A Ras-GTPase-activating protein SH3-domain-binding protein.

We report the purification of a Ras-GTPase-activating protein (GAP)-binding protein, G3BP, a ubiquitously expressed cytosolic 68-kDa protein that coimmunoprecipitates with GAP. G3BP physically associates with the SH3 domain of GAP, which previously had been shown to be essential for Ras signaling. The G3BP cDNA revealed that G3BP is a novel 466-amino-acid protein that shares several features with heterogeneous nuclear RNA-binding proteins, including ribonucleoprotein (RNP) motifs RNP1 and RNP2, an RG-rich domain, and acidic sequences. Recombinant G3BP binds effectively to the GAP SH3 domain G3BP coimmunoprecipitates with GAP only when cells are in a proliferating state, suggesting a recruitment of a GAP-G3BP complex when Ras is in its activated conformation.     

Grb2 dominantly associates with dynamin II in human hepatocellular carcinoma HepG2 cells.

The two SH3 domains and one SH2 domain containing adaptor protein Grb2 is an essential element of the Ras signaling pathway in multiple systems. The SH2 domain of Grb2 recognizes and interacts with phosphotyrosine residues on activated tyrosine kinases, whereas the SH3 domains bind to several proline-rich domain-containing proteins such as Sos1. To define the difference in Grb2-associated proteins in hepatocarcinoma cells, we performed coprecipitation analysis using recombinant GST-Grb2 fusion proteins and found that several protein components (p170, p125, p100, and p80) differently associated with GST-Grb2 proteins in human Chang liver and hepatocarcinoma HepG2 cells. Sos1 and p80 proteins dominantly bind to Grb2 fusion proteins in Chang liver, whereas p100 remarkably associate with Grb2 in HepG2 cells. Also GST-Grb2 SH2 proteins exclusively bound to the p46(Shc), p52(Shc), and p66(Shc) are important adaptors of the Ras pathway in HepG2 cells. The p100 protein has been identified as dynamin II. We observed that the N-SH3 and C-SH3 domains of Grb2 fusion proteins coprecipitated with dynamin II besides Sos1. These results suggest that dynamin II may be a functional molecule involved in Grb2-mediated signaling pathway on Ras activation for tumor progression and differentiation of hepatocarcinoma cells.     

The Ras-GTPase-activating protein SH3 domain is required for Cdc2 activation and mos induction by oncogenic Ras in Xenopus oocytes independently of mitogen-activated protein kinase activation.

The Ras-GTPase-activating protein (RasGAP) is an important modulator of p21ras - dependent signal transduction in Xenopus oocytes and in mammalian cells. We investigated the role of the RasGAP SH3 domain in signal transduction with a monoclonal antibody against the SH3 domain of RasGaP. This antibody prevented the activation of the maturation-promoting factor complex (cyclin B-p34cdc2) by oncogenic Ras. The antibody appears to be specific because as little as 5 ng injected per oocyte reduced the level of Cdc2 activation by 50% whereas 100 ng of nonspecific immunoglobulin G did not affect Cdc2 activation. The antibody blocked the Cdc2 activation induced by oncogenic Ras but not that induced by progesterone, which acts independently of Ras. A peptide corresponding to positions 317 to 326 of a sequence in the SH3 domain of human RasGAP blocked Cdc2 activation, whereas a peptide corresponding to positions 273 to 305 of a sequence in the N-terminal moiety of the SH3 domain of RasGAP had no effect. The antibody did not block the mitogen-activated protein (MAP) kinase cascade (activation of MAPK/ERK kinase [MEK], MAP kinase, and S6 kinase p90rsk). Surprisingly, injection of the negative MAP kinase mutant protein ERK2 K52R (containing a K-to-R mutation at position 52) blocked the Cdc2 activation induced by oncogenic Ras as well as blocking the activation of MAP kinase. Thus, MAP kinase is also implicated in the regulation of Cdc2 activity. In this study, we further investigated the regulation of the synthesis of the c-mos oncogene product, which is necessary for the activation of Cdc2. We report that the synthesis of the c-mos oncogene product, which is necessary for the activation antibody to the SH3 domain of RasGAP and by injecting the negative MAP kinase mutant protein ERK2 K52R. These results suggest that oncogenic Ras activates two signaling mechanisms: the MAP kinase cascade and a signaling pathway implicating the SH3 domain of RasGAP. These mechanisms might control Mos protein expression implicated in Cdc2 activation.     

Asparagine 26, glutamic acid 31, valine 45, and tyrosine 64 of Ras proteins are required for their oncogenicity.

Ras and Rap1 proteins are related GTP-dependent signal transducers which require Gly-12, the effector domain (residues 32-40), and Ala-59 for stimulation of their GTPase activities by GAP1 and GAP3, respectively. The replacement of Gly-12 by Val or Ala-59 by Thr potentiates the Ras oncogenicity and Rap1A antioncogenicity. However, the mutations in the effector domain, in particular the replacement of Thr-35 by Ala, abolish both Ras oncogenicity and Rap1A antioncogenicity, indicating that the effector domain is involved in interactions of these signal transducers with their targets as well as the GAPs. In this paper, we demonstrate that (i) replacement of Tyr-64 of the Ha-Ras protein or Phe-64 of the Rap1A protein by Glu or other non-hydrophobic amino acids reduces their intrinsic GTPase activities and abolishes their stimulation by GAP1 or GAP3, respectively, (ii) replacement of Tyr-64 by Gly and other non-hydrophobic amino acids results in complete loss of the oncogenicity of the v-Ha-Ras protein, indicating that the hydrophobic residue 64, in addition to the known effector domain, is essential for the Ras protein to interact with its target as well as GAP1. In addition we have found that Asn-26, Glu-31, and Val-45 of the v-Ha-Ras protein are required for its oncogenicity. Replacement of the Ras residues at either positions 26, 31, or 45 by the corresponding Rap1A residues abolishes the Ras oncogenicity.     

Identification of residues in GTPase-activating protein Src homology 2 domains that control binding to tyrosine phosphorylated growth factor receptors and p62.

Ras GTPase-activating protein (GAP) contains two Src homology 2 (SH2) domains which are implicated in binding to tyrosine-phosphorylated sites in specific activated growth factor receptors and to a cytoplasmic tyrosine-phosphorylated protein, p62. We have used site-directed mutagenesis of the two GAP SH2 domains (SH2-N and SH2-C) to identify residues involved in receptor and p62 binding. A bacterial fusion protein containing the precise SH2-N domain, as defined by sequence homology, associated with both the activated beta platelet-derived growth factor receptor and epidermal growth factor receptor, and p62 in vitro. However, short deletions at either the N or C termini of the SH2-N domain abolished binding, suggesting that the entire SH2 sequence is required for formation of an active domain. Conservative substitutions of 2 highly conserved basic residues in the SH2-N domain, an arginine and a histidine, resulted in complete loss of receptor and p62 binding, whereas other basic residues, and residues at variable SH2 sites, were more tolerant of substitution. The conserved arginine and histidine therefore appear critical for association with phosphotyrosine-containing proteins, possibly through an interaction with phosphotyrosine. The GAP SH2-C domain, unlike SH2-N, does not bind efficiently to activated receptors or p62 in vitro. The SH2-C domain lacks 3 residues which are otherwise well conserved, and contribute to high affinity SH2-N binding. Replacement of 1 of these residues, a cysteine, with the consensus glycine, conferred SH2-C binding activity toward tyrosine-phosphorylated p62 and epidermal growth factor receptor. Loss-of-function and gain-of-function mutations in the GAP SH2 domains can therefore be used to identify residues that are critical for receptor and p62 binding.     

The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion.

Ras GTPase activating protein (GAP) possesses a C-terminal domain that interacts with GTP-bound Ras, and an N-terminal region containing two SH2 domains and an SH3 domain. In addition to its association with Ras, GAP binds stably to autophosphorylated beta PDGF receptors, and to two cytoplasmic phosphoproteins: p62, an RNA binding protein, and p190, which possesses GAP activity towards small guanine nucleotide binding proteins in the Rho/Rac family. To define the region of GAP that mediates these interactions with cellular phosphoproteins, and to investigate the biological significance of these complexes, a truncated GAP polypeptide (GAP-N) containing residues 1-445 was stably expressed in Rat-2 fibroblasts. GAP-N contains the SH2 and SH3 domains, but lacks the Ras GTPase activating domain. Stimulation of cells expressing GAP-N with PDGF induced association of GAP-N with the beta PDGF receptor, and phosphorylation of GAP-N on tyrosine, consistent with the notion that GAP SH2 domains direct binding to the autophosphorylated beta PDGF receptor in vivo. GAP-N bound constitutively to p190 in both serum-deprived and growth factor-stimulated cells. This GAP-N-p190 complex had Rho GAP activity in vitro. The expression of GAP-N in Rat-2 cells correlated with changes in the cytoskeleton and in cell adhesion, typified by the disruption of action stress fibres, a reduction in focal contacts, and an impaired ability to adhere to fibronectin. These results suggest that the N-terminal domain of GAP can direct interactions with cellular phosphoproteins in vivo, and thereby exert an effector function which modulates the cytoskeleton and cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)     

A RasGAP SH3 peptide aptamer inhibits RasGAP-Aurora interaction and induces caspase-independent tumor cell death

The Ras GTPase-activating protein RasGAP catalyzes the conversion of active GTP-bound Ras into inactive GDP-bound Ras. However, RasGAP also acts as a positive effector of Ras and exerts an anti-apoptotic activity that is independent of its GAP function and that involves its SH3 (Src homology) domain. We used a combinatorial peptide aptamer approach to select a collection of RasGAP SH3 specific ligands. We mapped the peptide aptamer binding sites by performing yeast two-hybrid mating assays against a panel of RasGAP SH3 mutants. We examined the biological activity of a peptide aptamer targeting a pocket delineated by residues D295/7, L313 and W317. This aptamer shows a caspase-independent cytotoxic activity on tumor cell lines. It disrupts the interaction between RasGAP and Aurora B kinase. This work identifies the above-mentioned pocket as an interesting therapeutic target to pursue and points its cognate peptide aptamer as a promising guide to discover RasGAP small-molecule drug candidates.     

Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain.

Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected.     

Revisiting G3BP1 as a RasGAP binding protein: sensitization of tumor cells to chemotherapy by the RasGAP 317-326 sequence does not involve G3BP1

RasGAP is a multifunctional protein that controls Ras activity and that is found in chromosomal passenger complexes. It also negatively or positively regulates apoptosis depending on the extent of its cleavage by caspase-3. RasGAP has been reported to bind to G3BP1 (RasGAP SH3-domain-binding protein 1), a protein regulating mRNA stability and stress granule formation. The region of RasGAP (amino acids 317–326) thought to bind to G3BP1 corresponds exactly to the sequence within fragment N2, a caspase-3-generated fragment of RasGAP, that mediates sensitization of tumor cells to genotoxins. While assessing the contribution of G3BP1 in the anti-cancer function of a cell-permeable peptide containing the 317–326 sequence of RasGAP (TAT-RasGAP_317–326), we found that, in conditions where G3BP1 and RasGAP bind to known partners, no interaction between G3BP1 and RasGAP could be detected. TAT-RasGAP_317–326 did not modulate binding of G3BP1 to USP10, stress granule formation or c-myc mRNA levels. Finally, TAT-RasGAP_317–326 was able to sensitize G3BP1 knock-out cells to cisplatin-induced apoptosis. Collectively these results indicate that G3BP1 and its putative RasGAP binding region have no functional influence on each other. Importantly, our data provide arguments against G3BP1 being a genuine RasGAP-binding partner. Hence, G3BP1-mediated signaling may not involve RasGAP.     

Direct interaction of SOS1 Ras exchange protein with the SH3 domain of phospholipase C-gamma1

A recent report that microinjection of the SH3 domain of PLC-gamma1 could induce DNA synthesis raised the functional importance of the SH3 domain of PLC-gamma1 in mitogenic signaling. In this report, we provide evidence that SOS1, a p21Ras-specific guanine nucleotide exchange factor, directly binds to the SH3 domain of PLC-gamma1, and that the SH3 domain of PLC-gamma1 is involved in SOS1-mediated p21Ras activation. SOS1 was coprecipitated with the GST-fused SH3 domain of PLC-gamma1 in vitro. The interaction between SOS1 and the PLC-gamma1 SH3 domain is mediated by direct physical interaction. The carboxyl-terminal proline-rich domain of SOS1 is involved in the interaction with the PLC-gamma1 SH3 domain. Moreover, PLC-gamma1 could be co-immunoprecipitated with SOS1 antibody in cell lysates. From transient expression studies, we could demonstrate that the SH3 domain of PLC-gamma1 is necessary for the association with SOS1 in vivo. Intriguingly, overexpression of the SH3 domain of PLC-gamma1, lipase-inactive PLC-gamma1, or wild-type PLC-gamma1 elevated p21Ras activity and ERK activity when compared with vector transfected cells. The PLC-gamma1 mutant lacking the SH3 domain could not activate p21Ras. p21Ras activities in cell lines overexpressing either PLC-gamma1 or the SH2-SH2-SH3 domain of PLC-gamma1 were elevated about 2-fold compared to vector transfected cells. This study is the first to demonstrate that the PLC-gamma1 SH3 domain enhances p21Ras activity, and that the SH3 domain of PLC-gamma1 may be involved in the SOS1-mediated signaling pathway.     

Rsr1 and Rap1 GTPases are activated by the same GTPase-activating protein and require threonine 65 for their activation

The Rsr1 protein of Saccharomyces cerevisiae has been shown to be essential for bud site selection (Bender, A., and Pringle, J. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9976-9980). This protein of 272 amino acids shares approximately 50% sequence identity with both Ras and Rap GTPases. However, neither GTP binding nor GTPase activity of the Rsr1 protein has been reported. The Rsr1 protein shares with human Rap1 GTPases the four specific motifs, i.e. Gly-12, residues 32-40, Ala-59, and residues 64-70, that are required for GAP3-dependent activation of the Rap1 GTPases. In this paper we demonstrate that the intrinsic GTPase activity of the Rsr1 protein is stimulated by GAP3 purified from bovine brain cytosol. The Rsr1 GTPase is not activated by either GAP1 or GAP2 which are specific for the Ras and Rho GTPases, respectively. Thus, it appears that the Rsr1 GTPase is a new member of the Rap1 GTPase family. Replacement of Gly-12 by Val in the Rsr1 GTPase completely abolishes the GAP3-dependent activation. The chimeric GTPases, Ras(1-60)/Rsr1(61-168) and Rsr1(1-65)/Ras(66-189), are activated by GAP3 but not by GAP1. Replacement of Thr-65 by Ser in the latter chimeric GTPase completely abolishes the GAP3-dependent activation, indicating that Thr-65 is required for distinguishing GAP3 from GAP1. We have previously shown that Gln-61 and Ser-65 are sufficient to determine the GAP1 specificity. Replacement of Thr-35 by Ala in the common effector domain (residues 32-40) of the chimeric Ras/Rsr1 GTPases completely abolishes GAP3-dependent activation.     

人EEN蛋白的结构预测和功能分析

采用基于神经网络的算法预测了我们自行克隆的新的白血病相关蛋白ＥＥＮ（ｅｘｔｒａ ｅｌｅｖｅｎｎｉｎｅｔｅｅｎ, ＥＥＮ）全长分子的二级结构,结果表明：ＥＥＮ 蛋白可能有三个结构域,Ｎ 端由三段α螺旋和短β折叠组成,中间为四段α螺旋组成的四螺旋结构,Ｃ端为ＳＨ３结构域,类似于在受体酪氨酸激酶信号传导途径中起重要作用的ＳＥＭ－５／ＧＲＢ２ Ｃ端ＳＨ３结构域;利用同源蛋白结构模拟的方法,模拟了ＥＥＮ ＳＨ３结构域的三维结构,结果表明：ＥＥＮ ＳＨ３结构域与ＳＥＭ－５／ＧＲＢ２ ＳＨ３结构域具有相近的结构,构成脯氨酸结合区的氨基酸非常保守．上述结果提示：ＥＥＮ 蛋白可能为新的信号蛋白,可能涉及新的信号传导途径或新的信号传导旁路,ＳＨ３结构域是其功能区域．     

Activation of the JNK pathway is essential for transformation by the Met oncogene.

The Met/Hepatocyte Growth Factor (HGF) receptor tyrosine kinase is oncogenically activated through a rearrangement that creates a hybrid gene Tpr-Met. The resultant chimeric p65(Tpr-Met) protein is constitutively phosphorylated on tyrosine residues in vivo and associates with a number of SH2-containing signaling molecules including the p85 subunit of PI-3 kinase and the Grb2 adaptor protein, which couples receptor tyrosine kinases to the Ras signaling pathway. Mutation of the binding site for Grb2 impairs the ability of Tpr-Met oncoprotein to transform fibroblasts, suggesting that the activation of the Ras/MAP kinase signaling pathway through Grb2 may be essential for cellular transformation. To test this hypothesis dominant-negative mutants of Grb2 with deletions of the SH3 domains were introduced into Tpr-Met transformed fibroblasts. Cells overexpressing the mutants were found to be morphologically reverted and exhibited reduced growth in soft agar. Surprisingly, the Grb2 mutants blocked activation of the JNK/SAPK but not MAP kinase activity induced by the Tpr-Met oncoprotein. Additionally, cells expressing dominant-negative Grb2 mutants had reduced PI-3-kinase activity and dominant-negative mutants of Rac1 blocked both Tpr-Met-induced transformation and activation of JNK. These experiments reveal a novel link between Met and the JNK pathway, which is essential for transformation by this oncogene.     

Sam68 associates with the SH3 domains of Grb2 recruiting GAP to the Grb2-SOS complex in insulin receptor signaling

The 68 kDa Src substrate associated during mitosis (Sam68) is an RNA binding protein with Src homology (SH) 2 and 3 domain binding sites. We have recently found that Sam68 is a substrate of the insulin receptor (IR) that translocates from the nucleus to the cytoplasm and that Tyr-phosphorylated Sam68 associates with the SH2 domains of p85 PI3K and GAP, in vivo and in vitro. In the present work, we have further demonstrated the cytoplasmic localization of Sam68, which is increased in cells overexpressing IR. Besides, we sought to further study the association of Sam68 with the Ras-GAP pathway by assessing the interactions with SH3 domains of Grb2. We employed GST-fusion proteins containing the SH3 domains of Grb2 (N or C), and recombinant Sam68 for in vitro studies. In vivo studies of protein-protein interaction were assessed by co-immunoprecipitation experiments with specific antibodies against Sam68, GAP, Grb2, SOS, and phosphotyrosine; and by affinity precipitation with the fusion proteins (SH3-Grb2). Insulin stimulation of HTC-IR cells promotes phosphorylation of Sam68 and its association with the SH2 domains of GAP. Sam68 is constitutively associated with the SH3 domains of Grb2 and it does not change upon insulin stimulation, but Sam68 is Tyr-phosphorylated and promotes the association of GAP with the Grb2-SOS complex. In vitro studies with fusion proteins showed that Sam68 association with Grb2 is preferentially mediated by the C-terminal SH3 domains of Grb2. In conclusion, Sam68 is a substrate of the IR and may have a role as a docking protein in IR signaling, recruiting GAP to the Grb2-SOS complex, and in this way it may modulate Ras activity.     

In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase.

We have used a transient expression system and mutant platelet-derived growth factor (PDGF) receptors to study the binding specificities of the Src homology 2 (SH2) regions of the Ras GTPase-activator protein (GAP) and the p85 alpha subunit of phosphatidylinositol 3-kinase (PI3 kinase). A number of fusion proteins, each tagged with an epitope allowing recognition by a monoclonal antibody, were expressed at levels comparable to those of endogenous GAP. Fusion proteins containing the central SH2-SH3-SH2 region of GAP or the C-terminal region of p85 alpha, which includes two SH2 domains, bound to PDGF receptors in response to PDGF stimulation. Both fusion proteins showed the same requirements for tyrosine phosphorylation sites in the PDGF receptor as the full-length proteins from which they were derived, i.e., binding of the GAP fusion protein was reduced by mutation of Tyr-771, and binding of the p85 fusion protein was reduced by mutation of Tyr-740, Tyr-751, or both residues. Fusion proteins containing single SH2 domains from either GAP or p85 alpha did not bind detectably to PDGF receptors in this system, suggesting that two SH2 domains in a single polypeptide cooperate to raise the affinity of binding. The sequence specificities of individual SH2 domains were deduced from the binding properties of fusion proteins containing one SH2 domain from GAP and another from p85. The results suggest that the C-terminal GAP SH2 domain specifies binding to Tyr-771, the C-terminal p85 alpha SH2 domain binds to either Tyr-740 or Tyr-751, and each protein's N-terminal SH2 domain binds to unidentified phosphorylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

GTPase-activating protein SH2-SH3 domains induce gene expression in a Ras-dependent fashion.

The p21ras GTPase-activating protein (GAP) is thought to function as both a negative regulator and a downstream target of p21ras. Here, we have investigated the role of GAP by using a transient expression assay with a fos luciferase reporter plasmid. We used GAP deletion mutants that lack the domain involved in interaction with p21ras and encode essentially only the SH2-SH3 domains. When these GAP deletion mutants were expressed, we observed a marked induction of fos promoter activity similar to induction by activated p21ras. Expression of a full-length GAP construct had no effect on the activity of the fos promoter. Activation of the fos promoter by these GAP SH2-SH3 regions was inhibited by cotransfection of a dominant inhibitory mutant of p21ras, Ras(Asn-17). Thus, the induction of gene expression by GAP SH2-SH3 domains is dependent on p21ras activity. Moreover, induction of fos promoter activity by GAP SH2-SH3 domains is increased severalfold after cotransfection of an activated mutant of p21ras, Ras(Leu-61), or insulin stimulation of A14 cells, both leading to an increase in the levels of GTP-bound p21ras. The combined effect of Ras(Leu-61) and the GAP deletion mutants was not inhibited by Ras(Asn-17), indicating that GAP SH2-SH3 domains do not function to activate endogenous p21ras but cooperate with another signal coming from active p21ras. These data suggest that GAP SH2-SH3 domains serve to induce gene expression by p21ras but that additional signals coming from p21ras are required for them to function.     

The epidermal growth factor receptor phosphorylates GTPase-activating protein (GAP) at Tyr-460, adjacent to the GAP SH2 domains.

GTPase-activating protein (GAP) stimulates the ability of p21ras to hydrolyze GTP to GDP. Since GAP is phosphorylated by a variety of activated or oncogenic protein-tyrosine kinases, it may couple tyrosine kinases to the Ras signaling pathway. The epidermal growth factor (EGF) receptor cytoplasmic domain phosphorylated human GAP in vitro within a single tryptic phosphopeptide. The same GAP peptide was also apparently phosphorylated on tyrosine in EGF-stimulated rat fibroblasts. Circumstantial evidence suggested that residue 460 might be the site of GAP tyrosine phosphorylation. This possibility was confirmed by phosphorylation of a synthetic peptide corresponding to the predicted tryptic peptide containing Tyr-460. Alteration of Tyr-460 to phenylalanine by site-directed mutagenesis diminished the in vitro phosphorylation of a bacterial GAP polypeptide by the EGF receptor. We conclude that Tyr-460 is a site of GAP tyrosine phosphorylation by the EGF receptor in vitro and likely in vivo. GAP Tyr-460 is located immediately C terminal to the second GAP SH2 domain, suggesting that its phosphorylation might have a role in regulating protein-protein interactions.     

The residues of Ras and Rap proteins that determine their GAP specificities.

The oncogenic transformation of a normal fibroblast by mutated Ras genes can be reversed by overexpression of a Ras-related gene called Rap1A (or Krev1). Both Ras and Rap1A proteins are G proteins and appear to serve as signal transducers only in the GTP-bound form. Therefore, GAP1 and GAP3, which stimulate the intrinsic GTPase activities of normal Ras and Rap1A proteins, respectively, serve as attenuators of their signal transducing activities. In this paper, we describe the enzymatic properties of several mutated Rap1A and chimeric Ras/Rap1A (or -1B) proteins which lead to the following conclusions: (i) the GAP3-dependent activation of both Rap1A and -1B GTPases requires Gly12, but neither Thr61 nor Gln63; (ii) residues 64 to 70 of the Rap1 GTPases are sufficient to determine their specificities for GAP3; and (iii) residues 61 to 65 of the Ras GTPases are sufficient for determining their specificities for GAP1. Thus, the domains of the Ras or Rap1 proteins that determine whether their signals are attenuated by GAP1 or GAP3 are distinct from the N-terminal domain (residues 21 to 54) that determines whether their signals are oncogenic or antioncogenic. The Arg12 mutant of chimeric HaRas(1-54)/Rap1A(55-184) protein has been previously reported to be oncogenic (Zhang, K., Noda, M., Vass, W. C., Papageorge, A.G., and Lowy, D.R. (1990) Science 249, 162-165). In this paper, we show that the Val12 mutant of chimeric HaRas(1-54)/Rap1B(55-184) protein is also oncogenic, suggesting that the C-terminal geranylgeranylation of the Rap 1B protein can replace functionally the C-terminal farnesylation of the Ras protein to allow the G protein to be oncogenic.     

Tight association of GRB2 with receptor protein-tyrosine phosphatase alpha is mediated by the SH2 and C-terminal SH3 domains.

Receptor protein-tyrosine phosphatase alpha (RPTPalpha), a transmembrane member of the extensive family of protein-tyrosine phosphatases (PTPs), is constitutively phosphorylated on Tyr789, a consensus binding site for the SH2 domain of the SH3-SH2-SH3 adaptor protein GRB2. We have previously shown that GRB2 binds to P.Tyr789 in vivo and in vitro via its SH2 domain. Here, we report that not only the GRB2 SH2 domain, but also the C-terminal SH3 domain is involved in binding to RPTPalpha in vitro and in vivo. Although the N-terminal SH3 domain of GRB2 is essential for binding to the Ras guanine nucleotide exchange factor Son of Sevenless (Sos), an RPTPalpha-GRB2-Sos complex could not be detected. The inclusion of peptides encompassing an hSos1 proline-rich motif in cell lysates resulted in enhanced binding of RPTPalpha to GRB2 in vitro, suggesting that steric hindrance prohibits formation of the RPTPalpha-GRB2-Sos complex. In vitro binding experiments indicated that the binding of GRB2 to Sos/dynamin and RPTPalpha was mutually exclusive. Analysis of in vitro binding kinetics coupled with results from transient co-transfections demonstrated that RPTPalpha is tightly bound to GRB2. The site of interaction of the C-terminal SH3 domain of GRB2 with RPTPalpha was mapped using deletion mutants to an 18-residue region in the N-terminal PTP domain. Arg469, within this region, was identified as one of the residues that is involved in the interaction with the C-terminal SH3 domain of GRB2. RPTPalpha residues 469-486 are localized close to the catalytic site cleft in the structure of the N-terminal PTP-domain, suggesting that interaction with the C-terminal SH3 domain may block access to the catalytic site, thus inhibiting RPTPalpha activity.