Secondary literature sources for the RasGEF domain

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

Direct involvement of the small GTP-binding protein Rho in lbc oncogene function.

Zheng Y, Olson MF, Hall A, Cerione RA, Toksoz D
J Biol Chem. 1995; 270: 9031-4

The lbc oncogene is tumorigenic in nude mice, transforms NIH 3T3 fibroblasts, and encodes a Dbl homology domain found in several transforming gene products including the dbl oncogene product. While both lbc- and dbl-transformed NIH 3T3 foci exhibited a comparable gross appearance, lbc-transformed cell morphology was clearly distinct from that of dbl-transformed cells. Given these differences, we investigated the biochemical activity and target specificity of the Lbc oncoprotein both in vivo and in vitro. Here we show that Lbc associates specifically with the GTP-binding protein Rho in vivo, but not with the Ras, Rac, or Cdc42Hs GTP-binding proteins, and that recombinant, affinity-purified Lbc specifically catalyzes the guanine-nucleotide exchange activity of Rho in vitro. Consistent with an in vivo role for Lbc in Rho regulation, we further demonstrate that micro-injected onco-lbc potently induces actin stress fiber formation in quiescent Swiss 3T3 fibroblasts indistinguishable from that induced by Rho. Finally, lbc-induced NIH 3T3 focus formation is inhibited by co-transfection with a rho dominant-negative mutant. These results strongly indicate that the lbc oncogene encodes a specific guanine nucleotide exchange factor for Rho and causes cellular transformation through activation of the Rho signaling pathway.

Phosphatidylinositol 4,5-bisphosphate provides an alternative to guanine nucleotide exchange factors by stimulating the dissociation of GDP from Cdc42Hs.

Zheng Y, Glaven JA, Wu WJ, Cerione RA
J Biol Chem. 1996; 271: 23815-9

Members of the Rho subfamily of Ras-related GTP-binding proteins play important roles in the organization of the actin cytoskeleton and in the regulation of cell growth. We have shown previously that the dbl oncogene product, which represents a prototype for a family of growth regulatory proteins, activates Rho subfamily GTP-binding proteins by catalyzing the dissociation of GDP from their nucleotide binding site. In the present study, we demonstrate that the acidic phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2), provides an alternative mechanism for the activation of Cdc42Hs. Among a variety of lipids tested, only PIP2 was able to stimulate GDP release from Cdc42Hs in a dose-dependent manner, with a half-maximum effect at approximately 50 microM. Unlike the Dbl oncoprotein, which requires the presence of (free) guanine nucleotide in the medium to replace the GDP bound to Cdc42Hs, PIP2 stimulates GDP release from Cdc42Hs in the absence of free guanine nucleotide. PIP2, when incorporated into phosphatidylcholine carrier vesicles, binds tightly to the guanine nucleotide-depleted form of Cdc42Hs and weakly to the GDP-bound form of the GTP-binding protein but does not bind to GTP-bound Cdc42Hs, similar to what was observed for the Dbl oncoprotein. However, mutational analysis of Cdc42Hs indicates that the site that is essential for the functional interaction between PIP2 and Cdc42Hs is distinct from the Dbl-binding site and is located at the positively charged carboxyl-terminal end of the GTP-binding protein. The GDP-releasing activity of PIP2 is highly effective toward Cdc42Hs and Rho (and is similar to the reported effects of PIP2 on Arf (Terui, T., Kahn, R. A., and Randazzo, P. A., (1994) J. Biol. Chem. 269, 28130-28135)), is less effective with Rac, and is not observed with Ras, Rap1a, or Ran. The ability of PIP2 to activate Cdc42Hs (or Rho) and Arf provides a possible point of convergence for the biological pathways regulated by these different GTP-binding proteins and may be related to the synergism observed between Arf and Rho-subtype proteins in the stimulation of phospholipase D activity (Singer, W. D., Brown, H. A., Bokoch, G. M., and Sternweis, P. C. (1995) J. Biol. Chem. 270, 14944-14950).

Mechanistic aspects of signaling through Ras in NIH 3T3 cells.

Zhang K, Papageorge AG, Lowy DR
Science. 1992; 257: 671-4

Serum and growth factors can increase the proportion of Ras in the active guanosine triphosphate (GTP)-bound form. Growth factors might stimulate guanine nucleotide exchange or decrease the activity of the guanosine triphosphatase-activating proteins GAP and neurofibromin (NF1). In NIH 3T3 cells that overexpress the mutant Ras protein His116, which releases bound guanine nucleotide at a constitutively high rate and retains sensitivity to GAP and NF1, the proportion of GTP bound to the His116 protein was not altered by serum or platelet-derived growth factor. However, these mitogens increased the proportion of Ras in the GTP-bound form in cells that overexpressed control Ras proteins with a normal intrinsic rate of guanine nucleotide release. The amount of GTP-bound His116 or control Ras proteins was higher in cells at low density than in cells at high density, which have more GAP-like activity. The lower proportion of GTP-bound Ras in NIH 3T3 cells at high density may result from increased GAP-like activity. By contrast, serum and platelet-derived growth factors appear to stimulate guanine nucleotide exchange.

Structure of guanine-nucleotide-exchange factor human Mss4 and identification of its Rab-interacting surface.

Yu H, Schreiber SL
Nature. 1995; 376: 788-91

Guanine-nucleotide-exchange factors (GEFs) promote the exchange of GDP for GTP in Ras GTPases, and thereby positively regulate their functions. Members of the Sec4/Ypt1/Rab branch of the Ras superfamily are essential for vesicular transport. A GEF for a subset of Rab proteins, termed mammalian suppressor of Sec4 (Mss4), has been identified. Here we use multidimensional NMR to determine the structure of human Mss4 (hMss4), which is the first tertiary structure established for a protein with GEF activity. Mss4 contains a central beta-sheet sandwiched between two small sheets. It also binds a Zn2+ ion through Cys 23, Cys 26, Cys 94 and Cys 97. The Rab-binding surface of hMss4 has subsequently been delineated using chemical-shift perturbation experiments and site-directed mutagenesis. The active site of hMss4 involves the Zn(2+)-binding region and a neighbouring loop.

Crystal structure of Rac1 in complex with the guanine nucleotide exchange region of Tiam1.

Worthylake DK, Rossman KL, Sondek J
Nature. 2000; 408: 682-8

The principal guanine nucleotide exchange factors for Rho family G proteins contain tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains that catalyse nucleotide exchange and the activation of G proteins. Here we have determined the crystal structure of the DH and PH domains of the T-lymphoma invasion and metastasis factor 1 (Tiam1) protein in complex with its cognate Rho family G protein, Rac1. The two switch regions of Rac1 are stabilized in conformations that disrupt both magnesium binding and guanine nucleotide interaction. The resulting cleft in Rac1 is devoid of nucleotide and highly exposed to solvent. The PH domain of Tiam1 does not contact Rac1, and the position and orientation of the PH domain is markedly altered relative to the structure of the uncomplexed, GTPase-free DH/PH element from Sos1. The Tiam1/Rac1 structure highlights the interactions that catalyse nucleotide exchange on Rho family G proteins, and illustrates structural determinants dictating specificity between individual Rho family members and their associated Dbl-related guanine nucleotide exchange factors.

Stimulation of gene induction and cell growth by the Ras effector Rlf.

Wolthuis RM, de Ruiter ND, Cool RH, Bos JL
EMBO J. 1997; 16: 6748-61

Rlf is a ubiquitously expressed distinct relative of RalGDS that interacts with active Ras in vitro. We now demonstrate that Rlf, when co-expressed with Ras mutants, associates in vivo with RasV12 and the effector-domain mutant RasV12G37, but not with RasV12E38 or RasV12C40. Rlf exhibits guanine nucleotide exchange activity towards the small GTPase Ral and, importantly, Rlf-induced Ral activation is stimulated by active Ras. In addition, RasV12 and RasV12G37 synergize with Rlf in the transcriptional activation of the c-fos promoter. Rlf, when targeted to the plasma membrane using the Ras farnesyl attachment site (Rlf-CAAX), is constitutively active, inducing both Ral activation and c-fos promoter activity. Rlf-CAAX-induced gene expression is insensitive to dominant negative Ras and the MEK inhibitor PD98059, and involves activation of the serum response element. Furthermore, expression of Rlf-CAAX is sufficient to induce proliferation of NIH 3T3 cells under low-serum conditions. These data demonstrate that Rlf is an effector of Ras which functions as an exchange factor for Ral. Rlf mediates a distinct Ras-induced signalling pathway to gene induction. Finally, a constitutively active form of Rlf can stimulate transcriptional activation and cell growth.

Ras caught in another affair: the exchange factors for Ral.

Wolthuis RM, Bos JL
Curr Opin Genet Dev. 1999; 9: 112-7

The Ral guanine nucleotide exchange factors are direct targets of Ras, providing a mechanism for Ral activation by extracellular signals. In addition, Ral can be activated by a Ras-independent pathway. Ral guanine nucleotide exchange factors contribute to cellular transformation induced by oncogenic Ras through an Erk-independent mechanism which may involve activation of transcription.

Ras-effector interactions, the problem of specificity.

Wittinghofer A, Herrmann C
FEBS Lett. 1995; 369: 52-6

Ras plays the role of a molecular switch in many cellular signalling pathways. The Raf-kinase has been identified as the direct target molecule of Ras in mammalian cells. However, in recent reports other proteins have been characterised as putative Ras effectors which have neither a functional nor a structural relationship to each other. In addition it has been shown that also other members of the Ras family like Rap and R-Ras can interact with some of these proteins. To address the problem of specificity and of biological relevance of the interactions, they have to be carefully quantified and the cellular localisation of the proteins involved taken into account.

Three-dimensional structure and properties of wild-type and mutant H-ras-encoded p21.

Wittinghofer A, Franken SM, Scheidig AJ, Rensland H, Lautwein A, Pai EF, Goody RS
Ciba Found Symp. 1993; 176: 6-21

Ras (or p21) is the product of the ras proto-oncogene and is believed to be involved in growth-promoting signal transduction. The structure of the guanine nucleotide-binding domain of H-Ras (or p21H-ras) in the triphosphate conformation was determined at very high resolution (1.4 A). All the binding interactions between protein and Gpp[NH]p and Mg2+ can be resolved in great detail. The region around amino acids 61-65 is flexible and exists in two conformations, one of which seems to be important for catalysis. The properties and structures of several oncogenic and non-oncogenic mutant forms of Ras have also been determined. Since the structure of the GDP-bound form is also known, the nature of the conformational change from the GTP-bound to the GDP-bound form can be inferred from the 3-D structure. A mechanism for the intrinsic GTP hydrolysis has been proposed. Its implications for the GAP-stimulated GTPase reaction is discussed in the light of recent kinetic and mutational experiments.

A novel Dictyostelium RasGEF is required for normal endocytosis, cell motility and multicellular development.

Wilkins A, Chubb JR, Insall RH
Curr Biol. 2000; 10: 1427-37

BACKGROUND: Dictyostelium possesses a surprisingly large number of Ras proteins and little is known about their activators, the guanine nucleotide exchange factors (GEFs). It is also unclear, in Dictyostelium or in higher eukaryotes, whether Ras pathways are linear, with each Ras controlled by its own GEF, or networked, with multiple GEFs acting on multiple Ras proteins. RESULTS: We have identified the Dictyostelium gene that encodes RasGEFB, a protein with homology to known RasGEFs such as the Son-of-sevenless (Sos) protein. Dictyostelium cells in which the gene for RasGEFB was disrupted moved unusually rapidly, but lost the ability to perform macropinocytosis and therefore to grow in liquid medium. Crowns, the sites of macropinocytosis, were replaced by polarised lamellipodia. Mutant cells were also profoundly defective in early development, although they eventually formed tiny but normally proportioned fruiting bodies. This defect correlated with loss of discoidin Igamma mRNA, a starvation-induced gene, although other genes required for development were expressed normally or even precociously. RasGEFB was able to rescue a Saccharomyces CDC25 mutant, indicating that it is a genuine GEF for Ras proteins. CONCLUSIONS: RasGEFB appears to be the principal activator of the RasS protein, which regulates macropinocytosis and cell speed, but it also appears to regulate one or more other Ras proteins.

Signal transduction pathways involving Ras. Mini review.

Wiesmuller L, Wittinghofer F
Cell Signal. 1994; 6: 247-67

Expression cloning of lfc, a novel oncogene with structural similarities to guanine nucleotide exchange factors and to the regulatory region of protein kinase C.

Whitehead I, Kirk H, Tognon C, Trigo-Gonzalez G, Kay R
J Biol Chem. 1995; 270: 18388-95

In order to identify cDNAs that can induce oncogenic transformation, a retroviral vector was used to transfer a library of cDNAs from the murine 32D hemopoietic cell line into NIH 3T3 fibroblasts. We have identified and recovered a provirus containing a 1.8-kilobase pair cDNA whose expression causes morphological transformation in NIH 3T3 cells. The transforming cDNA contains a complete open reading frame that encodes a protein (designated Lfc) with a region of sequence similarity to the product of the lbc oncogene. This region includes a domain that is characteristic of the CDC24 family of guanine nucleotide exchange factors in tandem with a pleckstrin homology (PH) domain. The Lfc protein is distinguished from Lbc by a 150-amino acid NH2-terminal extension that contains a cysteine- and histidine-rich domain similar to the diacylglycerol-binding site (zinc butterfly) found in protein kinase C. NH2- and COOH-terminal deletion analysis revealed that both the PH and putative guanine nucleotide exchange factor domains are required, but the zinc butterfly is dispensable, for transformation. Although the removal of the PH domain of the Lfc protein completely eliminated its ability to transform NIH 3T3 cells, replacement of this domain with an isoprenylation site restored all of its transforming activity. This suggests that a PH domain-dependent recruitment of the Lfc protein to the cellular membrane is a necessary step for cellular transformation. The lfc gene is expressed in a broad range of tissues as well as in a variety of hemopoietic and non-hemopoietic cell lines. Lfc appears to be a new member of a growing family of proteins that are likely to act as activators of Ras-like proteins in a developmental or cell-lineage specific manner.

Retroviral transduction and oncogenic selection of a cDNA encoding Dbs, a homolog of the Dbl guanine nucleotide exchange factor.

Whitehead I, Kirk H, Kay R
Oncogene. 1995; 10: 713-21

A retroviral vector was used to transfer a large library of cDNAs from the 32D murine hemopoietic cell line to NIH3T3 fibroblasts, for the purpose of selecting cDNAs that induce oncogenic transformation. One highly transformed colony arising in the infected NIH3T3 cell culture contained a provirus with a 1900 bp cDNA insert. After recovery and reincorporation into a retroviral vector, this cDNA induced rapid morphological transformation and proliferation when expressed in NIH3T3 or C3H10T1/2 fibroblast cell lines. The transforming cDNA encoded a protein, designated Dbs, which had a region of high sequence similarity to the Dbl proto-oncogene. This region included motifs characteristic of the CDC24 family of guanine nucleotide exchange factors, and an adjacent pleckstrin homology domain. Dbs was distinguished from Dbl by an N-terminal extension and the presence of an SH3 domain at its C terminus. Deletions of the Dbs-encoding cDNA demonstrated that transformation of NIH3T3 cells required intact exchange factor and pleckstrin homology domains, but did not require the SH3 domain. In contrast to Dbl, the N-terminal sequences of Dbs did not suppress its transforming activity. The Dbs gene was expressed at low levels in several murine hemopoietic cell lines and in thymus and spleen, and at higher levels in other tissues, particularly in brain. Dbs may be one of a large family of exchange factors which provide cell-type specific pathways for regulating proliferating via the activation of Ras-like proteins.

A novel membrane factor stimulates guanine nucleotide exchange reaction of ras proteins.

West M, Kung HF, Kamata T
FEBS Lett. 1990; 259: 245-8

A factor with a molecular weight of 100 kDa, which markedly enhanced the guanine nucleotide exchange reaction of ras p21 proteins, was partially purified from bovine brain tissues. The factor was predominantly associated with the plasma membrane. When the partially purified factor and excess cold GTP were added to [3H]GDP.Gly12 p21 or Val12 in the presence of 2 mM MgCl2, the nucleotide exchange rate was stimulated up to 25-fold. The stimulation of the p21-nucleotide exchange reaction by the factor was completely blocked by the Y13-259 ras-neutralizing antibody. Taken together, these results suggest that the factor may control the rate limiting GDP/GTP exchange step in recycling of p21 in ras-mediated signal transduction.

Cloning and analysis of human cDNAs encoding a 140-kDa brain guanine nucleotide-exchange factor, Cdc25GEF, which regulates the function of Ras.

Wei W, Das B, Park W, Broek D
Gene. 1994; 151: 279-84

Ras proteins bound to GDP are biologically inactive while those bound to GTP are active. Ras-specific guanine nucleotide-exchange factors (GEFs) have been shown to activate Ras proteins. We used oligodeoxyribonucleotide primers with sequences similar to the cDNAs of rat and mouse cdc25 (encoding a Ras-GEF) to amplify, by the PCR, sequences with the potential to encode a 1275-amino-acid protein homologous to the rodent Cdc25GEF proteins. Northern blot analysis detected a brain-specific 5-kb transcript. We provide evidence for a novel alternately spliced transcript of cdc25 and show that these alternately spliced transcripts are differentially expressed in various regions of the adult nervous system. Antibodies raised against the C terminus of the protein recognize a 140-kDa protein in brain extracts of human, rat, guinea pig and cow; the 140-kDa protein is associated predominantly, if not exclusively, with a crude membrane fraction of brain. The C terminus of human Cdc25GEF can complement the loss of CDC25 function in Saccharomyces cerevisiae. A glutathione S-transferase fusion protein containing the C terminus of the cdc25 product can stimulate guanine nucleotide exchange on H-Ras in vitro. Further, the Cdc25-fusion protein binds tightly to the nucleotide-free form of H-Ras in vitro, and this binding is reversed by the addition of GTP.

Insulin-stimulated disassociation of the SOS-Grb2 complex.

Waters SB, Yamauchi K, Pessin JE
Mol Cell Biol. 1995; 15: 2791-9

Insulin stimulation of differentiated 3T3-L1 adipocytes or Chinese hamster ovary cells expressing high levels of the insulin receptor resulted in a time-dependent decrease in the electrophoretic mobility of SOS on sodium dodecyl sulfate-polyacrylamide gels. The reduction in SOS mobility was completely reversed by alkaline phosphatase treatment, and the in vitro phosphorylation of SOS by mitogen-activated protein kinase resulted in a decrease of electrophoretic mobility identical to that following in vivo insulin stimulation. Immunoprecipitation of Grb2 followed by SOS immunoblotting demonstrated a disassociation of the SOS-Grb2 complex that paralleled the decrease in SOS electrophoretic mobility. Similarly, SOS immunoprecipitation followed by Grb2 immunoblotting also indicated an uncoupling of the SOS-Grb2 complex. Further, incubation of whole-cell extracts with glutathione-S-transferase-Grb2 fusion proteins demonstrated that insulin stimulation resulted in a decreased affinity of SOS for Grb2. In contrast, the dissociation of SOS from Grb2 did not affect the interactions between Grb2 and tyrosine-phosphorylated Shc. In addition to insulin, several other agents which activate the mitogen-activated protein kinase pathway (platelet-derived growth factor, serum, and phorbol ester) also resulted in the uncoupling of the SOS-Grb2 complex. Consistent with these results, expression of v-ras and v-raf resulted in a constitutive decrease in the association between SOS and Grb2. Together, these data suggest a molecular mechanism accounting for the transient activation of ras due to the uncoupling of the SOS-Grb2 complex following SOS phosphorylation.

Desensitization of Ras activation by a feedback disassociation of the SOS-Grb2 complex.

Waters SB, Holt KH, Ross SE, Syu LJ, Guan KL, Saltiel AR, Koretzky GA, Pessin JE
J Biol Chem. 1995; 270: 20883-6

Activation of Ras by the exchange of bound GDP for GTP is predominantly catalyzed by the guanylnucleotide exchange factor SOS. Receptor tyrosine kinases increase Ras-GTP loading by targeting SOS to the plasma membrane location of Ras through the small adaptor protein Grb2. However, despite the continuous stimulation of receptor tyrosine kinase activity, Ras activation is transient and, in the case of insulin, begins returning to the GDP-bound state within 5 min. We report here that the cascade of serine kinases activated directly by Ras results in a mitogen-activated protein kinase kinase (MEK)-dependent phosphorylation of SOS and subsequent disassociation of the Grb2-SOS complex, thereby interrupting the ability of SOS to catalyze nucleotide exchange on Ras. These data demonstrate a molecular feedback mechanism accounting for the desensitization of Ras-GTP loading following insulin stimulation.

Insulin and epidermal growth factor receptors regulate distinct pools of Grb2-SOS in the control of Ras activation.

Waters SB, Chen D, Kao AW, Okada S, Holt KH, Pessin JE
J Biol Chem. 1996; 271: 18224-30

Insulin and epidermal growth factor (EGF) stimulate a rapid but transient increase in the amount of GTP bound to Ras that returns to the basal GDP-bound state within 10-30 min. Although insulin stimulation resulted in a dissociation of the Grb2.SOS complex, EGF did not affect the Grb2.SOS complex but instead induced dissociation of Grb2-SOS from tyrosine-phosphorylated Shc. The dissociation of Grb2-SOS from Shc was not due to dephosphorylation as Shc remained persistently tyrosine-phosphorylated during this time. Furthermore, there was no decrease in the extent of insulin receptor substrate 1, insulin receptor, or EGF receptor tyrosine phosphorylation. Surprisingly, however, despite the EGF-induced decrease in the amount of Grb2-SOS bound to Shc, the extent of Grb2 associated with Shc remained constant, and there was a concomitant increase in the amount of SOS associated with Grb2. In addition, after the insulin-stimulated dissociation of Grb2 from SOS, EGF treatment induced the reassociation of the Grb2.SOS complex. Quantitative immunoprecipitation demonstrated that only a small fraction of the total cellular pool of Grb2 was associated with SOS. Similarly, only a small fraction of SOS and Grb2 were co-immunoprecipitated with Shc. Together, these data suggest the presence of distinct Grb2-SOS pools that are independently utilized by insulin and EGF in their recruitment to tyrosine-phosphorylated Shc.

The Grb2 binding domain of mSos1 is not required for downstream signal transduction.

Wang W, Fisher EM, Jia Q, Dunn JM, Porfiri E, Downward J, Egan SE
Nat Genet. 1995; 10: 294-300

Cellular Ras proteins are activated primarily by specific guanine-nucleotide releasing factors such as the Son of Sevenless (Sos) proteins. This activation event is thought to occur in response to plasma membrane localization of a complex containing Sos and a small adapter protein Grb2. We have isolated a dominant mutant allele of mSos1 which transforms Rat1 cells, yet is no longer able to bind Grb2. Biochemical experiments reveal that the subcellular distribution of this truncated Sos protein is not altered with respect to the wild type Sos protein. These data argue against a role for Grb2 in the direct recruitment of Sos proteins to the plasma membrane and suggest that Grb2 may function to overcome negative regulation of Sos by its C terminus.

Discriminatory residues in Ras and Rap for guanine nucleotide exchange factor recognition.

van den Berghe N, Cool RH, Wittinghofer A
J Biol Chem. 1999; 274: 11078-85

The inability of the S17N mutant of Rap1A to sequester the catalytic domain of the Rap guanine nucleotide exchange factor C3G (van den Berghe, N., Cool, R. H., Horn, G., and Wittinghofer, A. (1997) Oncogene 15, 845-850) prompted us to study possible fundamental differences in the way Rap1 interacts with C3G compared with the interaction of Ras with the catalytic domain of the mouse Ras guanine nucleotide exchange factor Cdc25(Mm). A variety of mutants in both Ras and Rap1A were designed, and both the C3G and Cdc25(Mm) catalyzed release of guanine nucleotide from these mutants was studied. In addition, we could identify regions in Rap2A that are responsible for the lack of recognition by C3G and induce high C3G activity by replacement of these residues with the corresponding Rap1A residues. The different Ras and Rap mutants showed that many residues were equally important for both C3G and Cdc25(Mm), suggesting that they interact similarly with their substrates. However, several residues were also identified to be important for the exchange reaction with only C3G (Leu70) or only Cdc25(Mm) (Gln61 and Tyr40). These results are discussed in the light of the structure of the Ras-Sos complex and suggest that some important differences in the interaction of Rap1 with C3G and Ras with Cdc25(Mm) indeed exist and that marker residues have been identified for the different structural requirements.

Receptor-tyrosine-kinase- and G beta gamma-mediated MAP kinase activation by a common signalling pathway.

van Biesen T, Hawes BE, Luttrell DK, Krueger KM, Touhara K, Porfiri E, Sakaue M, Luttrell LM, Lefkowitz RJ
Nature. 1995; 376: 781-4

Mitogen-activated protein (MAP) kinases mediate the phosphorylation and activation of nuclear transcription factors that regulate cell growth. MAP kinase activation may result from stimulation of either tyrosine-kinase (RTK) receptors, which possess intrinsic tyrosine kinase activity, or G-protein-coupled receptors (GPCR). RTK-mediated mitogenic signalling involves a series of SH2- and SH3-dependent protein-protein interactions between tyrosine-phosphorylated receptor, Shc, Grb2 and Sos, resulting in Ras-dependent MAP kinase activation. The beta gamma subunits of heterotrimeric G proteins (G beta gamma) also mediate Ras-dependent MAP kinase activation by an as-yet unknown mechanism. Here we demonstrate that activation of MAP kinase by Gi-coupled receptors is preceded by the G beta gamma-mediated tyrosine phosphorylation of Shc, leading to an increased functional association between Shc, Grb2 and Sos. Moreover, disruption of the Shc-Grb2-Sos complex blocks G beta gamma-mediated MAP kinase activation, indicating that G beta gamma does not mediate MAP kinase activation by a direct interaction with Sos. These results indicate that G beta gamma-mediated MAP kinase activation is initiated by a tyrosine phosphorylation event and proceeds by a pathway common to both GPCRs and RTKs.

Cloning and characterization of the S. pombe gene efc25+, a new putative guanine nucleotide exchange factor.

Tratner I, Fourticq-Esqueoute A, Tillit J, Baldacci G
Gene. 1997; 193: 203-10

We report the cloning and characterization of a new S. pombe gene, efc25+, for 'exchange factor Cdc25-like'. The C-terminal region of the predicted product of this gene displays high sequence homology with a number of guanine nucleotide exchange factors for Ras. These include Cdc25 of Saccharomyces cerevisiae, Cdc25 of Saccharomyces kluyveri, Csc25 of Candida albicans, Sdc25 of S. cerevisiae and Ste6 of Schizosaccharomyces pombe. Disruption of efc25+ resulted in cells with a spherical shape reminiscent of the abnormal morphological phenotype of ras1 deletion mutants. However, unlike ras1 null mutants, strains deleted for efc25+ were proficient for mating and sporulation. This differs from the only other Ras1 exchange factor characterized so far in S. pombe, the Ste6 protein, whose deletion results in defects in mating and sporulation but not in cell shape. We hypothesize that Efc25 is an exchange factor for Ras1 and that it is involved in a signaling pathway different from that involving Ste6.

Guanine nucleotide exchange factors CalDAG-GEFI and CalDAG-GEFII are colocalized in striatal projection neurons.

Toki S, Kawasaki H, Tashiro N, Housman DE, Graybiel AM
J Comp Neurol. 2001; 437: 398-407

CalDAG-GEFI and CalDAG-GEFII (identical to RasGRP) are novel, brain-enriched guanine nucleotide exchange factors (GEFs) that can be stimulated by calcium and diacylglycerol and that can activate small GTPases, including Ras and Rap1, molecules increasingly recognized as having signaling functions in neurons. Here, we show that CalDAG-GEFI and CalDAG-GEFII mRNAs, detected by in situ hybridization analysis, have sharply contrasting forebrain-predominant distributions in the mature brain: CalDAG-GEFI is expressed mainly in the striatum and olfactory structures and deep cortical layers, whereas CalDAG-GEFII is expressed widely in the forebrain. Within the striatum, however, the two CalDAG-GEF mRNAs have nearly identical distributions: they are coexpressed in striatal projection neurons that give rise to the direct and indirect pathways of the basal ganglia. Subcellular fractionation analysis of the substantia nigra with monoclonal antibodies against CalDAG-GEFI suggests that CalDAG-GEFI protein is present not only in the cell bodies of striatal projection neurons but also in their axons and axon terminals. These results suggest that the CalDAG-GEFs may be key intracellular regulators whereby calcium and diacylglycerol signals can regulate cellular functions through small GTPases in the basal ganglia circuits. Copyright 2001 Wiley-Liss, Inc.

Structure of TCTP reveals unexpected relationship with guanine nucleotide-free chaperones.

Thaw P, Baxter NJ, Hounslow AM, Price C, Waltho JP, Craven CJ
Nat Struct Biol. 2001; 8: 701-4

The translationally controlled tumor-associated proteins (TCTPs) are a highly conserved and abundantly expressed family of eukaryotic proteins that are implicated in both cell growth and the human acute allergic response but whose intracellular biochemical function has remained elusive. We report here the solution structure of the TCTP from Schizosaccharomyces pombe, which, on the basis of sequence homology, defines the fold of the entire family. We show that TCTPs form a structural superfamily with the Mss4/Dss4 family of proteins, which bind to the GDP/GTP free form of Rab proteins (members of the Ras superfamily) and have been termed guanine nucleotide-free chaperones (GFCs). Mss4 also acts as a relatively inefficient guanine nucleotide exchange factor (GEF). We further show that the Rab protein binding site on Mss4 coincides with the region of highest sequence conservation in the TCTP family. This is the first link to any other family of proteins that has been established for the TCTP family and suggests the presence of a GFC/GEF at extremely high abundance in eukaryotic cells.

Guanine-nucleotide exchange protein C3G activates JNK1 by a ras-independent mechanism. JNK1 activation inhibited by kinase negative forms of MLK3 and DLK mixed lineage kinases.

Tanaka S, Hanafusa H
J Biol Chem. 1998; 273: 1281-4

Recently we have reported that the adaptor protein Crk transmits signals to c-Jun kinase (JNK) through C3G, a guanine-nucleotide exchange protein for the Ras family of small G proteins. Transient expression of C3G in 293T cells induced JNK1 activation without a significant effect on extracellular signal-related kinase 1 (ERK1), whereas mSos1 activated equally both JNK1 and ERK1. Coexpression of the dominant negative form of Ras-N17 did not suppress C3G-induced JNK1 activation but reduced the activity of JNK1 induced by mSos1, suggesting that Ras is not required for JNK activation by C3G. Ras-independent activation of JNK was supported by the finding that C3G-induced JNK activation was not inhibited by the dominant negative forms of Rac or Pak, which are components of the signaling pathway from Ras leading to JNK activation. In contrast, C3G-induced JNK1 activation was strongly inhibited by coexpression of the kinase negative forms of the mixed lineage kinase (MLK) family of proteins, MLK3 and dual leucine zipper kinase (DLK). In addition, MLK3-induced JNK1 activation was found to be suppressed by the kinase negative form of DLK, which bound to MLK3. These results suggest that C3G activates JNK1 through a pathway involving the MLK family of proteins.

Functional involvement of mSos in interleukin-3 and thrombin stimulation of the Ras, mitogen-activated protein kinase pathway in BaF3 murine hematopoietic cells.

Tago K, Kaziro Y, Satoh T
J Biochem (Tokyo). 1998; 123: 659-67

Stimulation of the interleukin (IL)-3 receptor provokes rapid activation of the Ras pathway in various hematopoietic cell lines. Also, a wide range of G-protein-coupled receptors induce Ras activation following ligand stimulation. In this report, we investigate the mechanism underlying Ras activation upon stimulation of these two types of receptors in hematopoietic cells. Thrombin, a G-protein-coupled receptor ligand, was found to stimulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) in IL-3-dependent BaF3 cells, suggesting a significant function of thrombin receptor-mediated signaling. We show that the Ras-guanine nucleotide exchange factor mSos is indispensable for activation of the Ras pathway in IL-3- or thrombin-stimulated BaF3 cells. The activation of Ras in response to IL-3 as defined by accumulation of the GTP-bound form was impaired by conditional overexpression of a dominant-negative mutant of mSos (DeltamSos1). Furthermore, following induction of DeltamSos1, IL-3 enhancement of the kinase activities of c-Raf-1, ERK2, and JNK1 downstream of Ras was almost completely blocked. Similarly, thrombin-induced Ras-dependent ERK2 activation was diminished by DeltamSos1. However, the tyrosine phosphorylation pattern of cellular substrates upon thrombin stimulation was entirely different from the pattern of IL-3-induced tyrosine phosphorylation. Collectively, these results provide evidence that mSos plays a crucial role in both IL-3 and thrombin activation of the Ras pathway in hematopoietic cells, although molecules (including tyrosine kinases) mediating the signal to mSos are likely to be different between the two types of receptors.

GEFs: master regulators of G-protein activation.

Sprang S
Trends Biochem Sci. 2001; 26: 266-7

The ADP ribosylation factor-nucleotide exchange factors Gea1p and Gea2p have overlapping, but not redundant functions in retrograde transport from the Golgi to the endoplasmic reticulum.

Spang A, Herrmann JM, Hamamoto S, Schekman R
Mol Biol Cell. 2001; 12: 1035-45

The activation of the small ras-like GTPase Arf1p requires the action of guanine nucleotide exchange factors. Four Arf1p guanine nucleotide exchange factors have been identified in yeast: Sec7p, Syt1p, Gea1p, and its homologue Gea2p. We identified GEA2 as a multicopy suppressor of a sec21-3 temperature-sensitive mutant. SEC21 encodes the gamma-subunit of coatomer, a heptameric protein complex that together with Arf1p forms the COPI coat. GEA1 and GEA2 have at least partially overlapping functions, because deletion of either gene results in no obvious phenotype, whereas the double null mutant is inviable. Conditional mutants defective in both GEA1 and GEA2 accumulate endoplasmic reticulum and Golgi membranes under restrictive conditions. The two genes do not serve completely overlapping functions because a Deltagea1 Deltaarf1 mutant is not more sickly than a Deltaarf1 strain, whereas Deltagea2 Deltaarf1 is inviable. Biochemical experiments revealed similar distributions and activities for the two proteins. Gea1p and Gea2p exist both in membrane-bound and in soluble forms. The membrane-bound forms, at least one of which, Gea2p, can be visualized on Golgi structures, are both required for vesicle budding and protein transport from the Golgi to the endoplasmic reticulum. In contrast, Sec7p, which is required for protein transport within the Golgi, is not required for retrograde protein trafficking.

The SH2/SH3 domain-containing protein GRB2 interacts with tyrosine-phosphorylated IRS1 and Shc: implications for insulin control of ras signalling.

Skolnik EY, Lee CH, Batzer A, Vicentini LM, Zhou M, Daly R, Myers MJ Jr, Backer JM, Ullrich A, White MF, et al.
EMBO J. 1993; 12: 1929-36

GRB2, a small protein comprising one SH2 domain and two SH3 domains, represents the human homologue of the Caenorhabditis elegans protein, sem-5. Both GRB2 and sem-5 have been implicated in a highly conserved mechanism that regulates p21ras signalling by receptor tyrosine kinases. In this report we show that in response to insulin, GRB2 forms a stable complex with two tyrosine-phosphorylated proteins. One protein is the major insulin receptor substrate IRS-1 and the second is the SH2 domain-containing oncogenic protein, Shc. The interactions between GRB2 and these two proteins require ligand activation of the insulin receptor and are mediated by the binding of the SH2 domain of GRB2 to phosphotyrosines on both IRS-1 and Shc. Although GRB2 associates with IRS-1 and Shc, it is not tyrosine-phosphorylated after insulin stimulation, implying that GRB2 is not a substrate for the insulin receptor. Furthermore, we have identified a short sequence motif (YV/IN) present in IRS-1, EGFR and Shc, which specifically binds the SH2 domain of GRB2 with high affinity. Interestingly, both GRB2 and phosphatidylinositol-3 (PI-3) kinase can simultaneously bind distinct tyrosine phosphorylated regions on the same IRS-1 molecule, suggesting a mechanism whereby IRS-1 could provide the core for a large signalling complex. We propose a model whereby insulin stimulation leads to formation of multiple protein--protein interactions between GRB2 and the two targets IRS-1 and Shc. These interactions may play a crucial role in activation of p21ras and the control of downstream effector molecules.

An SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to sevenless and Sos proteins in vitro.

Simon MA, Dodson GS, Rubin GM
Cell. 1993; 73: 169-77

Activation of the sevenless protein-tyrosine kinase is required for the proper specification of R7 photoreceptors in the Drosophila eye. The activation of a Ras protein, p21Ras1, is a crucial early event in the signaling pathway, and constitutive activation of p21Ras1 is sufficient to induce all of the effects of sevenless action. Here we report that another gene, E(sev)2B, required for proper signaling by sevenless encodes a protein of the structure SH3-SH2-SH3. We further provide evidence that the E(sev)2B protein is required for activation of p21Ras1 but not for any subsequent events, and that this protein can bind in vitro to sevenless and to Son of sevenless (Sos), a putative guanine nucleotide exchange factor for p21Ras1. These results suggest that the E(sev)2B protein may act to stimulate the ability of Sos to catalyze p21Ras1 activation by linking sevenless and Sos in a signaling complex. We have renamed the E(sev)2B locus downstream of receptor kinases (drk).

Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase.

Simon MA, Bowtell DD, Dodson GS, Laverty TR, Rubin GM
Cell. 1991; 67: 701-16

We have conducted a genetic screen for mutations that decrease the effectiveness of signaling by a protein tyrosine kinase, the product of the Drosophila melanogaster sevenless gene. These mutations define seven genes whose wild-type products may be required for signaling by sevenless. Four of the seven genes also appear to be essential for signaling by a second protein tyrosine kinase, the product of the Ellipse gene. The putative products of two of these seven genes have been identified. One encodes a ras protein. The other locus encodes a protein that is homologous to the S. cerevisiae CDC25 protein, an activator of guanine nucleotide exchange by ras proteins. These results suggest that the stimulation of ras protein activity is a key element in the signaling by sevenless and Ellipse and that this stimulation may be achieved by activating the exchange of GTP for bound GDP by the ras protein.

Differential response of the Ras exchange factor, Ras-GRF to tyrosine kinase and G protein mediated signals.

Shou C, Wurmser A, Suen KL, Barbacid M, Feig LA, Ling K [corrected to Suen KL]
Oncogene. 1995; 10: 1887-93

Ras-GRF, a guanine-nucleotide exchange factor that activates Ras p21, was tested for its ability to couple to either tyrosine kinase or heterotrimeric G protein signal transduction pathways. Ras-GRF failed to bind the SH2 and SH3 containing adaptor protein Grb2, either in vitro or in vivo. Furthermore, Ras-GRF did not form a stable complex with activated EGF receptor. However, as has been shown previously (Cen et al., 1994), the presence of Ras-GRF in NIH3T3 cells enhanced the activation of Ras induced by serum stimulation. A similar effect was not observed with PDGF stimulation. Moreover, serum stimulation lead to the hyperphosphorylation of Ras-GRF. Both the serum induced super-activation of Ras, and the hyperphosphorylation of Ras-GRF were blocked by pretreatment of cells with the Gi,o inhibitor pertussis toxin, but not by pretreatment with the tyrosine kinase inhibitor genistein. These results suggest that Ras-GRF has the capacity to mediate Ras activation initiated by signals using heterotrimeric G proteins.

Residues crucial for Ras interaction with GDP-GTP exchangers.

Segal M, Willumsen BM, Levitzki A
Proc Natl Acad Sci U S A. 1993; 90: 5564-8

Cdc25 is essential for Ras-mediated activation of adenylyl cyclase in the yeast Saccharomyces cerevisiae. This protein acts by catalyzing GDP-GTP exchange on yeast Ras. Harvey (Ha) ras expressed in S. cerevisiae is also recognized by both Cdc25 and Sdc25, a yeast homolog of Cdc25. Thus it is feasible to examine molecular aspects of mammalian Ras modulation by Cdc25 using the RAS/cAMP pathway in yeast as a model system. Here, we describe mutational analysis of Ha-ras for the identification of residues critical for the ability of Ras to interact with Cdc25 and related guanine nucleotide-release proteins. Mutations within codons 97-108 impaired Ras-mediated activation of adenylyl cyclase in the presence but not in the absence of mammalian GTPase-activating protein. Such mutations, therefore, affected the ability of Ras to undergo GDP-GTP exchange catalyzed by the guanine nucleotide exchanger without preventing Ras activation of the effector. Similar mutations were previously shown to impair the ability of c-ras to transform mammalian cells while having a less drastic effect on v-ras.

An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.

Scita G, Tenca P, Areces LB, Tocchetti A, Frittoli E, Giardina G, Ponzanelli I, Sini P, Innocenti M, Di Fiore PP
J Cell Biol. 2001; 154: 1031-44

Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.

EPS8 and E3B1 transduce signals from Ras to Rac.

Scita G, Nordstrom J, Carbone R, Tenca P, Giardina G, Gutkind S, Bjarnegard M, Betsholtz C, Di Fiore PP
Nature. 1999; 401: 290-3

The small guanine nucleotide (GTP)-binding protein Rac regulates mitogen-induced cytoskeletal changes and c-Jun amino-terminal kinase (JNK), and its activity is required for Ras-mediated cell transformation. Epistatic analysis placed Rac as a key downstream target in Ras signalling; however, the biochemical mechanism regulating the cross-talk among these small GTP-binding proteins remains to be elucidated. Eps8 (relative molecular mass 97,000) is a substrate of receptors with tyrosine kinase activity which binds, through its SH3 domain, to a protein designated E3b1/Abi-1. Here we show that Eps8 and E3b1/Abi-1 participate in the transduction of signals from Ras to Rac, by regulating Rac-specific guanine nucleotide exchange factor (GEF) activities. We also show that Eps8, E3b1 and Sos-1 form a tri-complex in vivo that exhibits Rac-specific GEF activity in vitro. We propose a model in which Eps8 mediates the transfer of signals between Ras and Rac, by forming a complex with E3b1 and Sos-1.

Identification of a human guanine nucleotide-releasing factor (H-GRF55) specific for Ras proteins.

Schweighoffer F, Faure M, Fath I, Chevallier-Multon MC, Apiou F, Dutrillaux B, Sturani E, Jacquet M, Tocque B
Oncogene. 1993; 8: 1477-85

A critical step in the activation of cellular Ras is the release of bound GDP. Oligonucleotide primers derived from a mouse cDNA sequence homologous to the Saccharomyces cerevisiae CDC25 gene product were used to screen a human brain cDNA library. The cloning led to the isolation of a 2.8-kb cDNA predicted to encode a protein of 488 amino acids. This protein was produced in Escherichia coli as a glutathione S-transferase fusion protein and functioned in vitro as a specific guanine nucleotide-releasing factor. Polyclonal antibodies raised against the last 281 amino acids of the protein allowed a protein in the molecular weight range of 55 kDa to be identified in human cortex homogenates. Analysis by Northern blotting led to the identification of a 5.5-kb mRNA in brain poly(A)+ RNA. The functionality of the encoded protein was evaluated after expression in different cells: (i) in Saccharomyces cerevisiae the effects of the cdc25.5 and RAS2 Ala-22 mutations were reversed; (ii) in chinese hamster ovary cells, a RAS-responsive element was transactivated as demonstrated by the expression of a CAT reporter gene under the control of the polyomavirus enhancer. Finally, in situ hybridization on of human chromosomes revealed a localization on band 15q2.4.

Phosphorylation-dependent and constitutive activation of Rho proteins by wild-type and oncogenic Vav-2.

Schuebel KE, Movilla N, Rosa JL, Bustelo XR
EMBO J. 1998; 17: 6608-21

We show here that Vav-2, a member of the Vav family of oncoproteins, acts as a guanosine nucleotide exchange factor (GEF) for RhoG and RhoA-like GTPases in a phosphotyrosine-dependent manner. Moreover, we show that Vav-2 oncogenic activation correlates with the acquisition of phosphorylation-independent exchange activity. In vivo, wild-type Vav-2 is activated oncogenically by tyrosine kinases, an effect enhanced further by co-expression of RhoA. Likewise, the Vav-2 oncoprotein synergizes with RhoA and RhoB proteins in cellular transformation. Transient transfection assays in NIH-3T3 cells show that phosphorylated wild-type Vav-2 and the Vav-2 oncoprotein induce cytoskeletal changes resembling those observed by the activation of the RhoG pathway. In contrast, the constitutive expression of the Vav-2 oncoprotein in rodent fibroblasts leads to major alterations in cell morphology and to highly enlarged cells in which karyokinesis and cytokinesis frequently are uncoupled. These results identify a regulated GEF for the RhoA subfamily, provide a biochemical explanation for vav family oncogenicity, and establish a new signaling model in which specific Vav-like proteins couple tyrosine kinase signals with the activation of distinct subsets of the Rho/Rac family of GTPases.

Shc is the predominant signaling molecule coupling insulin receptors to activation of guanine nucleotide releasing factor and p21ras-GTP formation.

Sasaoka T, Draznin B, Leitner JW, Langlois WJ, Olefsky JM
J Biol Chem. 1994; 269: 10734-8

Insulin stimulates tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and She in Rat1 fibroblasts overexpressing wild type insulin receptors. We investigated the relative role of IRS-1 and She in insulin activation of guanine nucleotide releasing factor (GNRF) and p21ras-GTP formation. The time course of insulin-stimulated tyrosine phosphorylation of IRS-1 was rapid, whereas Shc phosphorylation was relatively slow. Growth factor receptor bound protein-2 (Grb2) associated with IRS-1 rapidly and gradually dissociated after 5 min, whereas Grb2 association with Shc was slower and reached a maximum at 10 min after insulin stimulation. Thus, the kinetics of Grb2 association with IRS-1 and She corresponded closely to the time course of tyrosine phosphorylation of IRS-1 and Shc, respectively. Importantly, 3-13-fold more Grb2 was associated with Shc than with IRS-1. In addition, the kinetics of insulin-stimulated GNRF activity and p21ras-GTP formation corresponded more closely to the time course of Shc phosphorylation than to the kinetics of IRS-1 phosphorylation. Furthermore, immunoprecipitation of Shc proteins from cell lysates of insulin-stimulated cells removed 67% of the GNRF activity, whereas precipitation of IRS-1 had a negligible effect on GNRF activity. Thus, although both IRS-1 and Shc associate with Grb2, the current results indicate that Shc plays a more important role than IRS-1 in insulin stimulation of GNRF activity and subsequent p21ras-GTP formation.

A dominant-negative mutant of mSOS1 inhibits insulin-induced Ras activation and reveals Ras-dependent and -independent insulin signaling pathways.

Sakaue M, Bowtell D, Kasuga M
Mol Cell Biol. 1995; 15: 379-88

The role of the Grb2-SOS complex in insulin signal transduction was investigated with a deletion mutant of mSOS1 that lacks the guanine nucleotide exchange domain of the wild-type protein. Cells over-expressing either wild-type (CHO-IR/SOS cells) or mutant (CHO-IR/delta SOS cells) mSOS1 were established by transfection of Chinese hamster ovary cells that express human insulin receptors (CHO-IR cells) with the appropriate expression plasmid. The mutant mSOS1 protein did not contain the guanine nucleotide exchange activity in vitro and associated with Grb2 both in vivo and in vitro. In both CHO-IR and CHO-IR/SOS cells, insulin rapidly stimulated the formation of GTP-bound Ras and the phosphorylation of mitogen-activated protein (MAP) kinase; both these effects of insulin were markedly inhibited in CHO-IR/delta SOS cells. Insulin-induced glycogen synthase and 70-kDa S6 kinase activities were not affected by expression of either wild-type or mutant mSOS1. These results show that the mutant mSOS1 acts in a dominant-negative manner and suggest that the Grb2-SOS complex mediates, at least in part, insulin-induced activation of Ras in intact cells. The data also indicate that Ras activation is not required for insulin-induced stimulation of glycogen synthase and 70-kDa S6 kinase.

Sed4p functions as a positive regulator of Sar1p probably through inhibition of the GTPase activation by Sec23p.

Saito-Nakano Y, Nakano A
Genes Cells. 2000; 5: 1039-48

BACKGROUND: Sar1p belongs to a unique subfamily of small GTPases and is essential for formation of the transport vesicles from the endoplasmic reticulum (ER) that are destined to the Golgi apparatus. To understand how the GTPase cycle of Sar1p is regulated, we screened for multicopy suppressors of sar1 ts mutants and identified SED4. RESULTS: Although deletion of sed4 alone shows no growth defect, sar1 delta(sed4) double mutant cells are inviable. In the delta(sed4) background, the suppression activity of SAR1 towards sec12 and sec16 is lost. These observations suggest that SED4 is a very close partner of SAR1 and imply that Sed4p may act to increase the active Sar1p in the cell. Over-expression of SEC12 does not remedy the lethality of sar1 delta(sed4). The purified cytoplasmic domain of Sed4p does not show a guanine nucleotide exchange (GEF) activity toward Sar1p nor increase the GEF activity of Sec12p. On the contrary, over-expression of SED4 aggravates the ts growth of sec23 cells. The cytoplasmic domain of Sed4p weakly inhibits the GTPase-activating (GAP) activity of Sec23p toward Sar1p. In a microsome-based COPII binding assay, the binding of the GDP-form mutant Sar1p (D32G) is lower on the delta(sed4) microsomes than on the wild-type membranes. CONCLUSION: We propose a model that Sed4p counteracts the GAP action of Sec23p on to Sar1p.

The heterotrimeric G q protein-coupled angiotensin II receptor activates p21 ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes.

Sadoshima J, Izumo S
EMBO J. 1996; 15: 775-87

p21 ras plays as important role in cell proliferation, transformation and differentiation. Recently, the requirement of p21 ras has been suggested for cellular responses induced by stimulation of heterotrimeric G protein-coupled receptors. However, it remains to be determined how agonists for G protein-coupled receptors activate p21 ras in metazoans. We show here that stimulation of the G q protein-coupled angiotensin II (Ang II) receptor causes activation of p21 ras in cardiac myocytes. The p21 ras activation by Ang II is mediated by an increase in the guanine nucleotide exchange activity, but not by an inhibition of the GTPase-activating protein. Ang II causes rapid tyrosine phosphorylation of Shc and its association with Grb2 and mSos-1, a guanine nucleotide exchange factor of p21 ras. This leads to translocation of mSos-1 to the membrane fraction. Shc associates with the SH3 domain of Fyn whose tyrosine kinase activity is activated by Ang II with a similar time course as that of tyrosine phosphorylation of Shc. Ang II-induced increase in the guanine nucleotide exchange activity was inhibited by a peptide ligand specific to the SH3 domain of the Src family tyrosine kinases. These results suggest that an agonist for a pertussis toxin-insensitive G protein-coupled receptor may initiate the cross-talk with non-receptor-type tyrosine kinases, thereby activating p21 ras using a similar mechanism as receptor tyrosine kinase-induced p21 ras activation.

Thermodynamics of Ras/effector and Cdc42/effector interactions probed by isothermal titration calorimetry.

Rudolph MG, Linnemann T, Grunewald P, Wittinghofer A, Vetter IR, Herrmann C
J Biol Chem. 2001; 276: 23914-21

Proliferation, differentiation, and morphology of eucaryotic cells is regulated by a large network of signaling molecules. Among the major players are members of the Ras and Rho/Rac subfamilies of small GTPases that bind to different sets of effector proteins. Recognition of multiple effectors is important for communicating signals into different pathways, leading to the question of how an individual GTPase achieves tight binding to diverse targets. To understand the observed specificity, detailed information about binding energetics is expected to complement the information gained from the three-dimensional structures of GTPase/effector protein complexes. Here, the thermodynamics of the interaction of four closely related members of the Ras subfamily with four different effectors and, additionally, the more distantly related Cdc42/WASP couple were quantified by means of isothermal titration calorimetry. The heat capacity changes upon complex formation were rationalized in light of the GTPase/effector complex structures. Changes in enthalpy, entropy, and heat capacity of association with various Ras proteins are similar for the same effector. In contrast, although the structures of the Ras-binding domains are similar, the thermodynamics of the Ras/Raf and Ras/Ral guanine nucleotide dissociation stimulator interactions are quite different. The energy profile of the Cdc42/WASP interaction is similar to Ras/Ral guanine nucleotide dissociation stimulator, despite largely different structures and interface areas of the complexes. Water molecules in the interface cannot fully account for the observed discrepancy but may explain the large range of Ras/effector binding specificity. The differences in the thermodynamic parameters, particularly the entropy changes, could help in the design of effector-specific inhibitors that selectively block a single pathway.

Activation of the Ral and phosphatidylinositol 3' kinase signaling pathways by the ras-related protein TC21.

Rosario M, Paterson HF, Marshall CJ
Mol Cell Biol. 2001; 21: 3750-62

TC21 is a member of the Ras superfamily of small GTP-binding proteins that, like Ras, has been implicated in the regulation of growth-stimulating pathways. We have previously identified the Raf/mitogen-activated protein kinase pathway as a direct TC21 effector pathway required for TC21-induced transformation (M. Rosario, H. F. Paterson, and C. J. Marshall, EMBO J. 18:1270-1279, 1999). In this study we have identified two further effector pathways for TC21, which contribute to TC21-stimulated transformation: the phosphatidylinositol 3' kinase (PI-3K) and Ral signaling pathways. Expression of constitutively active TC21 leads to the activation of Ral A and the PI-3K-dependent activation of Akt/protein kinase B. Strong activation of the PI-3K/Akt pathway is seen even with very low levels of TC21 expression, suggesting that TC21 may be a key small GTPase-regulator of PI-3K. TC21-induced alterations in cellular morphology in NIH 3T3 and PC12 cells are also PI-3K dependent. On the other hand, activation of the Ral pathway by TC21 is required for TC21-stimulated DNA synthesis but not transformed morphology. We show that inhibition of Ral signaling blocks DNA synthesis in human tumor cell lines containing activating mutations in TC21, demonstrating for the first time that this pathway is required for the proliferation of human tumor cells. Finally, we provide mechanisms for the activation of these pathways, namely, the direct in vivo interaction of TC21 with guanine nucleotide exchange factors for Ral, resulting in their translocation to the plasma membrane, and the direct interaction of TC21 with PI-3K. In both cases, the effector domain region of TC21 is required since point mutations in this region can interfere with activation of downstream signaling.

Isoform-specific insertion near the Grb2-binding domain modulates the intrinsic guanine nucleotide exchange activity of hSos1.

Rojas JM, Subleski M, Coque JJ, Guerrero C, Saez R, Li BQ, Lopez E, Zarich N, Aroca P, Kamata T, Santos E
Oncogene. 1999; 18: 1651-61

Two human hSos1 isoforms (Isf I and Isf II; Rojas et al., Oncogene 12, 2291-2300, 1996) defined by the presence of a distinct 15 amino acid stretch in one of them, were compared biologically and biochemically using representative NIH3T3 transfectants overexpressing either one. We showed that hSos1-Isf II is significantly more effective than hSos1-Isf I to induce proliferation or malignant transformation of rodent fibroblasts when transfected alone or in conjunction with normal H-Ras (Gly12). The hSos1-Isf II-Ras cotransfectants consistently exhibited higher saturation density, lower cell-doubling times, increased focus-forming activity and higher ability to grow on semisolid medium and at low serum concentration than their hSos1-Isf I-Ras counterparts. Furthermore, the ratio of GTP/GDP bound to cellular p21ras was consistently higher in the hSos1-Isf II-transfected clones, both under basal and stimulated conditions. However, no significant differences were detected in vivo between Isf I- and Isf II-transfected clones regarding the amount, stability and subcellular localization of Sos1-Grb2 complex, or the level of hSos1 phosphorylation upon cellular stimulation. Interestingly, direct Ras guanine nucleotide exchange activity assays in cellular lysates showed that Isf II transfectants consistently exhibited about threefold higher activity than Isf I transfectants under basal, unstimulated conditions. Microinjection into Xenopus oocytes of purified peptides corresponding to the C-terminal region of both isoforms (encompassing the 15 amino acid insertion area and the first Grb2-binding motif) showed that only the Isf II peptide, but not its corresponding Isf I peptide, was able to induce measurable rates of meiotic maturation, and synergyzed with insulin, but not progesterone, in induction of GVBD. Our results suggest that the increased biological potency displayed by hSos1-Isf II is due to higher intrinsic guanine nucleotide exchange activity conferred upon this isoform by the 15 a.a. insertion located in proximity to its Grb2 binding region.

Functional roles of the Shc phosphotyrosine binding and Src homology 2 domains in insulin and epidermal growth factor signaling.

Ricketts WA, Rose DW, Shoelson S, Olefsky JM
J Biol Chem. 1996; 271: 26165-9

Shc is involved in the activation of Ras in response to many growth factors. Shc contains two phosphotyrosine binding domains, an Src homology 2 (SH2) domain in the carboxyl terminus of the protein and a phosphotyrosine binding (PTB) domain in the amino terminus. Since functional roles for these two domains have not been established, we microinjected glutathione S-transferase fusion proteins of either the Shc PTB or SH2 domains into fibroblasts expressing insulin and epidermal growth factor receptors and measured their effects on DNA synthesis. We found that the Shc PTB was necessary for insulin-induced mitogenic signaling, whereas the SH2 domain was not. In contrast, for epidermal growth factor signaling, the Shc SH2 was functionally more important. These differential modes of signal transduction may be an important factor in determining the specificity of the response of a cell to external stimuli.

Leukemia-associated Rho guanine nucleotide exchange factor, a Dbl family protein found mutated in leukemia, causes transformation by activation of RhoA.

Reuther GW, Lambert QT, Booden MA, Wennerberg K, Becknell B, Marcucci G, Sondek J, Caligiuri MA, Der CJ
J Biol Chem. 2001; 276: 27145-51

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) was originally identified as a fusion partner with mixed-lineage leukemia in a patient with acute myeloid leukemia. LARG possesses a tandem Dbl homology and pleckstrin homology domain structure and, consequently, may function as an activator of Rho GTPases. In this study, we demonstrate that LARG is a functional Dbl protein. Expression of LARG in cells caused activation of the serum response factor, a known downstream target of Rho-mediated signaling pathways. Transient overexpression of LARG did not activate the extracellular signal-regulated kinase or c-Jun NH(2)-terminal kinase mitogen-activated protein kinase cascade, suggesting LARG is not an activator of Ras, Rac, or Cdc42. We performed in vitro exchange assays where the isolated Dbl homology (DH) or DH/pleckstrin homology domains of LARG functioned as a strong activator of RhoA, but exhibited no activity toward Rac1 or Cdc42. We found that LARG could complex with RhoA, but not Rac or Cdc42, in vitro, and that expression of LARG caused an increase in the levels of the activated GTP-bound form of RhoA, but not Rac1 or Cdc42, in vivo. Thus, we conclude that LARG is a RhoA-specific guanine nucleotide exchange factor. Finally, like activated RhoA, we determined that LARG cooperated with activated Raf-1 to transform NIH3T3 cells. These data demonstrate that LARG is the first functional Dbl protein mutated in cancer and indicate LARG-mediated activation of RhoA may play a role in the development of human leukemias.

Differential requirement for p21ras activation in the metabolic signaling by insulin.

Reusch JE, Bhuripanyo P, Carel K, Leitner JW, Hsieh P, DePaolo D, Draznin B
J Biol Chem. 1995; 270: 2036-40

To evaluate the role of the "Ras pathway" in mediating metabolic signaling by insulin, we employed lovastatin to exhibit isoprenilation of Ras proteins in Rat-1 fibroblasts transfected with human insulin receptors (HIRc cells) and in differentiated 3T3-L1 adipocytes. Lovastatin blocked an ability of insulin to activate p21ras and mitogen-activated protein kinase. Lovastatin also significantly (p < 0.01) reduced insulin effects on thymidine incorporation and glucose incorporation into glycogen. Nevertheless, an effect of insulin on glucose uptake remained unaffected. It appears that in contrast to its mitogenic action and to its effect on glycogenesis, an effect of insulin on glucose uptake does not require p21ras activation.

Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for Rac and Rho GTPases.

Ren Y, Li R, Zheng Y, Busch H
J Biol Chem. 1998; 273: 34954-60

The Rho-related small GTPases are critical elements involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. The Dbl-like guanine nucleotide exchange factors (GEF) have been implicated in direct activation of these GTPases. Here we have identified a new member of the Dbl family, GEF-H1, by screening a human HeLa cell cDNA library. GEF-H1 encodes a 100-kDa protein containing the conserved structural array of a Dbl homology domain in tandem with a pleckstrin homology domain and is most closely related to the lfc oncogene, but additionally it contains a unique coiled-coil domain at the carboxyl terminus. Biochemical analysis reveals that GEF-H1 is capable of stimulating guanine nucleotide exchange of Rac and Rho but is inactive toward Cdc42, TC10, or Ras. Moreover, GEF-H1 binds to Rac and Rho proteins in both the GDP- and guanosine 5'-3-O-(thio)triphosphate-bound states without detectable affinity for Cdc42 or Ras. Immunofluorescence reveals that GEF-H1 colocalizes with microtubules through the carboxyl-terminal coiled-coil domain. Overexpression of GEF-H1 in COS-7 cells results in induction of membrane ruffles. These results suggest that GEF-H1 may have a direct role in activation of Rac and/or Rho and in bringing the activated GTPase to specific target sites such as microtubules.

SH3 domains of the adapter molecule Grb2 complex with two proteins in T cells: the guanine nucleotide exchange protein Sos and a 75-kDa protein that is a substrate for T cell antigen receptor-activated tyrosine kinases.

Reif K, Buday L, Downward J, Cantrell DA
J Biol Chem. 1994; 269: 14081-7

In T lymphocytes activated via the T cell antigen receptor (TCR), the SH2- and SH3-containing adapter molecule Grb2 forms a complex with the Ras guanine nucleotide exchange protein Sos and tyrosine phospho-proteins. The interaction of Sos with Grb2 is mediated via the Grb2 SH3 domains. In this study, it is shown that a 75-kDa protein is also complexed with the Grb2 SH3 domains in T cells, but not in Rat-1 fibroblasts. The identity of the p75 protein is not known, but immunoblot analysis with phosphotyrosine antibodies indicated that it is rapidly tyrosine-phosphorylated in TCR-activated T cells. This characteristic clearly distinguishes p75 from Sos since Sos is not a phosphotyrosine protein. In vitro binding studies indicated that the p75 phosphotyrosine protein binds to a glutathione S-transferase fusion protein of intact Grb2, but not to a Grb2 fusion protein mutated in its SH3 domains. p75 can also bind to the single COOH-terminal Grb2 SH3 domain, whereas Sos has an in vitro binding preference for the NH2-terminal Grb2 SH3 domain. Collectively, these data indicate that in T cells, two proteins can complex with the Grb2 SH3 domains: Sos and a p75 molecule that is tyrosine-phosphorylated in TCR-activated cells. The significance of p75 association with Grb2 is not clear, but by analogy with Sos, p75 is a potential candidate for a Grb2 effector protein. Data are presented showing that the interaction of the Grb2 SH2 domains with tyrosine phosphoproteins may be regulated by conformational restraints imposed by different molecules complexing with the Grb2 SH3 domains. It is thus possible to speculate that the interaction of either p75 or Sos with the Grb2 SH3 domain may influence the interaction of the Grb2 SH2 domain with tyrosine phosphoproteins.

Identification of guanine nucleotide exchange factors (GEFs) for the Rap1 GTPase. Regulation of MR-GEF by M-Ras-GTP interaction.

Rebhun JF, Castro AF, Quilliam LA
J Biol Chem. 2000; 275: 34901-8

Although the Ras subfamily of GTPases consists of approximately 20 members, only a limited number of guanine nucleotide exchange factors (GEFs) that couple extracellular stimuli to Ras protein activation have been identified. Furthermore, no novel downstream effectors have been identified for the M-Ras/R-Ras3 GTPase. Here we report the identification and characterization of three Ras family GEFs that are most abundantly expressed in brain. Two of these GEFs, MR-GEF (M-Ras-regulated GEF, KIAA0277) and PDZ-GEF (KIAA0313) bound specifically to nucleotide-free Rap1 and Rap1/Rap2, respectively. Both proteins functioned as Rap1 GEFs in vivo. A third GEF, GRP3 (KIAA0846), activated both Ras and Rap1 and shared significant sequence homology with the calcium- and diacylglycerol-activated GEFs, GRP1 and GRP2. Similarly to previously identified Rap GEFs, C3G and Smg GDS, each of the newly identified exchange factors promoted the activation of Elk-1 in the LNCaP prostate tumor cell line where B-Raf can couple Rap1 to the extracellular receptor-activated kinase cascade. MR-GEF and PDZ-GEF both contain a region immediately N-terminal to their catalytic domains that share sequence homology with Ras-associating or RalGDS/AF6 homology (RA) domains. By searching for in vitro interaction with Ras-GTP proteins, PDZ-GEF specifically bound to Rap1A- and Rap2B-GTP, whereas MR-GEF bound to M-Ras-GTP. C-terminally truncated MR-GEF, lacking the GEF catalytic domain, retained its ability to bind M-Ras-GTP, suggesting that the RA domain is important for this interaction. Co-immunoprecipitation studies confirmed the interaction of M-Ras-GTP with MR-GEF in vivo. In addition, a constitutively active M-Ras(71L) mutant inhibited the ability of MR-GEF to promote Rap1A activation in a dose-dependent manner. These data suggest that M-Ras may inhibit Rap1 in order to elicit its biological effects.

Interaction of Shc with Grb2 regulates the Grb2 association with mSOS.

Ravichandran KS, Lorenz U, Shoelson SE, Burakoff SJ
Ann N Y Acad Sci. 1995; 766: 202-3

Interaction of Shc with the zeta chain of the T cell receptor upon T cell activation.

Ravichandran KS, Lee KK, Songyang Z, Cantley LC, Burn P, Burakoff SJ
Science. 1993; 262: 902-5

The shc oncogene product is tyrosine-phosphorylated by Src family kinases and after its phosphorylation interacts with the adapter protein Grb2 (growth factor receptor-bound protein 2). In turn, Grb2 interacts with the guanine nucleotide exchange factor for Ras, mSOS. Because several Src family kinases participate in T cell activation and Shc functions upstream of Ras, the role of Shc in T cell signaling was examined. Shc was phosphorylated on tyrosine after activation through the T cell receptor (TCR), and subsequently interacted with Grb2 and mSOS. The Src homology region 2 (SH2) domain of Shc directly interacted with the tyrosine-phosphorylated zeta chain of the TCR. Thus, Shc may couple TCR activation to the Ras signaling pathway.

Membrane-targeting potentiates guanine nucleotide exchange factor CDC25 and SOS1 activation of Ras transforming activity.

Quilliam LA, Huff SY, Rabun KM, Wei W, Park W, Broek D, Der CJ
Proc Natl Acad Sci U S A. 1994; 91: 8512-6

Growth factor-triggered activation of Ras proteins is believed to be mediated by guanine nucleotide exchange factors (CDC25/GRF and SOS1/2) that promote formation of the active Ras GTP-bound state. Although the mechanism(s) of guanine nucleotide exchange factor regulation is unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. To evaluate this model, we generated constructs that encode the catalytic domains of human CDC25 or mouse SOS1, either alone (designated cCDC25 and cSOS1, respectively) or terminating in the carboxyl-terminal CAAX membrane-targeting sequence from K-Ras4B (designated cCDC25-CAAX and cSOS1-CAAX, respectively; in CAAX, C is Cys, A is an aliphatic amino acid, and X is Ser or Met). We then compared the transforming potential of cCDC25 and cSOS1 with their membrane-targeted counterparts. We observed that addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their focus-forming activity (10- to 50-fold) in NIH 3T3 transfection assays. Similarly, we observed that the membrane-targeted versions showed a 5- to 10-fold enhanced ability to induce transcriptional activation from the Ets/AP-1 Ras-responsive element. Furthermore, whereas cells that stably expressed cCDC25 or cSOS1 exhibited the same morphologies as untransformed NIH 3T3 cells, cells expressing cCDC25-CAAX or cSOS1-CAAX displayed transformed morphologies that were indistinguishable from the elongated and refractile morphology of oncogenic Ras-transformed cells. Thus, these results suggest that membrane translocation alone is sufficient to potentiate guanine nucleotide exchange factor activation of Ras.

Involvement of the switch 2 domain of Ras in its interaction with guanine nucleotide exchange factors.

Quilliam LA, Hisaka MM, Zhong S, Lowry A, Mosteller RD, Han J, Drugan JK, Broek D, Campbell SL, Der CJ
J Biol Chem. 1996; 271: 11076-82

While Ras proteins are activated by stimulated GDP release, which enables acquisition of the active GTP-bound state, little is known about how guanine nucleotide exchange factors (GEFs) interact with Ras to promote this exchange reaction. Here we report that mutations within the switch 2 domain of Ras (residues 62-69) inhibit activation of Ras by the mammalian GEFs, Sos1, and GRF/CDC25Mm. While mutations in the 62-69 region blocked upstream activation of Ras, they did not disrupt Ras effector functions, including transcriptional activation and transformation of NIH 3T3 cells. Biochemical analysis indicated that the loss of GEF responsiveness of a Ras(69N) mutant was due to a loss of GEF binding, with no change in intrinsic nucleotide exchange activity. Furthermore, structural analysis of Ras(69N) using NMR spectroscopy indicated that mutation of residue 69 had a very localized effect on Ras structure that was limited to alpha-helix 2 of the switch 2 domain. Together, these results suggest that the switch 2 domain of Ras forms a direct interaction with GEFs.

Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras.

Pronk GJ, de Vries-Smits AM, Buday L, Downward J, Maassen JA, Medema RH, Bos JL
Mol Cell Biol. 1994; 14: 1575-81

Shc proteins are phosphorylated on tyrosine residues and associate with growth factor receptor-bound protein 2 (Grb2) upon treatment of cells with epidermal growth factor (EGF) or insulin. We have studied the role of Shc in insulin- and EGF-induced activation of p21ras in NIH 3T3 cells overexpressing human insulin receptors (A14 cells). A14 cells are equally responsive to insulin and EGF with respect to activation of p21ras. Analysis of Shc immunoprecipitates revealed that (i) both insulin and EGF treatment resulted in Shc tyrosine phosphorylation and (ii) Shc antibodies coimmunoprecipitated both Grb2 and mSOS after insulin and EGF treatment. The induction of tyrosine phosphorylation of Shc and the presence of Grb2 and mSOS in Shc immunoprecipitates followed similar time courses, with somewhat higher levels after EGF treatment. In mSOS immunoprecipitates, Shc could be detected as well. Furthermore, Shc immune complexes contained guanine nucleotide exchange activity toward p21ras in vitro. From these results, we conclude that after insulin and EGF treatment, Shc associates with both Grb2 and mSOS and therefore may mediate, at least in part, insulin- and EGF-induced activation of p21ras. In addition, we investigated whether the Grb2-mSOS complex associates with the insulin receptor or with insulin receptor substrate 1 (IRS1). Although we observed association of Grb2 with IRS1, we did not detect complex formation between mSOS and IRS1 in experiments in which the association of mSOS with Shc was readily detectable. Furthermore, whereas EGF treatment resulted in the association of mSOS with the EGF receptor, insulin treatment did not result in the association of mSOS with the insulin receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of epidermal growth factor receptor signaling by phosphorylation of the ras exchange factor hSOS1.

Porfiri E, McCormick F
J Biol Chem. 1996; 271: 5871-7

In response to stimulation with epidermal growth factor (EGF), the guanine nucleotide exchange factor human SOS1 (hSOS1) promotes the activation of Ras by forming a complex with Grb2 and the human EGF receptor (hEGFR). hSOS1 was phosphorylated in cells stimulated with EGF or phorbol 12-myristate 13-acetate or following co-transfection with activated Ras or Raf. Co-transfection with dominant negative Ras resulted in a decrease of EGF-induced hSOS1 phosphorylation. The mitogen-activated protein kinase (MAPK) phosphorylated hSOS1 in vitro within the carboxyl-terminal proline-rich domain. The same region of hSOS1 was phosphorylated in vivo, in cells stimulated with EGF. Tryptic phosphopeptide mapping showed that MAPK phosphorylated hSOS1 in vitro on sites which were also phosphorylated in vivo. Phosphorylation by MAPK did not affect hSOS1 binding to Grb2 in vitro. However, reconstitution of the hSOS1-Grb2-hEGFR complex showed that phosphorylation by MAPK markedly reduced the ability of hSOS1 to associate with the hEGFR through Grb2. Similarly, phosphorylated hSOS1 was unable to form a complex with Shc through Grb2. Thus phosphorylation of hSOS1, by affecting its interaction with the hEGFR or Shc, down-regulates signal transduction from the hEGFR to the Ras pathway.

Prenylation of Ras proteins is required for efficient hSOS1-promoted guanine nucleotide exchange.

Porfiri E, Evans T, Chardin P, Hancock JF
J Biol Chem. 1994; 269: 22672-7

We have studied whether hSOS1, a mammalian guanine nucleotide exchange factors responsible for activating Ras in response to growth factor stimulation, requires post-translational processing of Ras proteins to promote guanine nucleotide exchange. Our results showed that full-length hSOS1 catalyzed guanine nucleotide exchange on prenylated K-Ras(4B) but with a much lower efficiency on unprocessed K-Ras(4B). The apparent Km of hSOS1 for prenylated K-Ras(4B) was 225 (+/- 25) nM with a Vmax of 0.7 (+/- 0.1) mmol/min/mmol. The activity of hSOS1 against unprocessed K-Ras(4B) was too low to measure Km and Vmax. Consistent with these observations, full-length hSOS1 formed a complex with nucleotide-depleted prenylated K-Ras(4B) but not with unprocessed K-Ras(4B). A geranylgeranylated mutant of K-Ras(4B) was an equally good substrate for hSOS1 as wild-type farnesylated K-Ras. Similarly hSOS1 promoted guanine nucleotide exchange on prenylated Ha-Ras but showed minimal activity toward unprocessed Ha-Ras. Neither the polybasic domain of K-Ras (4B) or palmitoylation of Ha-Ras were required for hSOS1-promoted guanine nucleotide exchange. We attempted to identify a minimal region of hSOS1 capable of promoting guanine nucleotide exchange on both prenylated and unprocessed K-Ras. However, a truncated form of hSOS1 comprising only the CDC25 homology domain retained preferential catalytic activity against prenylated K-Ras, whereas the cognate domain from CDC25 was more active against unprocessed K-Ras.

No Sec7-homology domain in guanine-nucleotide-exchange factors that act on Ras and Rho.

Ponting CP, Bork P, Schultz J, Aravind L
Trends Biochem Sci. 1999; 24: 177-8

Grb2 interaction with MEK-kinase 1 is involved in regulation of Jun-kinase activities in response to epidermal growth factor.

Pomerance M, Multon MC, Parker F, Venot C, Blondeau JP, Tocque B, Schweighoffer F
J Biol Chem. 1998; 273: 24301-4

Epidermal growth factor (EGF) receptor was shown to be involved in the activation pathway of the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) cascade not only by EGF, but also by UV radiation or osmotic stress. This paper describes a specific interaction between the COOH-terminal SH3 domain of Grb2 and the NH2-terminal regulatory domain of MEKK1 in ER22 cells overexpressing the EGF receptor. This interaction results in the formation of a constitutive complex between Grb2 and MEKK1 in both proliferating and resting cells. EGF stimulation causes this complex to be rapidly and transiently recruited by Shc proteins. The subsequent release of the Grb2-MEKK1 complex from Shc proteins correlates with JNK activation. Transfection of the NH2-terminal regulatory domain of MEKK1 specifically inhibits EGF-dependent JNK activation indicating that Grb2 is involved in MEKK1 activation. Thus, adaptor proteins have a new role in the regulation of the SAPK/JNK cascade after EGF stimulation.

The guanine nucleotide exchange factor CNrasGEF activates ras in response to cAMP and cGMP.

Pham N, Cheglakov I, Koch CA, de Hoog CL, Moran MF, Rotin D
Curr Biol. 2000; 10: 555-8

Small GTPase proteins such as Ras are key regulators of cellular proliferation and are activated by guanine nucleotide exchange/releasing factors (GEFs/GRFs). Three classes of Ras GRFs have been identified to date, represented by Sos1/2, Ras-GRF1/2 and Ras-GRP. Here, we describe a novel candidate Ras activator, cyclic nucleotide rasGEF (CNrasGEF), which contains CDC25, Ras exchange motif (REM), Ras-association (RA), PDZ and cNMP (cAMP/cGMP) binding (cNMP-BD) domains, two PY motifs and a carboxy-terminal SxV sequence. CNrasGEF can activate Ras in vitro, and it binds cAMP directly via its cNMP-BD. In cells, CNrasGEF activates Ras in response to elevation of intracellular cAMP or cGMP, or treatment with their analogues 8-Br-cAMP or 8-Br-cGMP, independently of protein kinases A and G (PKA and PKG). This activation is prevented in CNrasGEF lacking its CDC25 domain or cNMP-BD. CNrasGEF can also activate the small GTPase Rap1 in cells, but this activation is constitutive and independent of cAMP. CNrasGEF is expressed mainly in the brain and is localized at the plasma membrane, a localization dependent on the presence of intact PDZ domain but not the SxV sequence. These results suggest that CNrasGEF may directly connect cAMP-generating pathways or cGMP-generating pathways to Ras.

Novel aspects of Ras proteins biology: regulation and implications.

Perez-Sala D, Rebollo A
Cell Death Differ. 1999; 6: 722-8

The importance of Ras proteins as crucial crossroads in cellular signaling pathways has been well established. In spite of the elucidation of the mechanism of RAS activation by growth factors and the delineation of MAP kinase cascades, the overall framework of Ras interactions is far from being complete. Novel regulators of Ras GDP/GTP exchange have been identified that may mediate the activation of Ras in response to changes in intracellular calcium and diacylglycerol. The direct activation of Ras by free radicals such as nitric oxide also suggests potential regulation of Ras function by the cellular redox state. In addition, the array of Ras effectors continues to expand, uncovering links between Ras and other cellular signaling pathways. Ras is emerging as a dual regulator of cellular functions, playing either positive or negative roles in the regulation of proliferation and apoptosis. The signals transmitted by Ras may be modulated by other pathways triggered in parallel, resulting in the final order for proliferation or apoptosis. The diversity of ras-mediated effects may be related in part to differential involvement of Ras homologues in distinct cellular processes. The study of Ras posttranslational modifications has yielded a broad battery of inhibitors that have been envisaged as anti-cancer agents. Although an irreversible modification, Ras isoprenylation appears to be modulated by growth factors and by the activity of the isoprenoid biosynthetic pathway, which may lead to changes in Ras activity.

A direct repeat sequence at the Rasgrf1 locus and imprinted expression.

Pearsall RS, Plass C, Romano MA, Garrick MD, Shibata H, Hayashizaki Y, Held WA
Genomics. 1999; 55: 194-201

Genomic imprinting is an epigenetic modification that can lead to parental-specific monoallelic expression of specific autosomal genes. While methylation of CpG dinucleotides is thought to be a strong candidate for this epigenetic modification, little is known about the establishment or maintenance of parental origin-specific methylation patterns. We have recently identified a portion of mouse chromosome 9 containing a paternally methylated region associated with a paternally expressed imprinted gene, Ras protein-specific guanine nucleotide-releasing factor 1 (Rasgrf1). This area of chromosome 9 also contains a short, direct tandem repeat in close proximity to a paternally methylated NotI site 30 kb upstream of Rasgrf1. Short, direct tandem repeats have been found associated with other imprinted genes and may act as important regulatory structures. Here we demonstrate that two rodent species (Mus and Rattus) contain a similar direct repeat structure associated with a region of paternal-specific methylation. In both species, the Rasgrf1 gene shows paternal-specific monoallelic expression in neonatal brain. A more divergent rodent species (Peromyscus) appears to lack a similar repeat structure based on Southern Blot analysis. Peromyscus animals show biallelic expression of Rasgrf1 in neonatal brain. These results suggest that direct repeat elements may play an important role in the imprinting process.

Alternate mechanisms of ras activation are complementary and favor and formation of ras-GTP.

Patel G, MacDonald MJ, Khosravi-Far R, Hisaka MM, Der CJ
Oncogene. 1992; 7: 283-8

The mechanisms of ras activation by mutations in residue 61 and in the NKXD guanine nucleotide-binding consensus sequence (ras residues 116-119) have been evaluated. Weakly transforming mutations that either reduce intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities (61P) or enhance guanine nucleotide exchange rates (116H, 119E) were combined into the same H-ras proteins. The resulting double-mutant proteins exhibited significantly stronger transforming forming activities than are observed with each individual mutation, suggesting that the consequences of these two different mechanisms of activation favor maintenance of ras in the active form, which is GTP bound. In vivo nucleotide association analysis demonstrated a direct relationship between ras-GTP formation and transforming activity. Although both 61P and 61L mutations result in reduced intrinsic GTPase activity and loss of GAP stimulation in vitro, only H-ras(61L) exhibits strong transforming activity. While H-ras(61L) is found predominantly in the GTP-bound form, H-ras(61P) is predominantly complexed with GDP in vivo. Thus, in vitro GAP stimulation of GTPase activity does not directly correlate with transforming potential, suggesting that other ras-specific regulatory components may also be important in regulating the cycling of ras between CDP- and GTP-bound states.

Co-ordination and specificity of the action of GTPase-activating proteins and GDP/GTP-exchange factors on Ras.

Parrini MC, Giglione C, Parmeggiani A
Biochem Soc Trans. 1997; 25: 997-1001

Regulation of GTP-binding state in RalA through Ca2+ and calmodulin.

Park JB
Exp Mol Med. 2001; 33: 54-8

RalA GTPase, a member of Ras superfamily proteins, shows alternative forms between the active GTP-binding and the inactive GDP-binding states. Ral-specific guanine nucleotide exchange factor such as RalGDS interacts with activated Ras and cooperates with Ras indicating that Ral can be activated through Ras signaling pathway. Another activation path for Ral are through Ca2+-dependent but Ras-independent manner. In this study, studies were carried out to examine possible effects of Ca2+ and calmodulin, Ca2+-binding protein, directly on the GTP/GDP-binding state to recombinant unprenylated GST-RalA proteins. The results showed that Ca2+ stimulated the binding of GTP to RalA, whereas it reduced the binding of GDP to RalA. However, it does not involve a high affinity association of Ca2+ with RalA. Ca2+/calmodulin stimulated the GTPase activity of RalA. These results indicate that Ca2+ alone activates RalA by stimulating GTP-binding to RalA and Ca2+/calmodulin inactivates RalA by increasing the activity of RalGTPase.

Association of the DF3/MUC1 breast cancer antigen with Grb2 and the Sos/Ras exchange protein.

Pandey P, Kharbanda S, Kufe D
Cancer Res. 1995; 55: 4000-3

The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in human breast carcinomas. Although the precise functional role of this protein remains unclear, the cytoplasmic tail contains potential tyrosine phosphorylation sites for binding to Src homology 2 (SH2) domains. In the present studies using human MCF-7 breast cancer cells, we show that tyrosine phosphorylated DF3 directly interacts with the SH2 domain of the adaptor protein Grb2. The findings indicate that a pYTNP site in DF3 is responsible for this interaction. The results also demonstrate that the DF3/Grb2 complex associates with the guanine nucleotide exchange protein Sos. Because Sos binds to the SH3 domains of Grb2 and, thereby, associates with Ras at the cell membrane, formation of a DF3/Grb2/Sos complex supports a role for DF3 in intracellular signaling.

Guanine nucleotide exchange factors: activators of Ras superfamily proteins.

Overbeck AF, Brtva TR, Cox AD, Graham SM, Huff SY, Khosravi-Far R, Quilliam LA, Solski PA, Der CJ
Mol Reprod Dev. 1995; 42: 468-76

Members of the Ras superfamily of proteins function as regulated GDP/GTP switches that cycle between active GTP-complexed and inactive GDP-complexed states. Guanine nucleotide exchange factors (GEFs) stimulate formation of the GTP-bound state, whereas GTPase activating proteins (GAPs) catalyze the formation of the GDP-bound state. We describe three studies that evaluate the mechanism of action of GEFs for Ras (SOS1 and RasGRF/CDC25) or Ras-related Rho (Dbl and Vav) proteins. Growth factor-mediated activation of Ras is believed to be mediated by activation of Ras GEFs (CDC25/GRF and SOS1/2). Although the mechanisms of Ras GEF regulation are unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. Our observation that the addition of the Ras plasma membrane-targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their transforming and transactivation activities (10-50 fold and 5-10 fold, respectively) suggests that membrane translocation alone is sufficient to potentiate GEF activation of Ras. We have determined that two Ras-related proteins, designated R-Ras and R-Ras2/TC21, can trigger the malignant transformation of NIH 3T3 cells via activation of the Ras signal transduction pathway. Furthermore, like Ras and R-Ras, we observed that TC21 GTPase activity was stimulated by Ras GAPs. However, we observed that both SOS1 and CDC25 were activators of normal TC21, but not R-Ras, transforming activities. Therefore, TC21, but not R-Ras, may be activated by the same extracellular signaling events that activate Ras proteins. Dbl family proteins are believed to function as GEFs and activators of the Ras-related Rho family of proteins. However, one Dbl family oncogene, designated Vav, has been reported to be a GEF for Ras proteins. Therefore we were interested in determining whether Dbl family oncogenes cause transformation by triggering the constitutive activation of Rho or Ras proteins. Our results suggest that Dbl oncogenes cause transformation via a Ras-independent activation of MAP kinases and Rho family proteins.

Guanine nucleotide exchange factors for the Rho GTPases: a role in human disease?

Olson MF
J Mol Med. 1996; 74: 563-71

The Rho family of low molecular weight GTP-binding proteins control important aspects of cell shape, adhesion, movement and growth. The DBL-homology (DH) protein family of upstream regulators of Rho GTPases has recently been identified, and deregulated expression of these proteins can have dramatic cellular consequences. This review examines the possible role of DH proteins and Rho GTPases in oncogenesis, metastasis and development.

A Drosophila SH2-SH3 adaptor protein implicated in coupling the sevenless tyrosine kinase to an activator of Ras guanine nucleotide exchange, Sos.

Olivier JP, Raabe T, Henkemeyer M, Dickson B, Mbamalu G, Margolis B, Schlessinger J, Hafen E, Pawson T
Cell. 1993; 73: 179-91

A Drosophila gene (drk) encodes a widely expressed protein with a single SH2 domain and two flanking SH3 domains, which is homologous to the Sem-5 protein of C. elegans and mammalian GRB2. Genetic analysis suggests that drk function is essential for signaling by the sevenless receptor tyrosine kinase. Drk biological activity correlates with binding of its SH2 domain to activated receptor tyrosine kinases and concomitant localization of drk to the plasma membrane. In vitro, drk also binds directly to the C-terminal tail of Sos, a Ras guanine nucleotide-releasing protein (GNRP), which, like Ras1 and drk, is required for sevenless signaling. These results suggest that drk binds autophosphorylated receptor tyrosine kinases with its SH2 domain and the Sos GNRP through its SH3 domains, thereby coupling receptor tyrosine kinases to Ras activation. The conservation of these signaling proteins during evolution indicates that this is a general mechanism for linking tyrosine kinases to Ras.

Interactions between Src homology (SH) 2/SH3 adapter proteins and the guanylnucleotide exchange factor SOS are differentially regulated by insulin and epidermal growth factor.

Okada S, Pessin JE
J Biol Chem. 1996; 271: 25533-8

Co-immunoprecipitation of whole cell extracts demonstrated that the guanylnucleotide exchange factor SOS was associated with the small adapter proteins Grb2, CrkII, and Nck. In vitro binding indicated a similar binding affinity of SOS for all three adapter proteins but with a slightly lower Kd for Grb2 (approximately 2.5-fold) compared with Nck and CrkII. Insulin stimulation resulted in co-immunoprecipitation of tyrosine-phosphorylated IRS1 with Grb2 and to a lesser extent CrkII. Although Grb2 also associated with tyrosine-phosphorylated Shc, there was no detectable interaction of CrkII with Shc. In contrast, EGF stimulation resulted in the predominant co-immunoprecipitation of Grb2 with the EGF receptor, whereas CrkII primarily associated with an unidentified 120-130-kDa protein. Similar to the ability of insulin to induce the dissociation of the Grb2-SOS complex, there was a concomitant time-dependent dissociation of the CrkII-SOS and Nck-SOS complexes. However, EGF stimulation had no effect on the association state of the Grb2-SOS or the Nck-SOS complexes but did result in a time-dependent dissociation of the CrkII from SOS. Together, these data demonstrate that different cellular pools of SOS associate with different adapter proteins forming various signaling complexes, each undergoing distinct patterns of assembly/disassembly following growth factor stimulation.

Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2-SOS and CrkII-C3G complexes.

Okada S, Matsuda M, Anafi M, Pawson T, Pessin JE
EMBO J. 1998; 17: 2554-65

Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor resulted in a time-dependent decrease in the amount of GTP bound to Rap1. The inactivation of Rap1 was associated with an insulin-stimulated decrease in the amount of Rap1 that was bound to Raf1. In parallel with the dissociation of Raf1 from Rap1, there was an increased association of Raf1 with Ras. Concomitant with the inactivation of Rap1 and decrease in Rap1-Raf1 binding, we observed a rapid insulin-stimulated dissociation of the CrkII-C3G complex which occurred in a Ras-independent manner. The dissociation of the CrkII-C3G was recapitulated in vitro using a GST-C3G fusion protein to precipitate CrkII from whole cell detergent extracts. The association of GST-C3G with CrkII was also dose dependent and demonstrated that insulin reduced the affinity of CrkII for C3G without any effect on CrkII protein levels. Furthermore, the reduction in CrkII binding affinity was reversible by tyrosine dephosphorylation with PTP1B and by mutation of Tyr221 to phenylalanine. Together, these data demonstrate that insulin treatment results in the de-repression of Rap1 inhibitory function on the Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade.

[Regulation of a small GTPase Rap2]

Ohba Y
Hokkaido Igaku Zasshi. 2001; 76: 13-20

Rap2 is a member of Ras-family G proteins and related most closely to Rap1; however little is known about the regulation of Rap2 activity. In this study, I have compared the regulation and function of Rap2 with those of Rap1. In 293T cells, Rap2 was regulated by the same set of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) as those which regulated Rap1. Rap2 was localized at both plasma membrane and intracellular membrane compartments, as Rap1 was. Rap2 bound to the Ras-binding domain of Raf and inhibited Ras-dependent activation of Elk1 transcription factor. I have found that the GTP-bound form exceeds 50% of total Rap2 in the cells. This observation suggests that Rap2 suppresses Ras-mediated activation of ERK/MAP kinase cascade in quiescent cells.

Sensitivity of wild type and mutant ras alleles to Ras specific exchange factors: Identification of factor specific requirements.

Nielsen KH, Gredsted L, Broach JR, Willumsen BM
Oncogene. 2001; 20: 2091-100

We have investigated the productive interaction between the four mammalian Ras proteins (H-, N-, KA- and KB-Ras) and their activators, the mammalian exchange factors mSos1, GRF1 and GRP, by using a modified Saccharomyces cerevisiae whose growth is dependent on activation of a mammalian Ras protein by its activator. All four mammalian Ras proteins were activated with similar efficiencies by the individual exchange factors. The H-Ras mutant V103E, which is competent for membrane localization, nucleotide binding, intrinsic and stimulated GTPase activity as well as intrinsic exchange, was defective for activation by all factors tested, suggesting that the integrity of this residue is necessary for catalyzed exchange. However, when other H-Ras mutants were studied, some distinct sensitivities to the exchange factors were observed. GRP-mediated, but not mSos1-mediated, exchange was blocked in additional mutants, suggesting different structural requirements for GRP. Analysis of Ras-mediated gene activation in murine fibroblasts confirmed these results.

p55-hGRF, a short natural form of the Ras-GDP exchange factor high yield production and characterization.

Meyer P, Janin J, Baudet-Nessler S
Eur J Biochem. 1999; 263: 806-16

p55-hGRF, a natural short form of the guanine-nucleotide-releasing factor for p21-Ras from human brain, was expressed at high level in Escherichia coli as well as an engineered truncated form, p39-hGRF. A T7 polymerase expression system was used, resulting in the formation of insoluble cytoplasmic protein aggregates. The recombinant products were resolubilized, renatured and purified to homogeneity. The exchange activity of the refolded hGRF samples on H-Ras was comparable with that published for the soluble catalytic domain of the mouse counterpart, CDC25 Mm. Both p55-hGRF and p39-hGRF form dimers. We established a procedure to prepare and purify the complex with Ras. The results of the characterization study are consistent with a stoichiometry of 1:1 and an equilibrium between dimeric and monomeric forms of the complex.

Functional roles for the pleckstrin and Dbl homology regions in the Ras exchange factor Son-of-sevenless.

McCollam L, Bonfini L, Karlovich CA, Conway BR, Kozma LM, Banerjee U, Czech MP
J Biol Chem. 1995; 270: 15954-7

Activation of p21ras by receptor tyrosine kinases is thought to result from recruitment of guanine nucleotide exchange factors such as Son-of-sevenless (Sos) to plasma membrane receptor substrates via adaptor proteins such as Grb2. This hypothesis was tested in the present studies by evaluating the ability of truncation and deletion mutants of Drosophila (d)Sos to enhance [32P]GTP loading of p21ras when expressed in 32P-labeled COS or 293 cells. The dSos catalytic domain (residues 758-1125), expressed without the dSos NH2-terminal (residues 1-757) or adaptor-binding COOH-terminal (residues 1126-1596) regions, exhibits intrinsic exchange activity as evidenced by its rescue of mutant Saccharomyces cerevisiae deficient in endogenous GTP/GDP exchange activity. Here we show that this dSos catalytic domain fails to affect GTP p21ras levels when expressed in cultured mammalian cells unless the NH2-terminal domain is also present. Surprisingly, the COOH-terminal, adaptor binding domain of dSos was not sufficient to confer p21ras exchange activity to the Sos catalytic domain in these cells in the absence of the NH2-terminal domain. This function of promoting catalytic domain activity could be localized by mutational analysis to the pleckstrin and Dbl homology sequences located just NH2-terminal to the catalytic domain. The results demonstrate a functional role for these pleckstrin and Dbl domains within the dSos protein, and suggest the presence of unidentified cellular elements that interact with these domains and participate in the regulation of p21ras.

CRK protein binds to two guanine nucleotide-releasing proteins for the Ras family and modulates nerve growth factor-induced activation of Ras in PC12 cells.

Matsuda M, Hashimoto Y, Muroya K, Hasegawa H, Kurata T, Tanaka S, Nakamura S, Hattori S
Mol Cell Biol. 1994; 14: 5495-500

It has been reported that growth factors activate Ras through a complex of an adaptor type SH2-containing molecule, Grb2, and a Ras guanine nucleotide-releasing protein (GNRP), mSos. We report on the involvement of another adaptor molecule, CRK, in the activation of Ras. Overexpression of wild-type CRK proteins CRK-I and CRK-II enhanced the nerve growth factor (NGF)-induced activation of Ras in PC12 cells, although the basal level of GTP-bound active Ras was not altered. In contrast, mutants with a single amino acid substitution in either the SH2 or SH3 domain of the CRK-I protein inhibited the NGF-induced activation of Ras. Two GNRPs for the Ras family, mSos and C3G, were coimmunoprecipitated with the endogenous Crk proteins in PC12 cells. The association between C3G and the CRK mutants was dependent upon the presence of intact SH3. The SH2 domain of CRK bound to the SHC protein phosphorylated on tyrosine residues by NGF stimulation. The results demonstrate that, in addition to Grb2, CRK participates in signaling from the NGF receptor and that two GNRPs appear to transmit signals from these adaptor molecules to Ras.

Vav2 is required for cell spreading.

Marignani PA, Carpenter CL
J Cell Biol. 2001; 154: 177-86

Vav2 is a widely expressed Rho family guanine nucleotide exchange factor highly homologous to Vav1 and Vav3. Activated versions of Vav2 are transforming, but the normal function of Vav2 and how it is regulated are not known. We investigated the pathways that regulate Vav2 exchange activity in vivo and characterized its function. Overexpression of Vav2 activates Rac as assessed by both direct measurement of Rac-GTP and cell morphology. Vav2 also catalyzes exchange for RhoA, but does not cause morphologic changes indicative of RhoA activation. Vav2 nucleotide exchange is Src-dependent in vivo, since the coexpression of Vav2 and dominant negative Src, or treatment with the Src inhibitor PP2, blocks both Vav2-dependent Rac activation and lamellipodia formation. A mutation in the pleckstrin homology (PH) domain eliminates exchange activity and this construct does not induce lamellipodia, indicating the PH domain is necessary to catalyze nucleotide exchange. To further investigate the function of Vav2, we mutated the dbl homology (DH) domain and asked whether this mutant would function as a dominant negative to block Rac-dependent events. Studies using this mutant indicate that Vav2 is not necessary for platelet-derived growth factor- or epidermal growth factor-dependent activation of Rac. The Vav2 DH mutant did act as a dominant negative to inhibit spreading of NIH3T3 cells on fibronectin, specifically by blocking lamellipodia formation. These findings indicate that in fibroblasts Vav2 is necessary for integrin, but not growth factor-dependent activation of Rac leading to lamellipodia.

Activation of Ras by receptor tyrosine kinases.

Margolis B, Skolnik EY
J Am Soc Nephrol. 1994; 5: 1288-99

Ras, a small GTP-binding protein, is an important component of the signal transduction pathway used by growth factors to initiate cell growth and differentiation. Cell activation with growth factors such as epidermal growth factor (EGF) induces Ras to move from an inactive GDP-bound state to an active GTP-bound state. Recently, a combination of genetic and biochemical studies has resulted in the elucidation of a signaling pathway that leads from growth factor receptors to Ras. After binding EGF, the EGF receptor tyrosine kinase is activated, leading to receptor autophosphorylation on multiple tyrosine residues. Signaling proteins with Src homology 2 (SH2) domains then bind to these tyrosine-phosphorylated residues, initiating multiple signaling cascades. One of these SH2 domain proteins, Grb2, exists in the cytoplasm in a preformed complex with a second protein, Son of Sevenless (Sos), which can catalyze Ras GTP/GDP exchange. After growth factor stimulation, the tyrosine phosphorylated EGF receptor binds the Grb2/Sos complex, translocating it to the plasma membrane. This translocation is thought to bring Sos into close proximity with Ras, leading to the activation of Ras. In contrast, the insulin receptor does not bind Grb2 directly but rather induces the tyrosine phosphorylation of two proteins, insulin receptor substrate-1 and Shc, that bind the Grb2/Sos complex. Once Ras is activated, it proceeds to stimulate a cascade of protein kinases that are important in a myriad of growth factor responses.

Function and regulation of ras.

Lowy DR, Willumsen BM
Annu Rev Biochem. 1993; 62: 851-91

CAPRI regulates Ca(2+)-dependent inactivation of the Ras-MAPK pathway.

Lockyer PJ, Kupzig S, Cullen PJ
Curr Biol. 2001; 11: 981-6

Ca(2+) is a universal second messenger that is critical for cell growth and is intimately associated with many Ras-dependent cellular processes such as proliferation and differentiation [1]. Ras is a small GTP binding protein that operates as a molecular switch regulating the control of gene expression, cell growth, and differentiation through a pathway from receptors to mitogen-activated protein kinases (MAPKs) [2]. A role for intracellular Ca(2+) in the activation of Ras has been previously demonstrated, e.g., via the nonreceptor tyrosine kinase PYK2 [3] and by Ca(2+)/calmodulin-dependent guanine nucleotide exchange factors (GEFs) such as Ras-GRF [4]; however, there is no Ca(2+)-dependent mechanism for direct inactivation. An important advance toward greater understanding of the complex coordination within the Ras-signaling network is the spatio-temporal analysis of signaling events in vivo. Here, we describe the identification of CAPRI (Ca(2+)-promoted Ras inactivator), a Ca(2+)-dependent Ras GTPase-activating protein (GAP) that switches off the Ras-MAPK pathway following a stimulus that elevates intracellular Ca(2+). Analysis of the spatio-temporal dynamics of CAPRI indicates that Ca(2+) regulates the GAP by a fast C2 domain-dependent translocation mechanism.

Macrophage-stimulating protein activates Ras by both activation and translocation of SOS nucleotide exchange factor.

Li BQ, Wang MH, Kung HF, Ronsin C, Breathnach R, Leonard EJ, Kamata T
Biochem Biophys Res Commun. 1995; 216: 110-8

Macrophage-stimulating protein (MSP) is a chemotactic factor that activates the receptor tyrosine kinase RON. The involvement of Ras in MSP-induced signal transduction was investigated. Here we demonstrate that, in RON-transfected MDCK cells, an active GTP-bound form of Ras was rapidly accumulated by MSP treatment and the Ras-guanine nucleotide exchange activity in SOS immunoprecipitates was concomitantly increased. GAP activity was not changed under the same conditions used. Furthermore, the SH2 domain of adaptor protein GRB2, but not Shc, associated with the activated RON-beta chain, and GRB2-SOS complexes translocated from the cytosol to the membrane upon MSP treatment. These results strongly suggest that MSP activates Ras through RON, and that MSP-induced activation of Ras might be controlled by both the enhancement of catalytic exchange activity of SOS and its translocation to the membrane where its target Ras is localized.

Nerve growth factor stimulation of the Ras-guanine nucleotide exchange factor and GAP activities.

Li BQ, Kaplan D, Kung HF, Kamata T
Science. 1992; 256: 1456-9

The biological activity of Ras proteins is thought to be controlled by the guanine nucleotide exchange factor and the guanosine triphosphatase activating protein (GAP). Treatment of rat pheochromocytoma PC-12 cells with nerve growth factor (NGF) increased the amount of active Ras guanosine triphosphate complex and stimulated the activities of both the guanine nucleotide exchange factor and GAP. In PC-12 cells that overexpressed the tyrosine kinase encoded by the trk proto-oncogene (a component of the high-affinity NGF receptor), the NGF-induced activation of the regulatory proteins was potentiated. These results suggest that the NGF receptor system enhances the activities of both the guanine nucleotide exchange factor and GAP and that the activation of Ras might be controlled by the balance in activity between these two regulatory proteins.

Rasp21 sequences opposite the nucleotide binding pocket are required for GRF-mediated nucleotide release.

Leonardsen L, DeClue JE, Lybaek H, Lowy DR, Willumsen BM
Oncogene. 1996; 13: 2177-87

The substrate requirements for the catalytic activity of the mouse Cdc25 homolog Guanine nucleotide Release Factor, GRF, were determined using the catalytic domain of GRF expressed in insect cells and E. coli expressed H-Ras mutants. We found a requirement for the loop 7 residues in Ras (amino acids 102 to 105) for stimulation of guanine nucleotide release. The dependence on the switch I and II regions of Rasp21 (encompassing the residues that shift position in the GTP- versus GDP-bound protein), which had been seen with Sdc25-mediated exchange, was also found for GRF. In addition, the sensitivity of H-Ras to GRF was abolished when residues 130-139 were replaced by proline-aspartic acid-glutamine, whereas substitution of the entire loop 8 (residues 123-130 replaced by leucine-isoleucine-arginine) had no effect on the stimulation of guanine nucleotide release by GRF. Substrate activity of Ras proteins were independent of their post-translational processing, GDP release was stimulated threefold more effectively by GRF than was GTP release, and no major differences were found between the mammalian N-, H- and K-Ras proteins. Examining the responsiveness of the Ras protein from S. pombe and the human Ras like proteins RhoA, Rap1A, Rac1 and G25K revealed a strict Ras specificity; of these only S. pombe Ras was GRF sensitive.

Analysis of intrinsic and CDC25-stimulated guanine nucleotide exchange of p21ras-nucleotide complexes by fluorescence measurements.

Lenzen C, Cool RH, Wittinghofer A
Methods Enzymol. 1995; 255: 95-109

Independent binding of peptide ligands to the SH2 and SH3 domains of Grb2.

Lemmon MA, Ladbury JE, Mandiyan V, Zhou M, Schlessinger J
J Biol Chem. 1994; 269: 31653-8

Grb2, composed entirely of SH2 and SH3 domains, serves as an adaptor protein in signaling from growth factor-activated tyrosine kinase receptors. It interacts via its SH2 domain with the autophosphorylated carboxyl-terminal tail of activated epidermal growth factor (EGF) receptor and via its SH3 domains with proline-rich sequences in the Ras guanine nucleotide releasing factor, Son of sevenless (Sos). Recruitment of the Grb2-Sos complex to the receptor upon its stimulation leads to Ras activation. A major question remains as to whether SH2-mediated binding of Grb2 to the activated receptor results in conformational changes that influence its SH3-mediated association with Sos, thereby affecting Sos activity. This question is addressed through studies of the binding to intact Grb2 of an EGF receptor-derived phosphotyrosine-containing peptide and a Sos-derived proline-rich peptide using isothermal titration calorimetry and surface plasmon resonance measurements. The phosphopeptide binds to Grb2 in a 1:1 complex, with a KD of 0.4 microns. The Sos proline-rich peptide binds to Grb2 in a 2:1 complex, with a KD of 22 microns. Saturation of the SH2 domain of Grb2 with the EGFR phosphopeptide was found not to affect its subsequent binding to the Sos peptide. Thus we detected no influence of SH2 binding upon SH3-mediated interactions, suggesting that the domains do not communicate, and that recruitment itself of Sos to the cell surface is sufficient for Ras signaling.

Negative feedback regulation and desensitization of insulin- and epidermal growth factor-stimulated p21ras activation.

Langlois WJ, Sasaoka T, Saltiel AR, Olefsky JM
J Biol Chem. 1995; 270: 25320-3

Insulin and epidermal growth factor receptors transmit signals for cell proliferation and gene regulation through formation of active GTP-bound p21ras mediated by the guanine nucleotide exchange factor Sos. Sos is constitutively bound to the adaptor protein Grb2 and growth factor stimulation induces association of the Grb2/Sos complex with Shc and movement of Sos to the plasma membrane location of p21ras. Insulin or epidermal growth factor stimulation induces a rapid increase in p21ras levels, but after several minutes levels decline toward basal despite ongoing hormone stimulation. Here we show that deactivation of p21ras correlates closely with phosphorylation of Sos and dissociation of Sos from Grb2, and that inhibition of mitogen-activated protein (MAP) kinase kinase (also known as extracellular signal-related kinase (ERK) kinase, or MEK) blocks both events, resulting in prolonged p21ras activation. These data suggest that a negative feedback loop exists whereby activation of the Raf/MEK/MAP kinase cascade by p21ras causes Sos phosphorylation and, therefore, Sos/Grb2 dissociation, limiting the duration of p21ras activation by growth factors. A serine/threonine kinase downstream of MEK (probably MAP kinase) mediates this desensitization feedback pathway.

Insulin and epidermal growth factor influence guanine nucleotide-releasing factor by distinct mechanisms.

Langlois WJ, Medh J, Leitner JW, Sasaoka T, Olefsky JM, Draznin B
Endocrinology. 1994; 135: 2412-7

Both insulin and epidermal growth factor (EGF) significantly increased the activation of p21Ras in Rat-1 fibroblasts transfected with human insulin receptors. As both growth factors have been shown to stimulate guanine nucleotide exchange on p21Ras in permeabilized cells, we examined their effects on total cellular guanine nucleotide exchange factor (GEF) activity in a direct assay, using the rate of dissociation of [3H]GDP from p21Ras as a measure of GEF activity. Insulin increased GEF activity in a time-dependent manner, whereas EGF had no effect on GEF activity at any time point studied. To assess whether EGF stimulates guanine nucleotide exchange by recruiting Grb-2-Sos complexes to the plasma membrane, we measured GEF activity in both the cytosol and the plasma membrane fractions of the insulin- and EGF-treated cells. Insulin increased GEF activity in both fractions (48 +/- 12% [3H]GDP released vs. 24 +/- 6% in control plasma membranes, and 65 +/- 13% vs. 13 +/- 4% in control cytosolic fractions), whereas EGF enhanced only the plasma membrane-associated activity (43 +/- 12% of [3H]GDP release in the plasma membrane fraction and 10 +/- 2% in the cytosol). Western blotting of the subcellular fractions with Grb-2 and Sos antibodies revealed translocation of these elements to the plasma membrane after stimulation of cells with either insulin or EGF. Thus, whereas insulin stimulates guanine nucleotide exchange on Ras by both translocating Grb-2-Sos complexes to the plasma membrane and increasing the GEF activity of Sos, EGF does so by causing a translocation of Grb-2-Sos without increasing GEF activity.

Nitric oxide-stimulated guanine nucleotide exchange on p21ras.

Lander HM, Ogiste JS, Pearce SF, Levi R, Novogrodsky A
J Biol Chem. 1995; 270: 7017-20

The protooncogene p21ras, a monomeric G protein family member, plays a critical role in converting extracellular signals into intracellular biochemical events. Here, we report that nitric oxide (NO) activates p21ras in human T cells as evidenced by an increase in GTP-bound p21ras. In vitro studies using pure recombinant p21ras demonstrate that the activation is direct and reversible. Circular dichroism analysis reveals that NO induces a profound conformational change in p21ras in association with GDP/GTP exchange. The mechanism of activation is due to S-nitrosylation of a critical cysteine residue which stimulates guanine nucleotide exchange. Furthermore, we demonstrate that p21ras is essential for NO-induced downstream signaling, such as NF-kappa B activation, and that endogenous NO can activate p21ras in the same cell. These studies identify p21ras as a target of the same cell. These studies identify p21ras as a target of NO in T cells and suggest that NO activates p21ras by an action which mimics that of guanine nucleotide exchange factors.

Ras interaction with RalGDS effector targets.

Koyama S, Kikuchi A
Methods Enzymol. 2001; 332: 127-38

The Dictyostelium Bcr/Abr-related protein DRG regulates both Rac- and Rab-dependent pathways.

Knetsch ML, Schafers N, Horstmann H, Manstein DJ
EMBO J. 2001; 20: 1620-9

Dictyostelium discoideum DdRacGap1 (DRG) contains both Rho-GEF and Rho-GAP domains, a feature it shares with mammalian Bcr and Abr. To elucidate the physiological role of this multifunctional protein, we characterized the enzymatic activity of recombinant DRG fragments in vitro, created DRG-null cells, and studied the function of the protein in vivo by analysing the phenotypic changes displayed by DRG-depleted cells and DRG-null cells complemented with DRG or DRG fragments. Our results show that DRG-GEF modulates F-actin dynamics and cAMP-induced F-actin formation via Rac1-dependent signalling pathways. DRG's RacE-GAP activity is required for proper cytokinesis to occur. Additionally, we provide evidence that the specificity of DRG is not limited to members of the Rho family of small GTPases. A recombinant DRG-GAP accelerates the GTP hydrolysis of RabD 30-fold in vitro and our complementation studies show that DRG-GAP activity is required for the RabD-dependent regulation of the contractile vacuole system in Dictyostelium.

Divergent mechanisms for homologous desensitization of p21ras by insulin and growth factors.

Klarlund JK, Cherniack AD, Czech MP
J Biol Chem. 1995; 270: 23421-8

Previous work suggested that desensitization of p21ras in response to growth factors such as epidermal growth factor (EGF) results from receptor down-regulation. Here we show that p21ras is desensitized by insulin in 3T3-L1 adipocytes in the continued presence of activated insulin receptors, while loss of epidermal growth factor and platelet-derived growth factor (PDGF) receptors in response to their ligands correlates with p21ras desensitization. Furthermore, elevated amounts of Grb2/Shc complexes persisted throughout p21ras desensitization by insulin. However, immunoblotting of anti-Son-of-sevenless (Sos) 1 and 2 immunoprecipitates with anti-Grb2 antisera revealed that p21ras desensitization in response to insulin and PDGF, but not EGF, is associated with a marked decrease in cellular complexes containing Sos and Grb2 proteins. Nonetheless, the desensitization of p21ras in response to these stimuli was homologous, in that each peptide could reactivate [32P]GTP loading of p21ras after desensitization by any of the others. Taken together, these data indicate that insulin, EGF, and PDGF all cause disassembly of Sos proteins from signaling complexes during p21ras desensitization, but at least two mechanisms are involved. Insulin elicits dissociation of Sos from Grb2 SH3 domains, whereas EGF signaling is reversed by receptor down-regulation and Shc dephosphorylation, releasing Grb2 SH2 domains. PDGF action triggers both mechanisms of Grb2 disassembly, which probably operate in concert with GAP to attenuate p21ras signaling.

CrkII signals from epidermal growth factor receptor to Ras.

Kizaka-Kondoh S, Matsuda M, Okayama H
Proc Natl Acad Sci U S A. 1996; 93: 12177-82

A rat fibroblast mutant defective in oncogenic transformation and signaling from epidermal growth factor receptor to Ras has been isolated. The mutant contains dominant negative-type point mutations in the C-terminal SH3 domain of one crkII gene. Among the adapters tested, the mutant is complemented only by crkII cDNA. Expression of the mutated crkII in parent cells generates the phenotype indistinguishable from the mutant cell. Yet overexpression or reduced expression of Grb2 in the mutant before and after complementation with crkII have little effect on its phenotype. We conclude that adapter molecules are highly specific and that the oncogenic growth signal from epidermal growth factor receptor to Ras is predominantly mediated by CrkII in rat fibroblast.

Collybistin, a newly identified brain-specific GEF, induces submembrane clustering of gephyrin.

Kins S, Betz H, Kirsch J
Nat Neurosci. 2000; 3: 22-9

The formation of postsynaptic GABAA and glycine receptor clusters requires the receptor-associated peripheral membrane protein gephyrin. Here we describe two splice variants of a novel gephyrin-binding protein, termed collybistin I and II, which belong to the family of dbl-like GDP/GTP exchange factors (GEFs). Co-expression of collybistin II with gephyrin induced the formation of submembrane gephyrin aggregates that accumulate hetero-oligomeric glycine receptors. Our data suggest that collybistin II regulates the membrane deposition of gephyrin by activating a GTPase of the Rho/Rac family. Therefore, this protein may be an important determinant of inhibitory postsynaptic membrane formation and plasticity.

ralGDS family members interact with the effector loop of ras p21.

Kikuchi A, Demo SD, Ye ZH, Chen YW, Williams LT
Mol Cell Biol. 1994; 14: 7483-91

Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.

Dbl and Vav mediate transformation via mitogen-activated protein kinase pathways that are distinct from those activated by oncogenic Ras.

Khosravi-Far R, Chrzanowska-Wodnicka M, Solski PA, Eva A, Burridge K, Der CJ
Mol Cell Biol. 1994; 14: 6848-57

Vav and Dbl are members of a novel class of oncogene proteins that share significant sequence identity in a approximately 250-amino-acid domain, designated the Dbl homology domain. Although Dbl functions as a guanine nucleotide exchange factor (GEF) and activator of Rho family proteins, recent evidence has demonstrated that Vav functions as a GEF for Ras proteins. Thus, transformation by Vav and Dbl may be a consequence of constitutive activation of Ras and Rho proteins, respectively. To address this possibility, we have compared the transforming activities of Vav and Dbl with that of the Ras GEF, GRF/CDC25. As expected, GRF-transformed cells exhibited the same reduction in actin stress fibers and focal adhesions as Ras-transformed cells. In contrast, Vav- and Dbl-transformed cells showed the same well-developed stress fibers and focal adhesions observed in normal or RhoA(63L)-transformed NIH 3T3 cells. Furthermore, neither Vav- or Dbl-transformed cells exhibited the elevated levels of Ras-GTP (60%) observed with GRF-transformed cells. Finally, GRF, but not Vav or Dbl, induced transcriptional activation from Ras-responsive DNA elements (ets/AP-1, fos promoter, and kappa B). However, like Ras- and GRF-transformed cells, both Vav- and Dbl-transformed cells exhibited constitutively activated mitogen-activated protein kinases (MAPKs) (primarily p42MAPK/ERK2). Since kinase-deficient forms of p42MAPK/ERK2 and p44MAPK/ERK1 inhibited Dbl transformation, MAPK activation may be an important component of its transforming activity. Taken together, our observations indicate that Vav and Dbl transformation is not a consequence of Ras activation and instead may involve the constitutive activation of MAPKs.

A family of cAMP-binding proteins that directly activate Rap1.

Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE, Graybiel AM
Science. 1998; 282: 2275-9

cAMP (3',5' cyclic adenosine monophosphate) is a second messenger that in eukaryotic cells induces physiological responses ranging from growth, differentiation, and gene expression to secretion and neurotransmission. Most of these effects have been attributed to the binding of cAMP to cAMP-dependent protein kinase A (PKA). Here, a family of cAMP-binding proteins that are differentially distributed in the mammalian brain and body organs and that exhibit both cAMP-binding and guanine nucleotide exchange factor (GEF) domains is reported. These cAMP-regulated GEFs (cAMP-GEFs) bind cAMP and selectively activate the Ras superfamily guanine nucleotide binding protein Rap1A in a cAMP-dependent but PKA-independent manner. Our findings suggest the need to reformulate concepts of cAMP-mediated signaling to include direct coupling to Ras superfamily signaling.

Inhibition of the action of a stimulatory GDP/GTP exchange protein for smg p21 by acidic membrane phospholipids.

Kawamura S, Kaibuchi K, Hiroyoshi M, Fujioka H, Mizuno T, Takai Y
Jpn J Cancer Res. 1991; 82: 758-61

A stimulatory GDP/GTP exchange protein for smg p21 (smg p21 GDS) stimulated the dissociation of GDP from smg p21B. This reaction was inhibited by acidic membrane phospholipids such as phosphatidylinositol, phosphatidylinositol-4-monophosphate, phosphatidylinositol-4,5-bisphosphate, phosphatidic acid, and phosphatidylserine but not by phosphatidylcholine or phosphatidylethanolamine. These acidic phospholipids inhibited the smg p21 GDS action in a manner competitive with both smg p21 GDS and smg p21B. smg p21 GDS has other actions to inhibit the binding of smg p21B to membranes and to induce the dissociation of prebound smg p21B from the membranes. The acidic phospholipids also inhibited these two actions of smg p21 GDS. smg p21B has a polybasic region and an isoprenoid moiety in its C-terminal region which are necessary for its membrane-binding activity and its sensitivity to the smg p21 GDS actions. Therefore, it is possible that acidic membrane phospholipids interact with this polybasic region and thereby inhibit the smg p21 GDS actions.

Activation of the guanine nucleotide exchange factor Dbl following ACK1-dependent tyrosine phosphorylation.

Kato J, Kaziro Y, Satoh T
Biochem Biophys Res Commun. 2000; 268: 141-7

Signals triggered by diverse receptors modulate the activity of Rho family proteins, although the regulatory mechanism remains largely unknown. On the basis of their biochemical activity as guanine nucleotide exchange factors (GEFs), Dbl family proteins are believed to be implicated in the regulation of Rho family GTP-binding proteins in response to a variety of extracellular stimuli. Here we show that GEF activity of full-length proto-Dbl is enhanced upon tyrosine phosphorylation. When transiently coexpressed with the activated form of the non-receptor tyrosine kinase ACK1, a downstream target of Cdc42, Dbl became tyrosine-phosphorylated. In vitro GEF activity of Dbl toward Rho and Cdc42 was augmented following tyrosine phosphorylation. Moreover, accumulation of the GTP-bound form of Rho and Rac within the cell paralleled ACK-1-dependent tyrosine phosphorylation of Dbl. Consistently, activation of c-Jun N-terminal kinase downstream of Rho family GTP-binding proteins was also enhanced when Dbl was tyrosine-phosphorylated. Collectively, these findings suggest that the tyrosine kinase ACK1 may act as a regulator of Dbl, which in turn activates Rho family proteins.

In vivo functional analysis of the Ras exchange factor son of sevenless.

Karlovich CA, Bonfini L, McCollam L, Rogge RD, Daga A, Czech MP, Banerjee U
Science. 1995; 268: 576-9

The Son of sevenless (Sos) protein functions as a guanine nucleotide transfer factor for Ras and interacts with the receptor tyrosine kinase Sevenless through the protein Drk, a homolog of mammalian Grb2. In vivo structure-function analysis revealed that the amino terminus of Sos was essential for its function in flies. A molecule lacking the amino terminus was a potent dominant negative. In contrast, a Sos fragment lacking the Drk binding sites was functional and its activity was dependent on the presence of the Sevenless receptor. Furthermore, membrane localization of Sos was independent of Drk. A possible role for Drk as an activator of Sos is discussed and a Drk-independent interaction between Sos and Sevenless is proposed that is likely mediated by the pleckstrin homology domain within the amino terminus.

Regulation of RNA processing and transport by a nuclear guanine nucleotide release protein and members of the Ras superfamily.

Kadowaki T, Goldfarb D, Spitz LM, Tartakoff AM, Ohno M
EMBO J. 1993; 12: 2929-37

The RCC1 gene of mammals encodes a guanine nucleotide release protein (GNRP). RCC1 and a homolog in Saccharomyces cerevisiae (MTR1/PRP20/SRM1) have previously been implicated in control of mRNA metabolism and export from the nucleus. We here demonstrate that a temperature-sensitive fission yeast mutant which has a mutation in a homologous gene, and two of three additional (mtr1/prp20/srm1) mutants accumulate nuclear poly(A)+ RNA at 37 degrees C. In S.cerevisiae, maturation of rRNA and tRNA is also inhibited at 37 degrees C. Nevertheless, studies with the corresponding BHK-21 cell mutant indicate that protein import into the nucleus continues. MTR1 homologs regulate RNA processing at a point which is distinct from their regulation of chromosome condensation since: (i) poly(A)+ RNA accumulation in the fission yeast mutant precedes chromosome condensation, and (ii) unlike chromosome condensation, accumulation of nuclear poly(A)+ RNA does not require p34cdc28 kinase activation or protein synthesis. Moreover, experiments involving inhibition of DNA synthesis indicate that the S.cerevisiae homolog does not govern cell cycle checkpoint control. Since RCC1p acts as GNRP for Ran, a small nuclear GTPase of the ras superfamily, we have identified two homologs of Ran in S.cerevisiae (CNR1 and CNR2). Only CNR1 is essential, but both code for proteins extremely similar to Ran and can suppress mtr1 mutations in allele-specific fashion. Thus, MTR1 and its homologs appear to act as GNRPs for a family of conserved GTPases in controlling RNA metabolism and transport. Their role in governing checkpoint control appears to be restricted to higher eukaryotes.

Ras-GRF activates Ha-Ras, but not N-Ras or K-Ras 4B, protein in vivo.

Jones MK, Jackson JH
J Biol Chem. 1998; 273: 1782-7

Human cells contain four homologous Ras proteins, but it is unknown whether these homologues have different biological functions. As a first step in determining if Ras homologues might participate in distinct signaling cascades, we assessed whether a given Ras guanine nucleotide exchange factor could selectively activate a single Ras homologue in vivo. We found that Ras-GRF/Cdc25Mm activates Ha-Ras, but does not activate N-Ras or K-Ras 4B, protein in vivo. Moreover, our results suggested that residues within the C-terminal hypervariable domains of Ras proteins may dictate, at least in part, the specificity of Ras-GRF/CDC25Mm for Ha-Ras protein. Our studies represent the first biochemical evidence that a Ras GEF can selectively activate a single Ras homologue in vivo. Selective activation of a single Ras homologue by Ras-GRF/Cdc25Mm or other Ras guanine nucleotide exchange factors could potentially enable each of the Ras homologues to participate in different signal transduction pathways.

Inhibition of mSOS-activity by binding of phosphatidylinositol 4,5-P2 to the mSOS pleckstrin homology domain.

Jefferson AB, Klippel A, Williams LT
Oncogene. 1998; 16: 2303-10

There are several recently reported examples of inositol phospholipids binding to pleckstrin homology (PH) domains of proteins. The PH domain of SOS, a guanine nucleotide exchange factor for Ras, binds to phosphatidylinositol 4,5 bisphosphate (PtdIns4,5P2). We found that binding of PtdIns4,5P2 to 6-his-tagged recombinant mSOS in vitro inhibits the ability of SOS to catalyze the association of GTP on p21RAS. This inhibition was specific for PtdIns4,5P2: a number of other phosphatidylinositols and phosphatidylserine failed to inhibit Ras GTP-association. We confirmed that the specificity of binding of PtdIns's to recombinant GST-SOS-PH domain is the same as the specificity of PtdIns's for inhibition of SOS activity: namely, that only PtdIns4,5P2 binds significantly to the SOS-PH domain. In addition, the inhibition of Ras GTP-binding is not blocked by excess free inositols suggesting that SOS binds to PtdIns4,5P2 with higher affinity than it binds to free inositols. Addition of SOS-PH domain protein prevented the inhibition of SOS by PtdIns4,5P2 as did addition of the high affinity PtdIns4,5P2-binding drug neomycin. This confirmed that SOS inhibition is mediated by the SOS-PH domain binding to the inositol moiety of PtdIns4,5P2. Binding of Grb2 to SOS did not prevent the inhibition of SOS by PtdIns4,5P2 suggesting that there must be another mechanism for regulating this inhibition. These findings show that the phospholipid PtdIns4,5P2 can suppress the activity of an enzyme involved in signal transduction and suggest that this inhibitory effect must be relieved when SOS is activated.

There are GAPS and there are GAPS.

Iyengar R
Science. 1997; 275: 42-3

Activation of the Drosophila C3G leads to cell fate changes and overproliferation during development, mediated by the RAS-MAPK pathway and RAP1.

Ishimaru S, Williams R, Clark E, Hanafusa H, Gaul U
EMBO J. 1999; 18: 145-55

The cellular signal transduction pathways by which C3G, a RAS family guanine nucleotide exchange factor, mediates v-crk transformation are not well understood. Here we report the identification of Drosophila C3G, which, like its human cognate, specifically binds to CRK but not DRK/GRB2 adaptor molecules. During Drosophila development, constitutive membrane binding of C3G, which also occurs during v-crk transformation, results in cell fate changes and overproliferation, mimicking overactivity of the RAS-MAPK pathway. The effects of C3G overactivity can be suppressed by reducing the gene dose of components of the RAS-MAPK pathway and of RAP1. These findings provide the first in vivo evidence that membrane localization of C3G can trigger activation of RAP1 and RAS resulting in the activation of MAPK, one of the hallmarks of v-crk transformation previously thought to be mediated through activation of SOS.

The aimless RasGEF is required for processing of chemotactic signals through G-protein-coupled receptors in Dictyostelium.

Insall RH, Borleis J, Devreotes PN
Curr Biol. 1996; 6: 719-29

BACKGROUND: Ras proteins are small GTP-binding proteins that play an essential role in a wide range of processes, particularly in mammalian growth control. They act as molecular switches, being inactive when GDP is bound, and active when associated with GTP. Activation is accomplished by guanine nucleotide exchange factors (RasGEFs); when RasGEFs interact with Ras proteins, GDP is allowed to escape, and is replaced by GTP. Dictyostelium responds to chemoattractants through typical seven transmembrane domain receptors and heterotrimeric G proteins. There are at least five different Dictyostelium Ras genes, whose functions are not yet known. RESULTS: We have isolated the aimless gene, which encodes the Dictyostelium homologue of RasGEFs, during a screen for insertional mutants that fail to aggregate. We found that aimless null mutants grew at a normal rate, but were severely impaired in both chemotaxis and activation of adenylyl cyclase, both of which are critical for the early stages of development. Although coupling between receptors and their G proteins is unaffected, and several cyclic AMP (cAMP)-mediated responses appear normal, activation of adenylyl cyclase by receptors and GTP gamma S (a non-hydrolyzable GTP analogue) is reduced by up to 95%. The motility of mutant cells appears normal, suggesting a true defect in gradient sensing. CONCLUSIONS: The discovery of the aimless gene adds an interesting new member to the family of RasGEFs. Our data suggest an unforeseen role for a RasGEF, and therefore presumably a complete Ras pathway, in the processing of chemotactic signals through G-protein-coupled receptors.

Characterization of GFR, a novel guanine nucleotide exchange factor for Rap1.

Ichiba T, Hoshi Y, Eto Y, Tajima N, Kuraishi Y
FEBS Lett. 1999; 457: 85-9

Three groups of Rap1-specific guanine nucleotide exchange factors including C3G, CalDAG-GEFI, and Epac/cAMP-GEFI/II have been identified to date. In the present study, we report a new Rap1 guanine nucleotide exchange factor which we have named GFR (guanine nucleotide exchange factor for Rap1). GFR shows close sequence similarity to EPAC/cAMP-GEFI/II although GFR lacks a cAMP binding domain and contains a nuclear localization signal. We demonstrated that GFR can activate Rap1 but not H-Ras in 293T cells and that the cdc25 domain of GFR is required for the activation of Rap1. Northern blot analysis suggested that GFR mRNA is strongly expressed in the brain. In transfected HeLa cells, GFR has been found to be localized in the nuclei.

Inhibition of SDC25 C-domain-induced guanine-nucleotide exchange by guanine ring binding domain mutants of v-H-ras.

Hwang YW, Zhong JM, Poullet P, Parmeggiani A
J Biol Chem. 1993; 268: 24692-8

Guanine-nucleotide exchange is the reaction that controls the activation of H-ras p21. This reaction is stimulated by the guanine-nucleotide exchange factor. In this study we show that H-ras p21 harboring guanine ring binding domain (the conserved NKXD motif) mutations, such as N116I or K117E, are potent inhibitors of H-ras p21 guanine-nucleotide exchange reaction promoted by SDC25C (Saccharomyces cerevisiae SDC25 C-domain gene product), a guanine-nucleotide exchange factor. The inhibition is due to the formation of a stable but catalytically inactive complex consisting of the H-ras mutant and SDC25C. By examining the interaction of v-H-ras(N116I) or v-H-ras(K117E) with SDC25C at different concentrations of guanine-nucleotide, we demonstrate that the mechanism of SDC25C-promoted guanine-nucleotide exchange proceeds through the following pathway. First, SDC25C binds to H-ras and forms an intermediate H-ras.SDC25C complex; subsequently, an incoming guanine-nucleotide dissociates the complex, releasing SDC25C from H-ras and causes guanine-nucleotide exchange. This mechanism is similar to the one proposed for Escherichia coli elongation factor Ts-catalyzed guanine-nucleotide exchange.

Purification of a factor capable of stimulating the guanine nucleotide exchange reaction of ras proteins and its effect on ras-related small molecular mass G proteins.

Huang YK, Kung HF, Kamata T
Proc Natl Acad Sci U S A. 1990; 87: 8008-12

We have previously identified a membrane factor capable of stimulating guanine nucleotide exchange activity for ras p21 proteins. The ras guanine nucleotide exchange factor (rGEF) was purified from bovine brain to near homogeneity by successive chromatographies on DE52 DEAE-cellulose, Sepharose 6B, hydroxylapatite, and FPLC phenyl-Superose resins. SDS/polyacrylamide gel electrophoresis of the purified rGEF showed a single major protein with a molecular mass of 35 kDa. rGEF increased the exchange rate of GDP in normal [Gly12]p21 or oncogenic [Val12]p21 up to 30- to 40-fold under physiological concentrations of Mg2+. Since the factor was free from GDP/GTP binding activity and nonspecific GDP hydrolytic activity, we propose that rGEF may regulate GDP/GTP exchange reaction of ras proteins in response to external growth signals. Moreover, rGEF enhanced the dissociation of bound GDP from some of ras-like G proteins, R-ras, rap1-A, rab1-B, and rho proteins, raising the possibility that rGEF may affect the activities of these proteins.

Identification of amino acids in p21ras involved in exchange factor interaction.

Howe LR, Marshall CJ
Oncogene. 1993; 8: 2583-90

Through the genetic analysis of vulva development in C. elegans several different sites of mutation have been identified in the let-60 ras protein which have been postulated to affect the function of normal but not oncogenic p21ras (Beitel, G. J., S. G. Clark, and H. R. Horvitz. 1990. Nature 348: 503-509). We have introduced these mutations into mammalian Ha-ras and determined their effect on the function of cellular ras (c-ras), an oncogenically activated variant D12ras and the dominant negative N17ras. From these studies we conclude that two of the mutations S89-->F89 and delta 103-108 destabilise ras when it is in the GDP-bound form. However mutations at A66 and G75 lead to stable proteins on which a ras exchange factor SCD25 is unable to promote the formation of ras-GTP. The mutations at A66 show for the first time that helix alpha 2 of p21ras is involved in the stimulation of guanine nucleotide exchange by exchange factors.

A novel oncogene, ost, encodes a guanine nucleotide exchange factor that potentially links Rho and Rac signaling pathways.

Horii Y, Beeler JF, Sakaguchi K, Tachibana M, Miki T
EMBO J. 1994; 13: 4776-86

Transfection of NIH3T3 cells with an osteosarcoma expression cDNA library led to the appearance of foci of morphologically transformed cells which were found to harbor a novel oncogene, ost. The ost product was activated by truncation of the N-terminal domain of the ost proto-oncogene and was highly tumorigenic in nude mouse assays. The proto-ost cDNA, isolated subsequently, encodes a predicted protein of 100 kDa containing DH (Db1 homology) and PH (pleckstrin homology) domains. Ost is mainly phosphorylated on serine and localized in the cytoplasm. Purified Ost protein catalyzed guanine nucleotide exchange on RhoA and Cdc42 among the Rho and Ras family members tested, indicating that Ost can activate these small GTP-binding proteins. Ost did not detectably associate with RhoA or Cdc42, but interacted specifically with the GTP-bound form of Rac1, suggesting that Ost can function as an effector of Rac1. These results suggest that Ost is a critical regulatory component which links pathways that signal through Rac1, RhoA and Cdc42. Of the tissues examined, expression of ost was the highest in brain and could be localized to neurons and alpha-tanycytes, suggesting that Ost may participate in axonal transport in these specialized cells.

Epidermal growth factor receptor targeting prevents uncoupling of the Grb2-SOS complex.

Holt KH, Waters SB, Okada S, Yamauchi K, Decker SJ, Saltiel AR, Motto DG, Koretzky GA, Pessin JE
J Biol Chem. 1996; 271: 8300-6

Insulin stimulates the Ras/Raf/MEK/ERK pathway leading to feedback phosphorylation of the Ras guanylnucleotide exchange protein SOS and dissociation of Grb2 from SOS. Even though epidermal growth factor (EGF) also stimulates ERK activity and phosphorylation of SOS similar to insulin, EGF induces a dissociation of the Grb2-SOS complex from Shc. To determine the molecular basis for this difference, we examined the signaling properties of a mutant EGF receptor lacking the five major autophosphorylation sites. Although EGF stimulation of the mutant EGF receptor activates ERK and phosphorylation of both Shc and SOS, it fails to directly associate with either Shc or Grb2. However, under these conditions EGF induces a dissociation of the Grb2-SOS complex suggesting a role for receptor and/or plasma membrane targeting in the stabilization of Grb2-SOS interaction. Consistent with this hypothesis, expression of an SH2 domain Grb2 mutant which is unable to mediate plasma membrane targeting of the Grb2-SOS complex results in both insulin- and EGF-stimulated uncoupling of Grb2 from SOS. Furthermore, a plasma membrane-bound Grb2 fusion protein remains constitutively associated with SOS. Together, these data demonstrate that EGF stimulation prevents the feedback uncoupling of Grb2 from SOS by inducing a persistent plasma membrane receptor targeting of the Grb2-SOS complex.

Signal transduction in T lymphocytes using a conditional allele of Sos.

Holsinger LJ, Spencer DM, Austin DJ, Schreiber SL, Crabtree GR
Proc Natl Acad Sci U S A. 1995; 92: 9810-4

While Ras activation has been shown to play an important role in signal transduction by the T-lymphocyte antigen receptor, the mechanism of its activation in T cells is unclear. Membrane localization of the guanine nucleotide exchange factor Sos, but not Vav or Dbl, was sufficient for Ras-mediated signaling in T lymphocytes. Activation of Sos appears to involve membrane recruitment and not allosteric changes, because interaction of Sos with the linking molecule Grb-2 was not required for Ras activation. To extend this analysis, we constructed a modified Sos that could be localized to the membrane inducibly by using a rationally designed chemical inducer of dimerization, FK1012. The role of Grb-2 in signaling was mimicked with this technique, which induced the association of a modified Sos with the membrane, resulting in rapid activation of Ras-induced signaling. In contrast, inducible localization of Grb-2 to the membrane did not activate signaling and suggests that the interaction of Grb-2 with Sos in T cells is subject to regulation. This conditional allele of Sos demonstrates that membrane localization of Sos is sufficient for Ras activation in T cells and indicates that the role of Grb-2 is to realize the biologic advantages of linker-mediated dimerization: enhanced specificity and favorable kinetics for signaling. This method of generating conditional alleles may also be useful in dissecting other signal transduction pathways regulated by protein localization or protein-protein interactions.

A Grb2-associated docking protein in EGF- and insulin-receptor signalling.

Holgado-Madruga M, Emlet DR, Moscatello DK, Godwin AK, Wong AJ
Nature. 1996; 379: 560-4

The protein Grb2 plays a central role in signalling by receptor protein-tyrosine kinases, where its SH2 domain binds to the receptor and its two SH3 domains link to effectors. One target effector is Sos, so Grb2 links receptor protein-tyrosine kinases with the Ras signalling pathway. The SH3 domains can also couple to other signalling proteins, including Vav, c-Abl and dynamin. We have identified several bands in glial and medulloblastoma tumours that are recognized by Grb2 but these did not correspond to any known protein. Here we use recombinant Grb2 to isolate a complementary DNA called Gab1 (for Grb2-associated binder-1). Gab1 shares amino-acid homology and several structural features with IRS-1 (insulin-receptor substrate-1; refs 6,7), is a substrate of the EGF and insulin receptors, and can act as a docking protein for several SH2-containing proteins. Over-expression of Gab1 enhances cell growth and results in transformation. We conclude that Gab1 is a new protein in EGF and insulin receptor signalling which could integrate signals from different systems.

Differential interaction of the ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor.

Herrmann C, Horn G, Spaargaren M, Wittinghofer A
J Biol Chem. 1996; 271: 6794-800

The interactions of H-Ras, R-Ras, and Rap1A with the Ras-binding domains (RBD) of the c-Raf kinase and of the Ral guanine nucleotide exchange factor (RGF) was studied biochemically in solution. From deletion cloning the RGF-RBD was defined as a 97-amino acid-long fragment from the C-terminal end of the human RGF, which is an independent folding domain with high stability. Interestingly, whereas H-Ras binds with high affinity (KD = 20 nM) to Raf-RBD and with low affinity (KD = 1 microM) to RGF-RBD, Rap1A shows the opposite behavior. The binding of both RBDs to R-Ras is weak and shows no specificity. The interaction between Rap1A and RGF-RBD shows similar characteristics to the Ras-Raf interaction because it is blocked by mutations in the effector region (D38A) and it inhibits the dissociation of guanine nucleotide, which is the basis for the quantitative measurements in this work. Furthermore, the binding of RGF-RBD inhibits the interaction between Rap1A and Rap-GAP. As long as the cellular localizations of the different proteins and their biological functions are not clarified, these biochemical data seem to indicate that Ral-guanine nucleotide exchange factors is an effector molecule of Rap1A rather than of H-Ras.

Yeast RAS2 mutations modulating the ras-guanine exchange factor interaction.

Hermann-Le Denmat S, Jacquet M
FEBS Lett. 1997; 403: 95-9

We have used a two-hybrid approach to test various forms of Saccharomyces cerevisiae Ras2p for their ability to interact with the human guanine nucleotide exchange factor HGRF55. We have previously shown that a strong two-hybrid interaction is found between the HGRF55p and the dominant negative Ras2p(G22A) form of ras [Camus et al. (1995) Oncogene 11, 951-959]. We show here that the substitution N123I which weakens the guanine nucleotide binding also promotes ras-GEF interaction. We demonstrate that the R80D substitution alone completely abolishes the interaction of Ras2p(G22A) with GEF, whereas substitutions at positions 81, 82 and 73 have only small effects. Since residue 73 is involved in the response of ras to GEF, we propose that it plays a role in the conformational change induced by the GEF rather than in its binding. Those results emphasize the role of the alpha2 helix of the switch II region in the recognition of the GEF family.

Ras-mediated transcription activation: analysis by transient cotransfection assays.

Hauser CA, Westwick JK, Quilliam LA
Methods Enzymol. 1995; 255: 412-26

Internalized epidermal growth factor receptors participate in the activation of p21(ras) in fibroblasts.

Haugh JM, Huang AC, Wiley HS, Wells A, Lauffenburger DA
J Biol Chem. 1999; 274: 34350-60

Regulated activation of the highly conserved Ras GTPase is a central event in the stimulation of cell proliferation, motility, and differentiation elicited by receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR). In fibroblasts, this involves formation and membrane localization of Shc.Grb2.Sos complexes, which increases the rate of Ras guanine nucleotide exchange. In order to control Ras-mediated cell responses, this activity is regulated by receptor down-regulation and a feedback loop involving the dual specificity kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK). We investigated the role of EGFR endocytosis in the regulation of Ras activation. Of fundamental interest is whether activated receptors in endosomes can participate in the stimulation of Ras guanine nucleotide exchange, because the constitutive membrane localization of Ras may affect its compartmentalization. By exploiting the differences in postendocytic signaling of two EGFR ligands, epidermal growth factor and transforming growth factor-alpha, we found that activated EGFR located at the cell surface and in internal compartments contribute equally to the membrane recruitment and tyrosine phosphorylation of Shc in NR6 fibroblasts expressing wild-type EGFR. Importantly, both the rate of Ras-specific guanine nucleotide exchange and the level of Ras-GTP were depressed to near basal values on the time scale of receptor trafficking. Using the selective MEK inhibitor PD098059, we were able to block the feedback desensitization pathway and maintain activation of Ras. Under these conditions, the generation of Ras-GTP was not significantly affected by the subcellular location of activated EGFR. In conjunction with our previous analysis of the phospholipase C pathway in the same cell line, this suggests a selective continuation of specific signaling activities and cessation of others upon receptor endocytosis.

[Activation of Rap1, antagonist to ras, by Crk-C3G]

Hattori S, Matsuda M
Gan To Kagaku Ryoho. 1997; 24: 1414-21

Rap1 was identified as gene whose overexpression suppressed transformation by ras. Rap1 belongs to the Ras family. The amino acid sequences of Rap1 and Ras show 55% identity to each other. Due to this high sequence similarity, Rap1 binds to effector molecules of Ras, however, Rap1 does not activate them. Thus, Rap1 functions are antagonistic to Ras in the cells. C3G was identified as a Crk SH3-binding guanine nucleotide exchange factor. Biochemical and cell biological analyses revealed that C3G is a Rap1 activator. Since it has been considered that Crk transduces signals from tyrosine kinases, this finding suggests that the activity of Rap1 is also under the control of tyrosine kinases. Overexpression of C3G in ras-transformed cells caused the morphology of the cells to revert to that of normal cells. Moreover, a mutant cell line that was resistant to EGF-dependent transformation was isolated. In the cell line a mutation was found in crk gene that was the cause of the resistance. These findings suggest that Crk-C3G-Rap1 pathway may function as an anti-transformation machinery.

[Heterogeneity of Ras GTPase-activating proteins]

Hattori S
Seikagaku. 1994; 66: 1331-5

Shc regulates epidermal growth factor-induced activation of the JNK signaling pathway.

Hashimoto A, Kurosaki M, Gotoh N, Shibuya M, Kurosaki T
J Biol Chem. 1999; 274: 20139-43

Two adaptor molecules, Grb2 and Shc, have been implicated in the extracellular signal-regulated kinase (ERK) activation by receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR). Here we show that the EGF-mediated ERK activation is abolished by loss of Grb2, whereas this response is not affected by loss of Shc. Conversely, the EGF-mediated c-Jun N-terminal kinase (JNK) activation is dependent on Shc, but not Grb2. These findings strongly support distinct roles for Grb2 and Shc in controlling ERK and JNK activation after EGF stimulation.

Ras-Cdc25 and Rho-Dbl binding assays: complex formation in vitro.

Hart M, Powers S
Methods Enzymol. 1995; 255: 129-35

Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13.

Hart MJ, Jiang X, Kozasa T, Roscoe W, Singer WD, Gilman AG, Sternweis PC, Bollag G
Science. 1998; 280: 2112-4

Signaling pathways that link extracellular factors to activation of the monomeric guanosine triphosphatase (GTPase) Rho control cytoskeletal rearrangements and cell growth. Heterotrimeric guanine nucleotide-binding proteins (G proteins) participate in several of these pathways, although their mechanisms are unclear. The GTPase activities of two G protein alpha subunits, Galpha12 and Galpha13, are stimulated by the Rho guanine nucleotide exchange factor p115 RhoGEF. Activated Galpha13 bound tightly to p115 RhoGEF and stimulated its capacity to catalyze nucleotide exchange on Rho. In contrast, activated Galpha12 inhibited stimulation by Galpha13. Thus, p115 RhoGEF can directly link heterotrimeric G protein alpha subunits to regulation of Rho.

Cellular transformation and guanine nucleotide exchange activity are catalyzed by a common domain on the dbl oncogene product.

Hart MJ, Eva A, Zangrilli D, Aaronson SA, Evans T, Cerione RA, Zheng Y
J Biol Chem. 1994; 269: 62-5

The dbl oncogene product contains a 238-amino acid domain, which is shared by an expanding family of growth regulatory proteins. These include the Saccharomyces cerevisiae cell division cycle protein, CDC24, the breakpoint cluster region protein, the ect2 and vav oncogene products, and the brain GDP-releasing factor for Ras. Previous studies have provided evidence that oncogenic Dbl or an associated protein stimulates GDP dissociation from the human species (Hs) homolog of CDC42. We show here that Dbl specifically complexes with the GDP-bound forms of CDC42Hs and RhoA, but not Rac1 or TC10, and that this specificity correlates with the ability of Dbl to act as a GDP-releasing factor. Small deletions throughout the Dbl domain, which inactivate transformation, eliminated the ability of Dbl to stimulate GDP dissociation, whereas deletions outside of this domain did not impair either function. Finally, the Dbl domain itself, when expressed and purified as a recombinant protein, was shown to stimulate GDP dissociation from purified, recombinant CDC42Hs. These findings establish that a minimal unit on Dbl that is critical to its transforming function directly regulates GDP-GTP exchange activity.

Structure-based mutagenesis reveals distinct functions for Ras switch 1 and switch 2 in Sos-catalyzed guanine nucleotide exchange.

Hall BE, Yang SS, Boriack-Sjodin PA, Kuriyan J, Bar-Sagi D
J Biol Chem. 2001; 276: 27629-37

Ras GTPases function as binary switches in signaling pathways controlling cell growth and differentiation. The guanine nucleotide exchange factor Sos mediates the activation of Ras in response to extracellular signals. We have previously solved the crystal structure of nucleotide-free Ras in complex with the catalytic domain of Sos (Boriack-Sjodin, P. A., Margarit, S. M., Bar-Sagi, D., and Kuriyan, J. (1998) Nature 394, 337-343). The structure demonstrates that Sos induces conformational changes in two loop regions of Ras known as switch 1 and switch 2. In this study, we have employed site-directed mutagenesis to investigate the functional significance of the conformational changes for the catalytic function of Sos. Switch 2 of Ras is held in a very tight embrace by Sos, with almost every external side chain coordinated by Sos. Mutagenesis of contact residues at the switch 2-Sos interface shows that only a small set of side chains affect binding, with the most important contact being mediated by tyrosine 64, which is buried in a hydrophobic pocket of Sos in the Ras.Sos complex. Substitutions of Ras and Sos side chains that are inserted into the Mg(2+)- and nucleotide phosphate-binding site of switch 2 (Ras Ala(59) and Sos Leu(938) and Glu(942)) have no effect on the catalytic function of Sos. These results indicate that the interaction of Sos with switch 2 is necessary for tight binding, but is not the critical driving force for GDP displacement. The structural distortion of switch 1 induced by Sos is mediated by a small number of specific contacts between highly conserved residues on both Ras and Sos. Mutations of a subset of these residues (Ras Tyr(32) and Tyr(40)) result in an increase in the intrinsic rate of nucleotide dissociation from Ras and impair the binding of Ras to Sos. Based on this analysis, we propose that the interactions of Sos with the switch 1 and switch 2 regions of Ras have distinct functional consequences: the interaction with switch 2 mediates the anchoring of Ras to Sos, whereas the interaction with switch 1 leads to disruption of the nucleotide-binding site and GDP dissociation.

G proteins and small GTPases: distant relatives keep in touch.

Hall A
Science. 1998; 280: 2074-5

Tyrosine phosphorylation and activation of Vav GTP/GDP exchange activity in antigen receptor-triggered B cells.

Gulbins E, Langlet C, Baier G, Bonnefoy-Berard N, Herbert E, Altman A, Coggeshall KM
J Immunol. 1994; 152: 2123-9

Ag receptor triggering in B cells stimulates the activity of receptor-associated tyrosine protein kinases (TPK), leading to tyrosine phosphorylation of several cellular substrates, one of which is the Vav proto-oncogene product. We have recently determined that Vav is a TPK-regulated guanine nucleotide exchange factor for Ras in T cells. Here, we show that B cell extracts or Vav immunoprecipitates contain a Ras GDP/GTP exchange activity that is stimulated upon surface Ig (slg) triggering. The receptor-mediated stimulation of Vav exchange activity was blocked by the TPK antagonist, herbimycin A. Furthermore, immunodepletion of Vav from the B cell extracts removed approximately 80% of the Ras GDP/GTP exchange activity. These findings indicate, first, that B cell-derived Vav possesses GDP/GTP exchange activity for Ras; second, that the exchange activity of Vav is accelerated by a slg-triggered, herbimycin A-sensitive TPK and, third, that Vav accounts for most of the receptor-stimulated Ras GDP/GTP exchange activity. Thus, Vav may serve as a critical component in slg-mediated signal transduction pathways by coupling receptor-associated TPK to the activation of Ras proteins.

Making a difference: the role of cell-cell interactions in establishing separate identities for equivalent cells.

Greenwald I, Rubin GM
Cell. 1992; 68: 271-81

Activation of R-Ras by Ras-guanine nucleotide-releasing factor.

Gotoh T, Niino Y, Tokuda M, Hatase O, Nakamura S, Matsuda M, Hattori S
J Biol Chem. 1997; 272: 18602-7

Ras-GRF/CDC25(Mm), mSos, and C3G have been identified as guanine nucleotide-releasing factors for Ras family proteins. We investigated in this study the guanine nucleotide-releasing activities of Ras-GRF, mSos, and C3G toward R-Ras, which shows high sequence similarity to Ras. Ras-GRF markedly stimulated the dissociation of GDP from R-Ras, and C3G also promoted the release of R-Ras-bound GDP. Under the same conditions, mSos little affected the reaction. When Ras-GRF and R-Ras were coexpressed in COS7 cells, the remarkable accumulation of the active GTP-bound form of R-Ras was observed. C3G also increased active R-Ras in COS7 cells, while mSos did not give any effect. These results indicated that Ras-GRF and C3G could activate R-Ras. Furthermore, the activation of R-Ras by Ras-GRF was enhanced when cells were treated with ionomycin, which is known to increase the intracellular calcium concentration. The examination of tissue distribution of R-Ras, Ras-GRF, and mSos by the reverse transcription-polymerase chain reaction revealed that Ras-GRF was expressed only in brain and testis, whereas R-Ras, C3G, and mSos were expressed rather ubiquitously. These findings raise the possibility that R-Ras is activated by Ras-GRF in brain and testis, and by C3G in other tissues, respectively.

Identification of Rap1 as a target for the Crk SH3 domain-binding guanine nucleotide-releasing factor C3G.

Gotoh T, Hattori S, Nakamura S, Kitayama H, Noda M, Takai Y, Kaibuchi K, Matsui H, Hatase O, Takahashi H, et al.
Mol Cell Biol. 1995; 15: 6746-53

C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide-releasing factor, shows sequence similarity to CDC25 and Sos family proteins (S. Tanaka, T. Morishita, Y. Hashimoto, S. Hattori, S. Nakamura, M. Shibuya, K. Matuoka, T. Takenawa, T. Kurata, K. Nagashima, and M. Matsuda, Proc. Natl. Acad. Sci. USA 91:3443-3447, 1994). The substrate specificity of C3G was examined by in vitro and in vivo experiments. C3G markedly stimulated dissociation of bound GDP from Rap1B but marginally affected the same reaction of other Ras family proteins (Ha-Ras, N-Ras, and RalA). C3G also stimulated binding of GTP-gamma S [guanosine 5'-3-O-(thio)triphosphate] to Rap1B. When C3G and Rap1A were expressed in COS7 cells, marked accumulation of the active GTP-bound form of Rap1A was observed, while Sos was not effective in the activation of Rap1A. These results clearly show that C3G is an activator for Rap1. Furthermore, expression of C3G with a membrane localization signal in a v-Ki-ras transformant, DT, induced a reversion of the cells to the flat form, possibly through the activation of endogenous Rap1.

Ral-specific guanine nucleotide exchange factor activity opposes other Ras effectors in PC12 cells by inhibiting neurite outgrowth.

Goi T, Rusanescu G, Urano T, Feig LA
Mol Cell Biol. 1999; 19: 1731-41

Ras proteins can activate at least three classes of downstream target proteins: Raf kinases, phosphatidylinositol-3 phosphate (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). In NIH 3T3 cells, activated Ral-GEFs contribute to Ras-induced cell proliferation and oncogenic transformation by complementing the activities of Raf and PI3 kinases. In PC12 cells, activated Raf and PI3 kinases mediate Ras-induced cell cycle arrest and differentiation into a neuronal phenotype. Here, we show that in PC12 cells, Ral-GEF activity acts opposite to other Ras effectors. Elevation of Ral-GEF activity induced by transfection of a mutant Ras protein that preferentially activates Ral-GEFs, or by transfection of the catalytic domain of the Ral-GEF Rgr, suppressed cell cycle arrest and neurite outgrowth induced by nerve growth factor (NGF) treatment. In addition, Rgr reduced neurite outgrowth induced by a mutant Ras protein that preferentially activates Raf kinases. Furthermore, inhibition of Ral-GEF activity by expression of a dominant negative Ral mutant accelerated cell cycle arrest and enhanced neurite outgrowth in response to NGF treatment. Ral-GEF activity may function, at least in part, through inhibition of the Rho family GTPases, CDC42 and Rac. In contrast to Ras, which was activated for hours by NGF treatment, Ral was activated for only approximately 20 min. These findings suggest that one function of Ral-GEF signaling induced by NGF is to delay the onset of cell cycle arrest and neurite outgrowth induced by other Ras effectors. They also demonstrate that Ras has the potential to promote both antidifferentiation and prodifferentiation signaling pathways through activation of distinct effector proteins. Thus, in some cell types the ratio of activities among Ras effectors and their temporal regulation may be important determinants for cell fate decisions between proliferation and differentiation.

A new function of p120-GTPase-activating protein. Prevention of the guanine nucleotide exchange factor-stimulated nucleotide exchange on the active form of Ha-ras p21.

Giglione C, Parrini MC, Baouz S, Bernardi A, Parmeggiani A
J Biol Chem. 1997; 272: 25128-34

This work studies the coordination of the action of GTPase-activating protein (GAP) and guanine nucleotide exchange factor (GEF) on activated human c-Ha-Ras p21. Purified human p120-GAP was obtained with a new efficient procedure. To distinguish the GTPase-activating effect of p120-GAP from other effects dependent on the interaction with activated Ha-Ras, the nonhydrolyzable GTP analogue guanosine 5'-O-(thiotriphosphate) (GTPgammaS) was used. The results showed that the GTPgammaS/GTPgammaS exchange enhanced by the C-terminal catalytic domain of the yeast GEF Sdc25p (C-Sdc25p) is prevented by p120-GAP. This effect is strictly specific for the activated form of Ha-Ras, the target of GAP; no effect on Ha-Ras.GDP was detectable. The GAP catalytic domain also inhibited C-Sdc25p but to a lower extent. The interfering effect by p120-GAP was also evident in a homologous mammalian system, using full-length mouse RasGEF, its C-terminal half-molecule, or C-terminal catalytic domain. As a consequence of this inhibition, presence of p120-GAP enhanced the regeneration of Ha-Ras.GTPgammaS by GEF at a GDP:GTPgammaS ratio mimicking the in vivo GDP:GTP ratio. Our work describes a novel function of p120-GAP and suggests a mechanism by which GAP protects Ha-Ras.GTP in vivo against unproductive exchanges. This constrain is likely involved in the regulation of the physiological GDP/GTP cycle of Ras and in the action of p120-GAP as downstream effector of Ras. Helix alpha3 is proposed as a Ras element playing a key-role in the interference between GAP and GEF on Ras.

Raf-1 is involved in the regulation of the interaction between guanine nucleotide exchange factor and Ha-ras. Evidences for a function of Raf-1 and phosphatidylinositol 3-kinase upstream to Ras.

Giglione C, Parmeggiani A
J Biol Chem. 1998; 273: 34737-44

The observation that activated c-Ha-Ras p21 interacts with diverse protein ligands suggests the existence of mechanisms that regulate multiple interactions with Ras. This work studies the influence of the Ras effector c-Raf-1 on the action of guanine nucleotide exchange factors (GEFs) on Ha-Ras in vitro. Purified GEFs (the catalytic domain of yeast Sdc25p and the full-length and catalytic domain of mouse CDC25Mm) and the Ras binding domains (RBDs) of Raf-1 (Raf (1-149) and Raf (51-131)) were used. Our results show that not only the intrinsic GTP/GTP exchange on Ha-Ras but also the GEF-stimulated exchange is inhibited in a concentration-dependent manner by the RBDs of Raf. Conversely, the scintillation proximity assay, which monitors the effect of GEF on the Ras.Raf complex, showed that the binding of Raf and GEF to Ha-Ras.GTP is mutually exclusive. The various GEFs used yielded comparable results. It is noteworthy that under more physiological conditions mimicking the cellular GDP/GTP ratio, Raf enhances the GEF-stimulated GDP/GTP exchange on Ha-Ras, in agreement with the sequestration of Ras.GTP by Raf. Consistent with our results, the GEF-stimulated exchange of Ha-Ras.GTP was also inhibited by another effector of Ras, the RBD (amino acid residues 133-314) of phosphatidylinositol 3-kinase p110alpha. Our data show that Raf-1 and phosphatidylinositol 3-kinase can influence the upstream activation of Ha-Ras. The interference between Ras effectors and GEF could be a regulatory mechanism to promote the activity of Ha-Ras in the cell.

Expression, purification, and crystallization of the RGS-like domain from the Rho nucleotide exchange factor, PDZ-RhoGEF, using the surface entropy reduction approach.

Garrard SM, Longenecker KL, Lewis ME, Sheffield PJ, Derewenda ZS
Protein Expr Purif. 2001; 21: 412-6

Lsc-homology domains are found in several eukaryotic nucleotide exchange factors which act on Rho-family GTPases. They show limited amino acid sequence similarity to RGS proteins, which down-regulate the cellular signaling by the alpha-subunits of trimeric G-proteins and have been shown to interact with Galpha12 and Galpha13. It is believed that the RGS-like (RGSL) domain constitutes the functional link between G-protein-coupled receptors and cytosolic Rho-GTPases. We report here the expression, purification, and crystallization of the RGSL domain from the PDZ-RhoGEF. To obtain X-ray-grade crystals we have used the recently proposed approach of crystallization by mutational surface entropy reduction, in which selected Lys --> Ala, Glu --> Ala, and/or combined point mutations are introduced into the target protein to reduce the cumulative conformational entropy of surface residues. Of the five mutants that were designed and prepared, the second one tried (K463A, E465A, E466A) yielded crystals suitable for further analysis and diffracted X-rays to 2.8 A resolution on a home source. The crystals exhibit hexagonal symmetry, space group P6(1) 22 or P6(5) 22, with unit cell parameters a = b = 63.1 A, c = 202.1 A, and contain one molecule in the asymmetric unit. Copyright 2001 Academic Press.

Interaction of a novel GDP exchange inhibitor with the Ras protein.

Ganguly AK, Wang YS, Pramanik BN, Doll RJ, Snow ME, Taveras AG, Remiszewski S, Cesarz D, del Rosario J, Vibulbhan B, Brown JE, Kirschmeier P, Huang EC, Heimark L, Tsarbopoulos A, Girijavallabhan VM, Aust RM, Brown EL, DeLisle DM, Fuhrman SA, Hendrickson TF, Kissinger CR, Love RA, Sisson WA, Webber SE, et al.
Biochemistry. 1998; 37: 15631-7

Mutated, tumorigenic Ras is present in a variety of human tumors. Compounds that inhibit tumorigenic Ras function may be useful in the treatment of Ras-related tumors. The interaction of a novel GDP exchange inhibitor (SCH-54292) with the Ras-GDP protein was studied by NMR spectroscopy. The binding of the inhibitor to the Ras protein was enhanced at low Mg2+ concentrations, which enabled the preparation of a stable complex for NMR study. To understand the enhanced inhibitor binding and the increased GDP dissociation rates of the Ras protein, the conformational changes of the Ras protein at low Mg2+ concentrations was investigated using two-dimensional 1H-15N HSQC experiments. The Ras protein existed in two conformations in slow exchange on the NMR time scale under such conditions. The conformational changes mainly occurred in the GDP binding pocket, in the switch I and the switch II regions, and were reversible. The Ras protein resumed its regular conformation after an excess amount of Mg2+ was added. A model of the inhibitor in complex with the Ras-GDP protein was derived from intra- and intermolecular NOE distance constraints, and revealed that the inhibitor bound to the critical switch II region of the Ras protein.

Grb2 mediates the EGF-dependent activation of guanine nucleotide exchange on Ras.

Gale NW, Kaplan S, Lowenstein EJ, Schlessinger J, Bar-Sagi D
Nature. 1993; 363: 88-92

Activation of receptor tyrosine kinases such as those for epidermal growth factor (EGF), platelet-derived growth factor, or nerve growth factor converts the inactive, GDP-bound form of Ras to the active, GTP-bound form, and a dominant negative mutant of Ras interferes with signalling from such receptors. The mechanisms by which receptor tyrosine kinases and Ras are coupled, however, are not well understood. Many cytoplasmic proteins regulated by such receptors contain Src-homology (SH) 2 and 3 domains, and the SH2- and SH3-containing protein Grb2, like its homologue from Caenorhabditis elegans, Sem-5, appears to play an important role in the control of Ras by receptor tyrosine kinases. Here we show that overexpression of Grb2 potentiates the EGF-induced activation of Ras and mitogen-activated protein kinase by enhancing the rate of guanine nucleotide exchange on Ras. Cellular Grb2 appears to form a complex with a guanine-nucleotide-exchange factor for Ras, which binds to the ligand-activated EGF receptor, allowing the tyrosine kinase to modulate Ras activity.

A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho.

Fukuhara S, Murga C, Zohar M, Igishi T, Gutkind JS
J Biol Chem. 1999; 274: 5868-79

Small GTP-binding proteins of the Rho family play a critical role in signal transduction. However, there is still very limited information on how they are activated by cell surface receptors. Here, we used a consensus sequence for Dbl domains of Rho guanine nucleotide exchange factors (GEFs) to search DNA data bases, and identified a novel human GEF for Rho-related GTPases harboring structural features indicative of its possible regulatory mechanism(s). This protein contained a tandem DH/PH domain closely related to those of Rho-specific GEFs, a PDZ domain, a proline-rich domain, and an area of homology to Lsc, p115-RhoGEF, and a Drosophila RhoGEF that was termed Lsc-homology (LH) domain. This novel molecule, designated PDZ-RhoGEF, activated biological and biochemical pathways specific for Rho, and activation of these pathways required an intact DH and PH domain. However, the PDZ domain was dispensable for these functions, and mutants lacking the LH domain were more active, suggesting a negative regulatory role for the LH domain. A search for additional molecules exhibiting an LH domain revealed a limited homology with the catalytic region of a newly identified GTPase-activating protein for heterotrimeric G proteins, RGS14. This prompted us to investigate whether PDZ-RhoGEF could interact with representative members of each G protein family. We found that PDZ-RhoGEF was able to form, in vivo, stable complexes with two members of the Galpha12 family, Galpha12 and Galpha13, and that this interaction was mediated by the LH domain. Furthermore, we obtained evidence to suggest that PDZ-RhoGEF mediates the activation of Rho by Galpha12 and Galpha13. Together, these findings suggest the existence of a novel mechanism whereby the large family of cell surface receptors that transmit signals through heterotrimeric G proteins activate Rho-dependent pathways: by stimulating the activity of members of the Galpha12 family which, in turn, activate an exchange factor acting on Rho.

Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G(12) family to Rho.

Fukuhara S, Chikumi H, Gutkind JS
FEBS Lett. 2000; 485: 183-8

A putative guanine nucleotide exchange factor (GEF), termed leukemia-associated RhoGEF (LARG), was recently identified upon fusion to the coding sequence of the MLL gene in acute myeloid leukemia. Although the function of LARG is still unknown, it exhibits a number of structural domains suggestive of a role in signal transduction, including a PDZ domain, a LH/RGS domain, and a Dbl homology/pleckstrin homology domain. Here, we show that LARG can activate Rho in vivo. Furthermore, we present evidence that LARG is an integral component of a novel biochemical route whereby G protein-coupled receptors (GPCRs) and heterotrimeric G proteins of the G alpha(12) family stimulate Rho-dependent signaling pathways.

SopE and SopE2 from Salmonella typhimurium activate different sets of RhoGTPases of the host cell.

Friebel A, Ilchmann H, Aepfelbacher M, Ehrbar K, Machleidt W, Hardt WD
J Biol Chem. 2001; 276: 34035-40

The bacterial enteropathogen Salmonella typhimurium employs a specialized type III secretion system to inject toxins into host cells, which trigger signaling cascades leading to cell death in macrophages, secretion of pro-inflammatory cytokines, or rearrangements of the host cell cytoskeleton and thus to bacterial invasion. Two of the injected toxins, SopE and the 69% identical protein SopE2, are highly efficient guanine nucleotide exchange factors for the RhoGTPase Cdc42 of the host cell. However, it has been a puzzle why S. typhimurium might employ two toxins with redundant function. We hypothesized that SopE and SopE2 might have different specificities for certain host cellular RhoGTPases. In vitro guanine nucleotide exchange assays and surface plasmon resonance measurements revealed that SopE is an efficient guanine nucleotide exchange factor for Cdc42 and Rac1, whereas SopE2 was interacting efficiently only with Cdc42, but not with Rac1. Affinity precipitation of Cdc42.GTP and Rac1.GTP from lysates and characteristic cytoskeletal rearrangements of infected tissue culture cells confirmed that SopE is highly efficient at activating Cdc42 and Rac1 in vivo, whereas SopE2 was efficiently activating Cdc42, but not Rac1. We conclude that the translocated effector proteins SopE and SopE2 allow S. typhimurium to specifically activate different sets of RhoGTPase signaling cascades.

Activation of the exchange factor Ras-GRF by calcium requires an intact Dbl homology domain.

Freshney NW, Goonesekera SD, Feig LA
FEBS Lett. 1997; 407: 111-5

Ras-GRF is a guanine nucleotide exchange factor that activates Ras proteins. Its activity on Ras in cells is enhanced upon calcium influx. Activation follows calcium-induced binding of calmodulin to an IQ motif near the N-terminus of Ras-GRF. Ras-GRF also contains a Dbl homology (DH) domain C-terminal to the IQ motif. In many proteins, DH domains act as exchange factors for Rho-GTPase family members. However, we failed to detect exchange activity of this domain on well characterized Rho family members. Instead, we found that mutations analogous to those that block exchange activity of Dbl prevented Ras-GRF activation by calcium/ calmodulin in vivo. All DH domains are followed immediately by a pleckstrin homology (PH) domain. We found that a mutation at a conserved site within the PH domain following the DH domain also prevented Ras-GRF activation by calcium in vivo. These results suggest that in addition to playing a role as activators of Rho proteins, DH domains can also contribute to the coupling of cellular signals to Ras activation.

Purification of baculovirus-expressed human Sos1 protein.

Frech M, Cussac D, Chardin P, Bar-Sagi D
Methods Enzymol. 1995; 255: 125-9

To fear or not to fear: what was the question? A potential role for Ras-GRF in memory.

Finkbeiner S, Dalva MB
Bioessays. 1998; 20: 691-5

Learning, making memories, and forgetting are thought to require changes in the strengths of connections between neurons. Such changes in synaptic strength occur in two phases: an early phase that is likely mediated by covalent modifications to existing proteins, and a delayed phase that depends on new gene expression and protein synthesis. However, the biochemical mechanisms by which neuronal activity leads to changes in synaptic strength are poorly understood. Recently, it has been shown that animals that lack Ras guanine nucleotide releasing factor (Ras-GRF), a Ca(2+)-dependent activator of the small GTP-binding protein, Ras, do not learn fear responses normally, although other types of learning appear normal. These animals show defects in the delayed phase of memory formation within the neuronal circuit that mediates fear conditioning. This paper suggests that Ras-GRF couples synaptic activity to the molecular mechanisms that consolidate changes in synaptic strength within specific neuronal circuits.

Expression of two different products of CDC25Mm, a mammalian Ras activator, during development of mouse brain.

Ferrari C, Zippel R, Martegani E, Gnesutta N, Carrera V, Sturani E
Exp Cell Res. 1994; 210: 353-7

The CDC25Mm gene codes for Ras-guanine nucleotide exchange factors. Four different full-length cDNA clones derived from the same gene and coding for proteins of different sizes that have in common the last 661 amino acids have been isolated from mouse brain. In order to investigate the expression of the products of this gene in different tissues we have prepared two polyclonal antibodies directed toward two different regions of the protein comprised in the last C-terminal 472 amino acids. While in most of the tested tissues we have been unable to definitely identify CDC25Mm products, in NIH3T3 fibroblasts we have found a poorly expressed 120-kDa protein. In the mouse brain we have identified two proteins of 140 and 58 kDa. While the former is expressed in the adult mouse, the latter is present in the embryo and persists for few days after birth. This finding suggests that differential expression of various forms of CDC25Mm may be involved in brain development.

Guanine-nucleotide exchange factors: a family of positive regulators of Ras and related GTPases.

Feig LA
Curr Opin Cell Biol. 1994; 6: 204-11

The past year has seen remarkable advances in our understanding of how members of the Ras superfamily of GTPases are activated. Not only have new guanine-nucleotide exchange factors (GEFs) that activate these GTPases been identified, but in some cases, the mechanisms by which GEFs are connected to events at the cell surface have also been described. We have also come to appreciate the possibilities that GEFs may be multifunctional and that they may contribute to tissue-specific regulation of their cognate GTPase.

Cloning of a Grb2 isoform with apoptotic properties.

Fath I, Schweighoffer F, Rey I, Multon MC, Boiziau J, Duchesne M, Tocque B
Science. 1994; 264: 971-4

Growth factor receptor-bound protein 2 (Grb2) links tyrosine-phosphorylated proteins to a guanine nucleotide releasing factor of the son of sevenless (Sos) class by attaching to the former by its Src homology 2 (SH2) moiety and to the latter by its SH3 domains. An isoform of grb2 complementary DNA (cDNA) was cloned that has a deletion in the SH2 domain. The protein encoded by this cDNA, Grb3-3, did not bind to phosphorylated epidermal growth factor receptor (EGFR) but retained functional SH3 domains and inhibited EGF-induced transactivation of a Ras-responsive element. The messenger RNA encoding Grb3-3 was expressed in high amounts in the thymus of rats at an age when massive negative selection of thymocytes occurs. Microinjection of Grb3-3 into Swiss 3T3 fibroblasts induced apoptosis. These findings indicate that Grb3-3, by acting as a dominant negative protein over Grb2 and by suppressing proliferative signals, may trigger active programmed cell death.

Calcium activation of Ras mediated by neuronal exchange factor Ras-GRF.

Farnsworth CL, Freshney NW, Rosen LB, Ghosh A, Greenberg ME, Feig LA
Nature. 1995; 376: 524-7

Tyrosine kinase receptors stimulate the Ras signalling pathway by enhancing the activity of the SOS nucleotide-exchange factor. This occurs, at least in part, by the recruitment of an SOS-GRB2 complex to Ras in the plasma membrane. Here we describe a different signalling pathway to Ras that involves activation of the Ras-GRF exchange factor in response to Ca2+ influx. In particular, we show that the ability of Ras-GRF to activate Ras in vivo is markedly enhanced by raised Ca2+ concentrations. Activation is mediated by calmodulin binding to an IQ motif in Ras-GRF, because substitutions in conserved amino acids in this motif prevent both calmodulin binding to Ras-GRF and Ras-GRF activation in vivo. So far, full-length Ras-GRF has been detected only in brain neurons. Our findings implicate Ras-GRF in the regulation of neuronal functions that are influenced by Ca2+ signals.

The exchange factor Ras-GRF2 activates Ras-dependent and Rac-dependent mitogen-activated protein kinase pathways.

Fan WT, Koch CA, de Hoog CL, Fam NP, Moran MF
Curr Biol. 1998; 8: 935-8

Ras and Rac are membrane-associated GTPases that function as molecular switches activating intracellular mitogen-activated protein kinase (MAPK) cascades and other effector pathways in response to extracellular signals [1]. Activation of Ras and Rac into their GTP-bound conformations is directly controlled by specific guanine-nucleotide exchange factors (GEFs), which catalyze GDP release. Several Ras-specific GEFs that are related to the budding yeast protein Cdc25p have been described, whereas GEFs for Rac-related GTPases contain a region that is homologous to the oncoprotein DbI [2-3]. The Ras-GRF1 and Ras-GRF2 proteins, which couple Ras activation to serpentine receptors and calcium signals, contain both Cdc25 and DbI homology (DH) regions [3-4]. Here, we demonstrate that Ras-GRF2 is a bifunctional signaling protein that is able to bind and activate Ras and Rac, and thereby coordinate the activation of the extracellular-signal-regulated kinase (ERK) and stress-activated protein kinase (SAPK) pathways.

Mapping of the Ras-GRF2 gene (GRF2) to mouse chromosome 13C3-D1 and human chromosome 5q13, near the Ras-GAP gene.

Fam NP, Zhang LJ, Rommens JM, Beatty BG, Moran MF
Genomics. 1997; 39: 118-20

Cloning and characterization of Ras-GRF2, a novel guanine nucleotide exchange factor for Ras.

Fam NP, Fan WT, Wang Z, Zhang LJ, Chen H, Moran MF
Mol Cell Biol. 1997; 17: 1396-406

Conversion of Ras proteins into an activated GTP-bound state able to bind effector proteins is catalyzed by specific guanine nucleotide exchange factors in response to a large number of extracellular stimuli. Here we report the isolation of mouse cDNAs encoding Ras-GRF2, a multidomain 135-kDa protein containing a COOH-terminal Cdc25-related domain that stimulates release of GDP from Ras but not other GTPases in vitro. Ras-GRF2 bound specifically to immobilized Ras lacking bound nucleotides, suggesting stabilization of the nucleotide-free form of Ras as a mechanism of catalyzing nucleotide exchange. The NH2-terminal region of Ras-GRF2 is predicted to contain features common to various signaling proteins including two pleckstrin homology domains and a Dbl homology region. Ras-GRF2 also contains an IQ motif which was required for its apparent constitutive association with calmodulin in epithelial cells ectopically expressing Ras-GRF2. Transient expression of Ras-GRF2 in kidney epithelial cells stimulated GTP binding by Ras and potentiated calcium ionophore-induced activation of mitogen-activated protein kinase (ERK1) dependent upon the IQ motif. Calcium influx caused Ras-GRF2 subcellular localization to change from cytosolic to peripheral, suggesting a possible mechanism for controlling Ras-GRF2 interactions with Ras at the plasma membrane. Epithelial cells overexpressing Ras-GRF2 are morphologically transformed and grow in a disorganized manner with minimal intercellular contacts. Northern analysis indicated a 9-kb GRF2 transcript in brain and lung, where p135 Ras-GRF2 is known to be expressed, and RNAs of 12 kb and 2.2 kb were detected in several tissues. Thus, Ras-GRF2 proteins with different domain structures may be widely expressed and couple diverse extracellular signals to Ras activation.

Building inhibitory synapses: exchange factors getting into the act? [ comment]

Fallon JR
Nat Neurosci. 2000; 3: 5-6

Gephyrin is a synaptic protein that is required for clustering of glycine and GABAA receptors. Gephyrin itself may be regulated by a newly identified protein named collybistin.

Prolactin activates Ras via signaling proteins SHC, growth factor receptor bound 2, and son of sevenless.

Erwin RA, Kirken RA, Malabarba MG, Farrar WL, Rui H
Endocrinology. 1995; 136: 3512-8

Identification of the signal transduction pathways used by PRL is essential for understanding the role of PRL receptors in growth and differentiation processes. Early cellular mediators of PRL receptor activation include tyrosine kinases of the Janus kinase (JAK) and SRC families, with rapid nuclear signaling via tyrosine phosphorylated signal transducers and activators of transcription. In the present study we provide the first demonstration of PRL-induced activation of Ras, an oncogenic protein that supports an alternative signaling route from the membrane to the nucleus. PRL stimulated Ras in rat Nb2-SP lymphoma cells, as detected by a 2.0-fold increase in the GTP-bound state of the molecule (P < 0.01). This activation was associated with marked tyrosine phosphorylation and increased membrane association of the 52-kilodalton form of SHC. Moreover, PRL induced binding of SHC to growth factor receptor bound 2 and the guanine-nucleotide exchange factor son of sevenless, a common method used by growth factor receptors to activate Ras. In contrast, no apparent regulation by PRL of Ras via VAV or p120 Ras-guanosine triphosphatase-activating protein was detected, based upon an absence of PRL-inducible tyrosine phosphorylation of these proteins. Collectively, these results provide a molecular bridge between activation of PRL receptor-associated tyrosine kinases and subsequent stimulation of the serine/threonine kinase Raf-1, an established Ras target that was recently shown to be activated by PRL in Nb2 cells. We conclude that PRL is able to activate Ras via recruitment of the signaling proteins SHC, growth factor receptor bound 2, and son of sevenless in Nb2 cells. Moreover, PRL induced tyrosine phosphorylation of SHC in two of three PRL-responsive human breast cancer cell lines, suggesting that SHC-mediated Ras activation is a commonly used signaling strategy by PRL.

Mechanism of activation of the Caenorhabditis elegans ras homologue let-60 by a novel, temperature-sensitive, gain-of-function mutation.

Eisenmann DM, Kim SK
Genetics. 1997; 146: 553-65

The Caenorhabditis elegans let-60 gene encodes a Ras protein that mediates induction of the hermaphrodite vulva. To better understand how mutations constitutively activate Ras and cause unregulated cell division, we have characterized ga89, a temperature-sensitive, gain-of-function mutation in let-60 ras. At 25 degrees, ga89 increases let-60 activity resulting in a multivulva phenotype. At 15 degrees, ga89 decreases let-60 activity resulting in a vulvaless phenotype in let-60(ga89)/Df animals. The ga89 mutation causes a leucine (L) to phenylalanine (F) substitution at amino acid 19, a residue conserved in all Ras proteins. We introduced the L19F change into human H-Ras protein and found that the in vitro GTPase activity of H-Ras became temperature-dependent. Genetic experiments suggest that LET-60 (L19F) interacts with GAP and GNEF, since mutations that decrease GAP and GNEF activity affect the multivulva phenotype of let-60(ga89) animals. These results suggest that the L19F mutation primarily affects the intrinsic rate of GTP hydrolysis by Ras, and that this effect may be sufficient to account for the activated-Ras phenotype caused by let-60(ga89). Our results suggest that a mutation in a human ras gene analogous to ga89 might contribute to oncogenic transformation.

A novel potential effector of M-Ras and p21 Ras negatively regulates p21 Ras-mediated gene induction and cell growth.

Ehrhardt GR, Korherr C, Wieler JS, Knaus M, Schrader JW
Oncogene. 2001; 20: 188-97

Here, we report the identification and characterization of a new member of the RalGDS-family, which is widely expressed and interacts strongly and selectively with the GTP-bound forms of M-Ras and p21 Ras. This Ras pathway modulator (RPM), also termed RGL3, exhibited Ras-binding and catalytic domains typical of the RalGDS-family of guanine nucleotide exchange factors, and was most similar to Rlf (RalGDS-like factor), but was distinguished by a unique proline-rich region with multiple candidate SH3-domain binding sites. RPM/RGL3 resembled AF-6 and Nore1 in interacting strongly with constitutively active M-Ras and p21 Ras. In contrast to Rlf, transiently expressed RPM/RGL3 did not activate an Elk-1-inducible reporter gene alone or in combination with activated p21 Ras, but strongly inhibited induction of this reporter gene by co-expression of activated H-Ras or MEKK-1. This inhibitory effect was independent of the Ras binding domain and required a second signal provided by p21 Ras or MEKK-1, but not Raf-1 or M-Ras. Expression of RPM/RGL3 also strongly inhibited cell growth of fibroblasts transformed by an activated Src Y527F. Thus, RPM/RGL3 is a novel potential effector of both p21 Ras and M-Ras with the novel function of negatively regulating Elk-1-dependent gene induction downstream of p21 Ras or MEKK-1.

Association of Sos Ras exchange protein with Grb2 is implicated in tyrosine kinase signal transduction and transformation.

Egan SE, Giddings BW, Brooks MW, Buday L, Sizeland AM, Weinberg RA
Nature. 1993; 363: 45-51

The proteins Grb2-Sem-5, Shc and Sos have been implicated in the signalling pathway from tyrosine kinase receptors to Ras. Grb2-Sem-5 binds directly to murine Sos1, a Ras exchange factor, through two SH3 domains. Sos is also associated with ligand-activated tyrosine kinase receptors which bind Grb2-Sem-5, and with the Grb2-Sem-5 binding protein, Shc. Ectopic expression of Drosophila Sos stimulates morphological transformation of rodent fibroblasts. These data define a pathway by which tyrosine kinases act through Ras to control cell growth and differentiation.

Activation of the Rap1 guanine nucleotide exchange gene, CalDAG-GEF I, in BXH-2 murine myeloid leukemia.

Dupuy AJ, Morgan K, von Lintig FC, Shen H, Acar H, Hasz DE, Jenkins NA, Copeland NG, Boss GR, Largaespada DA
J Biol Chem. 2001; 276: 11804-11

Here we report the recurrent proviral activation of the Rap1-specific guanine nucleotide exchange factor CalDAG-GEF I (Kawasaki, H., Springett, G. M., Toki, S., Canales, J. J., Harlan, P., Blumenstiel, J. P., Chen, E. J., Bany, I. A., Mochizuki, N., Ashbacher, A., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13278-13283; Correction (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 318) gene in BXH-2 acute myeloid leukemia. We also show that CalDAG-GEF I encodes two protein isoforms, a full-length isoform (CalDAG-GEF Ia) and a C-terminally truncated isoform (CalDAG-GEF Ib). Expression of the full-length CalDAG-GEF Ia isoform in Rat2 fibroblasts enhances growth in low serum, whereas expression in Swiss 3T3 cells causes morphological transformation and increased saturation density. In FDCP1 myeloid cells, CalDAG-GEF Ia expression increases growth and saturation density in the presence of the diacylglycerol analogs phorbol 12-myristate 13-acetate (PMA), which activates CalDAG-GEF Ia exchange activity. Likewise, in 32Dcl3 myeloblast cells, CalDAG-GEF Ia expression increases cell adherence to fibronectin in response to PMA and calcium ionophore and allows higher saturation densities and prolonged growth on fibronectin-coated plates. These effects were correlated with increased Rap1, but not Ras, protein activation following PMA and calcium ionophore treatment. Our results suggest that Rap1-GTP delivers signals that favor progression through the cell cycle and morphological transformation. The identification of CalDAG-GEF I as a proto-oncogene in BXH-2 acute myeloid leukemia is the first evidence implicating Rap1 signaling in myeloid leukemia.

Insulin activates p21Ras and guanine nucleotide releasing factor in cells expressing wild type and mutant insulin receptors.

Draznin B, Chang L, Leitner JW, Takata Y, Olefsky JM
J Biol Chem. 1993; 268: 19998-20001

Insulin stimulates the formation of p21RasGTP in Rat-1 fibroblasts overexpressing wild type (HIRc) or mutant (delta CT and Y/F2) insulin receptors. Maximal insulin effect was observed at 7 min in Y/F2 cells, at 10 min in delta CT cells, and at 15 min in HIRc cells. Mutant insulin receptors which display enhanced mitogenic signaling properties stimulated p21Ras.GTP formation to a greater extent than wild type receptors. The amount of p21Ras was not affected by insulin (Western blotting). Tyrosine kinase inhibitor, Lavendustin A (10 nM), completely prevented insulin-induced activation of p21Ras in all cell lines. Insulin did not lead to GAP phosphorylation, or a change in cellular GAP activity, but did result in marked stimulation of Ras guanine nucleotide releasing factor (GRF) activity (by 48% in HIRc, 71% in delta CT, and 120% in Y/F2 cells). These results indicated that p21Ras.GTP is an important signaling molecule in insulin's mitogenic pathway, but may not participate in metabolic signaling and that insulin's stimulatory effects on p21Ras.GTP formation are mediated through Ras GRF.

Arsenite blocks growth factor induced activation of the MAP kinase cascade, upstream of Ras and downstream of Grb2-Sos.

Doza YN, Hall-Jackson CA, Cohen P
Oncogene. 1998; 17: 19-24

Pretreatment of cells with 0.5 mM sodium arsenite (but not other activators of stress-activated MAP kinase cascades) prevents the activation of p21Ras and strongly suppresses the activation of c-Raf and the MAP kinase cascade by a variety of growth factors. Arsenite appears to exert its effect by preventing the guanine nucleotide exchange factor mSos from converting Ras to its active GTP-bound state. Exposure to arsenite may be a simple way of assessing whether Ras plays an essential role in mediating activation of the MAP kinase cascade by extracellular signals.

Control of ras activation.

Downward J
Cancer Surv. 1996; 27: 87-100

Ras proteins are active when bound to GTP and inactive when bound to GDP: the activation state of Ras proteins is regulated by two families of proteins. GTPase activating proteins (p120GAP, neurofibromin and GAP1) are negative regulators that stimulate hydrolysis of bound GTP to GDP, and guanine nucleotide exchange factors (Sos and Ras-GRF) are positive regulators that stimulate the exchange of GDP bound to Ras for fresh GTP from the cytosol. Ras is activated in response to a wide variety of extracellular stimuli. The principal mechanism used involves formation of complexes of autophosphorylated growth factor receptors with the SH2 and SH3 domain containing adaptor protein GRB2 and the exchange factor Sos. In addition, another adaptor protein, Shc, may bind to GRB2. This causes translocation of Sos to the plasma membrane where Ras is located and hence increases the rate of nucleotide exchange on Ras leading to its activation. The activity of GTPase activating proteins may also be regulated under some circumstances. A number of mechanisms exist to return the activation state of Ras to basal after stimulation.

Measurement of nucleotide exchange and hydrolysis activities in immunoprecipitates.

Downward J
Methods Enzymol. 1995; 255: 110-7

Signal transduction. New exchange, new target.

Downward J
Nature. 1998; 396: 416-7

SOS phosphorylation and disassociation of the Grb2-SOS complex by the ERK and JNK signaling pathways.

Dong Chen, Waters SB, Holt KH, Pessin JE
J Biol Chem. 1996; 271: 6328-32

Insulin activation of Ras is mediated by the plasma membrane targeting of the guanylnucleotide exchange factor SOS associated with the small adapter protein Grb2. SOS also lies in an insulin-stimulated feedback pathway in which the serine/threonine phosphorylation of SOS results in disassociation of the Grb2-SOS complex thereby limiting the extent of Ras activation. To examine the relative role of the mitogen-activated protein kinases in the feedback phosphorylation of SOS we determined the signaling specificity of insulin, osmotic shock, and anisomycin to activate the ERK (extracellular-signal regulated kinase) and JNK (c-Jun kinase) pathways. In Chinese hamster ovary cells expressing the human insulin receptor and murine 3T3L1 adipocytes, insulin specifically activated ERK with no significant effect on JNK, whereas anisomycin specifically activated JNK but was unable to activate ERK. In contrast, osmotic shock was equally effective in the activation of both kinase pathways. Insulin and osmotic shock, but not anisomycin, resulted in SOS phosphorylation and disassociation of the Grb2-SOS complex, demonstrating that the JNK pathway was not involved in the insulin-stimulated feedback uncoupling of the Grb2- SOS complex. Both the insulin and osmotic shock-induced activation of ERK was prevented by treatment of cells with the specific MEK inhibitor (PD98059). However, expression of dominant-interfering Ras (N17Ras) inhibited the insulin- but not osmotic shock-stimulated phosphorylation of ERK and SOS. These data demonstrate that activation of the ERK pathway, but not JNK, is responsible for the feedback phosphorylation and disassociation of the Grb2-SOS complex.

Measurements of GTP/GDP exchange in permeabilized fibroblasts.

de Vries-Smits AM, van der Voorn, Downward J, Bos JL
Methods Enzymol. 1995; 255: 156-61

Shc associates with an unphosphorylated form of the p21ras guanine nucleotide exchange factor mSOS.

de Vries-Smits AM, Pronk GJ, Medema JP, Burgering BM, Bos JL
Oncogene. 1995; 10: 919-25

Association of the p21ras guanine nucleotide exchange factor mSOS with tyrosine-phosphorylated Shc has been implicated in the activation of p21ras. In addition, after growth factor stimulation mSOS becomes phosphorylated as indicated by the appearance of a form of mSOS with reduced electrophoretic mobility. This phosphorylation is delayed with respect to Shc-Grb2-mSOS complex formation and activation of p21ras. To investigate the role of mSOS phosphorylation in further detail we have investigated the effect of phosphorylation on mSOS complex formation and p21ras activation. We found that Shc is associated with the unphosphorylated, faster migrating form of mSOS. Furthermore, although there is a correlation between the amount of complexes formed and the activation of p21ras, there is no such a correlation between mSOS phosphorylation and p21ras activation. In addition, inhibition of mSOS phosphorylation did not affect complex formation of mSOS with tyrosine phosphorylated Shc. Also, induction of mSOS phosphorylation prior to complex formation did not affect EGF-induced association of mSOS with Shc significantly, and Shc still associated predominantly with the faster migrating form of mSOS. From these results we conclude that the unphosphorylated form of mSOS is associated with Shc and that perhaps a phosphorylation-dephosphorylation step is part of the mSOS activation-inactivation cycle.

cDNA cloning, gene characterization and 13q14.3 chromosomal assignment of CHC1-L, a chromosome condensation regulator-like guanine nucleotide exchange factor.

Devilder MC, Cadoret E, Cherel M, Moreau I, Rondeau G, Bezieau S, Moisan JP
Genomics. 1998; 54: 99-106

We report the characterization of a new gene mapped at chromosome band 13q14.3 telomeric to the retinoblastoma gene. This gene, designated CHC1L (for chromosome condensation 1-like), is composed of 14 exons spanning 30 kb of genomic DNA and encodes a ubiquitously expressed 3-kb mRNA. The N-terminal half of the deduced amino acid sequence shows strong homology with the seven tandem repeat structure of the regulator of chromosome condensation RCC1, which acts as a guanine nucleotide exchange factor (GEF) protein for the Ras-related GTPase Ran. CHC1L appears to be a new member of the RCC1-related GEF family.

The yeast exchange assay, a new complementary method to screen for Dbl-like protein specificity: identification of a novel RhoA exchange factor.

De Toledo M, Colombo K, Nagase T, Ohara O, Fort P, Blangy A
FEBS Lett. 2000; 480: 287-92

The target Rho GTPases of many guanine nucleotide exchange factors (GEFs) of the Dbl family remain to be identified. Here we report a new method: the yeast exchange assay (YEA), a rapid qualitative test to perform a wide range screen for GEF specificity. In this assay based on the two-hybrid system, a wild type GTPase binds to its effector only after activation by a specific GEF. We validated the YEA by activating GTPases by previously reported GEFs. We further established that a novel GEF, GEF337, activates RhoA in the YEA. GEF337 promoted nucleotide exchange on RhoA in vitro and promoted F-actin stress fiber assembly in fibroblasts, characteristic of RhoA activation.

Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP.

de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL
Nature. 1998; 396: 474-7

Rap1 is a small, Ras-like GTPase that was first identified as a protein that could suppress the oncogenic transformation of cells by Ras. Rap1 is activated by several extracellular stimuli and may be involved in cellular processes such as cell proliferation, cell differentiation, T-cell anergy and platelet activation. At least three different second messengers, namely diacylglycerol, calcium and cyclic AMP, are able to activate Rap1 by promoting its release of the guanine nucleotide GDP and its binding to GTP. Here we report that activation of Rap1 by forskolin and cAMP occurs independently of protein kinase A (also known as cAMP-activated protein kinase). We have cloned the gene encoding a guanine-nucleotide-exchange factor (GEF) which we have named Epac (exchange protein directly activated by cAMP). This protein contains a cAMP-binding site and a domain that is homologous to domains of known GEFs for Ras and Rap1. Epac binds cAMP in vitro and exhibits in vivo and in vitro GEF activity towards Rap1. cAMP strongly induces the GEF activity of Epac towards Rap1 both in vivo and in vitro. We conclude that Epac is a GEF for Rap1 that is regulated directly by cAMP and that Epac is a new target protein for cAMP.

Ras binding triggers ubiquitination of the Ras exchange factor Ras-GRF2.

de Hoog CL, Koehler JA, Goldstein MD, Taylor P, Figeys D, Moran MF
Mol Cell Biol. 2001; 21: 2107-17

Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.

Distinct subclasses of small GTPases interact with guanine nucleotide exchange factors in a similar manner.

Day GJ, Mosteller RD, Broek D
Mol Cell Biol. 1998; 18: 7444-54

The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3-loop 7 [alpha3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and alpha3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and alpha3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.

HERC3 binding to and regulation by ubiquitin.

Cruz C, Ventura F, Bartrons R, Rosa JL
FEBS Lett. 2001; 488: 74-80

Members of the HERC (domain homologous to E6 associated protein carboxy-terminus and RCC1 domain protein) family may function both as guanine nucleotide exchange factors and E3 ubiquitin ligases. Here we identify an unstudied member, HERC3. This protein was recognized by specific antibodies in different cell types. HERC3 was located in the cytosol and in vesicular-like structures containing beta-COP, ARF and Rab5 proteins. Involvement of HERC3 in the ubiquitin system was suggested by its ability to interact with ubiquitin. The conserved cysteine in HECT proteins was not essential for this non-covalent binding. Moreover, HERC3 was a substrate of ubiquitination being degraded by the proteasome. These observations indicate a fine regulation of HERC3 and suggest a role in vesicular traffic and ubiquitin-dependent processes.

Distal switch II region of Ras2p is required for interaction with guanine nucleotide exchange factor.

Crechet JB, Bernardi A, Parmeggiani A
J Biol Chem. 1996; 271: 17234-40

The interaction of Saccharomyces cerevisiae Ras2p with the catalytic domain of the GDP/GTP exchange factors (GEFs) mouse CDC25(Mm), yeast Cdc25p, and Sdc25p was analyzed by introducing the substitution R80D/N81D into Ras2p S24N, a mutant that is shown to interfere with the Ras2p wild type (wt)-GEF interaction by forming a stable complex. The triple mutant, like Ras2p R80D/N81D, did not interfere with the action of GEF on Ras2p wt (or H-Ras p21) and was unable to form a stable complex with GEF. The GEF stimulation of the nucleotide dissociation of the triple mutant was virtually abolished and strongly decreased with the double mutant. The affinity of Ras2p S24N/R80D/N81D for GDP and GTP was decreased 3 and 4 orders of magnitude, respectively, like that of Ras2p S24N, whereas the double mutant behaved as Ras2p wt. Like Ras2p S24N and unlike Ras2p R80D/N81D, the GTP-bound triple mutant did not activate adenylyl cyclase. Thus, the triple mutant and Ras2p S24N have opposite properties toward the binding to GEF but similarly modified behaviors toward GDP, GTP, and adenylyl cyclase. This work emphasizes the determinant role of the distal switch II region of Ras2p for the interaction with GEF and the different structural background of the interaction with adenylyl cyclase.

Interaction of a receptor tyrosine kinase, EGF-R, with caveolins. Caveolin binding negatively regulates tyrosine and serine/threonine kinase activities.

Couet J, Sargiacomo M, Lisanti MP
J Biol Chem. 1997; 272: 30429-38

Caveolin, a 21-24-kDa integral membrane protein, is a principal component of caveolae membranes. We and others have suggested that caveolin functions as a scaffolding protein to organize and concentrate certain caveolin-interacting signaling molecules within caveolae membranes. In this regard, it has been shown that a 20-amino acid membrane-proximal region of the cytosolic NH2-terminal domain of caveolin is sufficient to mediate the interaction of caveolin with signaling proteins, namely G-proteins, Src-like kinases, eNOS, and H-Ras. This caveolin-derived protein domain has been termed the caveolin-scaffolding domain. Binding of the caveolin-scaffolding domain functionally suppresses the activity of G-protein alpha subunits, eNOS, and Src-like kinases, suggesting that caveolin binding may also play a negative regulatory role in signal transduction. Here, we report the direct interaction of caveolin with a growth factor receptor, EGF-R, a known caveolae-associated receptor tyrosine kinase. Two consensus caveolin binding motifs have been previously defined using phage display technology. One of these motifs is present within the conserved kinase domains of most known receptor tyrosine kinases (termed region IX). We now show that this caveolin binding motif within the kinase domain of the EGF-R can mediate the interaction of the EGF-R with the scaffolding domains of caveolins 1 and 3 but not with caveolin 2. In addition, the scaffolding domains of caveolins 1 and 3 both functionally inhibit the autophosphorylation of the EGF-R kinase in vitro. Importantly, this caveolin-mediated inhibition of the EGF-R kinase could be prevented by the addition of an EGF-R-derived peptide that (i) contains a well conserved caveolin binding motif and (ii) is located within the kinase domain of the EGF-R and most known receptor tyrosine kinases. Similar results were obtained with protein kinase C, a serine/threonine kinase, suggesting that caveolin may function as a general kinase inhibitor. The implications of our results are discussed within the context of caveolae-mediated signal transduction. In this regard, caveolae-coupled signaling might explain how linear signaling pathways can branch and interconnect extensively, forming a signaling module or network.

Regulation of Sos activity by intramolecular interactions.

Corbalan-Garcia S, Margarit SM, Galron D, Yang SS, Bar-Sagi D
Mol Cell Biol. 1998; 18: 880-6

The guanine nucleotide exchange factor Sos mediates the coupling of receptor tyrosine kinases to Ras activation. To investigate the mechanisms that control Sos activity, we have analyzed the contribution of various domains to its catalytic activity. Using human Sos1 (hSos1) truncation mutants, we show that Sos proteins lacking either the amino or the carboxyl terminus domain, or both, display a guanine nucleotide exchange activity that is significantly higher compared with that of the full-length protein. These results demonstrate that both the amino and the carboxyl terminus domains of Sos are involved in the negative regulation of its catalytic activity. Furthermore, in vitro Ras binding experiments suggest that the amino and carboxyl terminus domains exert negative allosteric control on the interaction of the Sos catalytic domain with Ras. The guanine nucleotide exchange activity of hSos1 was not augmented by growth factor stimulation, indicating that Sos activity is constitutively maintained in a downregulated state. Deletion of both the amino and the carboxyl terminus domains was sufficient to activate the transforming potential of Sos. These findings suggest a novel negative regulatory role for the amino terminus domain of Sos and indicate a cooperation between the amino and the carboxyl terminus domains in the regulation of Sos activity.

The minimal active domain of the mouse ras exchange factor CDC25Mm.

Coccetti P, Mauri I, Alberghina L, Martegani E, Parmeggiani A
Biochem Biophys Res Commun. 1995; 206: 253-9

The minimal active domain of the mouse CDC25Mm, a GDP/GTP exchange factor (GEF) active on H-ras protein, was determined by constructing several deletion mutants of the C-terminal domain of the protein. The functional activity of these fragments was analyzed for the ability to complement the yeast temperature sensitive mutation cdc25-1 and to catalyze the GDP/GTP exchange on Ras proteins in vitro. A C-terminal domain of 256 residues (CDC25Mm 1005-1260) was sufficient for full biological activity in vivo. Deletion of 27 C-terminal amino acids (CDC25Mm 1005-1233) abolished the complementing activity while deletion of 25 N-terminal residues (CDC25Mm 1030-1260 corresponding to the most conserved domain) led to a complete loss of expression. The results in vivo were supported by experiments in vitro. Highly purified CDC25Mm 1005-1260, expressed in E. coli using the pMAL system, enhanced the GDP release from both H-ras p21 and S. cerevisiae Ras2p and its activity was nearly as high as that of CDC25Mm 974-1260. Comparison with the Cdc25p protein yielded further evidence that the minimal active domain of CDC25Mm is shorter than the yeast one.

Characterization of RasGRP2, a plasma membrane-targeted, dual specificity Ras/Rap exchange factor.

Clyde-Smith J, Silins G, Gartside M, Grimmond S, Etheridge M, Apolloni A, Hayward N, Hancock JF
J Biol Chem. 2000; 275: 32260-7

Ras proteins operate as molecular switches in signal transduction pathways downstream of tyrosine kinases and G-protein-coupled receptors. Ras is switched from the inactive GDP-bound state to the active GTP-bound state by guanine nucleotide exchange factors (GEFs). We report here the cloning and characterization of RasGRP2, a longer alternatively spliced form of the recently cloned RapGEF, CalDAG-GEFI. A unique feature of RasGRP2 is that it is targeted to the plasma membrane by a combination of N-terminal myristoylation and palmitoylation. In vivo, RasGRP2 selectively catalyzes nucleotide exchange on N- and Ki-Ras, but not Ha-Ras. RasGRP2 also catalyzes nucleotide exchange on Rap1, but this RapGEF activity is less potent than that associated with CalDAG-GEFI. The nucleotide exchange activity of RasGRP2 toward N-Ras is stimulated by diacylglycerol and inhibited by calcium. The effects of diacylglycerol and calcium are additive but are not accompanied by any detectable change in the subcellular localization of RasGRP2. In contrast, CalDAG-GEFI is localized predominantly to the cytosol and lacks Ras exchange activity in vivo. However, prolonged exposure to phorbol esters, or growth in serum, results in localization of CalDAG-GEFI to the cell membrane and restoration of Ras exchange activity. Expression of RasGRP2 or CalDAG-GEFI in NIH3T3 cells transfected with wild type N-Ras results in an accelerated growth rate but not morphologic transformation. Thus, under appropriate growth conditions, CalDAG-GEFI and RasGRP2 are dual specificity Ras and Rap exchange factors.

Activation of heterotrimeric G-protein signaling by a ras-related protein. Implications for signal integration.

Cismowski MJ, Ma C, Ribas C, Xie X, Spruyt M, Lizano JS, Lanier SM, Duzic E
J Biol Chem. 2000; 275: 23421-4

Utilizing a functional screen in the yeast Saccharomyces cerevisiae we identified mammalian proteins that activate heterotrimeric G-protein signaling pathways in a receptor-independent fashion. One of the identified activators, termed AGS1 (for activator of G-protein signaling), is a human Ras-related G-protein that defines a distinct subgroup of the Ras superfamily. Expression of AGS1 in yeast and in mammalian cells results in specific activation of Galpha(i)/Galpha(o) heterotrimeric signaling pathways. In addition, the in vivo and in vitro properties of AGS1 are consistent with it functioning as a direct guanine nucleotide exchange factor for Galpha(i)/Galpha(o). AGS1 thus presents a unique mechanism for signal integration via heterotrimeric G-protein signaling pathways.

Dictyostelium: an ideal organism for genetic dissection of Ras signalling networks.

Chubb JR, Insall RH
Biochim Biophys Acta. 2001; 1525: 262-71

Signalling pathways based on the small GTPase Ras regulate a multitude of cellular events in eukaryotic cells. Dictyostelium expresses a large and varied family of Ras proteins. It also uses a range of known Ras regulators, in particular RasGEFs, and effectors. The genetic tractability of Dictyostelium, together with the wide range of Ras proteins and regulators, make it an ideal model for the genetic dissection of Ras pathways. This review highlights the recent advances in our understanding of Ras function in Dictyostelium, and considers the implications of these findings for our understanding of eukaryotic signal transduction.

Disassembly of Son-of-sevenless proteins from Grb2 during p21ras desensitization by insulin.

Cherniack AD, Klarlund JK, Conway BR, Czech MP
J Biol Chem. 1995; 270: 1485-8

Insulin receptor signaling acutely stimulates GTP loading of p21ras, apparently by mobilizing complexes of Grb2 and the guanine nucleotide exchangers Son-of-sevenless (Sos) 1 and 2 to associate with tyrosine-phosphorylated proteins in the plasma membrane. Here we show that in 32P-labeled 3T3-L1 adipocytes the elevated cellular concentrations of [32P]GTP-bound p21ras in response to insulin return to near basal levels after 20-30 min of hormone stimulation, while insulin receptors remain activated. Lysates of such desensitized cells were quantitatively immunoprecipitated with an antiserum recognizing both Sos1 and Sos2 proteins or a specific anti-Sos2 antiserum. Immunoblot analysis of these precipitates revealed that insulin causes a marked hyperphosphorylation of Sos1 and a 50% decrease in Grb2 associated with Sos proteins under these conditions. Similarly, anti-Grb2 immunoprecipitates of such lysates revealed the presence of decreased Sos1 protein due to insulin action. The disassembly of Grb2 from Sos proteins slightly precedes the time course of p21ras deactivation in response to insulin. These data are consistent with the hypothesis that the dissociation of Grb2 from Sos proteins caused by insulin in 3T3-L1 cells mediates p21ras deactivation and desensitization.

GEFs: structural basis for their activation of small GTP-binding proteins.

Cherfils J, Chardin P
Trends Biochem Sci. 1999; 24: 306-11

Small GTP-binding proteins of the Ras superfamily function as molecular switches in fundamental events such as signal transduction, cytoskeleton dynamics and intracellular trafficking. Guanine-nucleotide-exchange factors (GEFs) positively regulate these GTP-binding proteins in response to a variety of signals. GEFs catalyze the dissociation of GDP from the inactive GTP-binding proteins. GTP can then bind and induce structural changes that allow interaction with effectors. Representative structures of four main classes of exchange factors have been described recently and, in two cases, structures of the GTP-binding protein-GEF complex have been solved. These structures, together with biochemical studies, have allowed a deeper understanding of the mechanisms of activation of Ras-like GTP-binding proteins and suggested how they might represent targets for therapeutic intervention.

Ras-15A protein shares highly similar dominant-negative biological properties with Ras-17N and forms a stable, guanine-nucleotide resistant complex with CDC25 exchange factor.

Chen SY, Huff SY, Lai CC, Der CJ, Powers S
Oncogene. 1994; 9: 2691-8

We show that expression of Ras-15A, previously shown to be a dominant-negative mutant in yeast, is a potent inhibitor of endogenous Ras protein function in mammalian cells. Expression of Ras-15A did not inhibit the growth of cells containing an oncogenic ras gene nor did it interfere with the ability of transiently expressed oncogenic ras or raf genes to activate transcription from a Ras-responsive ets1/AP-1 promoter. In contrast, expression of Ras-15A completely blocked growth of normal cells and activation of the ets1/AP-1 promoter by transiently overexpressed SOS1 and normal Ras proteins. These results suggest that Ras-15A, like Ras-17N, blocks endogenous Ras function by interfering with upstream activation of Ras proteins rather than downstream effects. To test whether Ras-15A and Ras-17N interfere with Ras function by blocking GDP-GTP exchange proteins, we examined their physical interaction with the CDC25 exchange protein. All three proteins formed stable complexes with CDC25 in the absence of guanine-nucleotides, but only Ras-15A was not released from CDC25 by physiological concentrations of GDP or GTP. These results establish that Ras-15A blocks the activation of normal Ras proteins by sequestering GDP-GTP exchange factors into non-productive complexes. In contrast, it would appear that the similar biological properties of Ras-17N are mediated by a reversible, competitive sequestration of exchange factors.

A murine CDC25/ras-GRF-related protein implicated in Ras regulation.

Chen L, Zhang LJ, Greer P, Tung PS, Moran MF
Dev Genet. 1993; 14: 339-46

A partial cDNA encoding a novel putative p2, ras guanine nucleotide release-inducing factor (GRF), GRF2, was amplified from murine embryonic stem cells. The presumptive catalytic region of GRF2 is related to the yeast Ras GRF encoded by CDC25. GRF2 is 80% identical to murine CDC25Mm/ras-GRF, but is more similar to yeast CDC25 than to other ras GRFs related to the Drosophila son of sevenless gene product. A 9-kb GRF2 messenger RNA was highly expressed in brain, but GRF2-specific antibodies recognized apparent GRF2 proteins in various mouse tissues in addition to brain. Thus GRF2 represents a novel widely-expressed protein that is highly related to CDC25Mm/ras-GRF, at least in its catalytic domain. Both GRF2 and CDC25Mm/ras-GRF are expressed in murine embryonic stem cells, suggesting that different Ras activators may regulate ras-dependent proliferation and differentiation in early mouse development.

Mammalian Grb2 regulates multiple steps in embryonic development and malignant transformation.

Cheng AM, Saxton TM, Sakai R, Kulkarni S, Mbamalu G, Vogel W, Tortorice CG, Cardiff RD, Cross JC, Muller WJ, Pawson T
Cell. 1998; 95: 793-803

Proteins with SH2 and SH3 domains link tyrosine kinases to intracellular pathways. To investigate the biological functions of a mammalian SH2/SH3 adaptor, we have introduced a null mutation into the mouse gene for Grb2. Analysis of mutant embryonic stem cells, embryos, and chimeras reveals that Grb2 is required during embyrogenesis for the differentiation of endodermal cells and formation of the epiblast. Grb2 acts physiologically as an adaptor, since replacing the C terminus of the Ras activator Sos1 with the Grb2 SH2 domain yields a fusion protein that largely rescues the defects caused by the Grb2 mutation. Furthermore, Grb2 is rate limiting for mammary carcinomas induced by polyomavirus middle T antigen. These data provide genetic evidence for a mammalian Grb2-Ras signaling pathway, mediated by SH2/SH3 domain interactions, that has multiple functions in embryogenesis and cancer.

Oncostatin M induces association of Grb2 with Janus kinase JAK2 in multiple myeloma cells.

Chauhan D, Kharbanda SM, Ogata A, Urashima M, Frank D, Malik N, Kufe DW, Anderson KC
J Exp Med. 1995; 182: 1801-6

Oncostatin M (OSM) is a 28-kD glycoprotein recently identified as a growth factor for human multiple myeloma cells. It belongs to a family of distantly related cytokines that includes interleukin 6, ciliary neurotrophic factor, leukemia-inhibitory factor, and interleukin 11. These cytokines initiate signaling by inducing either homodimerization of gp130 or heterodimerization of gp130 with leukemia-inhibitory factor receptor beta components. Such dimerization in turn activates receptor-associated tyrosine kinases. In the present study using U266B1 human multiple myeloma cells, we show that OSM induces tyrosine phosphorylation and activation of JAK2, but not JAK1 or Tyk2, kinases. The results also demonstrate that OSM induces direct interaction of JAK2 kinase with Grb2, an SH2/SH3 domain containing adaptor protein. The SH2 domain of Grb2 is directly associated with tyrosine-phosphorylated JAK2. Furthermore, the presence of Sos in the JAK2-Grb2 complex suggests a role for Ras in OSM-transduced signaling.

Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2.

Chardin P, Camonis JH, Gale NW, van Aelst L, Schlessinger J, Wigler MH, Bar-Sagi D
Science. 1993; 260: 1338-43

A human complementary DNA was isolated that encodes a widely expressed protein, hSos1, that is closely related to Sos, the product of the Drosophila son of sevenless gene. The hSos1 protein contains a region of significant sequence similarity to CDC25, a guanine nucleotide exchange factor for Ras from yeast. A fragment of hSos1 encoding the CDC25-related domain complemented loss of CDC25 function in yeast. This hSos1 domain specifically stimulated guanine nucleotide exchange on mammalian Ras proteins in vitro. Mammalian cells overexpressing full-length hSos1 had increased guanine nucleotide exchange activity. Thus hSos1 is a guanine nucleotide exchange factor for Ras. The hSos1 interacted with growth factor receptor-bound protein 2 (GRB2) in vivo and in vitro. This interaction was mediated by the carboxyl-terminal domain of hSos1 and the Src homology 3 (SH3) domains of GRB2. These results suggest that the coupling of receptor tyrosine kinases to Ras signaling is mediated by a molecular complex consisting of GRB2 and hSos1.

Regulated and constitutive activity by CDC25Mm (GRF), a Ras-specific exchange factor.

Cen H, Papageorge AG, Vass WC, Zhang KE, Lowy DR
Mol Cell Biol. 1993; 13: 7718-24

Serum stimulates cells to increase their proportion of Ras protein in the active GTP-bound state. We have recently identified four types (I to IV) of apparently full-length cDNAs from a single mammalian gene, called CDC25Mm or GRF, which is homologous to the Ras-specific exchange factor CDC25 of S. cerevisiae. The largest cDNA (type IV) is brain specific, with the other three classes, although they have distinct 5' ends, essentially representing progressive N-terminal deletions of this cDNA. When placed in a retroviral expression vector, all four types of cDNAs induced morphologic transformation of NIH 3T3 cells and an increase in the basal level of GTP.Ras. Serum stimulation of these transformants lead to a further increase in GTP.Ras only in cells expressing the type IV cDNA. Each type of GRF protein was found in cytosolic and membrane fractions, and the protein in each fraction could stimulate guanine nucleotide release from GDP.Ras in vitro. When NIH 3T3 cells and cells expressing the type IV protein were transfected with two versions of a mutant ras gene, one encoding membrane-associated Ras protein and the other encoding a cytosolic Ras protein, the basal levels of GTP bound to both forms of the mutant Ras protein were significantly higher in the cells expressing the type IV protein. However, serum increased the level of GTP bound to the membrane-associated mutant Ras protein in NIH 3T3 cells and in cells expressing the type IV protein but not in cells expressing the cytosolic version of the Ras protein. We conclude that each type of CDC25Mm induces cell transformation via the ability of its C terminus to stimulate guanine nucleotide exchange on Ras, the presence of N-terminal sequences is associated with a serum-dependent change in GTP.Ras, and the serum-dependent increase in GTP.Ras by exogenous CDC25Mm or by endogenous exchange factors probably requires membrane association of both Ras and the exchange factor.

Identification of guanine exchange factor key residues involved in exchange activity and Ras interaction.

Camus C, Hermann-Le Denmat S, Jacquet M
Oncogene. 1995; 11: 951-9

We have carried out a functional analysis of the human HGRF55 exchange factor in the yeast Saccharomyces cerevisiae. Twelve residues conserved among most of all known guanine exchange factors (GEFs) have been independently changed to alanine. Taking advantage of the ability of Hgrf55p to replace the yeast Cdc25p exchange factor, and using the two-hybrid system with RAS2ala22 allele, we have identified key residues for the interaction with Ras and/or its activation. Substitution of arginine 392 to alanine leads to a complete loss of interaction with Ras, though the protein remains stable. Substitution of Asp266 or Arg359 to alanine results in inactive proteins at 39 degrees C, still able however to interact with Ras. The other charged-to-alanine substitutions led to no detectable phenotype when present alone but most of them dramatically increased the temperature sensitive phenotype observed with [Asp266Ala] substitution. Surprisingly, the cysteine to alanine substitution in the highly conserved PCVPF/Y motif proved to be without effect, suggesting that the sulfhydryl group is not essential for stability or interaction with Ras.

Vav cooperates with Ras to transform rodent fibroblasts but is not a Ras GDP/GTP exchange factor.

Bustelo XR, Suen KL, Leftheris K, Meyers CA, Barbacid M
Oncogene. 1994; 9: 2405-13

Vav is a proto-oncogene specifically expressed in cells of hematopoietic origin. Its gene product contains a series of structural motifs, including SH2 and SH3 domains, suggestive of a role in signal transduction. The Vav protein also possesses a Dbl-homology (DH) domain previously found in regulators of the Ras superfamily of small GTP-binding proteins. Recently, Vav has been reported to be the major Ras GDP/GTP exchange factor (GEF) in hematopoietic cells [Gulbins et al., Science 260, 822 (1993); J. Immunol. 152, 2123 (1994)]. The following observations are inconsistent with such a role: (i) Vav proteins do not exhibit Ras GEF activity in standard GDP/GTP exchange assays; (ii) Cells overexpressing Vav do not have increased levels of GTP-bound Ras proteins; (iii) Overexpression of Vav does not overcome the growth inhibitory activity of RasN17, a mutant that blocks Ras signaling by inhibiting Ras GEFs; (iv) Transformation of NIH3T3 cells by Vav oncoproteins is not inhibited by a farnesyl transferase inhibitor that completely blocks transformation by both Ras and its well characterized GEF, RasCDC25 and (v) The morphology of Vav-transformed NIH3T3 cells is dramatically different from that induced by Ras and RasCDC25. Whereas these observations make it unlikely that Vav functions either as a RasGEF or as an upstream regulatory element of Ras, we have observed that Vav can cooperate with normal Ras proteins to transform NIH3T3 cells. These results suggest that Vav and Ras may mediate signal transduction by distinct, but interactive mitogenic pathways.

A novel mammalian guanine nucleotide exchange factor (GEF) specific for rab proteins.

Burton J, De Camilli P
Adv Second Messenger Phosphoprotein Res. 1994; 29: 109-19

Amino acid residues in the Ras-like GTPase Rab3A that specify sensitivity to factors that regulate the GTP/GDP cycling of Rab3A.

Burstein ES, Brondyk WH, Macara IG
J Biol Chem. 1992; 267: 22715-8

Two cellular factors have been described, Rab3A-GAP (GTPase-activating protein) and Rab3A-GRF (guanine nucleotide releasing factor) which, respectively, accelerate the intrinsic GTPase activity of, or the rate of dissociation of GDP from, the Ras-related GTP-binding protein, p25 Rab3A. Mutational analysis of p25 Rab3A was undertaken to define amino acid residues important for interaction with these factors. Mutations in residues 51-59, which correspond to the effector domain of p21 Ras, completely abolished sensitivity of p25 Rab3A to Rab3A-GRF and decreased the affinity of p25 Rab3A for Rab3A-GRF. Surprisingly, only one mutant in this region was Rab3A-GAP-insensitive, while the others retained partial, complete, or significantly increased GAP responsiveness. Mutations in the first G-domain had only modest effects on intrinsic GTPase activity and little effect on either Rab3A-GRF or Rab3A-GAP interactions. Truncation of 34 residues from the carboxyl terminus had no effect Rab3A-GAP sensitivity but facilitated Rab3A-GRF stimulation. Mutation T36N, analogous to the dominant inhibitory mutation T17N in Ras, which has been hypothesized to sequester an upstream activator of Ras, conferred a 10-fold higher affinity upon p25 Rab3A for Rab3A-GRF.

A complex of Grb2 adaptor protein, Sos exchange factor, and a 36-kDa membrane-bound tyrosine phosphoprotein is implicated in ras activation in T cells.

Buday L, Egan SE, Rodriguez Viciana P, Cantrell DA, Downward J
J Biol Chem. 1994; 269: 9019-23

T lymphocytes contain both Grb2, an SH2 and SH3 domain containing adaptor protein, and Sos, a guanine nucleotide exchange factor for Ras. Immunoprecipitates of Sos from the lysates of T cells contain a 36-kDa protein which is phosphorylated on tyrosine residues in response to T cell receptor/CD3 cross-linking. In vitro studies using different bacterially synthesized GST-Sos fusion proteins confirm the formation of complexes containing p36 and the proline-rich COOH-terminal domain of Sos. The use of mutant GST-Grb2 proteins in which both SH3 domains have been mutationally inactivated shows that Grb2 binds to tyrosine phosphorylated p36 via its SH2 domain. In Jurkat cells phosphorylated p36 is localized exclusively in the particulate fraction. In addition, another SH2 domain-containing protein, p52Shc is tyrosine phosphorylated upon TCR.CD3 cross-linking and associates with a 150-kDa phosphotyrosine containing protein. Taken together these data suggest that activation of Ras in T cells via the TCR.CD3 complex might be controlled, at least in part, by mechanisms similar to those found in fibroblasts, involving in this case formation of a complex of Grb2, Sos, and a membrane-bound tyrosine phosphoprotein of molecular mass 36-kDa.

Mutants of Rab3A analogous to oncogenic Ras mutants. Sensitivity to Rab3A-GTPase activating protein and Rab3A-guanine nucleotide releasing factor.

Brondyk WH, McKiernan CJ, Burstein ES, Macara IG
J Biol Chem. 1993; 268: 9410-5

Rab3A is a Ras-like GTPase that is believed to function in regulated secretion. A GTPase activating protein (GAP) and a guanine nucleotide releasing factor (GRF) specific for Rab3A have recently been described. In this study we have described the biochemical activities of Rab3A mutants in codons 31, 81, 135, and 166, which correspond to codons 12, 61, 116, and 146 in Ras. The results demonstrate that simple extrapolations from the properties of Ras mutants are not valid for all small GTPases. S31V-Rab3A and Q81L-Rab3A had a reduced basal GTPase activity, but surprisingly were sensitive to the effects of Rab3A-GAP and insensitive to Rab3A-GRF. Q81L-Rab3A and wild-type Rab3A that were bound to a nonhydrolyzable GTP analog had similar affinities for Rab3A-GAP. In vivo, the percent of Q81L-Rab3A (46%) and wild-type Rab3A (43%) complexed to GTP was similar. These findings indicate that Rab3A-GRF and Rab3A-GAP interact with residues different from those of previously described GAPs and GRFs. Both A166V-Rab3A and N135I-Rab3A had increased intrinsic dissociation rates for GDP. However, the dissociation rate for N135I-Rab3A was > 100-fold higher than that for A166V-Rab3A suggesting that, in vivo, of the two, N135I-Rab3A would more likely be preferentially in the GTP-bound state.

Regulators and effectors of ras proteins.

Bollag G, McCormick F
Annu Rev Cell Biol. 1991; 7: 601-32

The Saccharomyces cerevisiae gene product SDC25 C-domain functions as an oncoprotein in NIH3T3 cells.

Barlat I, Schweighoffer F, Chevallier-Multon MC, Duchesne M, Fath I, Landais D, Jacquet M, Tocque B
Oncogene. 1993; 8: 215-8

Ras proteins in mammalian cells cycle between a GTP-bound 'on' state and a GDP-bound 'off' state. Activation of Ras p21 results from the dissociation of tightly bound GDP and the exchange of bound GDP for GTP. A guanine nucleotide exchange factor is required for this activation. Activation promotes interaction with effector molecules and allows the signal to be transduced. In Saccharomyces cerevisiae, the function of guanine nucleotide exchange has been ascribed to the product of the CDC25 gene. The C-terminus domain of SDC25, a homologue of CDC25, can substitute for the CDC25 protein in yeast. We have demonstrated that the SDC25 C-terminus domain promotes GTP binding to Ras p21 in CHO cells. In the present study, we found that the stable expression of the SDC25 C-terminus domain induced transformation of NIH3T3 cells. Ras proteins in these tumorigenic cells were GTP bound. In addition, the coexpression of wild-type Ha-Ras protein with the SDC25 C-terminus was found to enhance the tumorigenic properties of the NIH3T3 cells. These results imply that, in subsets of human tumours, cellular Ras p21 might be found in its GTP-bound active form as a consequence of an oncogenic activation of a mammalian Ras guanine nucleotide exchange factor.

Maintenance of CDC42 GDP-bound state by Rho-GDI inhibits MAP kinase activation by the exchange factor Ras-GRF. evidence for Ras-GRF function being inhibited by Cdc42-GDP but unaffected by CDC42-GTP.

Arozarena I, Matallanas D, Crespo P
J Biol Chem. 2001; 276: 21878-84

The function of the Ras guanine nucleotide exchange factor Ras-GRF/cdc25(Mn) is subject to tight regulatory processes. We have recently shown that the activation of the Ras/MAPK pathway by Ras-GRF is controlled by the Rho family GTPase Cdc42 through still unknown mechanisms. Here, we report that retaining Cdc42 in its GDP-bound state by overexpressing Rho-GDI inhibits Ras-GRF-mediated MAPK activation. Conversely, Ras-GRF basal and LPA- or ionomycin-stimulated activities were unaffected by a constitutively active GTP-bound Cdc42. Moreover, the Cdc42 downstream effectors MLK3, ACK1, PAK1, and WASP had no detectable influence on Ras-GRF-mediated MAPK activation. In contrast, promoting GDP release from Cdc42 with the Rho family GEF Dbl or with ionomycin suppressed the restraint exerted by Cdc42 on Ras-GRF activity. We conclude that Cdc42-GDP inhibits Ras-GRF-induced MAPK activation, but neither Cdc42-GTP nor the Cdc42 downstream effectors affect Ras-GRF performance. Interestingly, the loss of the GDP-bound state by Cdc42 abolishes its inhibitory effects on Ras-GRF function. These results suggest that the Cdc42 mechanism of action may not be solely restricted to activation of downstream signaling cascades when GTP-loaded. Furthermore, the GDP-bound form may be acting as an inhibitory molecule down-modulating parallel signaling routes such as the Ras/MAPK pathway.

Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway.

Aronheim A, Engelberg D, Li N, al-Alawi N, Schlessinger J, Karin M
Cell. 1994; 78: 949-61

Activation of growth factor receptors results in tyrosine autophosphorylation and recruitment of SH2 domain-containing effectors, including Grb2. Grb2 recruitment mediates activation of the Ras nucleotide exchanger Sos by an unknown mechanism. To examine the role of membrane recruitment, we prepared Sos derivatives containing either myristoylation or farnesylation signals. This resulted in plasma membrane targeting of Sos and stimulation of the Ras signaling pathway, including ERK and AP-1 activities leading to oncogenic transformation. Sos derivatives with nonfunctional myristoylation or farnesylation sequences were inactive. Farnesylation of Sos also activated Ras signaling in yeast. In both mammalian cells and yeast, membrane-targeted Sos derivatives lacking the C-terminal region were considerably more active. Therefore, targeting of Sos to the plasma membrane in the vicinity of Ras appears to be the primary mechanism leading to activation of the Ras pathway. A secondary mechanism could involve relief of the inhibitory effect of the Sos C-terminal region.

The Ras GDP/GTP cycle is regulated by oxidizing agents at the level of Ras regulators and effectors.

Accorsi K, Giglione C, Vanoni M, Parmeggiani A
FEBS Lett. 2001; 492: 139-45

Reactive oxygen species (ROS) have been found to play important roles in regulating cellular functions. Their action in vivo has been related to specific effects on signal transduction pathways, such as Ras pathway. In order to characterize which elements of Ras pathway are affected by ROS, we have analyzed the action of different oxidizing agents on the ability of GTPase activating protein GAP and nucleotide exchange factor GEF to enhance the intrinsic activities of Ras. The action of these agents on the binding between H-Ras and its effector c-Raf-1 was also investigated. No effects were observed on the intrinsic activities of H-Ras or Ras2p. On the other hand, reversible inhibitions of GEF and GAP actions on Ras were found, whose extent was dependent on the agent used. As tested with the scintillation proximity assay, these agents also inhibited the binding of c-Raf-1 to H-Ras. Our data reveal new potential targets for the action of ROS on Ras pathway and suggest that they can influence the Ras activation state indirectly via regulators and effectors.

Triple association of CDC25-, Dbl- and Sec7-related domains in mammalian guanine-nucleotide-exchange factors.

Abergel C, Chavrier P, Claverie JM
Trends Biochem Sci. 1998; 23: 472-3