SMART MODE:

NORMAL GENOMIC

• Ariyoshi M, Schwabe JW
• A conserved structural motif reveals the essential transcriptional repression function of Spen proteins and their role in developmental signaling.
• Genes Dev. 2003; 17: 1909-20
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• Spen proteins regulate the expression of key transcriptional effectors in diverse signaling pathways. They are large proteins characterized by N-terminal RNA-binding motifs and a highly conserved C-terminal SPOC domain. The specific biological role of the SPOC domain (Spen paralog and ortholog C-terminal domain), and hence, the common function of Spen proteins, has been unclear to date. The Spen protein, SHARP (SMRT/HDAC1-associated repressor protein), was identified as a component of transcriptional repression complexes in both nuclear receptor and Notch/RBP-Jkappa signaling pathways. We have determined the 1.8 A crystal structure of the SPOC domain from SHARP. This structure shows that essentially all of the conserved surface residues map to a positively charged patch. Structure-based mutational analysis indicates that this conserved region is responsible for the interaction between SHARP and the universal transcriptional corepressor SMRT/NCoR (silencing mediator for retinoid and thyroid receptors/nuclear receptor corepressor. We demonstrate that this interaction involves a highly conserved acidic motif at the C terminus of SMRT/NCoR. These findings suggest that the conserved function of the SPOC domain is to mediate interaction with SMRT/NCoR corepressors, and that Spen proteins play an essential role in the repression complex.

• de Chiara C et al.
• The AXH module: an independently folded domain common to ataxin-1 and HBP1.
• FEBS Lett. 2003; 551: 107-12
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• Ataxin-1 (ATX1), a human protein responsible for spinocerebellar ataxia type 1 in humans, shares a region of homology, named AXH module, with the apparently unrelated transcription factor HBP1. Here, we describe the first characterisation of the AXH module in terms of its structural properties and stability. By producing protein constructs spanning the AXH modules of ATX1 and HBP1 and by comparing their properties, we have identified the minimal region sufficient for forming independently folded units (domains). Knowledge of the AXH domain boundaries allows us to map many of the interactions of ATX1 with other molecules onto the AXH module. We further show that the AXH of ATX1 is a dimerisation domain and is able to recognise RNA with the same nucleotide preference previously described for the full-length protein. AXH is therefore a novel protein-protein and RNA binding motif.

• De Rycker M, Venkatesan RN, Wei C, Price CM
• Vertebrate tankyrase domain structure and sterile alpha motif (SAM)-mediated multimerization.
• Biochem J. 2003; 372: 87-96
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• Tankyrases 1 and 2 are two highly related poly(ADP-ribose) polymerases that interact with a variety of cytoplasmic and nuclear proteins. Both proteins have been implicated in telomere length regulation, insulin signalling and centrosome function. To learn more about their mode of action, we have isolated the chicken tankyrase homologues and examined their interaction partners and subcellular location. Cross-species sequence comparison indicated that tankyrase domain structure is highly conserved and supports division of the ankyrin domain into five subdomains, which are each separated by a highly conserved LLEAAR/K motif. Glutathione S-transferase pull-down experiments demonstrated that the ankyrin domains of both proteins interact with chicken telomere repeat factor 1 (TRF1). Analysis of total cellular and nuclear proteins revealed that cells contain approximately twice as much tankyrase 1 as tankyrase 2. Although > or = 90% of each protein is present in the cytoplasm, both tankyrase 1 and 2 were detected in the nucleus. The nuclear location together with its ability to interact with TRF1, point to tankyrase 2 having a telomeric function. Yeast two-hybrid and cross-linking experiments show that both tankyrases can multimerize through their sterile-alpha motif domains. These results indicate that tankyrases may be master scaffolding proteins, capable of regulating assembly of large protein complexes.

• Cogswell C, Price SJ, Hou X, Guay-Woodford LM, Flaherty L, Bryda EC
• Positional cloning of jcpk/bpk locus of the mouse.
• Mamm Genome. 2003; 14: 242-9
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• By positional cloning techniques, we have identified the gene that is disrupted in the jcpk and bpk mouse models for polycystic kidney disease. This gene is the mouse homolog of the Drosophila Bicaudal C gene. Both of these mutations have been mapped to a very short stretch of Chromosome (Chr) 10. By sequencing the bicaudal C gene, Bicc1, in these models, it was found that the jcpk mutation results in a shortened and abnormal transcript, whereas the bpk mutation results in an abnormal 3' coding region. In Drosophila, this gene encodes a protein known to influence developmental processes. The mammalian homolog contains three KH (K homology) domains and a SAM (sterile alpha motif) domain and is expressed in the developing embryo, indicating that it may be important in RNA-binding and/or protein interactions during embryogenesis.

• Birck C, Chen Y, Hulett FM, Samama JP
• The crystal structure of the phosphorylation domain in PhoP reveals a functional tandem association mediated by an asymmetric interface.
• J Bacteriol. 2003; 185: 254-61
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• PhoP from Bacillus subtilis belongs to the OmpR subfamily of response regulators. It regulates the transcription of several operons and participates in a signal transduction network that controls adaptation of the bacteria to phosphate deficiency. The receiver domains of two members of this subfamily, PhoB from Escherichia coli and DrrD from Thermotoga maritima, have been structurally characterized. These modules have similar overall folds but display remarkable differences in the conformation of the beta4-alpha4 and alpha4 regions. The crystal structure of the receiver domain of PhoP (PhoPN) described in this paper illustrates yet another geometry in this region. Another major issue of the structure determination is the dimeric state of the protein and the novel mode of association between receiver domains. The protein-protein interface is provided by two different surfaces from each protomer, and the tandem unit formed through this asymmetric interface leaves free interaction surfaces. This design is well suited for further association of PhoP dimers to form oligomeric structures. The interprotein interface buries 970 A(2) from solvent and mostly involves interactions between charged residues. As described in the accompanying paper, mutations of a single residue in one salt bridge shielded from solvent prevented dimerization of the unphosphorylated and phosphorylated response regulator and had drastic functional consequences. The three structurally documented members of the OmpR family (PhoB, DrrD, and PhoP) provide a framework to consider possible relationships between structural features and sequence signatures in critical regions of the receiver domains.

• Hall TM
• SAM breaks its stereotype.
• Nat Struct Biol. 2003; 10: 677-9

• Kim CA, Gingery M, Pilpa RM, Bowie JU
• The SAM domain of polyhomeotic forms a helical polymer.
• Nat Struct Biol. 2002; 9: 453-7
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• The polycomb group (PcG) proteins are important in the maintenance of stable repression patterns during development. Several PcG members contain a protein protein interaction module called a SAM domain (also known as SPM, PNT and HLH). Here we report the high-resolution structure of the SAM domain of polyhomeotic (Ph). Ph-SAM forms a helical polymer structure, providing a likely mechanism for the extension of PcG complexes. The structure of the polymer resembles that formed by the SAM domain of another transcriptional repressor, TEL. The formation of these polymer structures by SAM domains in two divergent repressors suggests a conserved mode of repression involving a higher order chromatin structure.

• Green JB, Edwards TA, Trincao J, Escalante CR, Wharton RP, Aggarwal AK
• Crystallization and characterization of Smaug: a novel RNA-binding motif.
• Biochem Biophys Res Commun. 2002; 297: 1085-8
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• During Drosophila embryogenesis, Smaug protein represses translation of Nanos through an interaction with a specific element in its 3(')UTR. The repression occurs in the bulk cytoplasm of the embryo; Nanos is, however, successfully translated in the specialized cytoplasm of the posterior pole. This generates a gradient of Nanos emanating from the posterior pole that is essential for organizing proper abdominal segmentation. To understand the structural basis of RNA binding and translational control, we have crystallized a domain of Drosophila Smaug that binds RNA. The crystals belong to the space group R3 with unit cell dimensions of a=b=129.3A, c=33.1A, alpha=beta=90 degrees, gamma=120 degrees and diffract to 1.80A with synchrotron radiation. Initial characterization of this domain suggests that it encodes a novel RNA-binding motif.

• Wang X, McLachlan J, Zamore PD, Hall TM
• Modular recognition of RNA by a human pumilio-homology domain.
• Cell. 2002; 110: 501-12
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• Puf proteins are developmental regulators that control mRNA stability and translation by binding sequences in the 3' untranslated regions of their target mRNAs. We have determined the structure of the RNA binding domain of the human Puf protein, Pumilio1, bound to a high-affinity RNA ligand. The RNA binds the concave surface of the molecule, where each of the protein's eight repeats makes contacts with a different RNA base via three amino acid side chains at conserved positions. We have mutated these three side chains in one repeat, thereby altering the sequence specificity of Pumilio1. Thus, the high affinity and specificity of the PUM-HD for RNA is achieved using multiple copies of a simple repeated motif.

• Ramachander R et al.
• Oligomerization-dependent association of the SAM domains from Schizosaccharomyces pombe Byr2 and Ste4.
• J Biol Chem. 2002; 277: 39585-93
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• SAM (sterile alpha motif) domains are protein-protein interaction modules found in a large number of regulatory proteins. Byr2 and Ste4 are two SAM domain-containing proteins in the mating pheromone response pathway of the fission yeast, Schizosaccharomyces pombe. Byr2 is a mitogen-activated protein kinase kinase kinase that is regulated by Ste4. Tu et al. (Tu, H., Barr, M., Dong, D. L., and Wigler, M. (1997) Mol. Cell. Biol. 17, 5876-5887) showed that the isolated SAM domain of Byr2 binds a fragment of Ste4 that contains both a leucine zipper (Ste4-LZ) domain as well as a SAM domain, suggesting that Byr2-SAM and Ste4-SAM may form a hetero-oligomer. Here, we show that the individual SAM domains of Ste4 and Byr2 are monomeric at low concentrations and bind to each other in a 1:1 stoichiometry with a relatively weak dissociation constant of 56 +/- 3 microm. Inclusion of the Ste4-LZ domain, which determines the oligomeric state of Ste4, has a dramatic effect on binding affinity, however. We find that the Ste4-LZ domain is trimeric and, when included with the Ste4-SAM domain, yields a 3:1 Ste4-LZ-SAM:Byr2-SAM complex with a tight dissociation constant of 19 +/- 4 nm. These results suggest that the Ste4-LZ-SAM protein may recognize multiple binding sites on Byr2-SAM, indicating a new mode of oligomeric organization for SAM domains. The fact that high affinity binding occurs only with the addition of an oligomerization domain suggests that it may be necessary to include ancillary oligomerization modules when searching for binding partners of SAM domains.

• Pascal JM, Hart PJ, Hecht NB, Robertus JD
• Crystal structure of TB-RBP, a novel RNA-binding and regulating protein.
• J Mol Biol. 2002; 319: 1049-57
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• The testis/brain-RNA-binding protein (TB-RBP) spatially and temporally controls the expression of specific mRNAs in developing male germ cells and brain cells, and is implicated in DNA recombination and repair events. We report the 2.65 A crystal structure of mouse TB-RBP. The structure is predominantly alpha-helical and exhibits a novel protein fold and mode of assembly. Crystal symmetry and molecular symmetry combine to form an octet of TB-RBP monomers in the shape of an elongated spherical particle with a large cavity at its center. Amino acid residues that affect RNA and DNA binding are located on the interior surface of the assembled particle, and a putative nucleotide-binding domain that controls RNA binding is located at a dimer interface. Other modes of assembly are suggested for TB-RBP based on our structure and recently reported electron microscopic reconstructions of human TB-RBP.

• Mohrmann L, Kal AJ, Verrijzer CP
• Characterization of the extended Myb-like DNA-binding domain of trithorax group protein Zeste.
• J Biol Chem. 2002; 277: 47385-92
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• Zeste is a Drosophila sequence-specific DNA-binding protein that performs a variety of functions during chromatin-directed gene regulation. Its DNA-binding domain (DBD) was previously identified, but no similarities to established DNA-binding structures are known. Here we present sequence comparisons suggesting that the Zeste-DBD is a novel variant of the tri-helical Myb-DBD. Using band shift assays, we mapped the Zeste-DBD to 76 residues, corresponding to a single Myb repeat of only 50 residues. All residues involved in formation of the hydrophobic core of the Myb domain are conserved in Zeste, suggesting it forms an extended Myb domain. Mutagenesis studies determined (T/C/g)GAGTG(A/G/c) as the consensus Zeste recognition sequence. Reconstituted transcription experiments established that deviations from this optimal consensus compromise transcriptional activation by Zeste. In addition, flanking DNA is critical because Zeste-DBD binding requires a DNA sequence of minimally 16 base pairs, which is much longer than the consensus site. The DNA flanking the consensus is contacted by Zeste through sequence-independent backbone contacts. Interestingly, hydroxyl radical footprinting revealed that the Zeste-DNA backbone contacts all map to one face of the DNA. We compare the DNA-binding properties of Zeste with those of classical tri-helical DBDs harboring a helix-turn-helix motif and suggest a model for Zeste-DNA recognition.

• Hubbard SR
• Autoinhibitory mechanisms in receptor tyrosine kinases.
• Front Biosci. 2002; 7: 33040-33040
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• Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic tyrosine kinase catalytic activity in their cytoplasmic domains. RTKs are critical components in signal transduction pathways involved in cellular proliferation, differentiation, migration, and metabolism. This large protein family includes the receptors for many growth factors and for insulin. Ligand binding to the extracellular portion of these receptors results in receptor dimerization, which facilitates trans-autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues enhance receptor catalytic activity and/or provide docking sites for downstream signaling proteins. Because of the critical roles played by RTKs in cellular signaling processes, their catalytic activity is normally under tight control by intrinsic regulatory mechanisms as well as by protein tyrosine phosphatases. This review will focus on the autoinhibitory mechanisms that modulate RTK catalytic activity.

• Callebaut I
• An EVH1/WH1 domain as a key actor in TGFbeta signalling.
• FEBS Lett. 2002; 519: 178-80
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• EVH1 (enabled VASP (vasodilator-stimulated protein) homology 1)/WH1 (WASP (Wiskott-Aldrich syndrome protein) homology 1) domains, present in Ena VASP and WASP, are protein interaction modules specialised in binding proline-rich ligands. An EVH1/WH1 domain is here identified in the recently cloned SMIF protein, a key protein in transforming growth factor-beta (TGFbeta) signalling which was not yet related to defined domains. The SMIF EVH1/WH1 domain interacts with the proline-rich Smad4 activation domain, leading to translocation of so-formed complex to the nucleus where SMIF possesses strong intrinsic TGFbeta-inducible transcriptional activity. This finding highlights the pivotal role that the EVH1/WH1 family of domains play in multiple eukaryotic signal transduction pathways.

• Till JH et al.
• Crystal structure of the MuSK tyrosine kinase: insights into receptor autoregulation.
• Structure (Camb). 2002; 10: 1187-96
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• Muscle-specific kinase (MuSK) is a receptor tyrosine kinase expressed selectively in skeletal muscle. During neuromuscular synapse formation, agrin released from motor neurons stimulates MuSK autophosphorylation in the kinase activation loop and in the juxtamembrane region, leading to clustering of acetylcholine receptors. We have determined the crystal structure of the cytoplasmic domain of unphosphorylated MuSK at 2.05 A resolution. The structure reveals an autoinhibited kinase domain in which the activation loop obstructs ATP and substrate binding. Steady-state kinetic analysis demonstrates that autophosphorylation results in a 200-fold increase in k(cat) and a 10-fold decrease in the K(m) for ATP. These studies provide a molecular basis for understanding the regulation of MuSK catalytic activity and suggest that an additional in vivo component may contribute to regulation via the juxtamembrane region.

• Smith PC, Firestein S, Hunt JF
• The crystal structure of the olfactory marker protein at 2.3 A resolution.
• J Mol Biol. 2002; 319: 807-21
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• Olfactory marker protein (OMP) is a highly expressed and phylogenetically conserved cytoplasmic protein of unknown function found almost exclusively in mature olfactory sensory neurons. Electrophysiological studies of olfactory epithelia in OMP knock-out mice show strongly retarded recovery following odorant stimulation leading to an impaired response to pulsed odor stimulation. Although these studies show that OMP is a modulator of the olfactory signal-transduction cascade, its biochemical role is not established. In order to facilitate further studies on the molecular function of OMP, its crystal structure has been determined at 2.3 A resolution using multiwavelength anomalous diffraction experiments on selenium-labeled protein. OMP is observed to form a modified beta-clamshell structure with eight antiparallel beta-strands. While OMP has no significant sequence homology to proteins of known structure, it has a similar fold to a domain found in a variety of existing structures, including in a large family of viral capsid proteins. The surface of OMP is mostly convex and lacking obvious small molecule binding sites, suggesting that it is more likely to be involved in modulating protein-protein interaction than in interacting with small molecule ligands. Three highly conserved regions have been identified as leading candidates for protein-protein interaction sites in OMP. One of these sites represents a loop known to mediate ligand interactions in the structurally homologous EphB2 receptor ligand-binding domain. This site is partially buried in the crystal structure but fully exposed in the NMR solution structure of OMP due to a change in the orientation of an alpha-helix that projects outward from the structurally invariant beta-clamshell core. Gating of this conformational change by molecular interactions in the signal-transduction cascade could be used to control access to OMP's equivalent of the EphB2 ligand-interaction loop, thereby allowing OMP to function as a molecular switch.

• Duval M, Hsieh TF, Kim SY, Thomas TL
• Molecular characterization of AtNAM: a member of the Arabidopsis NAC domain superfamily.
• Plant Mol Biol. 2002; 50: 237-48
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• The petunia NAM and ArabidopsisATAF1 and CUC2 genes define the conserved NAC domain. In petunia, loss-of-function nam mutants result in embryos that fail to elaborate shoot apical meristems (SAM), and nam seedlings do not develop shoots and leaves. We have isolated a NAC domain gene, AtNAM, from an Arabidopsis developing seed cDNA library. Expression of AtNAM mRNA is restricted primarily to the region of the embryo including the SAM. The AtNAM gene contains three exons and is located on Chromosome 1. In vivo assays in yeast demonstrate that AtNAM encodes a transcription factor and that the NAC domain includes a specific DNA binding domain (DBD). The AtNAM DBD is contained within a 60 amino acid region which potentially folds into a helix-turn-helix motif that specifically binds to the CaMV 35S promoter. The putative transcriptional activation domain is located in the C-terminal region of the protein, a highly divergent region among NAC domain-containing genes. The Arabidopsis genome contains 90 predicted NAC domain genes; we refer to these collectively as the AtNAC superfamily. The first two exons of all members of this superfamily encode the NAC domain. Most AtNAC genes contain three exons with the last exon encoding an activation domain. A subfamily of AtNAC genes contains additional terminal exons coding for protein domains whose functions are unknown.

• Trivier E, Ganesan TS
• RYK, a catalytically inactive receptor tyrosine kinase, associates with EphB2 and EphB3 but does not interact with AF-6.
• J Biol Chem. 2002; 277: 23037-43
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• RYK is an atypical orphan receptor tyrosine kinase that lacks detectable kinase activity. Nevertheless, using a chimeric receptor approach, we previously found that RYK can signal via the mitogen-activated protein kinase pathway. Recently, it has been shown that murine Ryk can bind to and be phosphorylated by the ephrin receptors EphB2 and EphB3. In this study, we show that human RYK associates with EphB2 and EphB3 but is not phosphorylated by them. This association requires both the extracellular and cytoplasmic domains of RYK and is not dependent on activation of the Eph receptors. It was also previously shown that AF-6 (afadin), a PDZ domain-containing protein, associates with murine Ryk. We show here that AF-6 does not bind to human RYK in vitro or in vivo. This suggests that there are significant functional differences between human and murine RYK. Further studies are required to determine whether RYK modulates the signaling of EphB2 and EphB3.

• Stoilov P, Rafalska I, Stamm S
• YTH: a new domain in nuclear proteins.
• Trends Biochem Sci. 2002; 27: 495-7
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• A novel 100-150-residue domain has been identified in the human splicing factor YT521-B and its Drosophila and yeast homologues. Homology searches show that the domain is typical for the eukaryotes and is particularly abundant in plants. It is predicted to adopt a mixed alpha-helix-beta-sheet fold and to bind to RNA. We propose the name YTH (for YT521-B homology) for the domain.

• Wybenga-Groot LE, Baskin B, Ong SH, Tong J, Pawson T, Sicheri F
• Structural basis for autoinhibition of the Ephb2 receptor tyrosine kinase by the unphosphorylated juxtamembrane region.
• Cell. 2001; 106: 745-57
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• The Eph receptor tyrosine kinase family is regulated by autophosphorylation within the juxtamembrane region and the kinase activation segment. We have solved the X-ray crystal structure to 1.9 A resolution of an autoinhibited, unphosphorylated form of EphB2 comprised of the juxtamembrane region and the kinase domain. The structure, supported by mutagenesis data, reveals that the juxtamembrane segment adopts a helical conformation that distorts the small lobe of the kinase domain, and blocks the activation segment from attaining an activated conformation. Phosphorylation of conserved juxtamembrane tyrosines would relieve this autoinhibition by disturbing the association of the juxtamembrane segment with the kinase domain, while liberating phosphotyrosine sites for binding SH2 domains of target proteins. We propose that the autoinhibitory mechanism employed by EphB2 is a more general device through which receptor tyrosine kinases are controlled.

• Copeland PR, Stepanik VA, Driscoll DM
• Insight into mammalian selenocysteine insertion: domain structure and ribosome binding properties of Sec insertion sequence binding protein 2.
• Mol Cell Biol. 2001; 21: 1491-8
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• The cotranslational incorporation of the unusual amino acid selenocysteine (Sec) into both prokaryotic and eukaryotic proteins requires the recoding of a UGA stop codon as one specific for Sec. The recognition of UGA as Sec in mammalian selenoproteins requires a Sec insertion sequence (SECIS) element in the 3' untranslated region as well as the SECIS binding protein SBP2. Here we report a detailed analysis of SBP2 structure and function using truncation and site-directed mutagenesis. We have localized the RNA binding domain to a conserved region shared with several ribosomal proteins and eukaryotic translation termination release factor 1. We also identified a separate and novel functional domain N-terminal to the RNA binding domain which was required for Sec insertion but not for SECIS binding. Conversely, we showed that the RNA binding domain was necessary but not sufficient for Sec insertion and that the conserved glycine residue within this domain was required for SECIS binding. Using glycerol gradient sedimentation, we found that SBP2 was stably associated with the ribosomal fraction of cell lysates and that this interaction was not dependent on its SECIS binding activity. This interaction also occurred with purified components in vitro, and we present data which suggest that the SBP2-ribosome interaction occurs via 28S rRNA. SBP2 may, therefore, have a distinct function in selecting the ribosomes to be used for Sec insertion.

• Rudolph MG, Huang M, Teyton L, Wilson IA
• Crystal structure of an isolated V(alpha) domain of the 2C T-cell receptor.
• J Mol Biol. 2001; 314: 1-8
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• The T-cell receptor (TCR) is a heterodimeric cell-surface protein consisting of two chains, alpha and beta, each of which is composed of a variable (V) and a constant (C) domain. Crystals of the isolated V(alpha) domain of the murine TCR 2C were grown by serendipity from a solution containing the extracellular domains of the intact TCR 2C and CD3 gamma epsilon-chains. The V(alpha) crystal structure shows how crystal packing can substitute for another V(alpha) domain in a different fashion from that observed in V(alpha)/V(alpha) homodimer and V(alpha)/V(beta) heterodimer structures. Significant conformational changes occur in the CDR3 and beta(3)beta(4) loops that normally form part of the dimer interface. The monomeric V(alpha) domain provides the unique opportunity to study the effect of dimerization on the conformation of the unliganded complementarity-determining regions (CDR) of a TCR. This structure of an individual V(alpha) module has implications for stability and bioengineering of isolated antibody and immunoglobulin domains.

• Yu HH, Zisch AH, Dodelet VC, Pasquale EB
• Multiple signaling interactions of Abl and Arg kinases with the EphB2 receptor.
• Oncogene. 2001; 20: 3995-4006
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• The Eph family of receptor tyrosine kinases and the Abl family of non-receptor tyrosine kinases have both been implicated in tissue morphogenesis. They regulate the organization of the actin cytoskeleton in the developing nervous system and participate in signaling pathways involved in axon growth. Both Eph receptors and Abl are localized in the neuronal growth cone, suggesting that they play a role in axon pathfinding. Two-hybrid screens identified regions of Abl and Arg that bind to the EphB2 and EphA4 receptors, suggesting a novel signaling connection involving the two kinase families. The association of full-length Abl and Arg with EphB2 was confirmed by co-immunoprecipitation and found to involve several distinct protein interactions. The SH2 domains of Abl and Arg bind to tyrosine-phosphorylated motifs in the juxtamembrane region of EphB2. A second, phosphorylation-independent interaction with EphB2 involves non-conserved sequences in the C-terminal tails of Abl and Arg. A third interaction between Abl and EphB2 is probably mediated by an intermediary protein because it requires tyrosine phosphorylation of EphB2, but not the binding sites for the Abl SH2 domain. The connection between EphB2 and Abl/Arg appears to be reciprocal. Activated EphB2 causes tyrosine phosphorylation of Abl and Arg, and vice versa. Interestingly, treatment of COS cells and B35 neuronal-like cells with ephrin-B1 to activate endogenous EphB2 decreased the kinase activity of endogenous Abl. These data are consistent with the opposite effects that Eph receptors and Abl have on neurite ougrowth and suggest that Eph receptors and Abl family kinases have shared signaling activities.

• White EK, Moore-Jarrett T, Ruley HE
• PUM2, a novel murine puf protein, and its consensus RNA-binding site.
• RNA. 2001; 7: 1855-66
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• Members of the Puf family of RNA-binding proteins from Drosophila, Caenorhabditis elegans, and Dictyostelium are known to function as translational repressors. To identify mammalian proteins that might regulate posttranscriptional gene expression, we have characterized a novel murine Puf protein, PUM2. Pum2 transcripts were expressed in all murine tissues examined, suggesting the gene influences processes common to many cell types. Like all Puf family members, PUM2 contains a C-terminal RNA-binding domain related to the Drosophila Pumilio homology domain (PUM-HD). Two features found in the amino-terminus of PUM2, regions rich in serine and glutamine/alanine-rich regions, were also identified in most Puf family members. RNA sequences capable of binding with high affinity (6.5 nM) to a 48-kDa recombinant protein containing the PUM2 PUM-HD were isolated by using an iterative amplification-selection protocol (SELEX). The consensus sequence [UGUANAUARNNNNBBBBSCCS] of the PUM2 binding element (PBE) is related to, but distinct from, the 3' end of the Drosophila Nanos response element. The characterization of PUM2 and potential RNA-binding site will assist efforts to assess the extent and mechanism by which mammalian genes are regulated at a posttranscriptional level.

• Pautsch A, Zoephel A, Ahorn H, Spevak W, Hauptmann R, Nar H
• Crystal structure of bisphosphorylated IGF-1 receptor kinase: insight into domain movements upon kinase activation.
• Structure (Camb). 2001; 9: 955-65
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• BACKGROUND: The insulin-like growth-factor-1 (IGF-1) receptor, which is widely expressed in cells that have undergone oncogenic transformation, is emerging as a novel target in cancer therapy. IGF-1-induced receptor activation results in autophosphorylation of cytoplasmic kinase domains and enhances their capability to phosphorylate downstream substrates. Structures of the homologous insulin receptor kinase (IRK) exist in an open, unphosphorylated form and a closed, trisphosphorylated form. RESULTS: We have determined the 2.1 A crystal structure of the IGF-1 receptor protein tyrosine kinase domain phosphorylated at two tyrosine residues within the activation loop (IGF-1RK2P) and bound to an ATP analog. The ligand is not in a conformation compatible with phosphoryl transfer, and the activation loop is partially disordered. Compared to the homologous insulin receptor kinase, IGF-1RK2P is trapped in a half-closed, previously unobserved conformation. Observed domain movements can be dissected into two orthogonal rotational components. CONCLUSIONS: Conformational changes upon kinase activation are triggered by the degree of phosphorylation and are crucially dependent on the conformation of the proximal end of the kinase activation loop. This IGF-1RK structure will provide a molecular basis for the design of selective antioncogenic therapeutic agents.

• Kim CA et al.
• Polymerization of the SAM domain of TEL in leukemogenesis and transcriptional repression.
• EMBO J. 2001; 20: 4173-82
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• TEL is a transcriptional repressor that is a frequent target of chromosomal translocations in a large number of hematalogical malignancies. These rearrangements fuse a potent oligomerization module, the SAM domain of TEL, to a variety of tyrosine kinases or transcriptional regulatory proteins. The self-associating property of TEL-SAM is essential for cell transformation in many, if not all of these diseases. Here we show that the TEL-SAM domain forms a helical, head-to-tail polymeric structure held together by strong intermolecular contacts, providing the first clear demonstration that SAM domains can polymerize. Our results also suggest a mechanism by which SAM domains could mediate the spreading of transcriptional repression complexes along the chromosome.

• Xu Y, Seet LF, Hanson B, Hong W
• The Phox homology (PX) domain, a new player in phosphoinositide signalling.
• Biochem J. 2001; 360: 513-30
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• Phosphoinositides are key regulators of diverse cellular processes. The pleckstrin homology (PH) domain mediates the action of PtdIns(3,4)P(2), PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), while the FYVE domain relays the pulse of PtdIns3P. The recent establishment that the Phox homology (PX) domain interacts with PtdIns3P and other phosphoinositides suggests another mechanism by which phosphoinositides can regulate/integrate multiple cellular events via a spectrum of PX domain-containing proteins. Together with the recent discovery that the epsin N-terminal homologue (ENTH) domain interacts with PtdIns(4,5)P(2), it is becoming clear that phosphoinositides regulate diverse cellular events through interactions with several distinct structural motifs present in many different proteins.

• Gomez A, Wellbrock C, Gutbrod H, Dimitrijevic N, Schartl M
• Ligand-independent dimerization and activation of the oncogenic Xmrk receptor by two mutations in the extracellular domain.
• J Biol Chem. 2001; 276: 3333-40
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• Overexpression of the oncogenic receptor tyrosine kinase ONC-Xmrk is the first step in the development of hereditary malignant melanoma in the fish Xiphophorus. However, overexpression of its proto-oncogene counterpart (INV-Xmrk) is not sufficient for the oncogenic function of the receptor. Compared with INV-Xmrk, the ONC-Xmrk receptor displays 14 amino acid changes, suggesting the presence of activating mutations. To identify such activating mutations, a series of chimeric and mutant receptors were studied. None of the mutations present in the intracellular domain was found to be involved in receptor activation. In the extracellular domain, we found two mutations responsible for activation of the receptor. One is the substitution of a conserved cysteine (C578S) involved in intramolecular disulfide bonding. The other is a glycine to arginine exchange (G359R) in subdomain III. Either mutation leads to constitutive dimer formation and thereby to activation of the ONC-Xmrk receptor. Besides, the presence of these mutations slows down the processing of the Xmrk receptor in the endoplasmic reticulum, which is apparent as an incomplete glycosylation.

• Wang X, Zamore PD, Hall TM
• Crystal structure of a Pumilio homology domain.
• Mol Cell. 2001; 7: 855-65
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• Puf proteins regulate translation and mRNA stability by binding sequences in their target RNAs through the Pumilio homology domain (PUM-HD), which is characterized by eight tandem copies of a 36 amino acid motif, the PUM repeat. We have solved the structure of the PUM-HD from human Pumilio1 at 1.9 A resolution. The structure reveals that the eight PUM repeats correspond to eight copies of a single, repeated structural motif. The PUM repeats pack together to form a right-handed superhelix that approximates a half doughnut. The distribution of side chains on the inner and outer faces of this half doughnut suggests that the inner face of the PUM-HD binds RNA while the outer face interacts with proteins such as Nanos, Brain Tumor, and cytoplasmic polyadenylation element binding protein.

• Roszmusz E, Patthy A, Trexler M, Patthy L
• Localization of disulfide bonds in the frizzled module of Ror1 receptor tyrosine kinase.
• J Biol Chem. 2001; 276: 18485-90
• Display abstract

• The frizzled (FRZ) module is a novel module type that was first identified in G-protein-coupled receptors of the frizzled and smoothened families and has since been shown to be present in several secreted frizzled-related proteins, in some modular proteases, in collagen XVIII, and in various receptor tyrosine kinases of the Ror family. The FRZ modules constitute the extracellular ligand-binding region of frizzled receptors and are known to mediate signals of WNT family members through these receptors. With an eye toward defining the structure of this important module family, we have expressed the FRZ domain of rat Ror1 receptor tyrosine kinase in Pichia pastoris. By proteolytic digestion and amino acid sequencing the disulfide bonds were found to connect the 10 conserved cysteines in a 1-5, 2-4, 3-8, 6-10, and 7-9 pattern. Circular dichroism and differential scanning calorimetry studies on the recombinant protein indicate that the disulfide-bonded FRZ module corresponds to a single, compact, and remarkably stable folding domain possessing both alpha-helices and beta-strands.

• Himanen JP, Rajashankar KR, Lackmann M, Cowan CA, Henkemeyer M, Nikolov DB
• Crystal structure of an Eph receptor-ephrin complex.
• Nature. 2001; 414: 933-8
• Display abstract

• The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are important in regulating cell-cell interactions as they initiate a unique bidirectional signal transduction cascade whereby information is communicated into both the Eph-expressing and the ephrin-expressing cells. Initially identified as regulators of axon pathfinding and neuronal cell migration, Ephs and ephrins are now known to have roles in many other cell-cell interactions, including those of vascular endothelial cells and specialized epithelia. Here we report the crystal structure of the complex formed between EphB2 and ephrin-B2, determined at 2.7 A resolution. Each Eph receptor binds an ephrin ligand through an expansive dimerization interface dominated by the insertion of an extended ephrin loop into a channel at the surface of the receptor. Two Eph-Ephrin dimers then join to form a tetramer, in which each ligand interacts with two receptors and each receptor interacts with two ligands. The Eph and ephrin molecules are precisely positioned and orientated in these complexes, promoting higher-order clustering and the initiation of bidirectional signalling.

• Wang WK, Bycroft M, Foster NW, Buckle AM, Fersht AR, Chen YW
• Structure of the C-terminal sterile alpha-motif (SAM) domain of human p73 alpha.
• Acta Crystallogr D Biol Crystallogr. 2001; 57: 545-51
• Display abstract

• p73 is a homologue of the tumour suppressor p53 and contains all three functional domains of p53. The alpha-splice variant of p73 (p73 alpha) contains near its C-terminus an additional structural domain known as the sterile alpha-motif (SAM) that is probably responsible for regulating p53-like functions of p73. Here, the 2.54 A resolution crystal structure of this protein domain is reported. The crystal structure and the published solution structure have the same five-helix bundle fold that is characteristic of all SAM-domain structures, with an overall r.m.s.d. of 1.5 A for main-chain atoms. The hydrophobic core residues are well conserved, yet some large local differences are observed. The crystal structure reveals a dimeric organization, with the interface residues forming a mini four-helix bundle. However, analysis of solvation free energies and the surface area buried upon dimer formation indicated that this arrangement is more likely to be an effect of crystal packing rather than reflecting a physiological state. This is consistent with the solution structure being a monomer. The p73 alpha SAM domain also contains several interesting structural features: a Cys-X-X-Cys motif, a 3(10)-helix and a loop that have elevated B factors, and short tight inter-helical loops including two beta-turns; these elements are probably important in the normal function of this domain.

• Sprague ER, Redd MJ, Johnson AD, Wolberger C
• Structure of the C-terminal domain of Tup1, a corepressor of transcription in yeast.
• EMBO J. 2000; 19: 3016-27
• Display abstract

• The Tup1-Ssn6 corepressor complex regulates the expression of several sets of genes, including genes that specify mating type in the yeast Saccharomyces cerevisiae. Repression of mating-type genes occurs when Tup1-Ssn6 is brought to the DNA by the Matalpha2 DNA-binding protein and assembled upstream of a- and haploid-specific genes. We have determined the 2.3 A X-ray crystal structure of the C-terminal domain of Tup1 (accesion No. 1ERJ), a 43 kDa fragment that contains seven copies of the WD40 sequence motif and binds to the Matalpha2 protein. Moreover, this portion of the protein can partially substitute for full-length Tup1 in bringing about transcriptional repression. The structure reveals a seven-bladed beta propeller with an N-terminal subdomain that is anchored to the side of the propeller and extends the beta sheet of one of the blades. Point mutations in Tup1 that specifically affect the Tup1-Matalpha2 interaction cluster on one surface of the propeller. We identified regions of Tup1 that are conserved among the fungal Tup1 homologs and may be important in protein-protein interactions with additional components of the Tup1-mediated repression pathways.

• Binns KL, Taylor PP, Sicheri F, Pawson T, Holland SJ
• Phosphorylation of tyrosine residues in the kinase domain and juxtamembrane region regulates the biological and catalytic activities of Eph receptors.
• Mol Cell Biol. 2000; 20: 4791-805
• Display abstract

• Members of the Eph family of receptor tyrosine kinases exhibit a striking degree of amino acid homology, particularly notable in the kinase and membrane-proximal regions. A mutagenesis approach was taken to address the functions of specific conserved tyrosine residues within these catalytic and juxtamembrane domains. Ligand stimulation of wild-type EphB2 in neuronal NG108-15 cells resulted in an upregulation of catalytic activity and an increase in cellular tyrosine phosphorylation, accompanied by a retraction of neuritic processes. Tyrosine-to-phenylalanine substitutions within the conserved juxtamembrane motif abolished these responses. The mechanistic basis for these observations was examined using the highly related EphA4 receptor in a continuous coupled kinase assay. Tandem mass spectrometry experiments confirmed autophosphorylation of the two juxtamembrane tyrosine residues and also identified a tyrosine within the kinase domain activation segment as a phosphorylation site. Kinetic analysis revealed a decreased affinity for peptide substrate upon substitution of activation segment or juxtamembrane tyrosines. Together, our data suggest that the catalytic and therefore biological activities of Eph receptors are controlled by a two-component inhibitory mechanism, which is released by phosphorylation of the juxtamembrane and activation segment tyrosine residues.

• Hennessy F, Cheetham ME, Dirr HW, Blatch GL
• Analysis of the levels of conservation of the J domain among the various types of DnaJ-like proteins.
• Cell Stress Chaperones. 2000; 5: 347-58
• Display abstract

• DnaJ-like proteins are defined by the presence of an approximately 73 amino acid region termed the J domain. This region bears similarity to the initial 73 amino acids of the Escherichia coli protein DnaJ. Although the structures of the J domains of E coli DnaJ and human heat shock protein 40 have been solved using nuclear magnetic resonance, no detailed analysis of the amino acid conservation among the J domains of the various DnaJ-like proteins has yet been attempted. A multiple alignment of 223 J domain sequences was performed, and the levels of amino acid conservation at each position were established. It was found that the levels of sequence conservation were particularly high in 'true' DnaJ homologues (ie, those that share full domain conservation with DnaJ) and decreased substantially in those J domains in DnaJ-like proteins that contained no additional similarity to DnaJ outside their J domain. Residues were also identified that could be important for stabilizing the J domain and for mediating the interaction with heat shock protein 70.

• Hubbard SR, Till JH
• Protein tyrosine kinase structure and function.
• Annu Rev Biochem. 2000; 69: 373-98
• Display abstract

• Tyrosine phosphorylation is one of the key covalent modifications that occurs in multicellular organisms as a result of intercellular communication during embryogenesis and maintenance of adult tissues. The enzymes that carry out this modification are the protein tyrosine kinases (PTKs), which catalyze the transfer of the phosphate of ATP to tyrosine residues on protein substrates. Phosphorylation of tyrosine residues modulates enzymatic activity and creates binding sites for the recruitment of downstream signaling proteins. Two classes of PTKs are present in cells: the transmembrane receptor PTKs and the nonreceptor PTKs. Because PTKs are critical components of cellular signaling pathways, their catalytic activity is strictly regulated. Over the past several years, high-resolution structural studies of PTKs have provided a molecular basis for understanding the mechanisms by which receptor and nonreceptor PTKs are regulated. This review will highlight the important results that have emerged from these structural studies.

• Bausenwein BS, Schmidt M, Mielke B, Raabe T
• In vivo functional analysis of the daughter of sevenless protein in receptor tyrosine kinase signaling.
• Mech Dev. 2000; 90: 205-15
• Display abstract

• One mechanism used by receptor tyrosine kinases to relay a signal to different downstream effector molecules is to use adaptor proteins that provide docking sites for a variety of proteins. The daughter of sevenless (dos) gene was isolated in a genetic screen for components acting downstream of the Sevenless (Sev) receptor tyrosine kinase. Dos contains a N-terminally located PH domain and several tyrosine residues within consensus binding sites for a number of SH2 domain containing proteins. The structural features of Dos and experiments demonstrating tyrosine phosphorylation of Dos upon Sev activation suggested that Dos belongs to the family of multisite adaptor proteins that include the Insulin Receptor Substrate (IRS) proteins, Gab1, and Gab2. Here, we studied the structural requirements for Dos function in receptor tyrosine kinase mediated signaling processes by expressing mutated dos transgenes in the fly. We show that mutant Dos proteins lacking the putative binding sites for the SH2 domains of Shc, PhospholipaseC-gamma (PLC-gamma) and the regulatory subunit of Phosphoinositide 3-kinase (PI3-K) can substitute the loss of endogenous Dos function during development. In contrast, tyrosine 801, corresponding to a predicted Corkscrew (Csw) tyrosine phosphatase SH2 domain binding site, is essential for Dos function. Furthermore, we assayed whether the Pleckstrin homology (PH) domain is required for Dos function and localization. Evidence is provided that deletion or mutation of the PH domain interferes with the function but not with localization of the Dos protein. The Dos PH domain can be replaced by the Gab1 PH domain but not by a heterologous membrane anchor, suggesting a specific function of the PH domain in regulating signal transduction.

• Murthy TV, Jayadeva Bhat P
• Disruption of galactokinase signature sequence in gal3p of Saccharomyces cerevisiae does not lead to loss of signal transduction function.
• Biochem Biophys Res Commun. 2000; 273: 824-8
• Display abstract

• Gal3p of Saccharomyces cerevisiae is a 520-amino-acid residue protein, which activates the GAL genes in the presence of galactose by relieving the repression of Gal80p. It shows significant amino acid sequence homology to galactokinases but does not possess galactokinase activity. Deletion mutants of Gal3p were generated to identify the role of N-terminal amino acid residues required for function. The mutant versions of Gal3p could be detected on a Western blot. The Gal3p mutant lacking N-terminal 50-amino-acid residues which is disrupted for galactokinase signature sequence was found to be functional. These results suggest that the evolutionarily conserved galactokinase signature sequence present in known galactokinases may not have a role in Gal3p function.

• Aravind L, Koonin EV
• The STAS domain - a link between anion transporters and antisigma-factor antagonists.
• Curr Biol. 2000; 10: 535-535

• Bell CA, Tynan JA, Hart KC, Meyer AN, Robertson SC, Donoghue DJ
• Rotational coupling of the transmembrane and kinase domains of the Neu receptor tyrosine kinase.
• Mol Biol Cell. 2000; 11: 3589-99
• Display abstract

• Ligand binding to receptor tyrosine kinases (RTKs) regulates receptor dimerization and activation of the kinase domain. To examine the role of the transmembrane domain in regulation of RTK activation, we have exploited a simplified transmembrane motif, [VVVEVVV](n), previously shown to activate the Neu receptor. Here we demonstrate rotational linkage of the transmembrane domain with the kinase domain, as evidenced by a periodic activation of Neu as the dimerization motif is shifted across the transmembrane domain. These results indicate that activation requires a specific orientation of the kinase domains with respect to each other. Results obtained with platelet-derived growth factor receptor-beta suggest that this rotational linkage of the transmembrane domain to the kinase domain may be a general feature of RTKs. These observations suggest that activating mutations in RTK transmembrane and juxtamembrane domains will be limited to those residues that position the kinase domains in an allowed rotational conformation.

• Savvides SN, Boone T, Andrew Karplus P
• Flt3 ligand structure and unexpected commonalities of helical bundles and cystine knots.
• Nat Struct Biol. 2000; 7: 486-91
• Display abstract

• Human Flt3 ligand (Flt3L) stimulates early hematopoiesis by activating a type III tyrosine kinase receptor on primitive bone marrow stem cells. The crystal structure of soluble Flt3L reveals that it is a homodimer of two short chain alpha-helical bundles. Comparisons of structure-function relationships of Flt3L with the homologous hematopoietic cytokines macrophage colony stimulating factor (MCSF) and stem cell factor (SCF) suggest that they have a common receptor binding mode that is distinct from the paradigm derived from the complex of growth hormone with its receptor. Furthermore, we identify recognition features common to all helical and cystine-knot protein ligands that activate type III tyrosine kinase receptors, and the closely related type V tyrosine kinase receptors.

• Verdecia MA, Huang H, Dutil E, Kaiser DA, Hunter T, Noel JP
• Structure of the human anti-apoptotic protein survivin reveals a dimeric arrangement.
• Nat Struct Biol. 2000; 7: 602-8
• Display abstract

• Survivin is a 16.5 kDa protein that is expressed during the G2/M phase of the cell cycle and is hypothesized to inhibit a default apoptotic cascade initiated in mitosis. This inhibitory function is coupled to survivin's localization to the mitotic spindle. To begin to address the structural basis of survivin's function, we report the X-ray crystal structure of a recombinant form of full length survivin to 2.58 A resolution. Survivin consists of two defined domains including an N-terminal Zn2+-binding BIR domain linked to a 65 A amphipathic C-terminal alpha-helix. The crystal structure reveals an extensive dimerization interface along a hydrophobic surface on the BIR domain of each survivin monomer. A basic patch acting as a sulfate/phosphate-binding module, an acidic cluster projecting off the BIR domain, and a solvent-accessible hydrophobic surface residing on the C-terminal amphipathic helix, are suggestive of functional protein-protein interaction surfaces.

• Swairjo MA, Morales AJ, Wang CC, Ortiz AR, Schimmel P
• Crystal structure of trbp111: a structure-specific tRNA-binding protein.
• EMBO J. 2000; 19: 6287-98
• Display abstract

• Trbp111 is a 111 amino acid Aquifex aeolicus structure-specific tRNA-binding protein that has homologous counterparts distributed throughout evolution. A dimer is the functional unit for binding a single tRNA. Here we report the 3D structures of the A.aeolicus protein and its Escherichia coli homolog at resolutions of 2.50 and 1.87 A, respectively. The structure shows a symmetrical dimer of two core domains and a central dimerization domain where the N- and C-terminal regions of Trbp111 form an extensive dimer interface. The core of the monomer is a classical oligonucleotide/oligosaccharide-binding (OB) fold with a five-stranded ss-barrel and a small capping helix. This structure is similar to that seen in the anticodon-binding domain of three class II tRNA synthetases and several other proteins. Mutational analysis identified sites important for interactions with tRNA. These residues line the inner surfaces of two clefts formed between the ss-barrel of each monomer and the dimer interface. The results are consistent with a proposed model for asymmetrical docking of the convex side of tRNA to the dimer.

• Ramos A et al.
• RNA recognition by a Staufen double-stranded RNA-binding domain.
• EMBO J. 2000; 19: 997-1009
• Display abstract

• The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem-loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.

• Li J, Lee GI, Van Doren SR, Walker JC
• The FHA domain mediates phosphoprotein interactions.
• J Cell Sci. 2000; 113: 4143-9
• Display abstract

• The forkhead-associated (FHA) domain is a phosphopeptide-binding domain first identified in a group of forkhead transcription factors but is present in a wide variety of proteins from both prokaryotes and eukaryotes. In yeast and human, many proteins containing an FHA domain are found in the nucleus and involved in DNA repair, cell cycle arrest, or pre-mRNA processing. In plants, the FHA domain is part of a protein that is localized to the plasma membrane and participates in the regulation of receptor-like protein kinase signaling pathways. Recent studies show that a functional FHA domain consists of 120-140 amino acid residues, which is significantly larger than the sequence motif first described. Although FHA domains do not exhibit extensive sequence similarity, they share similar secondary and tertiary structures, featuring a sandwich of two anti-parallel (beta)-sheets. One intriguing finding is that FHA domains may bind phosphothreonine, phosphoserine and sometimes phosphotyrosine, distinguishing them from other well-studied phosphoprotein-binding domains. The diversity of proteins containing FHA domains and potential differences in binding specificities suggest the FHA domain is involved in coordinating diverse cellular processes.

• Heinemann U
• Structural biology: the foundation of molecular medicine.
• J Mol Med. 2000; 78: 245-6

• Read D, Butte MJ, Dernburg AF, Frasch M, Kornberg TB
• Functional studies of the BTB domain in the Drosophila GAGA and Mod(mdg4) proteins.
• Nucleic Acids Res. 2000; 28: 3864-70
• Display abstract

• The BTB/POZ (BTB) domain is an approximately 120 residue sequence that is conserved at the N-terminus of many proteins in both vertebrates and invertebrates. We found that the protein encoded by a lethal allele of the DROSOPHILA: modifier of mdg4 [mod(mdg4)] gene has two mutated residues in its BTB domain. The identities of the residues at the positions of these mutations are highly conserved in the BTB domain family of proteins, and when the corresponding mutations were engineered into the BTB domain-containing GAGA protein, the activity of GAGA as a transcription activator in a transient transfection assay was severely reduced. The functional equivalence of the BTB domains was established by showing that the BTB domain of the mod(mdg4) protein can effectively substitute for that of GAGA.

• Crucs S, Chatterjee S, Gavis ER
• Overlapping but distinct RNA elements control repression and activation of nanos translation.
• Mol Cell. 2000; 5: 457-67
• Display abstract

• Spatially restricted synthesis of Nanos protein in the Drosophila embryo is essential for anterior-posterior patterning. Nanos translation is restricted to the posterior of the embryo by translational repression of nanos mRNA throughout the bulk cytoplasm and selective activation of posteriorly localized nanos mRNA. A 90-nucleotide translational control element (TCE) mediates translational repression. We show that TCE function requires formation of a bipartite secondary structure that is recognized by Smaug repressor and at least one additional factor. We also demonstrate that translational activation requires the interaction of localization factors with sequences that overlap TCE structural motifs. The identification of separate but overlapping recognition motifs for translational repressors and localization factors provides a molecular mechanism for the switch between translational repression and activation.

• Shewchuk LM et al.
• Structure of the Tie2 RTK domain: self-inhibition by the nucleotide binding loop, activation loop, and C-terminal tail.
• Structure Fold Des. 2000; 8: 1105-13
• Display abstract

• BACKGROUND: Angiogenesis, the formation of new vessels from the existing vasculature, is a critical process during early development as well as in a number of disease processes. Tie2 (also known as Tek) is an endothelium-specific receptor tyrosine kinase involved in both angiogenesis and vasculature maintenance. RESULTS: We have determined the crystal structure of the Tie2 kinase domain to 2.2 A resolution. The structure contains the catalytic core, the kinase insert domain (KID), and the C-terminal tail. The overall fold is similar to that observed in other serine/threonine and tyrosine kinase structures; however, several unique features distinguish the Tie2 structure from those of other kinases. The Tie2 nucleotide binding loop is in an inhibitory conformation, which is not seen in other kinase structures, while its activation loop adopts an "activated-like" conformation in the absence of phosphorylation. Tyr-897, located in the N-terminal domain, may negatively regulate the activity of Tie2 by preventing dimerization of the kinase domains or by recruiting phosphatases when it is phosphorylated. CONCLUSION: Regulation of the kinase activity of Tie2 is a complex process. Conformational changes in the nucleotide binding loop, activation loop, C helix, and the C-terminal tail are required for ATP and substrate binding.

• Lietzke SE et al.
• Structural basis of 3-phosphoinositide recognition by pleckstrin homology domains.
• Mol Cell. 2000; 6: 385-94
• Display abstract

• Lipid second messengers generated by phosphoinositide (PI) 3-kinases regulate diverse cellular functions through interaction with pleckstrin homology (PH) domains in modular signaling proteins. The PH domain of Grp1, a PI 3-kinase-activated exchange factor for Arf GTPases, selectively binds phosphatidylinositol 3,4,5-trisphosphate with high affinity. We have determined the structure of the Grp1 PH domain in the unliganded form and bound to inositol 1,3,4,5-tetraphosphate. A novel mode of phosphoinositide recognition involving a 20-residue insertion within the beta6/beta7 loop explains the unusually high specificity of the Grp1 PH domain and the promiscuous 3-phosphoinositide binding typical of several PH domains including that of protein kinase B. When compared to other PH domains, general determinants of 3-phosphoinositide recognition and specificity can be deduced.

• Nabirochkina EN, Grishchuk AL, Soldatov AV
• [Cloning and characterization of the homologue of the Saccharomyces cerevisiae gene in Drosophila melanogaster]
• Genetika. 1999; 35: 1012-5
• Display abstract

• RAD23 is an evolutionary conserved protein, which is essential for DNA excision repair. It is believed that this protein is present in all eukaryotic organisms from yeast to mammals. In this work, molecular cloning of the Drosophila melanogaster RAD23 gene and an analysis of the encoded protein are reported.

• Meng W, Sawasdikosol S, Burakoff SJ, Eck MJ
• Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase.
• Nature. 1999; 398: 84-90
• Display abstract

• 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.

• McTigue MA et al.
• Crystal structure of the kinase domain of human vascular endothelial growth factor receptor 2: a key enzyme in angiogenesis.
• Structure Fold Des. 1999; 7: 319-30
• Display abstract

• BACKGROUND: Angiogenesis is involved in tumor growth, macular degeneration, retinopathy and other diseases. Vascular endothelial growth factor (VEGF) stimulates angiogenesis by binding to specific receptors (VEGFRs) on the surface of vascular endothelial cells. VEGFRs are receptor tyrosine kinases that, like the platelet-derived growth factor receptors (PDGFRs), contain a large insert within the kinase domain. RESULTS: We report here the generation, kinetic characterization, and 2.4 A crystal structure of the catalytic kinase domain of VEGF receptor 2 (VEGFR2). This protein construct, which lacks 50 central residues of the 68-residue kinase insert domain (KID), has comparable kinase activity to constructs containing the entire KID. The crystal structure, determined in an unliganded phosphorylated state, reveals an overall fold and catalytic residue positions similar to those observed in other tyrosine-kinase structures. The kinase activation loop, autophosphorylated on Y1059 prior to crystallization, is mostly disordered; however, a portion of it occupies a position inhibitory to substrate binding. The ends of the KID form a beta-like structure, not observed in other known tyrosine kinase structures, that packs near to the kinase C terminus. CONCLUSIONS: The majority of the VEGFR2 KID residues are not necessary for kinase activity. The unique structure observed for the ends of the KID may also occur in other PDGFR family members and may serve to properly orient the KID for signal transduction. This VEGFR2 kinase structure provides a target for design of selective anti-angiogenic therapeutic agents.

• Sedkov Y et al.
• Molecular genetic analysis of the Drosophila trithorax-related gene which encodes a novel SET domain protein.
• Mech Dev. 1999; 82: 171-9
• Display abstract

• The products of the trithorax and Polycomb groups genes maintain the activity and silence, respectively, of many developmental genes including genes of the homeotic complexes. This transcriptional regulation is likely to involve modification of chromatin structure. Here, we report the cloning and characterization of a new gene, trithorax-related (trr), which shares sequence similarities with members of both the trithorax and Polycomb groups. The trr transcript is 9.6 kb in length and is present throughout development. The TRR protein, as predicted from the nucleotide sequence of the open reading frame, is 2431 amino acids in length and contains a PHD finger-like domain and a SET domain, two highly conserved protein motifs found in several trithorax and Polycomb group proteins, and in modifiers of position effect variegation. TRR is most similar in sequence to the human ALR protein, suggesting that trr is a Drosophila homologue of the ALR. TRR is also highly homologous to Drosophila TRITHORAX protein and to its human homologue, ALL-1/HRX. However, preliminary genetic analysis of a trr null allele suggests that TRR protein may not be involved in regulation of homeotic genes (i.e. not a member of the trithorax or Polycomb groups) or in position effect variegation.

• Thanos CD, Bowie JU
• p53 Family members p63 and p73 are SAM domain-containing proteins.
• Protein Sci. 1999; 8: 1708-10
• Display abstract

• Homologs of the tumor suppressor p53, called p63 and p73, have been identified. The p63 and p73 family members possess a domain structure similar to p53, but contain variable C-terminal extensions. We find that some of the C-terminal extensions contain Sterile Alpha Motif (SAM) domains. SAM domains are protein modules that are involved in protein-protein interactions. Consistent with this role, the C-terminal SAM domains of the p63 and p73 may regulate function by recruiting other protein effectors.

• Zamore PD, Bartel DP, Lehmann R, Williamson JR
• The PUMILIO-RNA interaction: a single RNA-binding domain monomer recognizes a bipartite target sequence.
• Biochemistry. 1999; 38: 596-604
• Display abstract

• Translational repression of hunchback (hb) mRNA in the posterior of the Drosophila embryo requires two copies of a bipartite sequence, the Nanos Response Element (NRE), located in the 3' untranslated region of the mRNA. The PUMILIO (PUM) protein is thought to bind the NREs and thereby repress hb translation. The RNA-binding domain of PUM defines an evolutionarily conserved family of RNA-binding proteins, the PUM-Homology Domain (PUM-HD) proteins, which have been identified in yeast, plants, and animals. The PUM RNA-binding domain, the Drosophila PUM-HD (DmPUM-HD), has been shown previously to recognize nucleotides in both the 5' and 3' halves of the NRE, suggesting that a dimer of PUM might recognize one NRE. Here, we analyze the RNA-binding affinity and stoichiometry of the DmPUM-HD and find that one DmPUM-HD monomer binds independently and with equal affinity to each NRE (KD approximately 0.5 nM). We detect no cooperative interactions between DmPUM-HD monomers bound at adjacent sites. Our results imply that a single DmPUM-HD protein recognizes nucleotides in both the 5' and 3' NRE half-sites. Based on our estimate of the intraembryonic concentration of PUM (>40 nM), we propose that in vivo nearly all NREs are occupied by a PUM monomer.

• Insall R, Machesky L
• PH domains in WASP - a bug in the system? Wiskott-Aldrich syndrome protein.
• Trends Cell Biol. 1999; 9: 211-2

• Steinbacher S et al.
• The crystal structure of the Physarum polycephalum actin-fragmin kinase: an atypical protein kinase with a specialized substrate-binding domain.
• EMBO J. 1999; 18: 2923-9
• Display abstract

• Coordinated temporal and spatial regulation of the actin cytoskeleton is essential for diverse cellular processes such as cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells. In plasmodia of Physarum polycephalum, the F-actin capping activity of the actin-fragmin complex is regulated by the phosphorylation of actin. This is mediated by a novel type of protein kinase with no sequence homology to eukaryotic-type protein kinases. Here we present the crystal structure of the catalytic domain of the first cloned actin kinase in complex with AMP at 2.9 A resolution. The three-dimensional fold reveals a catalytic module of approximately 160 residues, in common with the eukaryotic protein kinase superfamily, which harbours the nucleotide binding site and the catalytic apparatus in an inter-lobe cleft. Several kinases that share this catalytic module differ in the overall architecture of their substrate recognition domain. The actin-fragmin kinase has acquired a unique flat substrate recognition domain which is supposed to confer stringent substrate specificity.

• Dodelet VC, Pazzagli C, Zisch AH, Hauser CA, Pasquale EB
• A novel signaling intermediate, SHEP1, directly couples Eph receptors to R-Ras and Rap1A.
• J Biol Chem. 1999; 274: 31941-6
• Display abstract

• The Eph family of receptor tyrosine kinases has been implicated in many developmental patterning processes, including cell segregation, cell migration, and axon guidance. The cellular components involved in the signaling pathways of the Eph receptors, however, are incompletely characterized. Using a yeast two-hybrid screen, we have identified a novel signaling intermediate, SHEP1 (SH2 domain-containing Eph receptor-binding protein 1), which is expressed in the embryonic and adult brain. SHEP1 contains an Src homology 2 domain that binds to a conserved tyrosine-phosphorylated motif in the juxtamembrane region of the EphB2 receptor and may itself be a target of EphB2 kinase activity, since it becomes heavily tyrosine-phosphorylated in cells expressing activated EphB2. SHEP1 also contains a domain similar to Ras guanine nucleotide exchange factor domains and binds to the GTPases R-Ras and Rap1A, but not Ha-Ras or RalA. Thus, SHEP1 directly links activated, tyrosine-phosphorylated Eph receptors to small Ras superfamily GTPases.

• Hall M
• Vernon frederick ("Sam") hall
• BMJ. 1999; 318: 265-265

• Wolf I, Rohrschneider LR
• Fiz1, a novel zinc finger protein interacting with the receptor tyrosine kinase Flt3.
• J Biol Chem. 1999; 274: 21478-84
• Display abstract

• The receptor tyrosine kinase Flt3 has been shown to play a role in proliferation and survival of hematopoietic progenitor cells as well as differentiation of early B lymphoid progenitors. However, the signaling events that control growth or differentiation are not completely understood. In order to identify new signaling molecules interacting with the cytoplasmic domain of Flt3, we performed a yeast two-hybrid screen. In addition to several SH2 domain-containing proteins, we have isolated a novel Flt3 interacting zinc finger protein (Fiz1) with 11 C(2)H(2)-type zinc fingers. Fiz1 binds to the catalytic domain of Flt3 but not to the structurally related receptor tyrosine kinases Kit, Fms, and platelet-derived growth factor receptor. This association is independent of kinase activity. The interaction between Flt3 and Fiz1 detected in yeast was confirmed by in vitro and in vivo coprecipitation assays. Fiz1 mRNA is expressed in all murine cell lines and tissues tested. Anti-Fiz1 antibodies recognize a 60-kDa protein, which is localized in the nucleus as well as in the cytoplasm. Together, these results identified a novel class of interaction between a receptor tyrosine kinase and a signaling molecule which is independent of the well established SH2 domain/phosphotyrosine binding.

• Dahanukar A, Walker JA, Wharton RP
• Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila.
• Mol Cell. 1999; 4: 209-18
• Display abstract

• During Drosophila embryogenesis, a gradient of Nanos protein emanating from the posterior pole organizes abdominal segmentation. This gradient arises from translational regulation of nanos mRNA, which is activated in the specialized cytoplasm at the posterior pole of the embryo and repressed elsewhere. Previously, we have defined cis-acting elements in the mRNA that mediate this translational switch. In this report, we identify a factor named Smaug that binds to these elements and represses translation in the bulk cytoplasm. Smaug interacts gentically and biochemically with Oskar, a key component of the pole plasm for activation of nanos mRNA and specification of the germline precursors. These observations suggest that Smaug operates a translational switch that governs the distribution of Nanos protein.

• Katso RM, Russell RB, Ganesan TS
• Functional analysis of H-Ryk, an atypical member of the receptor tyrosine kinase family.
• Mol Cell Biol. 1999; 19: 6427-40
• Display abstract

• H-Ryk is an atypical receptor tyrosine kinase which differs from other members of this family at a number of conserved residues in the activation and nucleotide binding domains. Using a chimeric receptor approach, we demonstrate that H-Ryk has impaired catalytic activity. Despite the receptor's inability to undergo autophosphorylation or phosphorylate substrates, we demonstrate that ligand stimulation of the chimeric receptor results in activation of the mitogen-activated protein kinase pathway. The ability to transduce signals is abolished by mutation of the invariant lysine (K334A) in subdomain II of H-Ryk. Further, by in vitro mutagenesis, we show that the amino acid substitutions in the activation domain of H-Ryk account for the loss of catalytic activity. In addition to the essential aspartate residue, either phenylalanine or glycine is required in the activation domain to maintain proper conformation of the catalytic domain and thus ensure receptor autophosphorylation. Homology modelling of the catalytic domain of H-Ryk provides a rationale for these findings. Thus, the signalling properties of H-Ryk are divergent from those of other classical receptor tyrosine kinases.

• Meng A, Moore B, Tang H, Yuan B, Lin S
• A Drosophila doublesex-related gene, terra, is involved in somitogenesis in vertebrates.
• Development. 1999; 126: 1259-68
• Display abstract

• The Drosophila doublesex (dsx) gene encodes a transcription factor that mediates sex determination. We describe the characterization of a novel zebrafish zinc-finger gene, terra, which contains a DNA binding domain similar to that of the Drosophila dsx gene. However, unlike dsx, terra is transiently expressed in the presomitic mesoderm and newly formed somites. Expression of terra in presomitic mesoderm is restricted to cells that lack expression of MyoD. In vivo, terra expression is reduced by hedgehog but enhanced by BMP signals. Overexpression of terra induces rapid apoptosis both in vitro and in vivo, suggesting that a tight regulation of terra expression is required during embryogenesis. Terra has both human and mouse homologs and is specifically expressed in mouse somites. Taken together, our findings suggest that terra is a highly conserved protein that plays specific roles in early somitogenesis of vertebrates.

• Huse M, Chen YG, Massague J, Kuriyan J
• Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12.
• Cell. 1999; 96: 425-36
• Display abstract

• Activation of the type I TGFbeta receptor (TbetaR-I) requires phosphorylation of a regulatory segment known as the GS region, located upstream of the serine/threonine kinase domain in the cytoplasmic portion of the receptor. The crystal structure of a fragment of unphosphorylated TbetaR-I, containing both the GS region and the catalytic domain, has been determined in complex with the FK506-binding protein FKBP12. TbetaR-I adopts an inactive conformation that is maintained by the unphosphorylated GS region. FKBP12 binds to the GS region of the receptor, capping the TbetaR-II phosphorylation sites and further stabilizing the inactive conformation of TbetaR-I. Certain structural features at the catalytic center of TbetaR-I are characteristic of tyrosine kinases rather than Ser/Thr kinases.

• Herbst R, Zhang X, Qin J, Simon MA
• Recruitment of the protein tyrosine phosphatase CSW by DOS is an essential step during signaling by the sevenless receptor tyrosine kinase.
• EMBO J. 1999; 18: 6950-61
• Display abstract

• The pleckstrin homology (PH) domain-containing protein Daughter of Sevenless (DOS) is an essential component of the Sevenless receptor tyrosine kinase (SEV) signaling cascade, which specifies R7 photoreceptor development in the Drosophila eye. Previous results have suggested that DOS becomes tyrosine phosphorylated during SEV signaling and collaborates with the protein tyrosine phosphatase CSW. We have investigated this possibility by identifying tyrosine residues 801 and 854 of DOS as the phosphorylated binding sites for the CSW SH2 domains. We show that these sites become phosphorylated in response to SEV activation and that phosphorylation of both sites is required to allow CSW to bind DOS. Mutant DOS proteins in which either Y801 or Y854 of DOS has been changed to phenylalanine are unable to function during signaling by SEV and other receptor tyrosine kinases. In contrast, we find that a mutant DOS protein in which all tyrosine phosphorylation sites except Y801 and Y854 have been removed is able effectively to provide DOS function during SEV signaling and to rescue the lethality associated with dos loss-of-function mutations. These results indicate that a primary role for DOS during signaling by SEV and other receptor tyrosine kinases is to become phosphorylated at Y801 and Y854 and then recruit CSW.

• Smibert CA, Lie YS, Shillinglaw W, Henzel WJ, Macdonald PM
• Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro.
• RNA. 1999; 5: 1535-47
• Display abstract

• Proper deployment of Nanos protein at the posterior of the Drosophila embryo, where it directs posterior development, requires a combination of RNA localization and translational controls. These controls ensure that only the posteriorly-localized nanos mRNA is translated, whereas unlocalized nanos mRNA is translationally repressed. Here we describe cloning of the gene encoding Smaug, an RNA-binding protein that interacts with the sequences, SREs, in the nanos mRNA that mediate translational repression. Using an in vitro translation assay, we demonstrate that SRE-dependent repression occurs in extracts from early stage embryos. Immunodepletion of Smaug from the extracts eliminates repression, consistent with the notion that Smaug is involved. Smaug is a novel gene and the existence of potential mammalian Smaug homologs raises the possibility that Smaug represents a new class of conserved translational repressor.

• Johnstone BH et al.
• The widely conserved Era G-protein contains an RNA-binding domain required for Era function in vivo.
• Mol Microbiol. 1999; 33: 1118-31
• Display abstract

• Era is a small G-protein widely conserved in eubacteria and eukaryotes. Although essential for bacterial growth and implicated in diverse cellular processes, its actual function remains unclear. Several lines of evidence suggest that Era may be involved in some aspect of RNA biology. The GTPase domain contains features in common with all G-proteins and is required for Era function in vivo. The C-terminal domain (EraCTD) bears scant similarity to proteins outside the Era subfamily. On the basis of sequence comparisons, we argue that the EraCTD is similar to, but distinct from, the KH RNA-binding domain. Although both contain the consensus VIGxxGxxI RNA-binding motif, the protein folds are probably different. We show that bacterial Era binds RNA in vitro and can form higher-order RNA-protein complexes. Mutations in the VIGxxGxxI motif and other conserved residues of the Escherichia coli EraCTD decrease RNA binding in vitro and have corresponding effects on Era function in vivo, including previously described effects on cell division and chromosome partitioning. Importantly, mutations in L-66, located in the predicted switch II region of the E. coli Era GTPase domain, also perturb binding, leading us to propose that the GTPase domain regulates RNA binding in response to unknown cellular cues. The possible biological significance of Era RNA binding is discussed.

• Hall DR et al.
• The high-resolution crystal structure of the molybdate-dependent transcriptional regulator (ModE) from Escherichia coli: a novel combination of domain folds.
• EMBO J. 1999; 18: 1435-46
• Display abstract

• The molybdate-dependent transcriptional regulator (ModE) from Escherichia coli functions as a sensor of molybdate concentration and a regulator for transcription of operons involved in the uptake and utilization of the essential element, molybdenum. We have determined the structure of ModE using multi-wavelength anomalous dispersion. Selenomethionyl and native ModE models are refined to 1. 75 and 2.1 A, respectively and describe the architecture and structural detail of a complete transcriptional regulator. ModE is a homodimer and each subunit comprises N- and C-terminal domains. The N-terminal domain carries a winged helix-turn-helix motif for binding to DNA and is primarily responsible for ModE dimerization. The C-terminal domain contains the molybdate-binding site and residues implicated in binding the oxyanion are identified. This domain is divided into sub-domains a and b which have similar folds, although the organization of secondary structure elements varies. The sub-domain fold is related to the oligomer binding-fold and similar to that of the subunits of several toxins which are involved in extensive protein-protein interactions. This suggests a role for the C-terminal domain in the formation of the ModE-protein-DNA complexes necessary to regulate transcription. Modelling of ModE interacting with DNA suggests that a large distortion of DNA is not necessary for complex formation.

• Yasukawa H et al.
• The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop.
• EMBO J. 1999; 18: 1309-20
• Display abstract

• 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.

• Wiesmann C, Ultsch MH, Bass SH, de Vos AM
• Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TrkA receptor.
• Nature. 1999; 401: 184-8
• Display abstract

• Nerve growth factor (NGF) is involved in a variety of processes involving signalling, such as cell differentiation and survival, growth cessation and apoptosis of neurons. These events are mediated by NGF as a result of binding to its two cell-surface receptors, TrkA and p75. TrkA is a receptor with tyrosine kinase activity that forms a high-affinity binding site for NGF. Of the five domains comprising its extracellular portion, the immunoglobulin-like domain proximal to the membrane (TrkA-d5 domain) is necessary and sufficient for NGF binding. Here we present the crystal structure of human NGF in complex with human TrkA-d5 at 2.2 A resolution. The ligand-receptor interface consists of two patches of similar size. One patch involves the central beta-sheet that forms the core of the homodimeric NGF molecule and the loops at the carboxy-terminal pole of TrkA-d5. The second patch comprises the amino-terminal residues of NGF, which adopt a helical conformation upon complex formation, packing against the 'ABED' sheet of TrkA-d5. The structure is consistent with results from mutagenesis experiments for all neurotrophins, and indicates that the first patch may constitute a conserved binding motif for all family members, whereas the second patch is specific for the interaction between NGF and TrkA.

• Chi SW, Ayed A, Arrowsmith CH
• Solution structure of a conserved C-terminal domain of p73 with structural homology to the SAM domain.
• EMBO J. 1999; 18: 4438-45
• Display abstract

• p73 and p63 are two recently cloned genes with homology to the tumor suppressor p53, whose protein product is a key transcriptional regulator of genes involved in cell cycle arrest and apoptosis. While all three proteins share conserved transcriptional activation, DNA-binding and oligomerization domains, p73 and p63 have an additional conserved C-terminal region. We have determined the three-dimensional solution structure of this conserved C-terminal domain of human p73. The structure reveals a small five-helix bundle with striking similarity to the SAM (sterile alpha motif) domains of two ephrin receptor tyrosine kinases. The SAM domain is a putative protein-protein interaction domain found in a variety of cytoplasmic signaling proteins and has been shown to form both homo- and hetero-oligomers. However, the SAM-like C-terminal domains of p73 and p63 are monomeric and do not interact with one another, suggesting that this domain may interact with additional, as yet uncharacterized proteins in a signaling and/or regulatory role.

• Hillier BJ, Christopherson KS, Prehoda KE, Bredt DS, Lim WA
• Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex.
• Science. 1999; 284: 812-5
• Display abstract

• The PDZ protein interaction domain of neuronal nitric oxide synthase (nNOS) can heterodimerize with the PDZ domains of postsynaptic density protein 95 and syntrophin through interactions that are not mediated by recognition of a typical carboxyl-terminal motif. The nNOS-syntrophin PDZ complex structure revealed that the domains interact in an unusual linear head-to-tail arrangement. The nNOS PDZ domain has two opposite interaction surfaces-one face has the canonical peptide binding groove, whereas the other has a beta-hairpin "finger." This nNOS beta finger docks in the syntrophin peptide binding groove, mimicking a peptide ligand, except that a sharp beta turn replaces the normally required carboxyl terminus. This structure explains how PDZ domains can participate in diverse interaction modes to assemble protein networks.

• Chantalat L et al.
• Crystal structure of the human protein kinase CK2 regulatory subunit reveals its zinc finger-mediated dimerization.
• EMBO J. 1999; 18: 2930-40
• Display abstract

• Protein kinase CK2 is a tetramer composed of two alpha catalytic subunits and two beta regulatory subunits. The structure of a C-terminal truncated form of the human beta subunit has been determined by X-ray crystallography to 1.7 A resolution. One dimer is observed in the asymmetric unit of the crystal. The most striking feature of the structure is the presence of a zinc finger mediating the dimerization. The monomer structure consists of two domains, one entirely alpha-helical and one including the zinc finger. The dimer has a crescent shape holding a highly acidic region at both ends. We propose that this acidic region is involved in the interactions with the polyamines and/or catalytic subunits. Interestingly, conserved amino acid residues among beta subunit sequences are clustered along one linear ridge that wraps around the entire dimer. This feature suggests that protein partners may interact with the dimer through a stretch of residues in an extended conformation.

• Songyang Z
• Recognition and regulation of primary-sequence motifs by signaling modular domains.
• Prog Biophys Mol Biol. 1999; 71: 359-72

• Hubbard SR
• Structural analysis of receptor tyrosine kinases.
• Prog Biophys Mol Biol. 1999; 71: 343-58
• Display abstract

• Receptor tyrosine kinases (RTKs) are single-pass transmembrane receptors that possess intrinsic cytoplasmic enzymatic activity, catalyzing the transfer of the gamma-phosphate of ATP to tyrosine residues in protein substrates. RTKs are essential components of signal transduction pathways that affect cell proliferation, differentiation, migration and metabolism. Included in this large protein family are the insulin receptor and the receptors for growth factors such as epidermal growth factor, fibroblast growth factor and vascular endothelial growth factor. Receptor activation occurs through ligand binding, which facilitates receptor dimerization and autophosphorylation of specific tyrosine residues in the cytoplasmic portion. The phosphotyrosine residues either enhance receptor catalytic activity or provide docking sites for downstream signaling proteins. Over the past several years, structural studies employing X-ray crystallography have advanced our understanding of the molecular mechanisms by which RTKs recognize their ligands and are activated by dimerization and tyrosine autophosphorylation. This review will highlight the key results that have emerged from these structural studies.

• Delattre M, Briand S, Paces-Fessy M, Blanchet-Tournier MF
• The Suppressor of fused gene, involved in Hedgehog signal transduction in Drosophila, is conserved in mammals.
• Dev Genes Evol. 1999; 209: 294-300
• Display abstract

• The Suppressor of fused [Su(fu)] gene of Drosophila melanogaster encodes a protein containing a PEST sequence [sequence enriched in proline (P), glutamic acid (E), serine (S) and threonine (T)] which acts as an antagonist to the serine-threonine kinase Fused in Hedgehog (Hh) signal transduction during embryogenesis. The Su(fu) gene isolated from a distantly related Drosophila species, D. virilis, shows significantly high homology throughout its protein sequence with its D. melanogaster counterpart. We show that these two Drosophila homologs of Su(fu) are functionally interchangeable in enhancing the fused phenotype. We have also isolated mammalian homologs of Su(fu). The absence of the PEST sequence in the mammalian Su(fu) protein suggests a different regulation for this product between fly and vertebrates. Using the yeast two-hybrid method, we show that the murine Su(fu) protein can interact directly with the Fused and Cubitus interruptus proteins, known partners of Su(fu) in Drosophila. These data are discussed in the light of their evolutionary relationships.

• Kalo MS, Pasquale EB
• Multiple in vivo tyrosine phosphorylation sites in EphB receptors.
• Biochemistry. 1999; 38: 14396-408
• Display abstract

• Autophosphorylation regulates the function of receptor tyrosine kinases. To dissect the mechanism by which Eph receptors transmit signals, we have developed an approach using matrix-assisted laser desorption-ionization (MALDI) mass spectrometry to map systematically their in vivo tyrosine phosphorylation sites. With this approach, phosphorylated peptides from receptors digested with various endoproteinases were selectively isolated on immobilized anti-phosphotyrosine antibodies and analyzed directly by MALDI mass spectrometry. Multiple in vivo tyrosine phosphorylation sites were identified in the juxtamembrane region, kinase domain, and carboxy-terminal tail of EphB2 and EphB5, and found to be remarkably conserved between these EphB receptors. A number of these sites were also identified as in vitro autophosphorylation sites of EphB5 by phosphopeptide mapping using two-dimensional chromatography. Only two in vitro tyrosine phosphorylation sites had previously been directly identified for Eph receptors. Our data further indicate that in vivo EphB2 and EphB5 are also extensively phosphorylated on serine and threonine residues. Because phosphorylation at each site can affect receptor signaling properties, the multiple phosphorylation sites identified here for the EphB receptors suggest a complex regulation of their functions, presumably achieved by autophosphorylation as well as phosphorylation by other kinases. In addition, we show that MALDI mass spectrometry can be used to determine the binding sites for Src homology 2 (SH2) domains by identifying the EphB2 phosphopeptides that bind to the SH2 domain of the Src kinase.

• Buchert M et al.
• The junction-associated protein AF-6 interacts and clusters with specific Eph receptor tyrosine kinases at specialized sites of cell-cell contact in the brain.
• J Cell Biol. 1999; 144: 361-71
• Display abstract

• The AF-6/afadin protein, which contains a single PDZ domain, forms a peripheral component of cell membranes at specialized sites of cell-cell junctions. To identify potential receptor-binding targets of AF-6 we screened the PDZ domain of AF-6 against a range of COOH-terminal peptides selected from receptors having potential PDZ domain-binding termini. The PDZ domain of AF-6 interacts with a subset of members of the Eph subfamily of RTKs via its COOH terminus both in vitro and in vivo. Cotransfection of a green fluorescent protein-tagged AF-6 fusion protein with full-length Eph receptors into heterologous cells induces a clustering of the Eph receptors and AF-6 at sites of cell-cell contact. Immunohistochemical analysis in the adult rat brain reveals coclustering of AF-6 with Eph receptors at postsynaptic membrane sites of excitatory synapses in the hippocampus. Furthermore, AF-6 is a substrate for a subgroup of Eph receptors and phosphorylation of AF-6 is dependent on a functional kinase domain of the receptor. The physical interaction of endogenous AF-6 with Eph receptors is demonstrated by coimmunoprecipitation from whole rat brain lysates. AF-6 is a candidate for mediating the clustering of Eph receptors at postsynaptic specializations in the adult rat brain.

• Maurel-Zaffran C, Chauvet S, Jullien N, Miassod R, Pradel J, Aragnol D
• nessy, an evolutionary conserved gene controlled by Hox proteins during Drosophila embryogenesis.
• Mech Dev. 1999; 86: 159-63
• Display abstract

• From a library of DNA fragments associated with Ultrabithorax protein in vivo, we have isolated nessy, a new Drosophila gene that encodes a putative transmembrane protein conserved in evolution from Caenorhabditis elegans, to human. Zygotic expression occurs transiently in mesectodermal cells at gastrulation, proceeds in mesoderm and endoderm lineages during germ band movements and becomes then restricted to anterior and posterior domains in the visceral mesoderm. The Hox proteins Ultrabithorax, Antennapedia and AbdominalA are likely acting simultaneously to repress nessy in the other parts of the visceral mesoderm.

• Smith T
• Picture story. Ephrin receptors divided.
• Nat Struct Biol. 1999; 6: 17-17

• Kapeller R et al.
• Tyrosine phosphorylation of tub and its association with Src homology 2 domain-containing proteins implicate tub in intracellular signaling by insulin.
• J Biol Chem. 1999; 274: 24980-6
• Display abstract

• A mutation in the tub gene leads to maturity-onset obesity, insulin resistance, and progressive retinal and cochlear degeneration in mice. tub is a member of a growing family of genes that encode proteins of unknown function that are remarkably conserved across species. The absence of obvious transmembrane domain(s) or signal sequence peptide motif(s) suggests that Tub is an intracellular protein. Additional sequence analysis revealed the presence of putative tyrosine phosphorylation motifs and Src homology 2 (SH2)-binding sites. Here we demonstrate that in CHO-IR cells, transfected Tub is phosphorylated on tyrosine in response to insulin and insulin-like growth factor-1 and that in PC12 cells, insulin but not EGF induced tyrosine phosphorylation of endogenous Tub. In vitro, Tub is phosphorylated by purified insulin receptor kinase as well as by Abl and JAK 2 but not by epidermal growth factor receptor and Src kinases. Furthermore, upon tyrosine phosphorylation, Tub associated selectively with the SH2 domains of Abl, Lck, and the C-terminal SH2 domain of phospholipase Cgamma and insulin enhanced the association of Tub with endogenous phospholipase Cgamma in CHO-IR cells. These data suggest that Tub may function as an adaptor protein linking the insulin receptor, and possibly other protein-tyrosine kinases, to SH2-containing proteins.

• Thanos CD, Faham S, Goodwill KE, Cascio D, Phillips M, Bowie JU
• Monomeric structure of the human EphB2 sterile alpha motif domain.
• J Biol Chem. 1999; 274: 37301-6
• Display abstract

• The sterile alpha motif (SAM) domain is a protein module found in many diverse signaling proteins. SAM domains in some systems have been shown to self-associate. Previous crystal structures of an EphA4-SAM domain dimer (Stapleton, D., Balan, I., Pawson, T., and Sicheri, F. (1999) Nat. Struct. Biol. 6, 44-49) and a possible EphB2-SAM oligomer (Thanos, C. D., Goodwill, K. E., and Bowie, J. U. (1999) Science 283, 833-836) both revealed large interfaces comprising an exchange of N-terminal peptide arms. Within the arm, a conserved hydrophobic residue (Tyr-8 in the EphB2-SAM structure or Phe-910 in the EphA4-SAM structure) is anchored into a hydrophobic cleft on a neighboring molecule. Here we have solved a new crystal form of the human EphB2-SAM domain that has the same overall SAM domain fold yet has no substantial intermolecular contacts. In the new structure, the N-terminal peptide arm of the EphB2-SAM domain protrudes out from the core of the molecule, leaving both the arm (including Tyr-8) and the hydrophobic cleft solvent-exposed. To verify that Tyr-8 is solvent-exposed in solution, we made a Tyr-8 to Ala-8 mutation and found that the EphB2-SAM domain structure and stability were only slightly altered. These results suggest that Tyr-8 is not part of the hydrophobic core of the EphB2-SAM domain and is conserved for functional reasons. Cystallographic evidence suggests a possible role for the N-terminal arm in oligomerization. In the absence of a direct demonstration of biological relevance, however, the functional role of the N-terminal arm remains an open question.

• Zisch AH, Kalo MS, Chong LD, Pasquale EB
• Complex formation between EphB2 and Src requires phosphorylation of tyrosine 611 in the EphB2 juxtamembrane region.
• Oncogene. 1998; 16: 2657-70
• Display abstract

• The cellular components of the neuronal signaling pathways of Eph receptor tyrosine kinases are only beginning to be elucidated. Here we show that in vivo tyrosine phosphorylation sites of the Eph receptors EphA3, EphA4, and EphB2 in embryonic retina serve as binding sites for the Src-homology 2 (SH2) domain of Src kinase. Furthermore, tyrosine-phosphorylated EphB2 was detected in Src immunoprecipitates from transfected Cos cells, indicating that EphB2 and Src can physically associate. Interestingly, a form of Src with reduced electrophoretic mobility and increased tyrosine phosphorylation was detected in Cos cells expressing tyrosine-phosphorylated EphB2, suggesting a functional interaction between EphB2 and Src. Yeast two-hybrid analysis in conjunction with site-directed mutagenesis demonstrated that phosphorylated tyrosine 611 in the juxtamembrane region of EphB2 is crucial for the interaction with the SH2 domain of Src. In contrast, binding of the carboxy-terminal SH2 domain of phospholipase Cgamma was not abolished upon mutation of tyrosine 611 in EphB2. Phosphopeptide mapping of autophosphorylated full-length EphB2, and wild-type and tyrosine to phenylalanine mutants of the EphB2 cytoplasmic domain fused to LexA, showed tyrosine 611 in the sequence motif YEDP as a major site of autophosphorylation in EphB2. Our mutational analysis also indicated that tyrosines 605 and 611 are important for EphB2 kinase activity. We propose Src kinase as a downstream effector that mediates the neuron's response to Eph receptor activation.

• Lehmann M, Siegmund T, Lintermann KG, Korge G
• The pipsqueak protein of Drosophila melanogaster binds to GAGA sequences through a novel DNA-binding domain.
• J Biol Chem. 1998; 273: 28504-9
• Display abstract

• Pipsqueak (Psq) belongs to a family of proteins defined by a phylogenetically old protein-protein interaction motif. Like the GAGA factor and other members of this family, Psq is an important developmental regulator in Drosophila, having pleiotropic functions during oogenesis, embryonic pattern formation, and adult development. The GAGA factor controls the transcriptional activation of homeotic genes and other genes by binding to control elements containing the GAGAG consensus motif. Binding is associated with formation of an open chromatin structure that makes the control regions accessible to transcriptional activators. We show here that Psq contains a novel DNA-binding domain, which binds, like the GAGA factor zinc finger DNA-binding domain, to target sites containing the GAGAG consensus motif. Binding is suppressed, as in the GAGA factor and other proteins of the family, by the associated protein-protein interaction motif. The DNA-binding domain, which we call the Psq domain, is identical with a previously identified region consisting of four tandem repeats of a conserved 50-amino acid sequence, the Psq motif. The Psq domain seems to be structurally related to known DNA-binding domains, both in its repetitive character and in the putative three-alpha-helix structure of the Psq motif, but it lacks the conserved sequence signatures of the classical eukaryotic DNA-binding motifs. Psq may thus represent the prototype of a new family of DNA-binding proteins.

• Chandra NR, Muirhead H, Holbrook JJ, Bernstein BE, Hol WG, Sessions RB
• A general method of domain closure is applied to phosphoglycerate kinase and the result compared with the crystal structure of a closed conformation of the enzyme.
• Proteins. 1998; 30: 372-80
• Display abstract

• The occurrence of large domain motions associated with the mechanism of action of many proteins is well established. We present a general method of predicting domain closure applicable to proteins containing domains separated by an apparent hinge. The method attempts to allow for natural directional bias within the closing protein by repeatedly applying a weak pulling force over a short distance between pairs of atoms chosen at random in the two domains in question. Appropriate parameters governing the pulling function were determined empirically. The method was applied to the bi-lobal protein PGK and a closed-form activated ternary complex generated for Bacillus stearothermophilus PGK. This model was compared with the recently determined crystal structure of closed-form Trypanosoma brucei PGK. The model predicts the correct hinge regions, although the magnitude of movement at one hinge point was overestimated, and provides a reasonable representation of the closed-form ternary complex.

• Hock B et al.
• PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor.
• Proc Natl Acad Sci U S A. 1998; 95: 9779-84
• Display abstract

• Eph-related receptor tyrosine kinases (RTKs) have been implicated in intercellular communication during embryonic development. To elucidate their signal transduction pathways, we applied the yeast two-hybrid system. We could demonstrate that the carboxyl termini of the Eph-related RTKs EphA7, EphB2, EphB3, EphB5, and EphB6 interact with the PDZ domain of the ras-binding protein AF6. A mutational analysis revealed that six C-terminal residues of the receptors are involved in binding to the PDZ domain of AF6 in a sequence-specific fashion. Moreover, this PDZ domain also interacts with C-terminal sequences derived from other transmembrane receptors such as neurexins and the Notch ligand Jagged. In contrast to the association of EphB3 to the PDZ domain of AF6, the interaction with full-length AF6 clearly depends on the kinase activity of EphB3, suggesting a regulated mechanism for the PDZ-domain-mediated interaction. These data gave rise to the idea that the binding of AF6 to EphB3 occurs in a cooperative fashion because of synergistic effects involving different epitopes of both proteins. Moreover, in NIH 3T3 and NG108 cells endogenous AF6 is phosphorylated specifically by EphB3 and EphB2 in a ligand-dependent fashion. Our observations add the PDZ domain to the group of conserved protein modules such as Src-homology-2 (SH2) and phosphotyrosine-binding (PTB) domains that regulate signal transduction through their ability to mediate the interaction with RTKs.

• Saldanha J, Singh J, Mahadevan D
• Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases.
• Protein Sci. 1998; 7: 1632-5
• Display abstract

• In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in the hedgehog signalling pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases

• Van Hoof C, Janssens V, Dinishiotu A, Merlevede W, Goris J
• Functional analysis of conserved domains in the phosphotyrosyl phosphatase activator. Molecular cloning of the homologues from Drosophila melanogaster and Saccharomyces cerevisiae.
• Biochemistry. 1998; 37: 12899-908
• Display abstract

• Phosphotyrosyl phosphatase activator (PTPA), a 37 kDa cytosolic protein that specifically activates the phosphotyrosyl phosphatase activity of the dimeric form of PP2A, was cloned from Drosophila melanogaster and Saccharomyces cerevisiae. Sequence alignment of PTPA from yeast to human revealed highly conserved regions including the type B fragment of the putative PTPA ATP binding site. We generated PTPA deletion mutants of these conserved regions as well as point mutations within regions that were suggested to be functionally important. The recombinant proteins were expressed in E. coli and subsequently purified. Activity measurements, linked with immunological detection, revealed that most of the well-conserved regions are essential for PTPA activity. However, neither the type A fragment of the putative ATP binding site nor the cysteine-rich region, present in all but the Drosophila and yeast homologues, appeared to be essential for PTPA activity. Moreover, we observed that PTPA truncated at glycine266 behaves as a dominant negative mutant since it is inhibitory to the wild-type PTPA.

• Lackmann M et al.
• Distinct subdomains of the EphA3 receptor mediate ligand binding and receptor dimerization.
• J Biol Chem. 1998; 273: 20228-37
• Display abstract

• Eph receptor tyrosine kinases and their ligands (ephrins) are highly conserved protein families implicated in patterning events during development, particularly in the nervous system. In a number of functional studies, strict conservation of structure and function across distantly related vertebrate species has been confirmed. In this study we make use of the observation that soluble human EphA3 (HEK) exerts a dominant negative effect on somite formation and axial organization during zebrafish embryogenesis to probe receptor function. Based on exon structure we have dissected the extracellular region of EphA3 receptor into evolutionarily conserved subdomains and used kinetic BIAcore analysis, mRNA injection into zebrafish embryos, and receptor transphosphorylation analysis to study their function. We show that ligand binding is restricted to the N-terminal region encoded by exon III, and we identify an independent, C-terminal receptor-dimerization domain. Recombinant proteins encoding either region in isolation can function as receptor antagonists in zebrafish. We propose a two-step mechanism of Eph receptor activation with distinct ligand binding and ligand-independent receptor-receptor oligomerization events.

• Owen DJ, Evans PR
• A structural explanation for the recognition of tyrosine-based endocytotic signals.
• Science. 1998; 282: 1327-32
• Display abstract

• Many cell surface proteins are marked for endocytosis by a cytoplasmic sequence motif, tyrosine-X-X-(hydrophobic residue), that is recognized by the mu2 subunit of AP2 adaptors. Crystal structures of the internalization signal binding domain of mu2 complexed with the internalization signal peptides of epidermal growth factor receptor and the trans-Golgi network protein TGN38 have been determined at 2.7 angstrom resolution. The signal peptides adopted an extended conformation rather than the expected tight turn. Specificity was conferred by hydrophobic pockets that bind the tyrosine and leucine in the peptide. In the crystal, the protein forms dimers that could increase the strength and specificity of binding to dimeric receptors.

• Zhou R
• The Eph family receptors and ligands.
• Pharmacol Ther. 1998; 77: 151-81
• Display abstract

• The Eph family is the largest of all known tyrosine kinase receptor-ligand systems. They are expressed in distinct, but overlapping, spatial and temporal patterns during embryonic development and postnatal life, and function in a variety of morphogenic events. The best known function is their role in the guidance of migration of axons and cells in the nervous system through repulsive interactions. They may also play a role in angiogenesis, tissue patterning, and tumor formation.

• Di Fruscio M, Chen T, Bonyadi S, Lasko P, Richard S
• The identification of two Drosophila K homology domain proteins. Kep1 and SAM are members of the Sam68 family of GSG domain proteins.
• J Biol Chem. 1998; 273: 30122-30
• Display abstract

• Sam68 is a member of a growing family of RNA-binding proteins that contains an extended K homology (KH) domain embedded in a larger domain called the GSG (GRP33, Sam68, GLD1) domain. To identify GSG domain family members, we searched data bases for expressed sequence tags encoding related portions of the Sam68 KH domain. Here we report the identification of two novel Drosophila KH domain proteins, which we termed KEP1 (KH encompassing protein) and SAM. SAM bears sequence identity with mammalian Sam68 and may be the Drosophila Sam68 homolog. We demonstrate that SAM, KEP1, and the recently identified Drosophila Who/How are RNA-binding proteins that are able to self-associate into homomultimers. The GSG domain of KEP1 and SAM was necessary to mediate the RNA binding and self-association. To elucidate the cellular roles of these proteins, SAM, KEP1, and Who/How were expressed in mammalian and Drosophila S2 cells. KEP1 and Who/How were nuclear and SAM was cytoplasmic. The expression of KEP1 and SAM, but not Who/How, activated apoptotic pathways in Drosophila S2 cells. The identification of KEP1 and SAM implies that a large GSG domain protein family exists and helps redefine the boundaries of the GSG domain. Taken together, our data suggest that KEP1 and SAM may play a role in the activation or regulation of apoptosis and further implicate the GSG domain in RNA binding and oligomerization.

• Garman SC, Kinet JP, Jardetzky TS
• Crystal structure of the human high-affinity IgE receptor.
• Cell. 1998; 95: 951-61
• Display abstract

• Allergic responses result from the activation of mast cells by the human high-affinity IgE receptor. IgE-mediated allergic reactions may develop to a variety of environmental compounds, but the initiation of a response requires the binding of IgE to its high-affinity receptor. We have solved the X-ray crystal structure of the antibody-binding domains of the human IgE receptor at 2.4 A resolution. The structure reveals a highly bent arrangement of immunoglobulin domains that form an extended convex surface of interaction with IgE. A prominent loop that confers specificity for IgE molecules extends from the receptor surface near an unusual arrangement of four exposed tryptophans. The crystal structure of the IgE receptor provides a foundation for the development of new therapeutic approaches to allergy treatment.

• Burke CL, Stern DF
• Activation of Neu (ErbB-2) mediated by disulfide bond-induced dimerization reveals a receptor tyrosine kinase dimer interface.
• Mol Cell Biol. 1998; 18: 5371-9
• Display abstract

• Receptor dimerization is a crucial intermediate step in activation of signaling by receptor tyrosine kinases (RTKs). However, dimerization of the RTK Neu (also designated ErbB-2, HER-2, and p185(neu)), while necessary, is not sufficient for signaling. Earlier work in our laboratory had shown that introduction of an ectopic cysteine into the Neu juxtamembrane domain induces Neu dimerization but not signaling. Since Neu signaling does require dimerization, we hypothesized that there are additional constraints that govern signaling ability. With the importance of the interreceptor cross-phosphorylation reaction, a likely constraint was the relative geometry of receptors within the dimer. We have tested this possibility by constructing a consecutive series of cysteine substitutions in the Neu juxtamembrane domain in order to force dimerization along a series of interreceptor faces. Within the group that dimerized constitutively, a subset had transforming activity. The substitutions in this subset all mapped to the same face of a predicted alpha helix, the most likely conformation for the intramembrane domain. Furthermore, this face of interaction aligns with the projected Neu* V664E substitution and with a predicted amphipathic interface in the Neu juxtamembrane domain. We propose that these results identify an RTK dimer interface and that dimerization of this RTK induces an extended contact between juxtamembrane and intramembrane alpha helices.

• Weiss A, Schlessinger J
• Switching signals on or off by receptor dimerization.
• Cell. 1998; 94: 277-80

• Yaich L et al.
• Functional analysis of the Numb phosphotyrosine-binding domain using site-directed mutagenesis.
• J Biol Chem. 1998; 273: 10381-8
• Display abstract

• The Numb protein is involved in cell fate determination during Drosophila neural development. Numb has a protein domain homologous to the phosphotyrosine-binding domain (PTB) in the adaptor protein Shc. In Shc, this domain interacts with specific phosphotyrosine containing motifs on receptor tyrosine kinases and other signaling molecules. Residues N-terminal to the phosphotyrosine are also crucial for phosphopeptide binding to the Shc PTB domain. Several amino acid residues in Shc have been implicated by site-directed mutagenesis to be critical for Shc binding to receptor tyrosine kinases. We have generated homologous mutations in Numb to test whether, in vivo, these changes affect Numb function during Drosophila sensory organ development. Two independent amino acid changes that interfere with Shc binding to phosphotyrosine residues do not affect Numb activity in vivo. In contrast, a mutation shown to abrogate the ability of the Shc PTB domain to bind residues upstream of the phosphotyrosine virtually eliminates Numb function. Similar results were observed in vitro by examining the binding of the Numb PTB domain to proteins from Schneider S2 cells. Our data confirm the importance of the PTB domain for Numb function but strongly suggest that the Numb PTB domain is not involved in phosphotyrosine-dependent interactions.

• Davies C, Gerstner RB, Draper DE, Ramakrishnan V, White SW
• The crystal structure of ribosomal protein S4 reveals a two-domain molecule with an extensive RNA-binding surface: one domain shows structural homology to the ETS DNA-binding motif.
• EMBO J. 1998; 17: 4545-58
• Display abstract

• We report the 1.7 A crystal structure of ribosomal protein S4 from Bacillus stearothermophilus. To facilitate the crystallization, 41 apparently flexible residues at the N-terminus of the protein have been deleted (S4Delta41). S4Delta41 has two domains; domain 1 is completely alpha-helical and domain 2 comprises a five-stranded antiparallel beta-sheet with three alpha-helices packed on one side. Domain 2 is an insertion within domain 1, and it shows significant structural homology to the ETS domain of eukaryotic transcription factors. A phylogenetic analysis of the S4 primary structure shows that the likely RNA interaction surface is predominantly on one side of the protein. The surface is extensive and highly positively charged, and is centered on a distinctive canyon at the domain interface. The latter feature contains two arginines that are totally conserved in all known species of S4 including eukaryotes, and are probably crucial in binding RNA. As has been shown for other ribosomal proteins, mutations within S4 that affect ribosome function appear to disrupt the RNA-binding sites. The structure provides a framework with which to probe the RNA-binding properties of S4 by site-directed mutagenesis.

• Himanen JP, Henkemeyer M, Nikolov DB
• Crystal structure of the ligand-binding domain of the receptor tyrosine kinase EphB2.
• Nature. 1998; 396: 486-91
• Display abstract

• The Eph receptors, which bind a group of cell-membrane-anchored ligands known as ephrins, represent the largest subfamily of receptor tyrosine kinases (RTKs). They are predominantly expressed in the developing and adult nervous system and are important in contact-mediated axon guidance, axon fasciculation and cell migration. Eph receptors are unique among other RTKs in that they fall into two subclasses with distinct ligand specificities, and in that they can themselves function as ligands to activate bidirectional cell-cell signalling. We report here the crystal structure at 2.9 A resolution of the amino-terminal ligand-binding domain of the EphB2 receptor (also known as Nuk). The domain folds into a compact jellyroll beta-sandwich composed of 11 antiparallel beta-strands. Using structure-based mutagenesis, we have identified an extended loop that is important for ligand binding and class specificity. This loop, which is conserved within but not between Eph RTK subclasses, packs against the concave beta-sandwich surface near positions at which missense mutations cause signalling defects, localizing the ligand-binding region on the surface of the receptor.

• Masiakowski P, Yancopoulos GD
• The Wnt receptor CRD domain is also found in MuSK and related orphan receptor tyrosine kinases.
• Curr Biol. 1998; 8: 407-407

• Ng J, Li R, Morgan K, Simon J
• Evolutionary conservation and predicted structure of the Drosophila extra sex combs repressor protein.
• Mol Cell Biol. 1997; 17: 6663-72
• Display abstract

• The Drosophila extra sex combs (esc) protein, a member of the Polycomb group (PcG), is a transcriptional repressor of homeotic genes. Genetic studies have shown that esc protein is required in early embryos at about the time that other PcG proteins become engaged in homeotic gene repression. The esc protein consists primarily of multiple copies of the WD repeat, a motif that has been implicated in protein-protein interaction. To further investigate the domain organization of esc protein, we have isolated and characterized esc homologs from divergent insect species. We report that esc protein is highly conserved in housefly (72% identical to Drosophila esc), butterfly (55% identical), and grasshopper (56% identical). We show that the butterfly homolog provides esc function in Drosophila, indicating that the sequence similarities reflect functional conservation. Homology modeling using the crystal structure of another WD repeat protein, the G-protein beta-subunit, predicts that esc protein adopts a beta-propeller structure. The sequence comparisons and modeling suggest that there are seven WD repeats in esc protein which together form a seven-bladed beta-propeller. We locate the conserved regions in esc protein with respect to this predicted structure. Site-directed mutagenesis of specific loops, predicted to extend from the propeller surface, identifies conserved parts of esc protein required for function in vivo. We suggest that these regions might mediate physical interaction with esc partner proteins.

• Watson P, Davey J
• Analysis of Prk1, a putative receptor kinase from fission yeast.
• Biochem Soc Trans. 1997; 25: 444-444

• Smit L, Borst J
• The Cbl family of signal transduction molecules.
• Crit Rev Oncog. 1997; 8: 359-79
• Display abstract

• This review summarizes the current knowledge about the structure, expression, and signaling function of Cbl. A mutant Cbl form (v-Cbl) was first identified as the product of the Cas-NS-1 murine retrovirus that induces lymphomas and leukemias. Two members of the Cbl family have since been defined in mammals (c-Cbl and Cbl-b), one in C. elegans (Sli-1) and one in Drosophila (D-Cbl). There is high conservation between Cbl species in the amino-terminal region that contains a putative phosphotyrosine binding domain and a Ring finger motif. In the carboxy-terminal region, mammalian Cbl forms share a proline-rich stretch, conserved tyrosine residues, and a leucine zipper. Cbl participates in signaling by receptor protein tyrosine kinases, as well as antigen and cytokine receptors that signal via associated cytoplasmic protein tyrosine kinases. Cbl is recruited to the tyrosine kinase module of these receptors and tyrosine phosphorylated after cellular activation. It functions as a scaffold protein and associates with SH2 and SH3 domain containing molecules, including the Crk adaptor family and Vav. By analogy with the C. elegans homologue Sli-1, Cbl is proposed to be a negative regulator of receptor tyrosine kinase signaling. After deletion mutation in a region close to the Ring finger, c-Cbl becomes oncogenic. Such mutations are suggested to result in a structural alteration, allowing oncogenic mutants to displace wild-type c-Cbl from the receptor complex and to abrogate its negative regulatory function.

• Oishi I, Sugiyama S, Liu ZJ, Yamamura H, Nishida Y, Minami Y
• A novel Drosophila receptor tyrosine kinase expressed specifically in the nervous system. Unique structural features and implication in developmental signaling.
• J Biol Chem. 1997; 272: 11916-23
• Display abstract

• We report the identification and characterization of Dnrk (Drosophila neurospecific receptor kinase), a Drosophila gene encoding a putative receptor tyrosine kinase (RTK) highly related to the Trk and Ror families of RTKs. During Drosophila embryogenesis, the Dnrk gene is expressed specifically in the developing nervous system. The Dnrk protein possesses two conserved cysteine-containing domains and a kringle domain within its extracellular domain, resembling those observed in Ror family RTKs (Ror1, Ror2, and a Drosophila Ror, Dror). This protein contains the catalytic tyrosine kinase (TK) domain with two putative ATP-binding motifs, resembling those observed in another Drosophila RTK (Dtrk) that mediates homophilic cell adhesion. The TK domain of Dnrk, expressed in bacteria or mammalian cells, exhibits apparent autophosphorylation activities in vitro. The TK domain lacking the distal ATP-binding motif also exhibits autophosphorylation activity, yet to a lesser extent. In addition to its TK activity, there are several putative tyrosine-containing motifs that upon phosphorylation may interact with Src homology 2 regions of other signaling molecules. Collectively, these results suggest that Dnrk may play an important role in neural development during Drosophila embryogenesis.

• Dubois T et al.
• Structure and sites of phosphorylation of 14-3-3 protein: role in coordinating signal transduction pathways.
• J Protein Chem. 1997; 16: 513-22
• Display abstract

• The 14-3-3 family are homo- and heterodimeric proteins whose biological role has been unclear for some time, although they are now gaining acceptance as a novel type of 'adaptor' protein that modulates interactions between components of signal transduction pathways, rather than by direct activation or inhibition. It is becoming apparent that phosphorylation of the binding partner and possibly also the 14-3-3 proteins may regulate these interactions. 14-3-3 isoforms interact with a novel phosphoserine (Sp) motif on many proteins, RSX1,2SpXP. The two isoforms that interact with Raf-1 are phosphorylated in vivo on Ser185 in a consensus sequence motif for proline-directed kinases. The crystal structure of 14-3-3 indicates that this phosphorylation could regulate interaction of 14-3-3 with its target proteins. We have now identified a number of additional phosphorylation sites on distinct mammalian and yeast isoforms.

• Heinrichs V, Baker BS
• In vivo analysis of the functional domains of the Drosophila splicing regulator RBP1.
• Proc Natl Acad Sci U S A. 1997; 94: 115-20
• Display abstract

• The Drosophila splicing factor RBP1 participates together with TRA and TRA-2 in the regulation of alternative splicing of doublesex (dsx) pre-mRNA. It does so by recognizing RBP1 RNA target sequences in the dsx pre-mRNA. RBP1 belongs to the Ser-Arg-rich (SR) protein family of splicing factors, which have in common a N-terminal RNA recognition motif-type RNA binding domain, a Gly-rich region, and a C-terminal SR domain. Using a tissue culture transfection assay, we demonstrate that the Gly residues within the Gly-rich domain, the ribonucleoprotein motifs within the RNA recognition motif RNA binding domain, and the SR domain are required for regulation of dsx splicing by RBP1 in vivo. Furthermore, using a two-hybrid system, we show protein-protein interactions between RBP1 and itself and between RBP1 and TRA-2. The SR domain and the Gly residues within the Gly-rich domain of RBP1 were found to be involved in these protein-protein interactions. Our results suggest that RBP1 and TRA-2 function in regulation of dsx splicing by forming a complex.

• Hoffmann KM, Tonks NK, Barford D
• The crystal structure of domain 1 of receptor protein-tyrosine phosphatase mu.
• J Biol Chem. 1997; 272: 27505-8
• Display abstract

• Receptor-like protein-tyrosine phosphatases (RPTPs) play important roles in regulating intracellular processes. We have been investigating the regulation and function of RPTPmu, a receptor-like PTP related to the Ig superfamily of cell adhesion molecules. Recently, the crystal structure of a dimer of the membrane proximal domain of RPTPalpha (RPTPalpha D1) was described (Bilwes, A. M., den Hertog, J., Hunter, T., and Noel J. P. (1996) Nature 382, 555-559). Within this crystal structure, the catalytic site of each subunit of the dimer is sterically blocked by the insertion of the N-terminal helix-turn-helix segment of the dyad-related monomer. It was proposed that dimerization would lead to inhibition of catalytic activity and may provide a paradigm for the regulation of the RPTP family. We have determined the crystal structure, to 2.3 A resolution, of RPTPmu D1, which shares 46% sequence identity with that of RPTPalpha D1. Although the tertiary structures of RPTPalpha D1 and RPTPmu D1 are very similar, with a root mean square deviation between equivalent Calpha atoms of 1.1 A, the quaternary structures of these two proteins are different. Neither the catalytic site nor the N-terminal helix-turn-helix segment of RPTPmu D1 participates in protein-protein interactions. The catalytic site of RPTPmu D1 is unhindered and adopts an open conformation similar to that of the cytosolic PTP, PTP1B (Barford, D., Flint, A. J., and Tonks, N. K. (1994) Science 263, 1397-1404). We propose that dimerization-induced modulation of RPTP activity may not be a general feature of this family of enzymes.

• Peterson AJ, Kyba M, Bornemann D, Morgan K, Brock HW, Simon J
• A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.
• Mol Cell Biol. 1997; 17: 6683-92
• Display abstract

• The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph. Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analysis of site-directed point mutations identifies residues that are important for SPM domain function. These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo. We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

• Leon DA, Herberg FW, Banky P, Taylor SS
• A stable alpha-helical domain at the N terminus of the RIalpha subunits of cAMP-dependent protein kinase is a novel dimerization/docking motif.
• J Biol Chem. 1997; 272: 28431-7
• Display abstract

• The RIalpha subunit of cAMP-dependent protein kinase is maintained as an asymmetric dimer by a dimerization motif at the N terminus. Based on resistance to proteolysis and expression as a discrete domain in Escherichia coli, this motif is defined as residues 12-61. This motif is chemically, kinetically, and thermally stable. The two endogenous interchain disulfide bonds between Cys16 and Cys37 in RIalpha are extremely resistant to reduction even in 8 M urea, indicating that they are well shielded from the reducing environment of the cell. The disulfide bonds were present in recombinant RIalpha as well as when the dimerization domain alone was expressed in E. coli, emphasizing the unusual stability of this motif and the disulfide bonds. Although 100 mM dithiothreitol was sufficient to reduce the disulfide bonds, it did not abolish dimerization. In addition, a stable dimer also still formed when Cys37 was replaced with His, confirming unambiguously the original antiparallel alignment of the disulfide bonds. Thus, both in vitro and in vivo, disulfide bonds are not required for dimerization. Circular dichroism of the dimerization domain indicated a high content of a thermostable alpha-helix. Based on the CD data, trypsin resistance of the fragment, location of the disulfide bonds, and amphipathic helix predictions, potential models are discussed. A new alignment of the dimerization domains of RI, RII, and cGMP-dependent protein kinase elucidates fundamental similarities as well as significant differences among these three domains.

• Jousset C et al.
• A domain of TEL conserved in a subset of ETS proteins defines a specific oligomerization interface essential to the mitogenic properties of the TEL-PDGFR beta oncoprotein.
• EMBO J. 1997; 16: 69-82
• Display abstract

• TEL is a novel member of the ETS family of transcriptional regulators which is frequently involved in human leukemias as the result of specific chromosomal translocations. We show here by co-immunoprecipitation and GST chromatography analyses that TEL and TEL-derived fusion proteins form homotypic oligomers in vitro and in vivo. Deletion mutagenesis identifies the TEL oligomerization domain as a 65 amino acid region which is conserved in a subset of the ETS proteins including ETS-1, ETS-2, FLI-1, ERG-2 and GABP alpha in vertebrates and PNTP2, YAN and ELG in Drosophila. TEL-induced oligomerization is shown to be essential for the constitutive activation of the protein kinase activity and mitogenic properties of TEL-platelet derived growth factor receptor beta (PDGFR beta), a fusion oncoprotein characteristic of the leukemic cells of chronic myelomonocytic leukemia harboring a t(5;12) chromosomal translocation. Swapping experiments in which the TEL oligomerization domain was exchanged by the homologous domains of representative vertebrate ETS proteins including ETS-1, ERG-2 and GABP alpha show that oligomerization is a specific property of the TEL amino-terminal conserved domain. These results indicate that the amino-terminal domain conserved in a subset of the ETS proteins has evolved to generate a specialized protein-protein interaction interface which is likely to be an important determinant of their specificity as transcriptional regulators.

• Portman DS, O'Connor JP, Dreyfuss G
• YRA1, an essential Saccharomyces cerevisiae gene, encodes a novel nuclear protein with RNA annealing activity.
• RNA. 1997; 3: 527-37
• Display abstract

• The complexity of eukaryotic mRNA processing suggests a need for certain factors, called RNA chaperones, that can modulate RNA secondary structure as well as the interactions between pre-mRNA and trans-acting components. To identify factors that may fulfill this role in the yeast Saccharomyces cerevisiae, we fractionated whole-cell extracts and assayed for activity that could facilitate a specific RNA-RNA annealing reaction. We detected one strong RNA annealing activity and purified it to homogeneity. This previously undescribed factor, Yra1p, is localized to the nucleus; its sequence contains one RNP-motif RNA-binding domain. The YRA1 gene contains a 766-nt intron, the second-largest identified in this organism, and Yra1p serves an essential, nonredundant function. Taken together, our findings indicate that Yra1p is likely to have an important role in S. cerevisiae nuclear pre-mRNA metabolism.

• Jakobs R, de Lorenzo C, Spiess E, Strand D, Mechler BM
• Homo-oligomerization domains in the lethal(2)giant larvae tumor suppressor protein, p127 of Drosophila.
• J Mol Biol. 1996; 264: 484-96
• Display abstract

• The p127 tumor suppressor protein encoded by the lethal(2)giant larvae, l(2)gl, gene of Drosophila melanogaster forms high molecular mass complexes consisting predominantly of p127 molecules. To determine whether p127 can self-assemble in the absence of other binding factors, we analyzed the size of in vitro synthesized p127 by gel filtration and found that p127 is always recovered in a high molecular mass form, demonstrating that p127 can oligomerize on its own. Previous studies have revealed that p127 may contain three homo-oligomerization domains. To more accurately delineate these domains, we have generated a series of 32 chimaeric proteins made of defined portions of p127 fused to protein A, which behaves as a monomeric protein, and determined the level of oligomerization of the fused proteins. This study allowed us to map three discrete homo-oligomerization domains, each of approximately 50 amino acid residues in length. These domains, designated as HD-I, HD-II and HD-III, are located between amino acid residues 160 and 204, 247 and 298, and 706 and 749, respectively. Further analysis showed that the HD-I and HD-II domains can bind to themselves and to each other. We also mapped a domain in p127 between amino acid residues 377 and 438, which strongly reduces the degree of multimerization of chimaeric proteins containing HD-I and/or HD-II. Electron microscopy examination of negatively stained chimaeric proteins showed that protein A fused with either the domain HD-II or the domain HD-III forms discrete structures consistent with the formation of quaternary complexes, whereas protein A fused to a non-self binding domain of p127 appeared monomeric. Our results indicate that p127 alone is able to build quaternary structures forming a network with which other proteins associate. As revealed by the tumorous phenotype resulting from the inactivation of the l(2)gl gene, the organization of the p127 network and its association with other proteins play critical roles in the control of cell proliferation.

• Gregory SL, Kortschak RD, Kalionis B, Saint R
• Characterization of the dead ringer gene identifies a novel, highly conserved family of sequence-specific DNA-binding proteins.
• Mol Cell Biol. 1996; 16: 792-9
• Display abstract

• We reported the identification of a new family of DNA-binding proteins from our characterization of the dead ringer (dri) gene of Drosophila melanogaster. We show that dri encodes a nuclear protein that contains a sequence-specific DNA-binding domain that bears no similarity to known DNA-binding domains. A number of proteins were found to contain sequences homologous to this domain. Other proteins containing the conserved motif include yeast SWI1, two human retinoblastoma binding proteins, and other mammalian regulatory proteins. A mouse B-cell-specific regulator exhibits 75% identity with DRI over the 137-amino-acid DNA-binding domains of these proteins, indicating a high degree of conservation of this domain. Gel retardation and optimal binding site screens revealed that the in vitro sequence specificity of DRI is strikingly similar to that of many homeodomain proteins, although the sequence and predicted secondary structure do not resemble a homeodomain. The early general expression of dri and the similarity of DRI and homeodomain in vitro DNA-binding specificity compound the problem of understanding the in vivo specificity of action of these proteins. Maternally derived dri product is found throughout the embryo until germ band extension, when dri is expressed in a developmentally regulated set of tissues, including salivary gland ducts, parts of the gut, and a subset of neural cells. The discovery of this new, conserved DNA-binding domain offers an explanation for the regulatory activity of several important members of this class and predicts significant regulatory roles for the others.

• Ellis C, Kasmi F, Ganju P, Walls E, Panayotou G, Reith AD
• A juxtamembrane autophosphorylation site in the Eph family receptor tyrosine kinase, Sek, mediates high affinity interaction with p59fyn.
• Oncogene. 1996; 12: 1727-36
• Display abstract

• The large subfamily of receptor tyrosine kinases (RTKs) for which EPH is the prototype have likely roles in intercellular communication during normal mammalian development, but the biochemical signalling pathways utilised by this family are poorly characterised. We have now identified two in vitro autophosphorylation sites within the juxtamembrane domain of the Eph family member Sek, and a candidate binding protein for the activated Sek kinase. Specific antibodies defined Sek as a 130 kDa glycoprotein with protein kinase activity expressed in keratinocytes, whilst a bacterially expressed gst-Sek kinase domain fusion protein autophosphorylated exclusively on tyrosine residues, confirming that Sek encodes an authentic protein tyrosine kinase. Two dimensional phosphopeptide mapping and site-directed mutagenesis defined juxtamembrane residue Y602 as a major site of in vitro autophosphorylation in Sek, whilst Y596 was phosphorylated to a lower stoichiometry. Complimentary approaches of in vitro binding assays and BIAcore analysis revealed a high affinity association between the Y602 Sek autophosphorylation site and the cytoplasmic tyrosine kinase p59fyn, an interaction mediated through the SH2 domain of this intracellular signalling molecule. Moreover, these data identify the novel phosphotyrosyl motif pYEDP as mediating high affinity association with fyn-SH2, extending the previously defined consensus motif for this interaction. The extensive conservation of this fyn-binding motif within the juxtamembrane domain of Eph family RTKs suggests that signalling through fyn, or fyn-related, tyrosine kinases may be utilised by many members of this large subclass of transmembrane receptors.

• Smith SO, Smith CS, Bormann BJ
• Strong hydrogen bonding interactions involving a buried glutamic acid in the transmembrane sequence of the neu/erbB-2 receptor.
• Nat Struct Biol. 1996; 3: 252-8
• Display abstract

• The receptor tyrosine kinase encoded by the neu/erbB-2 proto-oncogene is constitutively activated by a single valine to glutamic acid substitution at position 664 in the predicted membrane-spanning sequence of the receptor. We have explored the structural changes involved in receptor activation with polarized FTIR and magic angle spinning NMR spectroscopy. The hydrophobic transmembrane sequence folds into a well-defined alpha-helical structure spanning the membrane bilayer. Measurements of the pKa and 13C chemical shift anisotropy of Glu 664 reveal that the side chain carboxyl group is protonated and strongly hydrogen bonded. These studies provide direct evidence for glutamate hydrogen-bonding interactions in the mechanism of receptor dimerization and activation.

• Bilwes AM, den Hertog J, Hunter T, Noel JP
• Structural basis for inhibition of receptor protein-tyrosine phosphatase-alpha by dimerization.
• Nature. 1996; 382: 555-9
• Display abstract

• Receptor-like protein-tyrosine phosphatases (RPTPs), like their non-receptor counterparts, regulate the level of phosphotyrosine-containing proteins derived from the action of protein-tyrosine kinases. RPTPs are type-I integral membrane proteins which contain one or two catalytic domains in their cytoplasmic region. It is not known whether extracellular ligands regulate the activity of RPTPs. Here we describe the crystal structure of the membrane-proximal catalytic domain (D1) of a typical RPTP, murine RPTP alpha. Significant structural deviations from the PTP1B fold reside within the amino-terminal helix-turn-helix segment of RPTPalphaD1 (residues 214 to 242) and a distinctive two-stranded beta-sheet formed between residues 211-213 and 458-461. The turn of the N-terminal segment inserts into the active site of a dyad-related D1 monomer. On the basis of two independent crystal structures, sequence alignments, and the reported biological activity of EGF receptor/CD45 chimaeras, we propose that dimerization and active-site blockage is a physiologically important mechanism for downregulating the catalytic activity of RPTPalpha and other RPTPs.

• Smibert CA, Wilson JE, Kerr K, Macdonald PM
• smaug protein represses translation of unlocalized nanos mRNA in the Drosophila embryo.
• Genes Dev. 1996; 10: 2600-9
• Display abstract

• nanos mRNA, which encodes the localized component of the Drosophila posterior body patterning determinant, is normally translated only at the posterior pole of the embryo, where the mRNA is concentrated. Here we identify two similar cis-acting sequences in the nanos mRNA 3' untranslated region that mediate translational repression. These sequences bind an embryonic protein of 135 kD, smaug, and we refer to them as smaug recognition elements (SREs). Analysis of point mutations in the SREs reveals a strong correlation between smaug binding and translational repression; mutants unable to bind smaug in vitro are not repressed translationally in vivo, whereas mutants that do bind smaug remain repressed translationally. These results strongly suggest that smaug acts in translational repression of unlocalized nanos mRNA. Translational repression is essential, as embryos expressing a nanos mRNA with mutated SREs develop with anterior body patterning defects and die, despite correct localization of the RNA.

• Kennedy D, Wood SA, Ramsdale T, Tam PP, Steiner KA, Mattick JS
• 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.
• Biomed Pept Proteins Nucleic Acids. 1996; 2: 93-9
• Display abstract

• 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.

• Bouvet P, Matsumoto K, Wolffe AP
• Sequence-specific RNA recognition by the Xenopus Y-box proteins. An essential role for the cold shock domain.
• J Biol Chem. 1995; 270: 28297-303
• Display abstract

• The Xenopus Y-box protein FRGY2 has a role in the translational silencing of masked maternal mRNA. Here, we determine that FRGY2 will recognize specific RNA sequences. The evolutionarily conserved nucleic acid-binding cold shock domain is required for sequence-specific interactions with RNA. However, RNA binding by FRGY2 is facilitated by N- and C-terminal regions flanking the cold shock domain. The hydrophilic C-terminal tail domain of FRGY2 interacts with RNA independent of the cold shock domain but does not determine sequence specificity. Thus, both sequence-specific and nonspecific RNA recognition domains are contained within the FRGY2 protein.

• Dura JM, Taillebourg E, Preat T
• The Drosophila learning and memory gene linotte encodes a putative receptor tyrosine kinase homologous to the human RYK gene product.
• FEBS Lett. 1995; 370: 250-4
• Display abstract

• The linotte mutant was isolated on the basis of its learning and memory deficit. Interestingly, linotte individuals carrying a null mutation are viable, indicating that the linotte gene is not required for vital functions. We show here that the linotte gene encodes a putative receptor tyrosine kinase, homologous to the human protein RYK. These products are unique among receptor tyrosine kinases, since they possess a short extra cellular domain, and a modified intracellular catalytic domain. In particular, the subdomains directly involved in ATP binding and phosphotransfer reaction display remarkable variations. These results suggest that linotte is part of a novel signal transduction cascade involved in learning and memory.

• Bycroft M, Grunert S, Murzin AG, Proctor M, St Johnston D
• NMR solution structure of a dsRNA binding domain from Drosophila staufen protein reveals homology to the N-terminal domain of ribosomal protein S5.
• EMBO J. 1995; 14: 3563-71
• Display abstract

• The double-stranded RNA binding domain (dsRBD) is an approximately 65 amino acid motif that is found in a variety of proteins that interact with double-stranded (ds) RNA, such as Escherichia coli RNase III and the dsRNA-dependent kinase, PKR. Drosophila staufen protein contains five copies of this motif, and the third of these binds dsRNA in vitro. Using multinuclear/multidimensional NMR methods, we have determined that staufen dsRBD3 forms a compact protein domain with an alpha-beta-beta-beta-alpha structure in which the two alpha-helices lie on one face of a three-stranded anti-parallel beta-sheet. This structure is very similar to that of the N-terminal domain of a prokaryotic ribosomal protein S5. Furthermore, the consensus derived from all known S5p family sequences shares several conserved residues with the dsRBD consensus sequence, indicating that the two domains share a common evolutionary origin. Using in vitro mutagenesis, we have identified several surface residues which are important for the RNA binding of the dsRBD, and these all lie on the same side of the domain. Two residues that are essential for RNA binding, F32 and K50, are also conserved in the S5 protein family, suggesting that the two domains interact with RNA in a similar way.

• Marengere LE, Pawson T
• Structure and function of SH2 domains.
• J Cell Sci Suppl. 1994; 18: 97-104
• Display abstract

• In order for cells to respond to their environment, a series of regulated molecular events has to take place. External signalling molecules bind to cellular receptors and thereby trigger the activation of multiple intracellular pathways, which modify cellular phenotypes. The cell-surface receptors for a wide range of polypeptide hormones possess protein tyrosine kinase activity, which is induced by binding of the appropriate extracellular ligand. Tyrosine phosphorylation can act as a molecular switch, by initiating the recruitment of cytoplasmic effector molecules containing Src homology (SH) 2 domains, to activated receptors. These SH2-containing proteins, in turn, regulate intracellular signalling pathways. Here, we discuss the role of tyrosine phosphorylation in triggering signalling pathways, as well as the functions of SH2 domains, which mediate these events through phosphotyrosine-dependent protein-protein interactions.

• Schacke H, Rinkevich B, Gamulin V, Muller IM, Muller WE
• Immunoglobulin-like domain is present in the extracellular part of the receptor tyrosine kinase from the marine sponge Geodia cydonium.
• J Mol Recognit. 1994; 7: 273-6
• Display abstract

• We have isolated and characterized two cDNAs from the marine sponge Geodia cydonium coding for a new member of a receptor tyrosine kinase of class II. The deduced amino acid sequence shows two characteristic domains: (i) the tyrosine kinase domain; and (ii) an immunoglobulin-like domain. The latter part shows high homology to the vertebrate C2 type immunoglobulin domain. This result demonstrates that immunoglobulin domains are not recent achievements of higher animals but exist also in those animals which have diverged from other organisms about 800 million years ago.

• Ordway RW, Pallanck L, Ganetzky B
• A TPR domain in the SNAP secretory proteins.
• Trends Biochem Sci. 1994; 19: 530-1

• Pawson T
• Tyrosine kinase signalling pathways.
• Princess Takamatsu Symp. 1994; 24: 303-22
• Display abstract

• Protein-tyrosine kinases act as receptors for a wide range of external signals that control the growth and differentiation of normal cells. Additionally, many retroviral and cellular oncogenes encode tyrosine kinase variants that are constitutively active. Recent evidence suggests that the intracellular targets of tyrosine kinases contain a protein module of approximately 100 amino acids, the Src homology 2 (SH2) domain. SH2 domains directly recognize tyrosine phosphorylation sites, and are thereby recruited to activated, autophosphorylated growth factor receptors. These interactions, in turn, stimulate the biochemical signalling pathways that control gene expression, cytoskeletal architecture, and cell metabolism. SH2-containing proteins frequently contain a distinct element of approximately 50 residues, the SH3 domain, that recognizes proline-rich motifs. Proteins with SH2 and SH3 domains can act as adaptors to couple tyrosine kinases to downstream targets with SH3-binding sites. A specific example of the synergistic action of SH2 and SH3 domains involves regulation of the Ras pathway by the adaptor protein Sem-5/drk/Grb2, which links tyrosine kinases to the Ras guanine nucleotide releasing protein Sos, which converts Ras to the active GTP-bound state.

• Schlessinger J
• Cellular signaling by receptor tyrosine kinases.
• Harvey Lect. 1993; 89: 105-23

• Johnson JD, Edman JC, Rutter WJ
• A receptor tyrosine kinase found in breast carcinoma cells has an extracellular discoidin I-like domain.
• Proc Natl Acad Sci U S A. 1993; 90: 10891-10891

• Inglese J, Freedman NJ, Koch WJ, Lefkowitz RJ
• Structure and mechanism of the G protein-coupled receptor kinases.
• J Biol Chem. 1993; 268: 23735-8

• Haynes SR
• The RNP motif protein family.
• New Biol. 1992; 4: 421-9
• Display abstract

• RNA-protein interactions play critical roles in a variety of important processes in the cell, such as mRNA splicing and translation. Many RNA binding proteins contain an approximately 80-aminoacid conserved sequence, the RNP motif, that has been shown in several cases to constitute the binding domain. As a class, RNP motif proteins bind many different RNAs with varying degrees of sequence specificity. A major area of current interest is the elucidation of the structure-function relationships in these proteins: the structure of the motif and the residues that are important for sequence-specific binding, the roles of the individual motifs in proteins with multiple RNP motifs, and the functions of other domains found in RNP motif proteins. Recent experiments have provided some insight into these issues and have paved the way for a more detailed understanding of this protein family.

• Shepherd JC, McGinnis W, Carrasco AE, De Robertis EM, Gehring WJ
• Fly and frog homoeo domains show homologies with yeast mating type regulatory proteins.
• Nature. 1984; 310: 70-1
• Display abstract

• Homoeotic genes in the bithorax and Antennapedia complexes of Drosophila melanogaster appear to specify the developmental fate of segments of the fly. Some of these genes (Ultrabithorax, Antennapedia and fushi tarazu) share homology due to their conservation of a 'homoeo domain'1,2 consisting of 60 amino acids. Cross-hybridization and cloning experiments show that the homoeo domain is conserved in a frog (Xenopus laevis) gene expressed in early development and may also be present in earthworm, beetle, chicken, mouse and human genomes. The extreme conservation found in the amino acid sequences between the Drosophila and Xenopus domains suggests that the domain has a vital function in the control of early development. Here we report the results of a search made in the Dayhoff sequence bank, which reveals a lesser but apparently significant homology between the homoeo domain and the amino acids coded from parts of the a 1 and alpha 2 mating type genes of the yeast Saccharomyces cerevisiae.

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