Crystal structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP

The biological functions of ras proteins are controlled by the bound guanine nucleotide GDP or GTP. The GTP-bound conformation is biologically active, and is rapidly deactivated to the GDP-bound conformation through interaction with GAP (GTPase Activating Protein). Most transforming mutants of ras proteins have drastically reduced GTP hydrolysis rates even in the presence of GAP. The crystal structures of the GDP complexes of ras proteins at 2.2 A resolution reveal the detailed interaction between the ras proteins and the GDP molecule. All the currently known transforming mutation positions are clustered around the bound guanine nucleotide molecule. The presumed "effector" region and the GAP recognition region are both highly exposed. No significant structural differences were found between the GDP complexes of normal ras protein and the oncogenic mutant with valine at position 12, except the side-chain of the valine residue. However, comparison with GTP-analog complexes of ras proteins suggests that the valine side-chain may inhibit GTP hydrolysis in two possible ways: (1) interacting directly with the gamma-phosphate and altering its orientation or the conformation of protein residues around the phosphates; and/or (2) preventing either the departure of gamma-phosphate on GTP hydrolysis or the entrance of a nucleophilic group to attack the gamma-phosphate. The structural similarity between ras protein and the bacterial elongation factor Tu suggests that their common structural motif might be conserved for other guanine nucleotide binding proteins.     

Ras GTPase-activating protein physically associates with mitogenically active phospholipids.

The physical interaction between GTPase-activating protein (GAP) and lipids has been characterized by two separate analyses. First, bacterially synthesized GAP molecules were found to associate with detergent-mixed micelles containing arachidonic but not with those containing arachidic acid. This association was detected by a faster elution time during molecular exclusion chromatography. Second, GAP molecules within a crude cellular lysate were specifically retained by a column on which certain lipids had been immobilized. The lipids able to retain GAP on such columns were identical to those which were shown previously to be most active in blocking GAP activity. The association between lipids and GAP was dependent upon magnesium ions. Lipids unable to inhibit GAP activity were also unable to physically associate with GAP. The tight association of GAP with these lipids was predicted by and helps to rationalize their ability to inhibit GAP activity.     

Coupling of ras p21 signalling and GTP hydrolysis by GTPase activating proteins.

Ras p21 proteins cycle between inactive, GDP-bound forms and active GTP-bound forms. Hydrolysis of bound GTP to GDP is mediated by proteins referred to as GAPs, two forms of which have been described. The first, p120-GAP, contains regions of homologies with tyrosine kinase oncogenes, and interacts with tyrosine phosphoproteins as well as with ras proteins; p120-GAP may therefore connect signalling pathways that involve tyrosine kinase and ras p21 proteins. The second type of GAP is the product of the neurofibromatosis type 1 gene (NF1-GAP). This is a protein of 325,000 Da that is defective in patients with NF1; NF1-GAP is regulated by signalling lipids, and may serve to connect ras p21 with phospholipid second messenger systems. The significance of ras p21 interaction with distinct GAPs is discussed.     

Serum stimulation of NIH 3T3 cells induces the production of lipids able to inhibit GTPase-activating protein activity.

Quiescent NIH 3T3 cells were stimulated with serum prior to the extraction of total cellular lipids. These lipids were fractionated on thin-layer chromatography plates, and individual fractions were tested for the ability to inhibit GTPase-activating protein (GAP) activity. Two separate GAP inhibitory lipids were produced. One behaved similarly to arachidonic acid during silica gel chromatography, whereas the other was related to a phosphoinositide. Further study of the arachidonic acid-related material indicated that it was produced between 1 and 5 min after serum addition but was never observed in high-density, contact-inhibited cultures. The identity of these lipids is under investigation. The possibility raised by these results, that a metabolite of arachidonic acid is involved in mitogenic signaling, was supported by the finding that several lipoxygenase products of arachidonic acid efficiently inhibited GAP activity. These results provide further support for the hypothesis that lipids, GAP, and ras activity function together in the control of cellular proliferation.     

A synthetic peptide corresponding to a sequence in the GTPase activating protein inhibits p21ras stimulation and promotes guanine nucleotide exchange

Amino acid sequence homology between the GTPase Activating Protein (GAP) and the GTP-binding regulatory protein, Gs alpha, suggests that a specific region of GAP primary structure (residues 891-898) may be involved in its stimulation of p21ras GTP hydrolytic activity (McCormick, F. [1989] Nature 340, 678-679). A peptide, designated p891, corresponding to GAP residues 891-906 (M891RTRVVSGFVFLRLIC906) was synthesized and tested for its ability to inhibit GAP-stimulated p21ras GTPase activity. At a concentration of 25 microM, p891 inhibited GAP activity approximately 50%. Unexpectedly, p891 also stimulated GTP binding to p21N-ras independent of GAP. This stimulation correlated with an enhancement of p21N-ras.GDP dissociation; an approximate 15-fold increase in the presence of 10 microM p891. In contrast, dissociation of the p21N-ras.GTP gamma S complex was unaffected by 10 microM p891. The p21N-ras.GDP complex was unresponsive to 100 microM mastoparan, a peptide toxin shown previously to accelerate GDP dissociation from the guanine nucleotide regulatory proteins, Gi and Go. p21H-ras, as well as the two p21H-ras effector mutants, Ala-38, and Ala-35, Leu-36, also exhibited increased rates of GDP dissociation in the presence of p891. Also tested were three ras-related GTP-binding proteins; rap, G25K and rac. The rap.-GDP complex was unaffected by 10 microM p891. Dissociation of the G25K- and rac.GDP complexes were enhanced slightly; approximately 1.3- and 1.8-fold over control, respectively. Thus, the inhibitory effect of p891 on GAP stimulation of p21ras suggests that amino acids within the region 891-906 of GAP may be essential for interaction with p21ras. In addition, p891 independently affects the nucleotide exchange properties of p21ras.     

Identification of amino acid residues required for Ras p21 target activation.

The Krev-1 gene has been shown to suppress ras-mediated transformation in vitro. Both ras and Krev-1 proteins have identical effector domains (ras residues 32 to 40), which are required for biological activity and for the interaction of Ras p21 with Ras GTPase-activating protein (GAP). In this study, five amino acid residues flanking the ras effector domain, which are not conserved with the Krev-1 protein, were shown to be required for normal protein-protein interactions and biological activity. The substitution of Krev-1 p21 residues 26, 27, 30, 31, and 45 with the corresponding amino acid residues from Ras p21 resulted in a Krev-1 protein which had ras function in both mammalian and yeast biological assays. Replacement of these residues in Ras p21 with the corresponding Krev-1 p21 amino acids resulted in ras proteins which were impaired biologically or reduced in their affinity for in vitro GAP binding. Evaluation of these mutant ras proteins have implications for Ras p21-GAP interactions in vivo.     

The catalytic domain of the neurofibromatosis type 1 gene product stimulates ras GTPase and complements ira mutants of S. cerevisiae

Sequencing of the neurofibromatosis gene (NF1) revealed a striking similarity among NF1, yeast IRA proteins, and mammalian GAP (GTPase-activating protein). Using both genetic and biochemical assays, we demonstrate that this homology domain of the NF1 protein interacts with ras proteins. First, expression of this NF1 domain suppressed the heat shock-sensitive phenotype of yeast ira1 and ira2 mutants. Second, this NF1 domain, after purification as a glutathione S-transferase (GST) fusion protein, strongly stimulated the GTPase activity of yeast RAS2 and human H-ras proteins. The GST-NF1 protein, however, did not stimulate the GTPase activity of oncogenic mutant ras proteins, H-rasVal-12 and yeast RAS2Val-19 mutants, or a yeast RAS2 effector mutant. These results establish that this NF1 domain has ras GAP activity similar to that found with IRA2 protein and mammalian GAP, and therefore may also regulate ras function in vivo.     

H-ras mutants lacking the epitope for the neutralizing monoclonal antibody Y13-259 show decreased biological activity and are deficient in GTPase-activating protein interaction.

We have generated deletion mutants of the H-ras p21 protein which lack residues 58 to 63 or 64 to 68 and contain either the normal glycine or an activating mutation, arginine, at position 12. None of the deleted proteins were recognized by monoclonal antibody Y13-259, and those mutants with activating mutations showed at least a 100-fold reduction in their transforming activities compared with the activities of their nondeleted counterparts. Alterations observed in the in vitro GTPase or GTP interchange properties of the deletion mutants were not consistent with the decrease in their transforming activities. Moreover, each mutant showed normal membrane localization, which is essential for its biological activity. Recently, a newly identified protein, designated GTPase-activating protein (GAP), was found to markedly increase GTPase activity of the normal ras p21 but not of p21 mutants bearing activating lesions (H. Adari, D. R. Lowy, B. M. Willumsen, C. J. Der, and F. McCormick, Science 240:518-521, 1988). We showed that GAP had no effect on the in vitro GTPase activity of the deletion mutants of the normal p21 protein. Since similar deletions in mutants with activating lesions at position 12 or 59 or both showed decreased transforming activity, our results suggest that the recognition site for Y13-259 within the ras p21 molecule influences directly or indirectly the interaction of ras p21 with GAP and that this interaction is critical for biological activity of ras proteins.     

The GTPase stimulatory activities of the neurofibromatosis type 1 and the yeast IRA2 proteins are inhibited by arachidonic acid.

Three proteins, GTPase activating protein (GAP), neurofibromatosis 1 (NF1) and the yeast inhibitory regulator of the RAS-cAMP pathway (IRA2), have the ability to stimulate the GTPase activity of Ras proteins from higher animals or yeast. Previous studies indicate that certain lipids are able to inhibit this activity associated with the mammalian GAP protein. Inhibition of GAP would be expected to biologically activate Ras protein. In these studies arachidonic acid is shown also to inhibit the activity of the catalytic fragments of the other two proteins, mammalian NF1 and the yeast IRA2 proteins. In addition, phosphatidic acid (containing arachidonic and stearic acid) was inhibitory for the catalytic fragment of NF1 protein, but did not inhibit the catalytic fragments of GAP or IRA2 proteins. These observations emphasize the biochemical similarity of these proteins and provide support for the suggestion that lipids might play an important role in their biological control, and therefore also in the control of Ras activity and cellular proliferation.     

How does p21ras transform cells?

Oncogenic forms of p21ras are found in a wide range of human tumors. However, the mechanism by which p21ras transforms remains obscure. Genetic evidence has identified a domain of p21ras that is involved with interaction with an effector molecule required for transformation. Two proteins, GAP and the tumor suppressor NF1, interact with p21ras in this region but it is an unresolved puzzle whether either of these is the an unresolved puzzle whether either of these is the effector. After interaction with an effector, two downstream events--activation of protein kinase C and another pathway--are necessary for induction of DNA synthesis by oncogenic p21ras; however, morphological transformation does not require activation of protein kinase C.     

Specificity of Inhibition of ras-p21 Signal Transduction by Peptides from GTPase Activating Protein (GAP) and the Son-of Sevenless (SOS) ras-Specific Guanine Nucleotide Exchange Protein

In previous studies, involving molecular modeling of wild-type and oncogenic forms of the ras-p21 protein bound to GTPase activating protein GAP and the ras-specific guanine nucleotide exchange-promoting protein, SOS, we identified specific domains of GAP and SOS proteins that differ in conformation when the computed average structures of the corresponding wild-type and oncogenic complexes are superimposed. Additionally, in these previous studies, we have synthesized peptides corresponding to these domains and found that all of them inhibit either or both oncogenic (Val 12-containing) p21- and insulin-activated wild-type p21-induced oocyte maturation. To document further the specificity of the inhibition of these peptides for the ras signal transduction pathway, we have now tested their effects on progesterone-induced maturation that occurs by a ras-independent pathway. None of these peptides, including a peptide corresponding to residues 980–989 of SOS that completely blocks oncogenic p21-induced maturation and also causes extensive inhibition of insulin-induced maturation, affects progesterone-induced maturation, suggesting that all of these peptides are specific for the ras pathway. Since our approach to the design of peptides that can inhibit oncogenic ras-p21 selectively is based on identifying domains that differ in conformation between oncogenic and wild-type complexes, we have now further synthesized peptides that correspond to domains of GAP (residues 903–910) and SOS (residues 792–804) that do not differ in conformation when the average structures are superimposed. These peptides do not inhibit either oncogenic p21- or insulin-induced oocyte maturation, supporting the overall strategy of using peptides from domains that change conformation as the ones most likely to inhibit oncogenic and/or wild-type ras-p21. These results further support the specificity of inhibition of the GAP and SOS peptides from the conformationally distinct domains of both proteins.     

Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.     

Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins

The protein products of the mammalian ras genes, p21ras, are regulatory guanine nucleotide binding proteins that are involved in the control of cell proliferation, though the exact biochemical processes regulated are unknown. Recently a cytoplasmic protein has been identified that interacts with and increases the GTPase activity of p21ras. It has been shown that this GTPase-activating protein, or GAP, interacts with the effector domain of ras, leading us and others to propose that GAP may be the target for regulation by p21ras. It has become apparent that ras is part of a much larger family of proteins, and at least 15 ras-related genes have now been identified in the mammalian genome. Each encodes a small (about 21 kDa) guanine nucleotide binding protein, but the functions of none of these regulatory molecules are known. We report here that mammalian cytoplasmic extracts contain GAP-like activity toward the products of two other ras-related genes, R-ras and rho. It appears that p23R-ras interacts with the same 125-kDa GAP protein as p21ras whereas p21rho interacts with a distinct 29-kDa protein, rho GAP.     

Regulation of tetradecanoyl phorbol acetate-induced responses in NIH 3T3 cells by GAP, the GTPase-activating protein associated with p21c-ras.

Proteins of the ras family of oncogenes have been implicated in signal transduction pathways initiated by protein kinase C (PKC) and by tyrosine kinase oncogenes and receptors, but the role that ras plays in these diverse signalling systems is poorly defined. The activity of ras proteins has been shown to be controlled in part by a cellular protein, GAP (GTPase-activating protein), that negatively regulates p21c-ras by enhancing its intrinsic GTPase activity. Thus, overexpression of GAP provides a tool for determining the step(s) in signal transduction dependent on p21c-ras activity. In this paper, we report that overexpression of GAP blocks the phorbol ester (tetradecanoyl phorbol acetate [TPA])-induced activation of p42 mitogen-activated protein kinase (p42mapk), c-fos expression, and DNA synthesis. GAP overexpression did not block responses to serum or fluoroaluminate. Moreover, not all biochemical events elicited by TPA were affected by GAP overexpression, as increased glucose uptake and phosphorylation of MARCKS, a major PKC substrate, occurred normally. Reduction of GAP expression to near normal levels restored the ability of the cells to activate p42mapk in response to TPA. These findings suggest that ras and GAP together play a key role in a PKC-dependent signal transduction pathway which leads to p42mapk activation and cell proliferation.     

Effector domain mutations dissociate p21ras effector function and GTPase-activating protein interaction.

The GTPase activity of p21ras is stimulated by GTPase-activating proteins (GAPs) such as p120GAP and the product of the neurofibromatosis 1 gene, which may negatively regulate p21 function. GAPs are also proposed effectors of ras. We have sought activating substitutions in c-H-ras in the region encoding the effector domain, on the rationale that such mutations would dissociate effector function from negative regulation by GAP. One such activating mutation, Pro-34-->Arg, encodes protein that is substantially bound to GTP in vivo. In vitro, this protein is not stimulated by GAPs, and its binding to p120GAP is grossly impaired. The results support the idea that the p21 structural requirements for effector function and GAP interaction are quite different and suggest that some molecule(s) other than p120GAP serves as the ras effector. In contrast to the results obtained with p120GAP, the Pro-34-->Arg p21 species is effectively coupled to the raf-1 product, as judged from electrophoretic mobility shifts of the Raf-1 phosphoprotein.     

Viral latency and transformation: the strategy of Epstein-Barr virus

Evidence that GAP is an effector of ras action can be summarized as follows: GAP interacts at a site on p21 defined genetically as the effector binding site. Regions of p21 that are nonessential for biological activity are nonessential for GAP interaction. GAP interacts with all known types of p21. (Upstream factors are expected to be specific for individual types). GAP interacts with p21 proteins (normal and mutant) in a GTP-dependent fashion. None of these constitute proof. It remains possible that GAP simply regulates p21-GTP levels, and binds to the same site as the true effector without transmitting a downstream signal. If indeed GAP mediates ras action, the question immediately arises as to the biochemical function of GAP itself. The requirement of ras proteins for membrane localization to exert their effects may be a valuable clue in the search for this function. Perhaps GAP is an enzyme (or is bound to an enzyme) that acts on membrane components in a p21-GTP-dependent manner and in doing so transmits signals to other downstream effectors. The ability of GAP to interact with many members of the ras family would allow many upstream signals to feed into this downstream pathway. Clearly, proof (or disproof) that GAP is downstream of ras is the next step toward clarification of this aspect of ras action; identification of biochemical activities associated with GAP (or the true ras effector) will, we hope, follow soon.     

Plasma membrane-targeted ras GTPase-activating protein is a potent suppressor of p21ras function.

Although p21ras is localized to the plasma membrane, proteins it interacts with, such as the GTPase-activating proteins (GAPs) ras GAP and neurofibromin (NF1), are not, suggesting that one function of p21ras GTP may be to target such proteins to the plasma membrane. To investigate the effects of targeting ras GAP to the plasma membrane, ras C-terminal motifs sufficient for plasma membrane localization of p21ras were cloned onto the C terminus of ras GAP. Plasma membrane-targeted ras GAP is growth inhibitory to NIH 3T3 fibroblasts and COS cells. This growth inhibition correlates with GAP catalytic activity, since the plasma membrane-targeted C-terminal catalytic domain or the GAP-related domain of neurofibromin is inhibitory, whereas the similarly targeted N-terminal domain is not. Moreover, the inhibition is abrogated by the inactivating mutation L902I, which abolishes ras GAP catalytic activity. Coexpression of oncogenic mutant ras rescues cell viability, but the majority of rescued colonies are phenotypically untransformed. Furthermore, in focus assays, targeted ras GAP suppresses transformation by oncogenic mutant ras, and in reversion assays, targeted ras GAP can revert cells transformed by oncogenic mutant ras. Neither the targeted or nontargeted N-terminal domain nor the L902I mutant of ras GAP has any transforming activity. These data demonstrate that ras GAP can function as a negative regulator of ras and that plasma membrane localization potentiates this activity. However, if ras GAP is involved in the effector functions of p21ras, it can only be part of the effector complex for cell transformation.     

Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s

ras p21 GTPase-activating protein (GAP) has been proposed to interact with the putative effector domain of ras p21s, and smg p21, a ras p21-like guanine nucleotide binding protein (G protein), has been shown to have the same amino acid sequence as ras p21s in this region. In the present studies, we examined the effects of ras p21 GAP on the GTPase activity of smg p21 purified from human platelets, of smg p21 on the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 purified from Escherichia coli, and of c-Ha-ras p21 on the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. ras p21 GAP stimulated the GTPase activity of c-Ha-ras p21 but not that of smg p21. The GTP-bound form of smg p21, however, inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21 in a dose-dependent manner. The half-maximum inhibition by smg p21 was obtained at 0.4 microM which was more potent than previously observed for ras p21 (2-200 microM). The GDP-bound form also inhibited the ras p21 GAP-stimulated GTPase activity of c-Ha-ras p21, but the efficiency was 40-50% that of the GTP-bound form. smg p21 GAP1 and -2 stimulated the GTPase activity of smg p21 but not that of c-Ha-ras p21. c-Ha-ras p21 did not inhibit the smg p21 GAP1- or -2-stimulated GTPase activity of smg p21. These results indicate that ras p21 GAP interacts with smg p21 without the subsequent stimulation of its GTPase activity.     

Transformation by pp60src or stimulation of cells with epidermal growth factor induces the stable association of tyrosine-phosphorylated cellular proteins with GTPase-activating protein.

GTPase-activating protein (GAP) is a cytosolic protein that stimulates the rate of hydrolysis of GTP (GTP to GDP) bound to normal p21ras, but does not catalyze the hydrolysis of GTP bound to oncogenic, activated forms of the ras protein. Transformation of cells with v-src or activated transforming variants of c-src or stimulation of cells with epidermal growth factor resulted in the stable association of GAP with two tyrosine-phosphorylated cellular proteins of 64 kDa (p64) and 190 kDa (p190). Analysis of GAP immune complexes isolated from extracts of metabolically labeled src-transformed cells and epidermal growth factor-stimulated cells indicated that tyrosine phosphorylation of p64 and p190 appeared to be coincident with the stable association of these proteins with GAP. Quantitation of the amount of p64 associated with GAP in v-src-transformed cells, however, indicated that only 15 to 25% of tyrosine-phosphorylated p64 was found in complex with GAP. Mutations within the SH2 region of pp60src that render activated pp60src defective for transformation inhibited the efficient formation of complexes between GAP and the tyrosine-phosphorylated forms of p64 and p190. From these data, we suggest that tyrosine phosphorylation and stable association of p64 with GAP is an important step in mediating cellular signaling through the p21ras-GAP pathway.     

A ras effector domain mutant which is temperature sensitive for cellular transformation: interactions with GTPase-activating protein and NF-1.

A series of v-rasH effector domain mutants were analyzed for their ability to transform rat 2 cells at either low or high temperatures. Three mutants were found to be significantly temperature sensitive: Ile-36 changed to Leu, Ser-39 changed to Cys (S39C), and Arg-41 changed to Leu. Of these, the codon 39 mutant (S39C) showed the greatest degree of temperature sensitivity. When the same mutation was analyzed in the proto-oncogene form of ras(c-rasH), this gene was also found to be temperature sensitive for transformation. Biochemical analysis of the proteins encoded by v-rasH(S39C) and c-rasH(S39C) demonstrated that the encoded p21ras proteins were stable and bound guanine nucleotides in vivo at permissive and nonpermissive temperatures. On the basis of these findings, it is likely that the temperature-sensitive phenotype results from an inability of the mutant (S39C) p21ras to interact properly with the ras target effector molecule(s) at the nonpermissive temperature. We therefore analyzed the interaction between the c-rasH(S39C) protein and the potential target molecules GTPase-activating protein (GAP) and the GAP-related domain of NF-1, on the basis of stimulation of the mutant p21ras GTPase activity by these molecules in vitro. Assays conducted across a range of temperatures revealed no temperature sensitivity for stimulation of the mutant protein, compared with that of authentic c-rasH protein. We conclude that for this mutant, there is a dissociation between the stimulation of p21ras GTPase activity by GAP and the GAP-related domain NF-1 and their potential target function. Our results are also consistent with the existence of a distinct, as-yet-unidentified effector for mammalian ras proteins.