pawson T
J Biol Chem. 1996; 271: 30472-8

Epidermal growth factor (EGF) stimulation leads to autophosphorylation of the epidermal growth factor receptor (EGFR) and tyrosine phosphorylation of Shc. The Grb2 SH2 domain binds to Tyr1068 of EGFR and Tyr317 of Shc while its SH3 domains bind to mSos1. Therefore, EGF treatment potentially results in the formation of several multimeric signaling complexes, including EGFR-Grb2-mSos1, EGFR-Shc-Grb2-mSos1, and Shc-Grb2-mSos1, linking the receptor to activation of the Ras GTPase. We have purified Grb2, mSos1, and the Grb2-mSos1 complex to high homogeneity, and used these isolated proteins to obtain binding affinities of mSos1 for Grb2 and of either Grb2 or Grb2-mSos1 for phosphotyrosine-containing peptides. mSos1 bound Grb2 with a KD of 0.4 microM; the stoichiometry of the Grb2-mSos1 complex was 1:1. An EGFR-derived phosphopeptide bound Grb2 with a KD of 0.7 microM, whereas the Shc-derived phosphopeptide bound Grb2 with a KD of 0.2 microM. Since Grb2 exists in a stable complex with mSos1, and both proteins can exist in a constitutive complex in unstimulated cells, we performed phosphopeptide binding studies on the Grb2-mSos1 complex to gain a better understanding of binding events in the intact cell. Grb2-mSos1 bound to both EGFR- and Shc-derived phosphopeptides with higher affinities (KD of 0.3 microM and 31 nM, respectively) than Grb2 alone. These findings suggest that the proximity of mSos1 to Grb2 in the complex can influence the interactions of the Grb2 SH2 domain with phosphopeptides and raise the possibility that in the Grb2-mSos1 complex the SH2 and SH3 domains of Grb2 are not independent of each other but may be indirectly linked by mSos1.

Phosphorylation at Tyr-838 in the kinase domain of EphA8 modulates Fyn binding to the Tyr-615 site by enhancing tyrosine kinase activity.

Choi S, Park S
Oncogene. 1999; 18: 5413-22

Eph-related receptors and their ephrin ligands are highly conserved protein families which play important roles in targeting axons and migrating cells. In this study we have examined the functional roles of two major autophosphorylation sites, Tyr-615 and Tyr-838, in the EphA8 receptor. Two-dimensional phosphopeptide mapping analysis demonstrated that Tyr-615 and Tyr-838 constitute major autophosphorylation sites in EphA8. Tyr-615 was phosphorylated to the highest stoichiometry, suggesting that phosphorylation at this site may have a physiologically important role. Upon conservative mutation of Tyr-838 located in the tyrosine kinase domain, the catalytic activity of EphA8 was strikingly reduced both in vitro and in vivo, whereas a mutation at Tyr-615 in the juxtamembrane domain did not impair the tyrosine kinase activity. In vitro binding experiments revealed that phosphorylation at Tyr-615 in EphA8 mediates the preferential binding to Fyn-SH2 domain rather than Src and Ras GTPase-activating protein (Ras GAP)-SH2 domains. Additionally, a high level of EphA8 was detected in Fyn immunoprecipitates in intact cells, indicating that EphA8 and Fyn can physically associate in vivo. In contrast, the association of full-length Fyn to EphA8 containing mutation at either Tyr-615 or Tyr-838 was greatly reduced. These data indicate that phosphorylation of Tyr-615 is critical for determining the association with Fyn whereas the integrity of Tyr-838 phosphorylation is required for efficient phosphorylation at Tyr-615 as well as other major sites. Finally, it was observed that cell attachment responses are attenuated by overexpression of wild type EphA8 receptor but to much less extent by EphA8 mutants lacking phosphorylation at either Tyr-615 or Tyr-838. Furthermore, transient expression of kinase-inactive Fyn in EphA8-overexpressing cells blocked cell attachment responses attenuated by the EphA8 signaling. We therefore propose that Fyn kinase is one of the major downstream targets for the EphA8 signaling pathway leading to a modification of cell adhesion, and that autophosphorylation at Tyr-838 is critical for positively regulating the EphA8 signaling event.

[Interaction of a synthetic fragment of the oncoprotein p21ras with cellular proteins]

Chertov OI, Khokhlachev AV, Deigin VI
Bioorg Khim. 1986; 12: 1157-63

A decapeptide corresponding to residues 35-44(-Thr-Ile-Glu-Asp-Ser-Tyr-Arg-Lys-Gln-Val-) of p21ras was synthesized. It was found that peptide causes precipitation of some proteins from the Triton X-100 lysate of NIH 3T3 EJ cells. SDS-PAGE demonstrated the presence of many proteins in this precipitate. The peptide labeled with [125I]Bolton-Hunter reagent specifically recognized four proteins of M. W. 27, 35, 50 and 85 kDa. The order of charged amino acid residues in the fragment 35-44 of p21ras is "complementary" to that of the substrate sequence of tyrosine-specific protein kinases (-Arg-X-X-Glu-Asp-X-X-Tyr-). It is suggested that p21ras proteins directly regulate phosphorylation of the target proteins of these kinases. A model for functioning of p21ras proteins predicts the presence in their structure of certain sites homologous to sequences recognizable by tyrosine-specific kinases. Indeed two such sites are present in the sequences of all p21ras proteins, namely the residues 88-92 and 104-108.

The conserved core of human immunodeficiency virus type 1 Nef is essential for association with Lck and for enhanced viral replication in T-lymphocytes.

Cheng H, Hoxie JP, Parks WP
Virology. 1999; 264: 5-15

The Nef protein of the primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), is a myristylated protein associated with increased viral replication and enhanced pathogenicity. Both the potentiation of T-lymphocyte activation and the enhanced serine-phosphorylation of HIV-1 capsid by Nef correlate with increased viral replication. We report the functional interactions of the Nef proteins with Src kinases. The Nef proteins from HIV-1 and SIV bind to Lck as well as Hck, Lyn, and Fyn. The SH3 and SH2 domains of Lck are sufficient for coprecipitation with non-tyrosine-phosphorylated Nef proteins. The conserved core region of HIV-1 Nef is essential for the interaction with Lck and is also important for enhanced HIV-1 replication in T-lymphocytes. In addition, we show that SIV and HIV-1 Nef proteins are differentially tyrosine-phosphorylated. The kinase-active Lck tyrosine-phosphorylates SIVmac239 Nef but does not phosphorylate HIV-1 Nef. These data suggest that the association of Nef and Lck is central to the enhanced viral replication of HIV-1 and SIV in T-lymphocytes.

Purification of bovine thymus cytosolic C-terminal Src kinase (CSK) and demonstration of differential efficiencies of phosphorylation and inactivation of p56lyn and pp60c-src by CSK.

Cheng HC, Bjorge JD, Aebersold R, Fujita DJ, Wang JH
Biochemistry. 1996; 35: 11874-87

The C-terminal src kinase (CSK) is a ubiquitously expressed, cytosolic enzyme capable of phosphorylating and inactivating several plasma membrane-bound src-family protein tyrosine kinases in vitro [Nada, S., Okada, M., MacAuley, A., Cooper, J.A., & Nakagawa, H. (1990) Nature 351, 69-72; Bergman, M., Mustelin, T., Oetken, C., Partanen, J., Flint, N.A., Amrein, K.E., Autero, M., Burn, P., & Alitalo, K. (1992) EMBO J. 11, 2919-2924]. We purified CSK to apparent homogeneity from bovine thymus cytosol to study in vitro how the purified enzyme recognizes the various src-family kinases as its substrates. A novel assay method was developed for assaying the ability of CSK to inactivate src-family tyrosine kinases. With this assay method, we demonstrated that CSK inactivated p56lyn with a significantly higher efficiency than pp60c-src. Phosphopeptide mapping of CSK-phosphorylated p56lyn and pp60c-src shows that the consensus tyrosine residue (also termed tail tyrosine) in the C-terminal regulatory domain of p56lyn was phosphorylated by CSK with an efficiency much higher than that of pp60c-src. Thus, the higher efficiency of inactivation of p56lyn by CSK is a result of the ability of p56lyn to serve as a better substrate of CSK. The synthetic peptides derived from the C-terminal portion of p56lyn and pp60c-src were much poorer substrates than the intact src-family kinases for CSK, indicating that the local structure around the tail tyrosine is not sufficient to direct efficient phosphorylation of p56lyn by CSK. Nevertheless, the slightly higher efficiency displayed by CSK in phosphorylating the peptide derived from the C-terminal portion of p56lyn than that from pp60c-src suggests that the structural differences between the C-terminal portions of p56lyn and pp60c-src contribute to the differential efficiencies displayed by CSK in phosphorylating the two kinases. Determination of the CSK-phosphorylation site in the src-C-terminal peptide by phosphopeptide mapping reveals that the whole C-terminal regulatory domain and an adjacent part of the protein kinase domain contain some of the structural determinants directing CSK to phosphorylate the consensus tail tyrosine of the src-family kinases.

Genomic organization and comparative analysis of the mouse tuberous sclerosis 1 (Tsc1) locus.

Cheadle JP, Dobbie L, Idziaszczyk S, Hodges AK, Smith AJ, Sampson JR, Young J
Mamm Genome. 2000; 11: 1135-8

Identification of a Src SH3 domain binding motif by screening a random phage display library.

Cheadle C, Ivashchenko Y, South V, Searfoss GH, French S, Howk R, Ricca GA, Jaye M
J Biol Chem. 1994; 269: 24034-9

A phage display library was constructed in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 10(7) different phage, was screened with a glutathione S-transferase (GST) fusion protein containing the Src homology 3 (Src SH3) domain and a protein kinase A phosphorylation site (GST/PKA/Src SH3). A family of proline-rich sequences was isolated following four cycles of enrichment and amplification. Phage containing these sequences were shown to specifically bind to the GST/PKA/Src SH3 protein but not to GST/PKA only. A comparison of the inferred amino acid sequence of the different phage clones revealed a consensus sequence, RPLPXXP, which conforms to a Src SH3 domain binding motif identified independently during an affinity screen of a lambda-lox mouse embryo cDNA library using a 32P-labeled Src SH3 protein fragment as the probe (Y. Ivashchenko, manuscript in preparation). Peptides based upon the 7-amino acid SH3 binding domain core motif displayed strong binding to both the Src and to the Fyn SH3 domains, but failed to bind to the SH3 domain of p21 Ras-GTPase-activating protein (Ras-GAP) and other proteins. We anticipate that further screening of the phage display library will be a useful tool for the rapid identification of additional SH3 domain binding sequences and will also help to establish the essential core motifs that define the specificity of interactions among the diverse proteins containing SH3 domains and those containing SH3 binding motifs.

Peptide ligands of pp60(c-src) SH2 domains: a thermodynamic and structural study.

Charifson PS, Shewchuk LM, Rocque W, Hummel CW, Jordan SR, Mohr C, Pacofsky GJ, Peel MR, Rodriguez M, Sternbach DD, Consler TG
Biochemistry. 1997; 36: 6283-93

Thermodynamic measurements, structural determinations, and molecular computations were applied to a series of peptide ligands of the pp60(c-src) SH2 domain in an attempt to understand the critical binding determinants for this class of molecules. Isothermal titration calorimetry (ITC) measurements were combined with structural data derived from X-ray crystallographic studies on 12 peptide-SH2 domain complexes. The peptide ligands studied fall into two general classes: (1) dipeptides of the general framework N-acetylphosphotyrosine (or phosphotyrosine replacement)-Glu or methionine (or S-methylcysteine)-X, where X represents a hydrophobic amine, and (2) tetra- or pentapeptides of the general framework N-acetylphosphotyrosine-Glu-Glu-Ile-X, where X represents either Glu, Gln, or NH2. Dipeptide analogs which featured X as either hexanolamine or heptanolamine were able to pick up new hydrogen bonds involving their hydroxyl groups within a predominantly lipophilic surface cavity. However, due to internal strain as well as the solvent accessibility of the new hydrogen bonds formed, no net increase in binding affinity was observed. Phosphatase-resistant benzylmalonate and alpha,alpha-difluorobenzyl phosphonate analogs of phosphotyrosine retained some binding affinity for the pp60(c-src) SH2 domain but caused local structural perturbations in the phosphotyrosine-binding site. In the case where a reversible covalent thiohemiacetal was formed between a formylated phosphotyrosine analog and the thiol side chain of Cys-188, deltaS was 25.6 cal/(mol K) lower than for the nonformylated phosphotyrosine parent. Normal mode calculations show that the dramatic decrease in entropy observed for the covalent thiohemiacetal complex is due to the inability of the phosphotyrosine moiety to transform lost rotational and translational degrees of freedom into new vibrational modes.

The Grb2 adaptor.

Chardin P, Cussac D, Maignan S, Ducruix A
FEBS Lett. 1995; 369: 47-51

Grb2 is an 'adaptor' protein made of one SH2 and two SH3 domains. The SH3 domains bind to prolinerich motifs in the C-terminal part of the ras exchange factor Sos. Binding of the Grb2 SH2 domain to phosphotyrosine motifs on receptors, or other adaptor proteins such as Shc, recruits this Grb2/Sos complex at the plasma membrane where Sos stimulates nucleotide exchange on ras, then ras activates raf and leads to MAP kinase activation. The structure of Grb2, the precise motifs recognised by its SH2 and SH3 domains, the way Grb2 performs its function, a possible regulation of its association with Sos, and its ability to complex with other proteins in vivo, are discussed.

Conserved intron elements repress splicing of a neuron-specific c-src exon in vitro.

Chan RC, Black DL
Mol Cell Biol. 1997; 17: 2970-2970

Disease specificity of kinase domains: the src-encoded catalytic domain converts erbB into a sarcoma oncogene.

Chang CM, Shu HK, Kung HJ
Proc Natl Acad Sci U S A. 1995; 92: 3928-32

src and erbB are two tyrosine kinase-encoding oncogenes carried by retroviruses, which have distinct disease specificities. The former induces predominantly sarcomas, and the latter, leukemias. Src and ErbB have similar catalytic domains but have very different regulatory domains. A wealth of information exists concerning how different regulatory domains [Src homology 2 (SH2) and SH3 domains and autophosphorylation sites] control substrate and disease specificities. Whether the catalytic domain helps determine these specificities remains to be explored. Here we show that the Src catalytic domain is enzymatically active when substituted into the ErbB backbone and interacts with the ErbB regulatory domain. This ErbB/Src chimera displays autophosphorylation and substrate phosphorylation patterns different from those of both Src and ErbB. Neither SH2 and SH3 nor autophosphorylation sites are required for the Src catalytic domain to exert its fibroblast transforming ability. Most significantly, the catalytic domain can convert erbB from a leukemogenic oncogene into a sarcomagenic oncogene, suggesting that the leukemogenic determinants in part reside within the ErbB catalytic domain.

Primary structure of the met protein tyrosine kinase domain.

Chan AM, King HW, Tempest PR, Deakin EA, Cooper CS, Brookes P
Oncogene. 1987; 1: 229-33

The primary structure of the protein tyrosine kinase domain of the human met gene has been determined from cDNA clones prepared from transcripts of the activated human met gene. These analyses reveal that the met kinase domain (located on human chromosome 7) possesses unique features that distinguish met from other members of the src family of protein tyrosine kinases. The results also demonstrate that the product of the activated met gene is a fusion protein and that the amino terminal end of this fusion protein, which is encoded by human chromosome 1, exhibits homology to laminin B1.

Specificity in protein-tyrosine kinase signaling.

Cantley LC, Songyang Z
Adv Second Messenger Phosphoprotein Res. 1997; 31: 41-8

Rescuing a destabilized protein fold through backbone cyclization.

Camarero JA, Fushman D, Sato S, Giriat I, Cowburn D, Raleigh DP, Muir TW
J Mol Biol. 2001; 308: 1045-62

We describe the physicochemical characterization of various circular and linear forms of the approximately 60 residue N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein. Structural, dynamic, thermodynamic, kinetic and biochemical studies reveal that backbone circularization does not prevent the adoption of the natural folded structure in any of the circular proteins. Both the folding and unfolding rate of the protein increased slightly upon circularization. Circularization did not lead to a significant thermodynamic stabilization of the full-length protein, suggesting that destabilizing enthalpic effects (e.g. strain) negate the expected favorable entropic contribution to overall stability. In contrast, we find circularization results in a dramatic stabilization of a truncated version of the SH3 domain lacking a key glutamate residue. The ability to rescue the destabilized mutant indicates that circularization may be a useful tool in protein engineering programs geared towards generating minimized proteins. Copyright 2001 Academic Press.

Peptide chemical ligation inside living cells: in vivo generation of a circular protein domain.

Camarero JA, Fushman D, Cowburn D, Muir TW
Bioorg Med Chem. 2001; 9: 2479-84

Here we describe the first example of a peptide chemical ligation reaction performed inside a living cell. A cell-based native chemical ligation approach was developed and used to generate a circular version of the N-terminal Src homology 3 (SH3) domain from the murine c-Crk adapter protein inside Escherichia coli cells. The in vivo cyclization reaction was extremely efficient and the resulting circular protein domain was fully biologically active and able to adopt the native SH3 folded structure. This work represents an important step towards the in vivo generation of small backbone cyclic peptides for use in basic biological research.

Studying receptor-ligand interactions using encoded amino acid scanning.

Camarero JA, Ayers B, Muir TW
Biochemistry. 1998; 37: 7487-95

A novel technique is described that allows the synthesis, functional analysis, and quantitative readout of defined arrays of polypeptide analogues in aqueous solution. Key to this approach is the use of a simple encoding-decoding system in which a unique Fmoc-amino acid tag is covalently attached to the C terminus of each member of a molecular array through a selectively cleavable bond. These tags can be cleanly removed from the molecules they encode, allowing single-step characterization and quantification of the entire mixture by HPLC. The utility of this technique is illustrated through the preparation of an array of proline-rich sequences based on the exchange factor C3G, one of the natural ligands of the N-terminal SH3 domain from the proto-oncogene, c-Crk. The array was designed to systematically modify those residues within the C3G peptide ligand thought to make key interactions with the c-Crk SH3 domain. Using competition binding experiments, it was possible to determine the relative ED50 values for the entire array of molecules simultaneously. These studies revealed that in order to maintain optimal binding to the SH3 domain, the P-3 side chain of the ligand must be positively charged and the P-0 side chain must be hydrophobic and extend beyond the gamma-carbon. The excellent correlation between these relative ED50 values and a series of relative Kd values determined from individual peptides suggests that this approach may be useful in determining, in a parallel fashion, the relative biological activities of arrays of polypeptides.

Conformationally constrained phosphotyrosyl mimetics designed as monomeric Src homology 2 domain inhibitors.

Burke TR Jr, Barchi JJ Jr, George C, Wolf G, Shoelson SE, Yan X
J Med Chem. 1995; 38: 1386-96

Inhibitors of specific src homology 2 (SH2) domain binding interactions could potentially afford new therapeutic approaches toward a variety of diseases, including several cancers. To date SH2 domain inhibitors have been confined to small phosphotyrosyl (pTyr)-containing peptides that appear to bind along the surface of SH2 domains with specific recognition features protruding into the protein. Among these protrusions is the pTyr residue itself, which is inserted into a well-formed binding pocket. In the present study monomeric pTyr mimetics were prepared having key aspects of their structure constrained to conformations of the bound pTyr residue observed in the previously reported X-ray structure of a pTyr-peptide bound to the Lck SH2 domain. The resulting constrained pTyr mimetics were examined for inhibitory potency in six SH2 domain constructs: Lck, Src, Grb2, and the C-terminal SH2 domains of PLC gamma (PLC gamma-C) and the p85 subunit of PI-3 kinase (p85-C), as well as the N-terminal SH2 domain of SH PTP2. Although inhibition constants were in the millimolar range, it was observed that capping pTyr as its N alpha-acetyl carboxamide [(L)-1] provided a roughly 2-3-fold increase in potency relative to free pTyr. Diastereomeric indanylglycine-based analogues (+/-)-3a,b were essentially inactive. Of note was methanobenzazocine (+/-)-2. While being racemic and a partial pTyr structure, this analogue retained full binding potency of the enantiomerically pure N alpha-acetyl pTyr amide (L)-1. Modification and elaboration of 2 could potentially result in small molecule inhibitors having greater potency.

Identification of Itk/Tsk Src homology 3 domain ligands.

Bunnell SC, Henry PA, Kolluri R, Kirchhausen T, Rickles RJ, Berg LJ
J Biol Chem. 1996; 271: 25646-56

The tyrosine kinase Itk/Tsk is a T cell specific analog of Btk, the tyrosine kinase defective in the human immunodeficiency X-linked agammaglobulinemia and in xid mice. T lymphocytes from Itk-deficient mice are refractory to mitogenic stimuli delivered through the T cell receptor (TCR). To gain insights into the biochemical role of Itk, the binding properties of the Itk SH3 domain were examined. An optimal Itk SH3 binding motif was derived by screening biased phage display libraries; peptides based on this motif bound with high affinity and selectivity to the Itk SH3 domain. Initial studies with T cell lysates indicated that the Itk SH3 domain bound Cbl, Fyn, and other tyrosine phosphoproteins from TCR-stimulated Jurkat cells. Under conditions of increased detergent stringency Sam 68, Wiskott-Aldrich Syndrome protein, and hnRNP-K, but not Cbl and Fyn, were bound to the Itk SH3 domain. By examining the ability of different SH3 domains to interact with deletion variants of Sam 68 and WASP, we demonstrated that the Itk-SH3 domain and the SH3 domains of Src family kinases bind to overlapping but distinct sets of proline-rich regions in Sam 68 and WASP.

Biochemical interactions integrating Itk with the T cell receptor-initiated signaling cascade.

Bunnell SC, Diehn M, Yaffe MB, Findell PR, Cantley LC, Berg LJ
J Biol Chem. 2000; 275: 2219-30

Itk, a Tec family tyrosine kinase, acts downstream of Lck and phosphatidylinositol 3'-kinase to facilitate T cell receptor (TCR)-dependent calcium influxes and increases in extracellular-regulated kinase activity. Here we demonstrate interactions between Itk and crucial components of TCR-dependent signaling pathways. First, the inositide-binding pocket of the Itk pleckstrin homology domain directs the constitutive association of Itk with buoyant membranes that are the primary site of TCR activation and are enriched in both Lck and LAT. This association is required for the transphosphorylation of Itk. Second, the Itk proline-rich region binds to Grb2 and LAT. Third, the Itk Src homology (SH3) 3 and SH2 domains interact cooperatively with Syk-phosphorylated SLP-76. Notably, SLP-76 contains a predicted binding motif for the Itk SH2 domain and binds to full-length Itk in vitro. Finally, we show that kinase-inactive Itk can antagonize the SLP-76-dependent activation of NF-AT. The inhibition of NF-AT activation depends on the Itk pleckstrin homology domain, proline-rich region, and SH2 domain. Together, these observations suggest that multivalent interactions recruit Itk to LAT-nucleated signaling complexes and facilitate the activation of LAT-associated phospholipase Cgamma1 by Itk.

Use of synthetic peptides and copolymers to study the substrate specificity and inhibition of the protein tyrosine kinase pp60c-src.

Budde RJ, Obeyesekere NU, Ke S, McMurray JS
Biochim Biophys Acta. 1995; 1248: 50-6

The ability of synthetic peptides and polypeptides to act as substrates and/or inhibitors of pp60c-src was examined. The random copolymer, poly(K4Y) had a threefold lower specificity than poly(E4Y). Peptides containing lysine vs. glutamate were also found to have a lower substrate specificity (Vmax:Km ratio). In order to assess the substrate specificity of acidic peptides, an assay protocol using DEAE-membranes was developed. Peptides containing a (YXE)5YXD motif (X = G, A, V, P, or norvaline) were tested as inhibitors and substrates of pp60c-src. The glycine-containing peptide was the best substrate having a specificity 16,000-fold higher than 5Val-angiotensin II, the most commonly used peptide substrate. Most of the peptides, except for the proline containing peptide, had Ki values of 20-100 microM. In a series of (XGE)5XGD peptides, where X = Y or F, tyrosine at position 10 was found to be the preferred site for accepting a phosphate. Analogs in which the glycine was replaced with alanine indicated that loss of flexibility around position 10 was detrimental to substrate specificity. Results suggest that conformational requirements of the peptides tested was important and substrate specificity was a more sensitive parameter than binding as measured by Ki values.

Isolation and identification of two proto-oncogene products related to c-fgr and fyn in a tyrosine-protein-kinase fraction of rat spleen.

Brunati AM, James P, Donella-Deana A, Matoskova B, Robbins KC, Pinna LA
Eur J Biochem. 1993; 216: 323-7

TPK-III, a tyrosine-protein-kinase fraction previously isolated from rat spleen [Brunati, A. M. & Pinna, L. A. (1988) Eur. J. Biochem. 172, 451-457] has been further purified and resolved by Mono-Q FPLC into two homogeneous compounds, Q1 and Q2, with molecular mass approximately 52 kDa, exhibiting high specific activities for phosphate incorporation into the phosphoacceptor substrate poly(Glu80Tyr20) (1194 nmol.min-1 x mg-1 and 579 nmol.min-1 x mg-1, respectively). Both Q1 and Q2 appear to be scr-related enzymes since they are readily recognised by anti-SEEP serum raised against the highly conserved segment at positions 330-345 of p60c-scr. Q1, but not Q2, interacts with a specific antibody raised against the N-terminal segment of p55c-fgr. Microsequence analysis of tryptic fragments generated from Q1 revealed five peptides which exactly overlap the expected segments of p55c-fgr. Two of these peptides were not entirely conserved in any of the other src-related tyrosine protein kinases. A sixth fragment is very similar, albeit not identical, to the C-terminus of p55c-fgr. Microsequence analysis of two tryptic fragments from the other TPK-III fraction, Q2, provided the sequences FQILNSSE and LTTQETGYIPSNY, which are identical to two segments of fyn not entirely conserved in any of the other src-related tyrosine protein kinases. These results provide evidence that the spleen tyrosine-protein-kinase fraction, conventionally designated TPK-III, is composed of products from two proto-oncogenes, one of which corresponds to fyn, while the other is either identical or very closely related to c-fgr.

NMR studies of the RRsrc peptide, a tyrosine kinase substrate.

Brockbank RL, Vogel HJ
Biochem Cell Biol. 1997; 75: 163-9

The proton and carbon-13 NMR resonances for the 13-residue synthetic RRsrc peptide were completely assigned using two-dimensional NMR spectroscopy. This peptide contains a tyrosine in position 9 that can be phosphorylated by many tyrosine protein kinases. On the basis of observed nuclear Overhauser enhancements and alpha-proton and alpha-carbon chemical shifts, the peptide appears to interconvert between extended and nascent helical structures. The helical conformation found in aqueous solution is compared with the corresponding structure calculated for the tyrosine 416 site of pp60src by homology modeling to the cAMP-dependent protein kinase (PKA) and also to the conformation modelled after the bound form of a PKA-inhibitor peptide.

SH2-kinase linker mutations release Hck tyrosine kinase and transforming activities in Rat-2 fibroblasts.

Briggs SD, Smithgall TE
J Biol Chem. 1999; 274: 26579-83

Biochemical and structural studies of Src and related kinases demonstrate that two intramolecular interactions suppress kinase activity. These interactions involve binding of the SH2 domain to a phosphotyrosine residue in the C-terminal tail and association of the SH3 domain with a polyproline type II helix formed by amino acids linking the SH2 and kinase domains. Recent studies have shown that high affinity interaction of the SH3 domain of Hck with the human immunodeficiency virus type I Nef protein activates Hck tyrosine kinase and biological activities, suggesting a mechanism that involves disruption of the SH3-linker interaction. To test the role of this interaction in the regulation of Hck kinase activity in living cells, we substituted alanines for prolines 225 and 228 in the linker region and observed that the resulting mutant (Hck-2PA) demonstrated strong transforming activity in a Rat-2 fibroblast focus-forming assay. Hck-2PA also exhibited elevated tyrosine kinase activity in terms of autophosphorylation, endogenous substrate phosphorylation, and in an in vitro kinase assay. The transforming and kinase activities of Hck-2PA were remarkably similar to those observed with a Hck mutant activated by Phe substitution of the conserved tail Tyr residue and with wild-type Hck following co-expression with human immunodeficiency virus Nef. Introduction of the 2PA and tail mutations into a single Hck expression construct did not increase kinase or transforming activity relative to the individual mutations. These data provide new evidence that SH3-linker interaction may represent the dominant mechanism controlling Hck tyrosine kinase activity in vivo.

SH3-mediated Hck tyrosine kinase activation and fibroblast transformation by the Nef protein of HIV-1.

Briggs SD, Sharkey M, Stevenson M, Smithgall TE
J Biol Chem. 1997; 272: 17899-902

Tyrosine kinases of the Src family are regulated via their Src homology 2 (SH2) and SH3 domains. The Nef protein of human immunodeficiency virus-1 (HIV-1) has previously been shown to bind with high affinity and specificity in vitro to the SH3 domain of Hck, a Src family member expressed primarily in myeloid cells. However, the effect of Nef on Hck activity in living cells is unknown. Here we show that Rat-2 fibroblasts co-expressing Hck and Nef rapidly developed transformed foci, whereas control cells expressing either protein alone did not. Nef formed a stable complex with Hck and stimulated its tyrosine kinase activity in vivo. Mutagenesis of the Nef proline-rich motif essential for SH3 binding completely blocked complex formation, kinase activation, and transformation, indicating that the Nef SH3-binding function is required for its effects on Hck. These results provide direct evidence that SH3 engagement is sufficient to activate a Src family kinase in vivo and suggest that Hck may be activated by Nef in HIV-infected macrophages.

HIV-1 Nef promotes survival of myeloid cells by a Stat3-dependent pathway.

Briggs SD, Scholtz B, Jacque JM, Swingler S, Stevenson M, Smithgall TE
J Biol Chem. 2001; 276: 25605-11

Human immunodeficiency virus Nef is a small myristylated protein that plays a critical role in AIDS progression. Nef binds with high affinity to the SH3 domain of the myeloid-restricted tyrosine kinase Hck in vitro, identifying this Src-related kinase as a possible cellular target for Nef in macrophages. Here we show that Nef activates endogenous Hck in the granulocyte-macrophage colony-stimulating factor-dependent myeloid cell line, TF-1. Unexpectedly, Nef induced cytokine-independent TF-1 cell outgrowth and constitutive activation of the Stat3 transcription factor. Induction of survival required the Nef SH3 binding and membrane-targeting motifs and was blocked by dominant-negative Stat3 mutants. Nef also stimulated Stat3 activation in primary human macrophages, providing evidence for Stat3 as a Nef effector in a target cell for human immunodeficiency virus.

Affinity of Src family kinase SH3 domains for HIV Nef in vitro does not predict kinase activation by Nef in vivo.

Briggs SD, Lerner EC, Smithgall TE
Biochemistry. 2000; 39: 489-95

Nef is an HIV accessory protein required for high-titer viral replication and AIDS progression. Previous studies have shown that the SH3 domains of Hck and Lyn bind to Nef via proline-rich sequences in vitro, identifying these Src-related kinases as potential targets for Nef in vivo. Association of Nef with Hck causes displacement of the intramolecular interaction between the SH3 domain and the SH2-kinase linker, leading to kinase activation both in vitro and in vivo. In this study, we investigated whether interaction with Nef induces activation of other Src family kinases (Lyn, Fyn, Src, and Lck) following coexpression with Nef in Rat-2 fibroblasts. Coexpression with Nef induced Hck kinase activation and fibroblast transformation, consistent with previous results. In contrast, coexpression of Nef with Lyn was without effect, despite equivalent binding of Nef to full-length Lyn and Hck. Furthermore, Nef was found to suppress the kinase and transforming activities of Fyn, the SH3 domain of which exhibits low affinity for Nef. Coexpression with Nef did not alter c-Src or Lck tyrosine kinase or transforming activity in this system. Differential modulation of Src family members by Nef may produce unique downstream signals depending on the profile of Src kinases expressed in a given cell type.

The Ras GTPase-activating protein (GAP) is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase, Hck.

Briggs SD, Bryant SS, Jove R, Sanderson SD, Smithgall TE
J Biol Chem. 1995; 270: 14718-24

The Ras GTPase-activating protein (GAP) is a target for protein tyrosine kinases of both the receptor and cytoplasmic classes and may serve to integrate tyrosine kinase and Ras signaling pathways. In this report, we provide evidence that GAP is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase Hck, which has been implicated in the regulation of myeloid cell growth, differentiation, and function. Wild-type (WT) or kinase-inactive (K269E) mutant Hck proteins were co-expressed with bovine GAP using the baculovirus/Sf-9 cell system. GAP was readily phosphorylated on tyrosine by WT but not K269E Hck. GAP was present in WT Hck immunoprecipitates from the co-infected cells, indicative of Hck.GAP complex formation. Unexpectedly, GAP also associated with the kinase-inactive mutant of Hck, suggesting that tyrosine autophosphorylation of Hck is not required for complex formation. The WT and K269E forms of Hck also associated with GAP mutants lacking either the C-terminal catalytic domain (delta CAT) or the Src homology region (delta SH), indicating that these GAP domains are dispensable for complex formation. Recombinant GST fusion proteins containing the Hck, Src, Fyn, or Lck SH3 domains associated with full-length GAP, delta CAT, and delta SH, all of which share an N-terminal proline-rich region resembling an SH3-binding motif (PPLPPPPPQLP). Deletion of the highly conserved YXY sequence from the Hck SH3 domain abolished binding. GAP-SH3 interaction was also inhibited by the proline-rich peptide GFPPLPPPPPQLPTLG, which corresponds to N-terminal amino acids 129-144 of bovine GAP. An N-terminal deletion mutant of GAP lacking this proline-rich region did not bind to the Hck SH3 domain. These data implicate the Hck SH3 domain in GAP interaction, and suggest a general function for the SH3 domains of Src family kinases in recognition of GAP via its proline-rich N-terminal domain.

pp60c-src binding to polyomavirus middle T-antigen (MT) requires residues 185 to 210 of the MT sequence.

Brewster CE, Glover HR, Dilworth SM
J Virol. 1997; 71: 5512-20

Interaction with the src family of tyrosine kinases is crucial to the transforming action of polyomavirus middle T-antigen (MT). Association with MT activates the tyrosine kinase activity of pp60(c-src) and, through subsequent MT phosphorylation, creates binding sites for signalling molecules whose stimulation culminates in cell transformation. Despite this importance, and many studies, little is known of the mechanisms by which pp60(c-src) binds to MT. We report here isolation of the first MT mutants that disrupt pp60(c-src) binding without affecting the interaction between MT and protein phosphatase 2A (PP2A). Through deletion analysis we established that interaction with pp60(c-src) requires the sequences between amino acids 185 and 210 of MT, but these residues have no effect on PP2A binding. Cells expressing these mutants showed few altered properties, indicating that the PP2A-MT interaction alone has little influence on cell phenotype. Subcellular location of these mutant MT molecules was indistinguishable by immunofluorescence analysis from that of wild-type MT but was altered markedly on loss of PP2A binding. This suggests a possible role for PP2A in specifying subcellular distribution.

Structure of a specific peptide complex of the carboxy-terminal SH2 domain from the p85 alpha subunit of phosphatidylinositol 3-kinase.

Breeze AL, Kara BV, Barratt DG, Anderson M, Smith JC, Luke RW, Best JR, Cartlidge SA
EMBO J. 1996; 15: 3579-89

We have determined the solution structure of the C-terminal SH2 domain of the p85 alpha subunit of human phosphatidylinositol (PI) 3-kinase (EC 2.7.1.137) in complex with a phosphorylated tyrosine pentapeptide sequence from the platelet-derived growth factor receptor using heteronuclear nuclear magnetic resonance spectroscopy. Overall, the structure is similar to other SH2 domain complexes, but displays different detail interactions within the phosphotyrosine binding site and in the recognition site for the +3 methionine residue of the peptide, the side chain of which inserts into a particularly deep and narrow pocket which is displaced relative to that of other SH2 domains. The contacts made within this +3 pocket provide the structural basis for the strong selection for methionine at this position which characterizes the SH2 domains of PI3-kinase. Comparison with spectral and structural features of the uncomplexed domain shows that the long BG loop becomes less mobile in the presence of the bound peptide. In contrast, extreme resonance broadening encountered for most residues in the beta D', beta E and beta F strands and associated connecting loops of the domain in the absence of peptide persists in the complex, implying conformational averaging in this part of the molecule on a microsecond-to-millisecond time scale.

A specific intermolecular association between the regulatory domains of a Tec family kinase.

Brazin KN, Fulton DB, Andreotti AH
J Mol Biol. 2000; 302: 607-23

Interleukin-2 tyrosine kinase (Itk), is a T-cell specific tyrosine kinase of the Tec family. We have examined a novel intermolecular interaction between the SH3 and SH2 domains of Itk. In addition to the interaction between the isolated domains, we have found that the dual SH3/SH2 domain-containing fragment of Itk self-associates in a specific manner in solution. Tec family members contain the SH3, SH2 and catalytic domains common to many kinase families but are distinguished by a unique amino-terminal sequence, which contains a proline-rich stretch. Previous work has identified an intramolecular regulatory association between the proline-rich region and the adjacent SH3 domain of Itk. The intermolecular interaction between the SH3 and SH2 domains of Itk that we describe provides a possible mechanism for displacement of this intramolecular regulatory sequence, a step that may be required for full Tec kinase activation. Additionally, localization of the interacting surfaces on both the SH3 and SH2 domains by chemical shift mapping has provided information about the molecular details of this recognition event. The interaction involves the conserved aromatic binding pocket of the SH3 domain and a newly defined binding surface on the SH2 domain. The interacting residues on the SH2 domain do not conform to the consensus motif for an SH3 proline-rich ligand. Interestingly, we note a striking correlation between the SH2 residues that mediate this interaction and those residues that, when mutated in the Tec family member Btk, cause the hereditary immune disorder, X-linked agamaglobulinemia.

Characterization of the human CSK locus.

Brauninger A, Karn T, Strebhardt K, Rubsamen-Waigmann H
Oncogene. 1993; 8: 1365-9

The CSK-gene encodes an intracellular protein-tyrosine kinase (PTK). In contrast to members of the src-family, an autophosphorylation site corresponding to Tyr416, as well as the equivalent of the regulatory Tyr527 in p60c-src are missing in the amino acid sequence deduced from the gene. CSK phosphorylates other members of the src-family of tyrosine kinases at their regulatory carboxy-terminus. By regulating the activity of these kinases, CSK may play an important role in cell growth and development. Here we describe the structure of the human CSK gene. The entire coding region spans a genomic distance of only 4.9 kb. It encompasses 12 exons ranging between 66 and 220 bp in size. The introns between coding exons vary between 76 and 920 bp in length. An exon coding for the 5'-untranslated region of CSK is separated from the first coding exon by an intron of more than 6400 bp. Based on comparisons of sequence homologies within the catalytic domains, the intracellular PTKs are divided into the src-family, the fes/fer- and the abl/arg-group. The genomic structure of four members of the SRC-family revealed nearly identical exon/intron boundaries within the catalytic domain of this family. They differ from those described for FES. Comparing the genomic structure of CSK with the exon/intron organisation of both, it is obvious that the exon/intron boundaries are in common either with those of the SRC-type or the FES boundaries. This intermediate exon/intron structure of CSK between FES and the SRC-family agrees with the position of CSK in a phylogenetic tree based on sequence homology within the kinase domain.

SH3-SPOT: an algorithm to predict preferred ligands to different members of the SH3 gene family.

Brannetti B, Via A, Cestra G, Cesareni G, Helmer-Citterich M
J Mol Biol. 2000; 298: 313-28

We have developed a procedure to predict the peptide binding specificity of an SH3 domain from its sequence. The procedure utilizes information extracted from position-specific contacts derived from six SH3/peptide or SH3/protein complexes of known structure. The framework of SH3/peptide contacts defined on the structure of the complexes is used to build a residue-residue interaction database derived from ligands obtained by panning peptide libraries displayed on filamentous phage.The SH3-specific interaction database is a multidimensional array containing frequencies of position-specific contacts. As input, SH3-SPOT requires the sequence of an SH3 domain and of a query decapeptide ligand. The array, that we call the SH3-specific matrix, is then used to evaluate the probability that the peptide would bind the given SH3 domain. This procedure is fast enough to be applied to the entire protein sequence database.Panning experiments were performed to search putative specific ligands of different SH3 domains in a database of decapeptides, or in a database of protein sequences. The procedure ranked some of the natural partners of interaction of a number of SH3 domains among the best ligands of the approximately 5. 6x10(9) different decapeptides in the SWISSPROT database. We expect the predictive power of the method to increase with the enrichment of the SH3-specific matrix by interaction data derived from new complex structures or from the characterization of new ligands. The procedure was developed using the SH3 domain family as test case but its application can easily be extended to other families of protein domains (such as, SH2, MHC, EH, PDZ, etc.).

Circular dichroic investigation of the native and non-native conformational states of the growth factor receptor-binding protein 2 N-terminal src homology domain 3: effect of binding to a proline-rich peptide from guanine nucleotide exchange factor.

Bousquet JA, Garbay C, Roques BP, Mely Y
Biochemistry. 2000; 39: 7722-35

SH3 (src homology domain 3) domains are small protein modules that interact with proline-rich peptides. The structure of the N-terminal SH3 domain from growth factor receptor-binding protein 2 (Grb2), an adapter protein in the intracellular signaling pathway to Ras, was investigated by circular dichroic (CD) spectroscopy. The compact native beta-barrel conformation, previously elucidated by NMR spectroscopy, was largely predominant at pH = 4.8, in the absence of salt. From the structural changes induced by varying pH, ionic strength, temperature, or hydrophobicity of the environment, evidence for the existence of distinct nonnative conformations was obtained in the far- and near-UV domains. Along the free energy scale, these appear to distribute into two conformational ensembles, depending on the extent of structural and thermodynamic differences compared to the native conformation. The first ensemble consists of non-native conformations with a nativelike secondary structure, and the second is composed of partially unfolded conformations having short alpha-helical fragments or turnlike motifs in their nonnative secondary structure. Most of the observed nonnative conformations exist in mild or nondenaturing conditions. They probably have distinct compactness of their inner structure, depending on the strength of nonlocal interactions, but only the native all-beta conformation possesses a condensed protein exterior, appropriate for the binding to the VPPPVPPRRR decapeptide from Sos. Upon binding, the native conformation undergoes a local tertiary structure change in a hydrophobic pocket at the binding site. This is accompanied by the PP-II helix folding of the proline-rich peptide. Interestingly, in the near-UV domain, a significant change in the spectral contribution of an aromatic exciton was observed, thus allowing quantitative tracking of the binding process.

Crystal structure of the cell cycle-regulatory protein suc1 reveals a beta-hinge conformational switch.

Bourne Y, Arvai AS, Bernstein SL, Watson MH, Reed SI, Endicott JE, Noble ME, Johnson LN, Tainer JA
Proc Natl Acad Sci U S A. 1995; 92: 10232-6

The Schizosaccharomyces pombe cell cycle-regulatory protein suc1, named as the suppressor of cdc2 temperature-sensitive mutations, is essential for cell cycle progression. To understand suc1 structure-function relationships and to help resolve conflicting interpretations of suc1 function based on genetic studies of suc1 and its functional homologs in both lower and higher eukaryotes, we have determined the crystal structure of the beta-interchanged suc1 dimer. Each domain consists of three alpha-helices and a four-stranded beta-sheet, completed by the interchange of terminal beta-strands between the two subunits. This beta-interchanged suc1 dimer, when compared with the beta-hairpin single-domain folds of suc1, reveals a beta-hinge motif formed by the conserved amino acid sequence HVPEPH. This beta-hinge mediates the subunit conformation and assembly of suc1: closing produces the intrasubunit beta-hairpin and single-domain fold, whereas opening leads to the intersubunit beta-strand interchange and interlocked dimer assembly reported here. This conformational switch markedly changes the surface accessibility of sequence-conserved residues available for recognition of cyclin-dependent kinase, suggesting a structural mechanism for beta-hinge-mediated regulation of suc1 biological function. Thus, suc1 belongs to the family of domain-swapping proteins, consisting of intertwined and dimeric protein structures in which the dual assembly modes regulate their function.

Recombinant Csk expressed in Escherichia coli is autophosphorylated on tyrosine residue(s).

Bougeret C, Rothhut B, Jullien P, Fischer S, Benarous R
Oncogene. 1993; 8: 1241-7

The C-terminal src kinase (Csk) is responsible for the phosphorylation of the carboxy-terminal tyrosine of several tyrosine kinases of the Src family. This phosphorylation site has a negative regulatory function. Csk is unique among the members of the protein tyrosine kinase family because it lacks the conserved tyrosine autophosphorylation site and has been thought to be devoid of autophosphorylation activity. Using the glutathione S-transferase (GST) bacterial expression system, we have produced large amounts of a chimeric rat Csk protein. We have determined that the GST-Csk fusion protein isolated from bacteria is autophosphorylated on tyrosine residue(s). GST-Csk and purified Csk are capable of undergoing autophosphorylation on tyrosine residue(s) in vitro. The GST-Csk fusion protein also phosphorylates exogenous substrates, including the heteropolymer poly-Glu/Tyr and enolase. This is the first indication that Csk is autophosphorylated on tyrosine residue(s) both in vivo in bacteria expressing Csk cDNA and in vitro. These findings suggest that the autophosphorylation of Csk might play a role in the regulation of its kinase activity as well as its binding to other cellular proteins.

Progesterone Receptor Contains a Proline-Rich Motif that Directly Interacts with SH3 Domains and Activates c-Src Family Tyrosine Kinases.

Boonyaratanakornkit V, Scott MP, Ribon V, Sherman L, Anderson SM, Maller JL, Miller WT, Edwards DP
Mol Cell. 2001; 8: 269-80

Steroid hormones have rapid nongenomic effects on cell-signaling pathways, but the receptor mechanisms responsible for this are not understood. We have identified a specific polyproline motif in the amino-terminal domain of conventional progesterone receptor (PR) that mediates direct progestin-dependent interaction of PR with SH3 domains of various cytoplasmic signaling molecules, including c-Src tyrosine kinases. Through this interaction, PR is a potent activator of Src kinases working by an SH3 domain displacement mechanism. By mutagenesis, we also show that rapid progestin-induced activation of Src and downstream MAP kinase in mammalian cells is dependent on PR-SH3 domain interaction, but not on the transcriptional activity of PR. Preliminary evidence for the biological significance of this PR signaling pathway through regulatory SH3 domains was shown with respect to an influence on progestin-induced growth arrest of breast epithelial cells and induction of Xenopus oocyte maturation.

X-Ray structure of citrate bound to Src SH2 leads to a high-affinity, bone-targeted Src SH2 inhibitor.

Bohacek RS, Dalgarno DC, Hatada M, Jacobsen VA, Lynch BA, Macek KJ, Merry T, Metcalf CA 3rd, Narula SS, Sawyer TK, Shakespeare WC, Violette SM, Weigele M
J Med Chem. 2001; 44: 660-3

Correlation of the phosphorylation states of pp60c-src with tyrosine kinase activity: the intramolecular pY530-SH2 complex retains significant activity if Y419 is phosphorylated.

Boerner RJ, Kassel DB, Barker SC, Ellis B, DeLacy P, Knight WB
Biochemistry. 1996; 35: 9519-25

Rapid digestion of pp60c-src tyrosine kinase (src TK) in combination with electrospray ionization mass spectrometry enabled the determination of the time course for autophosphorylation of three tyrosine sites (Y338, Y419, and Y530) and a correlation with src TK activity. A form of src TK was purified from baculovirus-infected cells which contains only Y338 partially phosphorylated. Incubation with MgATP increases the phosphorylation of all three sites. The autophosphorylation and dephosphorylation of Y419 are directly correlated with the level of src TK activity. The role of Y338 phosphorylation is unknown. Conditions resulting in complete autophosphorylation of Y530 were identified by electrospray ionization mass spectrometry. Surface plasmon resonance detection and size exclusion chromatography provide direct evidence for an intramolecular pY530-SH2 complex, supporting previous models [Matsuda, M., Mayer, B.J., Fukui, Y., & Hanafusa, H. (1990) Science 248, 1537-1539]. Contrary to these models, when the enzyme is fully phosphorylated on Y530, phosphorylated on Y419, and present only as the intramolecular pY530-SH2 complex, 20% of the kinase activity is retained. In addition, the k(m)'s for substrates are unaffected. Disruption of the pY530-SH2 interaction and activation of kinase activity by a high-affinity SH2 ligand yield a Kactivation which is 200-fold larger than the Kd for ligand binding to the uncomplexed src SH2 domain. These data suggest a Keq of 200 (unitless) for the intramolecular association of pY530 with the SH2 domain. We propose that the pY530-SH2 interaction modulates signal transduction by down-regulating src TK activity 5-fold, and perhaps more importantly by inhibiting protein-protein interactions with the SH2 domain. These results have significant implications relative to the development of SH2 ligands as therapeutics to control aberrant signal transduction. These ligands will be 200-fold less effective at inhibiting protein-protein interactions versus down-regulated src TK than versus activated src TK. This should minimize activation of src TK activity in normal cells and lead to an increased therapeutic index.

Catalytic activity of the SH2 domain of human pp60c-src; evidence from NMR, mass spectrometry, site-directed mutagenesis and kinetic studies for an inherent phosphatase activity.

Boerner RJ, Consler TG, Gampe RT Jr, Weigl D, Willard DH, Davis DG, Edison AM, Loganzo F Jr, Kassel DB, Xu RX, et al.
Biochemistry. 1995; 34: 15351-8

During solution structural studies it was apparent that the human recombinant pp60c-src SH2 domain (srcSH2, residues 144-249) possessed an inherent phosphatase (Pase) activity. Complexes of U[13C,15N]srcSH2 with unlabeled Ac-pYEEIE (I) were examined using 31P and 1H-detected isotope filtered NMR methods. The presence of a high-affinity complex in equimolar solutions of I and U[13C, 15N]-srcSH2 was demonstrated by chemical shift perturbations, line broadening, and the observation of intermolecular nuclear Overhauser effects from the pY and Ile side-chain protons of I to protons on amino acid residues present in the binding pocket of srcSH2. Solutions containing excess I relative to srcSH2 revealed a slow hydrolysis of I to produce Ac-YEEIE and inorganic phosphate. The hydrolysis rate determined from NMR and HPLC-electrospray ionization mass spectrometry data at 30 degrees C for solutions containing excess I was 0.002-0.003 h-1. srcSH2 also catalyzed the hydrolysis of p-nitrophenyl phosphate (pNPP). Isoelectric focusing gels of a number of mutant srcSH2s demonstrated that this activity comigrated with srcSH2. Km, kcat, and kcat/Km were 3.7 +/- 0.4 mM, 3.1 +/- 0.2 x 10(-2) min-1, and 8.4 +/- 0.4 M-1 min-1, respectively, toward pNPP. The C188A mutant of the srcSH2 domain displayed 15% of the activity displayed by wild-type srcSH2, demonstrating that this residue is not absolutely required for activity. Two additional mutations in the known pY binding site, R178K and R158K, also resulted in decreased pNPPase activity, suggesting that the activity resides in or near this site. The inhibitor profile and pH dependence suggest that this is a novel protein Pase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

1H and 15N NMR assignment and solution structure of the SH3 domain of spectrin: comparison of unrefined and refined structure sets with the crystal structure.

Blanco FJ, Ortiz AR, Serrano L
J Biomol NMR. 1997; 9: 347-57

The assignment of the 1H and 15N nuclear magnetic resonance spectra of the Src-homology region 3 domain of chicken brain alpha-spectrin has been obtained. A set of solution structures has been determined from distance and dihedral angle restraints, which provide a reasonable representation of the protein structure in solution, as evaluated by a principal component analysis of the global pairwise root-mean-square deviation (rmsd) in a large set of structures consisting of the refined and unrefined solution structures and the crystal structure. The solution structure is well defined, with a lower degree of convergence between the structures in the loop regions than in the secondary structure elements. The average pairwise rmsd between the 15 refined solution structures is 0.71 +/- 0.13 A for the backbone atoms and 1.43 +/- 0.14 A for all heavy atoms. The solution structure is basically the same as the crystal structure. The average rmsd between the 15 refined solution structures and the crystal structure is 0.76 A for the backbone atoms and 1.45 +/- 0.09 A for all heavy atoms. There are, however, small differences probably caused by intermolecular contacts in the crystal structure.

Characterization of two activated mutants of human pp60c-src that escape c-Src kinase regulation by distinct mechanisms.

Bjorge JD, Bellagamba C, Cheng HC, Tanaka A, Wang JH, Fujita DJ
J Biol Chem. 1995; 270: 24222-8

Two activated transforming mutants of human pp60c-src were found to possess single point mutations within the regulatory carboxyl terminus (E527K in CY CST201) and the kinase domain (E381G in WO CST1), respectively, that do not directly interfere with either the regulatory c-Src kinase (CSK) phosphorylation site (Tyr530) or the SH2/3 domains. In vivo, both mutant proteins are hypophosphorylated on their carboxyl-terminal regulatory tyrosines and are hyperactive. In an in vitro Src kinase inactivation assay, both mutant Src proteins exhibited resistance to inactivation by CSK relative to wild-type Src. Under these in vitro conditions, E381G c-Src was found to be phosphorylated by CSK to wild-type levels, while E527K c-Src was not detectably phosphorylated. The ability of CSK to phosphorylate a carboxyl-terminal peptide modelled against E527K c-Src was also impaired, suggesting that CSK is unable to recognize E527K c-Src as an efficient substrate. In the case of E381G c-Src, examination of whether its SH2/3 domains were accessible to the carboxyl-terminal regulatory phosphotyrosine revealed a highly reduced ability of autophosphorylated E381G c-Src to bind to a synthetic phosphopeptide modelled from the SH2-binding region of polyoma middle-T antigen which binds to Src SH2 with high affinity. This suggests that the E381G c-Src mutation results in an altered or reduced accessibility of the SH2 domain of the autophosphorylated form of E381G c-Src and may represent a previously undescribed mode of Src activation. Further study of these and other Src mutants may offer additional new insights into the regulation of "Src family" kinases.

SH2 and SH3-containing adaptor proteins: redundant or independent mediators of intracellular signal transduction.

Birge RB, Knudsen BS, Besser D, Hanafusa H
Genes Cells. 1996; 1: 595-613

Molecules which contain Src Homology 2 (SH2) and SH3 domains provide one of the principal ways by which signals are transduced in cells using protein-protein interactions between proline-rich motifs and SH3 domains and induced interactions between phosphotyrosine residues and SH2 domains. The simplest of SH2/SH3-containing proteins are the Crk, Grb2 and Nck adaptor proteins which contain SH2 and SH3 domains but no intrinsic catalytic activity. Whereas Grb2 connects activated receptor tyrosine kinases with Sos and activates p21ras, recent evidence suggests that this may not be the major mechanism by which Crk and Nck signal to downstream effectors. Identification of novel binding partners for Crk, Grb2 and Nck indicate that these adaptor proteins control distinct aspects of tyrosine kinase signalling.

Closing in on SH2 specificity.

Birge RB, Hanafusa H
Science. 1993; 262: 1522-4

Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides.

Bibbins KB, Boeuf H, Varmus HE
Mol Cell Biol. 1993; 13: 7278-87

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent.(ABSTRACT TRUNCATED AT 400 WORDS)

On the Role of Spectrin and Spectrin SH3 Domain in Integrin-induced Signaling.

Bialkowska K, Fox JE
Cell Mol Biol Lett. 2001; 6: 189-189

Atomic resolution structure of a mutant of the spectrin SH3 domain.

Berisio R, Viguera A, Serrano L, Wilmanns M
Acta Crystallogr D Biol Crystallogr. 2001; 57: 337-40

The crystal structure of an alpha-spectrin Src-homology 3 (SH3) domain mutant has been refined at 1.12 A resolution. This X-ray structure is at the highest resolution achieved so far for an SH3 domain. The structure allows the identification of a complete set of specific interactions and is useful for the elucidation of relations between structure and pH-dependent thermodynamic stability in a series of SH3 domain mutants.

Predicted secondary structure for the Src homology 3 domain.

Benner SA, Cohen MA, Gerloff D
J Mol Biol. 1993; 229: 295-305

A de novo secondary structure prediction has been prepared for Src homology domain 3, in advance of any crystallographic information concerning any member of this interesting protein family. The prediction can be compared with a crystal structure that will be published in Nature on October 29, 1992. The prediction is based on analysis of a multiple alignment of homologous proteins. The patterns of variation and conservation of amino acids across the alignment allow the determination of surface and internal positions, which then allow the assignment of secondary structure. The prediction is quite different both in method and, in this case, result from predictions based on propensities (e.g. Garnier-Osgurthorpe-Robson) of particular amino acids to appear in particular types of secondary structure.

The COOH-terminal Src kinase Csk is a tumor antigen in human carcinoma.

Benistant C, Bourgaux JF, Chapuis H, Mottet N, Roche S, Bali JP
Cancer Res. 2001; 61: 1415-20

The cytoplasmic tyrosine kinase cSrc is involved in the regulation of many important cellular functions including cell growth and transformation, and its activity is down-regulated by phosphorylation of the Tyr530 residue by the COOH-terminal Src tyrosine kinase, Csk. Because cSrc was previously found overexpressed, activated, and in some cases mutated in carcinoma, we investigated whether it could act as a tumor antigen. We show that whereas no autoantibodies were found against cSrc or its relative Fyn, up to 20% of patients with carcinoma had high-affinity autoantibodies against Csk. Immunity mainly resulted from a secondary response, as indicated by the presence of IgG1 in the sera. Antibodies were linked to the cancer because they were not detected in healthy subjects nor in patients with unrelated diseases, and their levels decreased in the sera of patients after surgical resection. Furthermore, they behaved as early markers of epithelial transformation because they were present in sera of patients with early-stage tumors and precancerous lesions such as colorectal polyps and in sera of patients that were scored negative for other cancer serological markers (CEA, CA15-3, CA19-9, p53 antibodies). Finally the presence of these antibodies was attributed, at least in part, to a substantial elevation of Csk protein levels in the corresponding tumors. However a strong increase in Src activity was also observed in these tissues, which suggested that Csk cannot regulate Src-like activity in carcinoma. Taken together, these data demonstrate that Csk acts as an autoantigen, and the detection of anti-Csk antibodies may have potential diagnostic usefulness in the early detection and postoperative follow-up of patients with carcinoma.

Ligands for the tyrosine kinase p56lck SH2 domain: discovery of potent dipeptide derivatives with monocharged, nonhydrolyzable phosphate replacements.

Beaulieu PL, Cameron DR, Ferland JM, Gauthier J, Ghiro E, Gillard J, Gorys V, Poirier M, Rancourt J, Wernic D, Llinas-Brunet M, Betageri R, Cardozo M, Hickey ER, Ingraham R, Jakes S, Kabcenell A, Kirrane T, Lukas S, Patel U, Proudfoot J, Sharma R, Tong L, Moss N
J Med Chem. 1999; 42: 1757-66

p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

SH2 domain protein interaction and possibilities for pharmacological intervention.

Beattie J
Cell Signal. 1996; 8: 75-86

SH2 domain containing proteins play a key role in the process of intracellular transmission of signalling events initiated at the cell surface. As a pre-requisite in the fulfillment of this function, these proteins bind to a variety of phospho-tyrosine (pY) containing target molecules. Delineation of these binding sites as essentially short linear peptides (both structurally and functionally) has led to the suggestion that the activity of these signalling complexes may be manipulated by the development of relatively simple peptide reagents. This review examines the range of possibilities open on this approach and the extent to which positive results have already been realised.

In vitro characterization of major ligands for Src homology 2 domains derived from protein tyrosine kinases, from the adaptor protein SHC and from GTPase-activating protein in Ramos B cells.

Baumann G, Maier D, Freuler F, Tschopp C, Baudisch K, Wienands J
Eur J Immunol. 1994; 24: 1799-807

Antigen receptors of B lymphocytes transmit their activation signal to the cell interior by associating with and activation of specific non-receptor tyrosine kinases. Most of these kinases as well as other cytoplasmic effectors contain at least one Src homology 2 (SH2) domain, known to bind tyrosine-phosphorylated proteins. We examined the binding specificity of SH2 domains from different signaling molecules in B cells and found that each of the SH2 domains tested bound distinct subsets of stimulation-dependent phosphoproteins in vitro. SH2 domains from Src-like tyrosine kinases bound predominantly to the HS1 phosphoprotein. The tandem SH2 domains of the ZAP-70 tyrosine kinase bound to phosphorylated Ig-beta but only weakly to Ig-alpha. Also the SHC-derived SH2 domain formed complexes with the tyrosine-phosphorylated Ig-alpha/beta heterodimer, while the C- and N-terminal SH2 domains of GTPase-activating protein displayed completely different binding preferences. These results suggest that cytoplasmic effector molecules can be recruited to the activated B cell receptor in an SH2-phosphotyrosine-mediated manner. The data also provide a possible explanation for the notion that Ig-alpha and Ig-beta might couple to different biochemical pathways.

The N-terminal domain of Homer/Vesl is a new class II EVH1 domain.

Barzik M, Carl UD, Schubert WD, Frank R, Wehland J, Heinz DW
J Mol Biol. 2001; 309: 155-69

Cellular activities controlled by signal transduction processes such as cell motility and cell growth depend on the tightly regulated assembly of multiprotein complexes. Adapter proteins that specifically interact with their target proteins are key components required for the formation of these assemblies. Ena/VASP-homology 1 (EVH1) domains are small constituents of large modular proteins involved in microfilament assembly that specifically recognize proline-rich regions. EVH1 domain-containing proteins are present in neuronal cells, like the Homer/Vesl protein family that is involved in memory-generating processes. Here, we describe the crystal structure of the murine EVH1 domain of Vesl 2 at 2.2 A resolution. The small globular protein consists of a seven-stranded antiparallel beta-barrel with a C-terminal alpha-helix packing alongside the barrel. A shallow groove running parallel with beta-strand VI forms an extended peptide-binding site. Using peptide library screenings, we present data that demonstrate the high affinity of the Vesl 2 EVH1 domain towards peptide sequences containing a proline-rich core sequence (PPSPF) that requires additional charged amino acid residues on either side for specific binding. Our functional data, substantiated by structural data, demonstrate that the ligand-binding of the Vesl EVH1 domain differs from the interaction characteristics of the previously examined EVH1 domains of the Evl/Mena proteins. Analogous to the Src homology 3 (SH3) domains that bind their cognate ligands in two distinct directions, we therefore propose the existence of two distinct classes of EVH1 domains.

NH2-terminal sequence of macrophage-expressed natural resistance-associated macrophage protein (Nramp) encodes a proline/serine-rich putative Src homology 3-binding domain.

Barton CH, White JK, Roach TI, Blackwell JM
J Exp Med. 1994; 179: 1683-7

The Lsh/Ity/Bcg locus on mouse chromosome 1 regulates macrophage (m phi) priming/activation for antimicrobial activity against intracellular pathogens. A candidate Bcg gene, designated natural resistance-associated m phi protein (Nramp), recently isolated from a pre-B cell cDNA library encodes a polytopic integral membrane protein with structural features common to prokaryotic and eukaryotic transporters. In the present study, an activated m phi cDNA library yielded new Nramp clones that differ in the 5' region from the published pre-B cell-derived clone sequence, resulting in addition of 64 amino acids at the NH2 terminus of the predicted protein. This new domain is rich in proline, serine, and basic amino acids, and includes three protein kinase C phosphorylation sites and a putative Src homology 3 binding domain. RNAs containing this domain are the only form found in the m phi. Hence, the protein encoded by this RNA is the candidate molecule mediating natural resistance to intra-m phi pathogens.

The peroxisomal membrane protein Pex13p shows a novel mode of SH3 interaction.

Barnett P, Bottger G, Klein AT, Tabak HF, Distel B
EMBO J. 2000; 19: 6382-91

Src homology 3 (SH3) domains are small non-catalytic protein modules capable of mediating protein-protein interactions by binding to proline-X-X-proline (P-X-X-P) motifs. Here we demonstrate that the SH3 domain of the integral peroxisomal membrane protein Pex13p is able to bind two proteins, one of which, Pex5p, represents a novel non-P-X-X-P ligand. Using alanine scanning, two-hybrid and in vitro interaction analysis, we show that an alpha-helical element in Pex5p is necessary and sufficient for SH3 interaction. Sup pressor analysis using Pex5p mutants located in this alpha-helical element allowed the identification of a unique site of interaction for Pex5p on the Pex13p-SH3 domain that is distinct from the classical P-X-X-P binding pocket. On the basis of a structural model of the Pex13p-SH3 domain we show that this interaction probably takes place between the RT- and distal loops. Thus, the Pex13p-SH3-Pex5p interaction establishes a novel mode of SH3 interaction.

An intramolecular SH3-domain interaction regulates c-Abl activity.

Barila D, Superti-Furga G
Nat Genet. 1998; 18: 280-2

The ABL1 proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine kinase (c-Abl) that has been implicated in processes of cell differentiation, cell division, cell adhesion and stress response. Alterations of ABL1 by chromosomal rearrangement or viral transduction can lead to malignant transformation. Activity of the c-Abl protein is negatively regulated by its SH3 domain through an unknown mechanism, and deletion of the SH3 domain turns ABL1 into an oncogene. We present evidence for an intramolecular inhibitory interaction of the SH3 domain with the catalytic domain and with the linker between the SH2 and catalytic domain (SH2-CD linker). Site-directed mutations in each of these three elements activate c-Abl. Mutations in the linker cause a conformational change of the molecule and increase binding of the SH3 domain to peptide ligands. Individual mutation of two charged residues in the SH3 and catalytic domain activates c-Abl, while inhibition is restored in the double reciprocal mutant. We propose that regulators of c-Abl will have opposite effects on its activity depending on their ability to favour or disrupt these intramolecular interactions.

A nuclear tyrosine phosphorylation circuit: c-Jun as an activator and substrate of c-Abl and JNK.

Barila D, Mangano R, Gonfloni S, Kretzschmar J, Moro M, Bohmann D, Superti-Furga G
EMBO J. 2000; 19: 273-81

The nuclear function of the c-Abl tyrosine kinase is not well understood. In order to identify nuclear substrates of Abl, we constructed a constitutively active and nuclear form of the protein. We found that active nuclear Abl efficiently phosphorylate c-Jun, a transcription factor not previously known to be tyrosine phosphorylated. After phosphorylation of c-Jun by Abl on Tyr170, both proteins interacted via the SH2 domain of Abl. Surprisingly, elevated levels of c-Jun activated nuclear Abl, resulting in activation of the JNK serine/threonine kinase. This phosphorylation circuit generates nuclear tyrosine phosphorylation and represents a reversal of previously known signalling models.

Wiskott-Aldrich syndrome protein (WASp) is a binding partner for c-Src family protein-tyrosine kinases.

Banin S, Truong O, Katz DR, Waterfield MD, Brickell PM, Gout I
Curr Biol. 1996; 6: 981-8

BACKGROUND. Receptor-mediated signal transduction requires the assembly of multimeric complexes of signalling proteins, and a number of conserved protein domains, such as the SH2, SH3 and PH domains, are involved in mediating protein-protein interactions in such complexes. The identification of binding partners for these domains has added considerably to our understanding of signal-transduction pathways, and the purpose of this work was to identify SH3-binding proteins in haematopoietic cells. RESULTS. We performed affinity-chromatography experiments with a panel of GST-SH3 fusion proteins (composed of glutathione-S-transferase appended to various SH3 domains) to search for SH3-binding proteins in a human megakaryocytic cell line. Protein microsequencing identified one of the SH3-binding proteins as WASp, the protein that is defective in Wiskott-Aldrich syndrome (WAS) and isolated X-linked thrombocytopenia. WASp bound preferentially in vitro to SH3 domains from c-Src family kinases, and analysis of proteins expressed in insect cells using a baculovirus vector demonstrated a specific interaction between WASp and the Fyn protein-tyrosine kinase. Finally, in vivo experiments showed that WASp and Fyn physically associate in human haematopoietic cells. CONCLUSIONS. Haematopoietic cells from individuals with WAS exhibit defects in cell morphology and signal transduction, including reduced proliferation and tyrosine phosphorylation in response to stimulatory factors. Members of the c Src family of protein-tyrosine kinases, including Fyn, are involved in a range of signalling pathways - such as those regulating cytoskeletal structure - in both haematopoietic and non-haematopoietic cells. Our data suggest that binding of Fyn to WASp may be a critical event in such signalling pathways in haematopoietic cells.

Structural and functional studies of the intracellular tyrosine kinase MATK gene and its translated product.

Avraham S, Jiang S, Ota S, Fu Y, Deng B, Dowler LL, White RA, Avraham H
J Biol Chem. 1995; 270: 1833-42

We recently cloned the cDNA which encodes a novel megakaryocyte-associated tyrosine kinase termed MATK. In this study, we have cloned and characterized the human MATK gene as well as the murine homolog of human MATK cDNA and performed functional studies of its translated product. Comparison of the deduced amino acid sequences of human and murine MATK cDNAs revealed 85% homology, indicating that MATK is highly conserved in mouse and human. The human gene consists of 13 exons interrupted by 12 introns. The genetic units which encode the SH3 and SH2 domains are located on separate exons. The putative ATP binding site (GXGXXG) is localized on exon 7, and the entire catalytic domain is subdivided into seven exons (7-13). Somatic cell hybrid analysis indicated that human MATK gene is located on chromosome 19 while the murine Matk gene is located on chromosome 10. The immediate 5'-flanking region was highly rich in GC sequences, and potential cis-acting elements were identified including several SP1, GATA-1, APRE, and APRE1. Antisense oligonucleotides directed against MATK mRNA sequences significantly inhibited megakaryocyte progenitor proliferation. Functional studies indicated that MATK can phosphorylate the carboxyl-terminal conserved tyrosine of the Src protein. These results support the notion that MATK acts as a regulator of p60c-src in megakaryocytic cells and participates in the pathways regulating growth of cells of this lineage.

The role of the Src homology 3-Src homology 2 interface in the regulation of Src kinases.

Arold ST, Ulmer TS, Mulhern TD, Werner JM, Ladbury JE, Campbell ID, Noble ME
J Biol Chem. 2001; 276: 17199-205

The regulatory fragment of Src kinases, comprising Src homology (SH) 3 and SH2 domains, is responsible for controlled repression of kinase activity. We have used a multidisciplinary approach involving crystallography, NMR, and isothermal titration calorimetry to study the regulatory fragment of Fyn (FynSH32) and its interaction with a physiological activator: a fragment of focal adhesion kinase that contains both phosphotyrosine and polyproline motifs. Although flexible, the preferred disposition of SH3 and SH2 domains in FynSH32 resembles the inactive forms of Hck and Src, differing significantly from LckSH32. This difference, which results from variation in the SH3-SH2 linker sequences, will affect the potential of the regulatory fragments to repress kinase activity. This surprising result implies that the mechanism of repression of Src family members may vary, explaining functional distinctions between Fyn and Lck. The interaction between FynSH32 and focal adhesion kinase is restricted to the canonical SH3 and SH2 binding sites and does not affect the dynamic independence of the two domains. Consequently, the interaction shows no enhancement by an avidity effect. Such an interaction may have evolved to gain specificity through an extended recognition site while maintaining rapid dissociation after signaling.

Characterization and molecular basis of the oligomeric structure of HIV-1 nef protein.

Arold S, Hoh F, Domergue S, Birck C, Delsuc MA, Jullien M, Dumas C
Protein Sci. 2000; 9: 1137-48

The Nef protein of human immunodeficiency virus type I (HIV-1) is an important determinant for the onset of AIDS disease. The self-association properties of HIV-1 Nef are analyzed by chemical cross-linking, dynamic light scattering, equilibrium analytical ultracentrifugation, and NMR spectroscopy. The experimental data show that the HIV-1 Nef core domain forms stable homo-dimers and trimers in solution, but not higher oligomers. These Nef homomers are not covalently linked by disulfide bridges, and the equilibrium between these forms is dependent on the Nef concentration. We further provide the molecular basis for the Nef core dimers and trimers obtained by analysis of crystallographic models. Oligomerization of biological polypeptides is a common tool used to trigger events in cellular signaling and endocytosis, both of which are targeted by Nef. The quaternary structure of Nef may be of physiological importance and may help to connect its cellular targets or to increase affinity of the viral molecule for its ligands. The herein described models for Nef dimers and trimers will allow further mutational studies to elucidate their role in vivo. These results provide novel insight into the structural and functional relationships of this important viral protein. Moreover, the oligomer interface may represent a novel target for the design of antiviral agents.

The c-src tyrosine kinase (CSK) gene, a potential antioncogene, localizes to human chromosome region 15q23----q25.

Armstrong E, Cannizzaro L, Bergman M, Huebner K, Alitalo K
Cytogenet Cell Genet. 1992; 60: 119-20

We have previously reported the cloning of a novel cytoplasmic tyrosine kinase, CSK. This tyrosine kinase has been shown to downregulate the tyrosine kinase activity of the c-src oncoprotein through tyrosine phosphorylation of the c-src carboxyl terminus. Cell transformation by src oncoproteins is caused by several oncogenic mechanisms, which interfere with this phosphorylation. The CSK gene could therefore potentially function as an antioncogene. We have here mapped the CSK gene to 15q23----q25 by in situ hybridization.

ConSurf: an algorithmic tool for the identification of functional regions in proteins by surface mapping of phylogenetic information.

Armon A, Graur D, Ben-Tal N
J Mol Biol. 2001; 307: 447-63

Experimental approaches for the identification of functionally important regions on the surface of a protein involve mutagenesis, in which exposed residues are replaced one after another while the change in binding to other proteins or changes in activity are recorded. However, practical considerations limit the use of these methods to small-scale studies, precluding a full mapping of all the functionally important residues on the surface of a protein. We present here an alternative approach involving the use of evolutionary data in the form of multiple-sequence alignment for a protein family to identify hot spots and surface patches that are likely to be in contact with other proteins, domains, peptides, DNA, RNA or ligands. The underlying assumption in this approach is that key residues that are important for binding should be conserved throughout evolution, just like residues that are crucial for maintaining the protein fold, i.e. buried residues. A main limitation in the implementation of this approach is that the sequence space of a protein family may be unevenly sampled, e.g. mammals may be overly represented. Thus, a seemingly conserved position in the alignment may reflect a taxonomically uneven sampling, rather than being indicative of structural or functional importance. To avoid this problem, we present here a novel methodology based on evolutionary relations among proteins as revealed by inferred phylogenetic trees, and demonstrate its capabilities for mapping binding sites in SH2 and PTB signaling domains. A computer program that implements these ideas is available freely at: http://ashtoret.tau.ac.il/ approximately rony Copyright 2001 Academic Press.

Chromosomal localization of the mouse Src-like adapter protein (Slap) gene and its putative human homolog SLA.

Angrist M, Wells DE, Chakravarti A, Pandey A
Genomics. 1995; 30: 623-5

Molecules containing Src-homology 2 (SH2) and Src-homology 3 (SH3) domains are critical components of signal transduction pathways that serve to relay signals originating from the cell surface to the interior of the cell. Src-like adapter protein (SLAP) is a recently described adapter protein that binds activated the Eck receptor protein-tyrosine kinase. Although SLAP bears a striking homology to the SH3 and SH2 domains of the Src family of nonreceptor tyrosine kinases, it does not contain a tyrosine kinase catalytic domain. In this report, the Slap gene was mapped by linkage analysis to mouse chromosome 15, while its putative human homolog (SLA) was identified and mapped to human 8q22.3-qter using a panel of somatic cell hybrids.

Computer-assisted re-design of spectrin SH3 residue clusters.

Angrand I, Serrano L, Lacroix E
Biomol Eng. 2001; 18: 125-34

We have developed a protein design computer program, called Perla, which performs searches in sequence space to uncover optimal amino acid sequences for desired protein three-dimensional structures. Optimal sequences are localised at the minima of a sequence-structure energy landscape defined using a complex scoring function (an all-atom molecular mechanics force field plus statistical terms including entropy and solvation) measured with respect to a reference state simulating a denatured protein. Sequence choices eventually optimise side chain packing, secondary structure propensities, and hydrogen bonding and electrostatics interactions. Perla was used to re-design clusters of residues of the SH3 domain of alpha-spectrin. Several mutant proteins were produced and characterised. Some of our designed proteins have significantly higher stabilities (stability enhancements about 0.25, 0.70 and 1.0 kcal mol(-1)) than the wild-type protein. These successful protein re-designs, and similar examples found in the literature, establish the quality of the structure-based computational approach to protein design.

Regulatory intramolecular association in a tyrosine kinase of the Tec family.

Andreotti AH, Bunnell SC, Feng S, Berg LJ, Schreiber SL
Nature. 1997; 385: 93-7

The T-cell-specific tyrosine kinase Itk is a member of the Tec family of non-receptor tyrosine kinases, and is required for signalling through the T-cell antigen receptor (TCR). The role of Itk in TCR signalling and the manner in which Itk activity is regulated are not well understood. Substrate binding and enzymatic activity of the structurally related Src kinases are regulated by an intramolecular interaction between the Src-homology-2 (SH2) domain and a phosphotyrosine. Although Itk also contains SH3, SH2 and tyrosine kinase domains, it lacks the corresponding regulatory phosphorylation site, and therefore must be regulated by an alternative mechanism. The proline-rich sequence adjacent to the SH3 domain of Tec family kinases contains an SH3 ligand, potentially allowing a different intramolecular interaction. By using multidimensional nuclear magnetic resonance we have determined the structure of a fragment of Itk, confirming that these domains interact intramolecularly. Formation of this intramolecular SH3-ligand complex prevents the Itk SH3 domain and proline-rich region from interacting with their respective protein ligands, Sam68 and Grb-2. We believe that this structure represents the first example of an intramolecular interaction between an SH3 domain and a proline-rich ligand, and has implications for the regulation of Tec family kinases.

Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src.

Anderson SK, Gibbs CP, Tanaka A, Kung HJ, Fujita DJ
Mol Cell Biol. 1985; 5: 1122-9

The nucleotide sequence of the 3' two-thirds of a highly conserved, molecularly cloned human cellular src gene (c-src) has been determined. This region of the c-src gene encodes the tyrosine kinase domain of the cellular src protein (pp60c-src) and corresponds to exons 6 through 12 of the chicken c-src gene, as well as nucleotides 545 to 1542 of the Rous sarcoma virus src gene (v-src). The human c-src sequence is very strongly conserved with respect to both the chicken c-src and the Rous sarcoma virus v-src genes, with nearly 90% nucleotide homology observed in this region. Amino acid sequence conservation in this region is even greater; 98% of the amino acids are conserved between human and chicken c-src. Furthermore, the exon sizes and the locations of the exon-intron boundaries are identical in the human and chicken c-src genes. However, sequences within the introns have not been conserved, and the introns within the human c-src gene are significantly larger than the corresponding introns within the chicken c-src gene. The strong amino acid conservation between the carboxy-terminal two-thirds of pp60c-src of species as divergent as humans and chickens suggests that this portion of the pp60c-src protein specifies one or more functional domains that are of great importance to some aspect of normal cellular growth or differentiation.

A potential SH3 domain-binding site in the Crk SH2 domain.

Anafi M, Rosen MK, Gish GD, Kay LE, Pawson T
J Biol Chem. 1996; 271: 21365-74

The Src homology 2 (SH2) domain of the mammalian adaptor protein Crk-II contains a proline-rich insert, predicted to lie within an extended DE loop, which is dispensable for phosphopeptide binding. Using the yeast two-hybrid system, this region of the Crk-II SH2 domain was found to interact with a subset of SH3 domains, notably the Abl SH3 domain. Furthermore, this proline-rich insert was found to modify the efficiency with which Crk-II was phosphorylated by the p140(c-abl) tyrosine kinase. In vitro, the interaction of full-length non-phosphorylated Crk-II with a glutathione S-transferase-Abl SH3 domain fusion protein was very weak. However, phosphorylation of Crk-II on Tyr-221 which induces an intramolecular association with the SH2 domain, or addition of a phosphopeptide corresponding to the Crk-II Tyr-221 phosphorylation site, stimulated association of Crk-II with the Abl SH3 domain. NMR spectroscopic analysis showed that binding of the Tyr-221 phosphopeptide to the Crk SH2 domain induced a chemical shift change in Val-71, located in the proline-rich insert, indicative of a change in the structure of the proline-rich loop in response of Crk SH2-pTyr-221 interaction. These results suggest that the proline-rich insert in the Crk SH2 domain constitutes an SH3 domain-binding site that can be regulated by binding of a phosphopeptide ligand to the Crk SH2 domain.

The formation of a covalent complex between a dipeptide ligand and the src SH2 domain.

Alligood KJ, Charifson PS, Crosby R, Consler TG, Feldman PL, Gampe RT Jr, Gilmer TM, Jordan SR, Milstead MW, Mohr C, Peel MR, Rocque W, Rodriguez M, Rusnak DW, Shewchuk LM, Sternbach DD
Bioorg Med Chem Lett. 1998; 8: 1189-94

The X-ray crystal structure of the src SH2 domain revealed the presence of a thiol residue (Cys 188) located proximal to the phosphotyrosine portion of a dipeptide ligand. An aldehyde bearing ligand (1) was designed to position an electrophilic carbonyl group in the vicinity of the thiol. X-ray crystallographic and NMR examination of the complex formed between (1) and the src SH2 domain revealed a hemithioacetal formed by addition of the thiol to the aldehyde group with an additional stabilizing hydrogen bond between the acetal hydroxyl and a backbone carbonyl.

Proline-rich sequences that bind to Src homology 3 domains with individual specificities.

Alexandropoulos K, Cheng G, Baltimore D
Proc Natl Acad Sci U S A. 1995; 92: 3110-4

To study the binding specificity of Src homology 3 (SH3) domains, we have screened a mouse embryonic expression library for peptide fragments that interact with them. Several clones were identified that express fragments of proteins which, through proline-rich binding sites, exhibit differential binding specificity to various SH3 domains. Src-SH3-specific binding uses a sequence of 7 aa of the consensus RPLPXXP, in which the N-terminal arginine is very important. The SH3 domains of the Src-related kinases Fyn, Lyn, and Hck bind to this sequence with the same affinity as that of the Src SH3. In contrast, a quite different proline-rich sequence from the Btk protein kinase binds to the Fyn, Lyn, and Hck SH3 domains, but not to the Src SH3. Specific binding of the Abl SH3 requires a longer, more proline-rich sequence but no arginine. One clone that binds to both Src and Abl SH3 domains through a common site exhibits reversed binding orientation, in that an arginine indispensable for binding to all tested SH3 domains occurs at the C terminus. Another clone contains overlapping yet distinct Src and Abl SH3 binding sites. Binding to the SH3 domains is mediated by a common PXXP amino acid sequence motif present on all ligands, and specificity comes about from other interactions, often ones involving arginine. The rules governing in vivo usage of particular sites by particular SH3 domains are not clear, but one binding orientation may be more specific than another.

Coordinate activation of c-Src by SH3- and SH2-binding sites on a novel p130Cas-related protein, Sin.

Alexandropoulos K, Baltimore D
Genes Dev. 1996; 10: 1341-55

To understand how protein-protein interactions mediated by the Src-SH3 domain affect c-Src signaling, we screened for proteins that interact with the Src-SH3. We found a novel protein, Sin (Src interacting or signal integrating protein), that binds to Src-SH3 with high affinity, contains numerous tyrosine residues in configurations suggestive of SH2-binding sites, and is related to the v-Src substrate p130Cas. In cotransfection assays, a small fragment of Sin retaining the Src-SH3-binding site and one tyrosine-containing motif induced c-Src activation as measured by a transcriptional reporter. Phosphorylation of the peptide on tyrosine by c-Src, as a consequence of Src-SH3 binding, was necessary for its stable interaction with c-Src in vivo and for transcriptional activation. Phosphorylation of multiple tyrosine-containing motifs found on Sin correlated with c-Crk and cellular phosphoprotein binding to Sin as well as increased c-Src activity. These data suggest that (1) SH2 and SH3 ligand sites on Sin cooperatively activate the signaling potential of c-Src, (2) Sin acts as both an activator and a substrate for c-Src, and (3) phosphorylated Sin may serve as a signaling effector molecule for Src by binding to multiple cellular proteins.

Molecular cloning of a novel protein-tyrosine phosphatase SH-PTP3 with sequence similarity to the src-homology region 2.

Adachi M, Sekiya M, Miyachi T, Matsuno K, Hinoda Y, Imai K, Yachi A
FEBS Lett. 1992; 314: 335-9

Protein-tyrosine phosphorylation and dephosphorylation are directly associated with cellular growth, signal transduction, and neoplastic transformation. Here we report the isolation of a complementary DNA (cDNA) clone encoding a novel protein-tyrosine phosphatase (PTP) from a human T cell PEER cDNA library. The predicted open reading frame encodes a approximately 68-kDa protein composed of 593 amino acids which contains two src-homology region 2's (SH2 domains) at the N terminus; this PTP is designated as SH-PTP3. Northern blot analysis revealed that SH-PTP3 mRNA was expressed throughout many tissues and the transcriptional size was consistent at about 6.0 kb. As with other SH2 domains in src-family kinases, the SH2 domains of SH-PTP3 may play a crucial role in interactions with tyrosine phosphorylated signaling proteins, including itself and protein tyrosine kinases (PTKs), to regulate targets' enzyme activity.

SH3 domains specifically regulate kinase activity of expressed Src family proteins.

Abrams CS, Zhao W
J Biol Chem. 1995; 270: 333-9

The Src homology 2 (SH2) and Src homology 3 (SH3) domain are approximately 50% conserved in various Src family kinase members. Several lines of evidence suggest that in Src these domains are sequence motifs that direct substrate recognition, regulate kinase activity, or control subcellular localization. We sought to investigate the function of the homology domains in human Lyn, and to determine whether the differences between various SH3 domains affect function. To do this, we generated variant forms of Lyn lacking SH2 and SH3 domains, and created chimeras in which the SH3 domains in human c-Src and Lyn were replaced with SH3 domains from other family members. In contrast to similar deletions in Src, forms of Lyn lacking SH2 or SH3 had decreased kinase activity. The SH3 chimeras all had individual characteristics. Insertion of the Blk SH3 domain into Lyn restored kinase activity, while insertion of the Fyn or Src SH3 into Lyn enhanced the kinase activity 2-3-fold. Insertion of the Lyn SH3 into Src also doubled kinase activity. Expression of the Lyn-Src SH3 chimera in mammalian cells induced cell transformation. This study 1) demonstrates that the regulation of Lyn is different than Src, and 2) provides new evidence that despite their homology, there are important functional differences between the SH3 domains of the various Src family members.

Phosphatidylinositol 3-kinase related kinases.

Abraham RT
Curr Opin Immunol. 1996; 8: 412-8

Studies in yeast, files and mammalian cells have uncovered a novel family of signal-transducing kinases which bear an evolutionary relationship to phosphatidylinositol 3-kinase. These phosphatidylinositol 3-kinase related enzymes play critical roles in DNA repair, V(D)J recombination and cell-cycle checkpoints, and their dysfunction leads to clinical manifestations ranging from immunodeficiency to cancer.