Dominant inhibitory Ras mutants selectively inhibit the activity of either cellular or oncogenic Ras.

Two dominant inhibitory Ras mutant proteins were analyzed by microinjection. One, [Asn-17]Ras, had a substitution in the putative Mg(2+)-binding site of Ha-Ras. The other, RAST, had a mutation in a yeast RAS protein that impaired its GTPase activity and increased its affinity for GAP. RAST also had a mutation that blocked its localization to the plasma membrane. In NIH 3T3 cells [Asn-17]Ras inhibited the function of normal Ras much more efficiently than that of oncogenic Ras. In contrast, RAST interfered with the transforming activity of oncogenic Ras more efficiently than that of normal Ras. These conclusions were based on two separate types of analysis. The inhibitory Ras mutant proteins were first microinjected into cells stably transformed either by oncogenic Ras or by high levels of expression of cellular Ras. Results obtained in stably transformed cells were then verified by coinjection of the inhibitory Ras mutant proteins together with transforming concentrations of either oncogenic or normal Ras protein. Whereas RAST was active in soluble form. [Asn-17]Ras required membrane localization for activity. Furthermore, mutations in the GAP/effector-binding domain reduced or eliminated the inhibitory activity of RAST but had no detectable effect on [Asn-17]Ras. These results are consistent with the possibility that [Asn-17]Ras functions by blocking the activation of endogenous Ras proteins, while RAST functions by blocking the ability of activated Ras to stimulate a downstream target within the cells. The properties of RAST suggest that interference with the GAP/effector-binding function of RAS represents a strategy for the preferential inactivation of oncogenic Ras in cells.     

Fluoride complexes of oncogenic Ras mutants to study the Ras-RasGap interaction

Down-regulation of Ras signalling is mediated by specific GTPase-activating proteins (GAPs), which stimulate the very slow GTPase reaction of Ras by 10(5)-fold. The basic features of the GAP activity involve the stabilisation of both switch regions of Ras in the transition state, and the insertion of an arginine finger. In the case of oncogenic Ras mutations, the features of the active site are disturbed. To understand these features in more detail, we have investigated the effects of oncogenic mutations of Ras and compared the GAP-stimulated GTPase reaction with the ability to form GAP-mediated aluminium or beryllium fluoride complexes. In general we find a correlation between the size of the amino acid at position 12, the GTPase activity and ability to form aluminium fluoride complexes. While Gly12 is very sensitive to even the smallest possible structural change, Gly13 is much less sensitive to steric hindrance, but is sensitive to charge. Oncogenic mutants of Ras defective in the GTPase activity can however form ground-state GppNHp complexes with GAP, which can be mimicked by beryllium fluoride binding. We show that beryllium fluoride complexes are less sensitive to structural changes and report on a state close to but different from the ground state of the GAP-stimulated GTPase reaction.     

Differential regulation of cellular activities by GTPase-activating protein and NF1.

The regulation of the GTPase activity of the Ras proteins is thought to be a key element of signal transduction. Ras proteins have intrinsic GTPase activity and are active in signal transduction when bound to GTP but not following hydrolysis of GTP to GDP. Three cellular Ras GTPase-activating proteins (Ras-gaps) which increase the GTPase activity of wild-type (wt) Ras but not activated Ras in vitro have been identified: type I and type II GAP and type I NF1. Mutations of wt Ras resulting in lowered intrinsic GTPase activity or loss of response to cellular Ras-gap proteins are thought to be the primary reason for the transforming properties of the Ras proteins. In vitro assays show type I and type II GAP and the GAP-related domain of type I NF1 to have similar biochemical properties with respect to activation of the wt Ras GTPase, and it appears as though both type I GAP and NF1 can modulate the GTPase function of Ras in cells. Here we report the assembling of a full-length coding clone for type I NF1 and the biological effects of microinjection of Ras and Ras-gap proteins into fibroblasts. We have found that type I GAP, type II GAP, and type I NF1 show markedly different biological activities in vivo. Coinjection of type I GAP or type I NF1, but not type II GAP, with wt Ras abolished the ability of wt Ras to induce expression from an AP-1-controlled reporter gene. We also found that serum-stimulated DNA synthesis was reduced by prior injection of cells with type I GAP but not type II GAP or type I NF1. These results suggest that type I GAP, type II GAP, and type I NF1 may have different activities in vivo and support the hypothesis that while type I forms of GAP and NF1 may act as negative regulators of wt Ras, they may do so with differential efficiencies.     

Mechanism of Activation of the Caenorhabditis Elegans Ras Homologue Let-60 by a Novel, Temperature-Sensitive, Gain-of-Function Mutation

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°, ga89 increases let-60 activity resulting in a multivulva phenotype. At 15°, 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.     

Characterization of a GTPase-activating protein for the Ras-related Ral protein

We have demonstrated the presence of a GTPase-activating protein (GAP) for the Ras-related Ral A protein in the cytosolic fraction of brain and testis. This protein, designated Ral-GAP, was distinguished from Ras-GAP by its behavior in two chromatography systems and by the fact that the two GAP proteins did not stimulate the GTPase activity of each others target GTP binding proteins. The lack of effect of Ral-GAP on Ras GTPase activity also distinguished it from the product of the neurofibromatosis gene NF-1. Ral-GAP also differed from Rho-GAP and Rap-GAP by virtue of its elution from a gel filtration column with proteins of Mr greater than 10(6). This was likely an overestimate of the protein's molecular mass, however, since it sedimented in sucrose gradients between standard proteins of 150 and 443 kDa. Ral-GAP failed to promote the GTPase activity of mutant Ral proteins containing amino acid substitutions that in Ras lead to GAP-insensitive proteins.     

Ral-GTPases mediate a distinct downstream signaling pathway from Ras that facilitates cellular transformation.

Ral proteins (RalA and RalB) comprise a distinct family of Ras-related GTPases (Feig and Emkey, 1993). Recently, Ral-GDS, the exchange factor that activates Ral proteins, has been shown to bind specifically to the activated forms of RasH, R-Ras and Rap1A, in the yeast two-hybrid system. Here we demonstrate that although all three GTPases have the capacity to bind Ral-GDS in mammalian cells, only RasH activates Ral-GDS. Furthermore, although constitutively activated Ra1A does not induce oncogenic transformation on its own, its expression enhances the transforming activities of both RasH and Raf. Finally, a dominant inhibitory form of RalA suppresses the transforming activities of both RasH and Raf. These results demonstrate that activation of Ral-GDS and thus its target, Ral, constitutes a distinct downstream signaling pathway from RasH that potentiates oncogenic transformation.     

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.     

Identification of amino acid residues of Ras protein that are essential for signal-transducing activity but not for enhancement of GTPase activity by GAP.

To determine the amino acid residues required for the signal-transducing activity of the human c-Ha-Ras protein, we introduced point mutations at residues 45-54 near the 'effector region' (residues 32-40). We transfected PC12 cells with these mutant genes and also micro-injected the mutant proteins, bound with an unhydrolyzable GTP analog, into PC12 cells. Both procedures showed that Val45----Glu and Gly48----Cys mutations impaired the ability of the Ras protein to induce morphological change of PC12 cells. These mutations did not affect the guanine nucleotide-binding activity or GTPase activity in the absence or presence of bovine GTPase-activating protein (GAP). Therefore, the Val45 and Gly48 residues should be included by definition in the effector region responsible for the signal transduction, while only a subset of the effector-region residues is required for enhancement of the GTPase activity by GAP.     

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.     

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.     

The role of Gln61 and Glu63 of Ras GTPases in their activation by NF1 and Ras GAP.

Two distinct GAPs of 120 and 235 kDa called GAP1 and NF1 serve as attenuators of Ras, a member of GTP-dependent signal transducers, by stimulating its intrinsic guanosine triphosphatase (GTPase) activity. The GAP1 (also called Ras GAP) is highly specific for Ras and does not stimulate the intrinsic GTPase activity of Rap1 or Rho. Using GAP1C, the C-terminal GTPase activating domain (residues 720-1044) of bovine GAP1, we have shown previously that the GAP1 specificity is determined by the Ras domain (residues 61-65) where Gln61 plays the primary role. The corresponding domain (residues 1175-1531) of human NF1 (called NF1C), which shares only 26% sequence identity with the GAP1C, also activates Ras GTPases. In this article, we demonstrate that the NF1C, like the GAP1C, is highly specific for Ras and does not activate either Rap1 or Rho GTPases. Furthermore, using a series of chimeric Ras/Rap1 and mutated Ras GTPases, we show that Gln at position 61 of the GTPases primarily determines that NF1C as well as GAP1C activates Ras GTPases, but not Rap1 GTPases, and Glu at position 63 of the GTPases is required for maximizing the sensitivity of Ras GTPases to both NF1C and GAP1C. Interestingly, replacement of Glu63 of c-HaRas by Lys reduces its intrinsic GTPase activity and abolishes the GTPase activation by both NF1C and GAP1C. Thus, the potentiation of oncogenicity by Lys63 mutation of c-HaRas appears primarily to be due to the loss of its sensitivity to the two major Ras signal attenuators (NF1 and GAP1).     

Relationship among guanine nucleotide exchange, GTP hydrolysis, and transforming potential of mutated ras proteins.

The effect of a series of mutations on the transforming potential of normal human rasH has been compared with their effects on GTPase and guanine nucleotide exchange rates of p21. The mutation Val-146 resulted in partial activation of transforming potential which could be attributed to a greater than 1,000-fold-increased rate of nucleotide exchange in the absence of an effect on GTPase. In contrast, the more modest enhancement of exchange rate (approximately 100-fold) which resulted from the mutation Met-14 did not affect biological activity. The partially activating mutation Thr-59 was found to result in both a 5-fold reduction in GTPase and a 10-fold increase in nucleotide exchange. However, the nontransforming mutant Ile-59 displayed a comparable decrease in GTPase without an effect on nucleotide exchange. The activating effect of the Thr-59 mutation may thus represent a combined effect of reduced GTPase and increased exchange. Similarly, the strongly activating mutation Leu-61 resulted in a fivefold increase in nucleotide exchange in addition to decreased GTPase, whereas weakly activating mutations at position 61 (Trp and Pro) resulted only in decreased GTPase without affecting nucleotide exchange rates. Finally, combining the two mutations Met-14 and Ile-59, which alone had no effect on biological activity, yielded a double mutant with a 20-fold increased transforming potential, demonstrating a synergistic effect of these two mutations. Overall, these results indicate that large increases in nucleotide exchange can activate ras transforming potential in the absence of decreased GTPase and that relatively modest increases in nucleotide exchange can act synergistically with decreased GTPase to contribute to ras activation.     

Guanine-nucleotide binding activity, interaction with GTPase-activating protein and solution conformation of the human c-Ha-Ras protein catalytic domain are retained upon deletion of C-terminal 18 amino acid residues

A trucated human c-Ha-Ras protein that lacks the C-terminal 18 amino acid residues and the truncated Ras protein with the amino acid substitution Gly Val in position 12 were prepared by anE. coli overexpression system. The truncated Ras protein showed the same guanine-nucleotide binding activity and GTPase activity as those of the full-length Ras protein. Further, the same extent of GTPase activity enhancement due to GTPase-activating protein was observed for the truncated and full-length Ras proteins. In fact, two-dimensional proton NMR analyses indicated that the tertiary structure of the truncated Ras protein (GDP-bound or GMPPNP-bound) was nearly the same as that of the corresponding catalytic domain of the full-length Ras protein. Moreover, a conformational change around the effector region upon GDP GMPPNP exchange occurred in the same manner for both proteins. These observations indicate that the C-terminal flanking region (18 amino acid residues) of the Ras protein does not appreciably interact with the catalytic domain. Therefore, the truncated Ras protein is suitable for studying the molecular mechanism involved in the GTPase activity and the interaction with the GTPase-activating protein. On the other hand, an active form of the truncated Ras protein, unlike that of the full-length Ras protein, did not induce neurite outgrowth of rat pheochromocytoma PC12 cells. Thus, membrane anchoring of the Ras protein through its C-terminal four residues is not required for the interaction of Ras and GAP, but may be essential for the following binding of the Ras-GAP complex with the putative downstream target.     

Compartmentalized Ras proteins transform NIH 3T3 cells with different efficiencies

Ras GTPases were long thought to function exclusively from the plasma membrane (PM). However, a current model suggests that Ras proteins can compartmentalize to regulate different functions, and an oncogenic H-Ras mutant that is restricted to the endomembrane can still transform cells. In this study, we demonstrated that cells transformed by endomembrane-restricted oncogenic H-Ras formed tumors in nude mice. To define downstream targets of endomembrane Ras pathways, we analyzed Cdc42, which concentrates in the endomembrane and has been shown to act downstream of Ras in Schizosaccharomyces pombe. Our data show that cell transformation induced by endomembrane-restricted oncogenic H-Ras was blocked when Cdc42 activity was inhibited. Moreover, H-Ras formed a complex with Cdc42 on the endomembrane, and this interaction was enhanced when H-Ras was GTP bound or when cells were stimulated by growth factors. H-Ras binding evidently induced Cdc42 activation by recruiting and/or activating Cdc42 exchange factors. In contrast, when constitutively active H-Ras was restricted to the PM by fusing to a PM localization signal from the Rit GTPase, the resulting protein did not detectably activate Cdc42 although it activated Raf-1 and efficiently induced hallmarks of Ras-induced senescence in human BJ foreskin fibroblasts. Surprisingly, PM-restricted oncogenic Ras when expressed alone could only weakly transform NIH 3T3 cells; however, when constitutively active Cdc42 was coexpressed, together they transformed cells much more efficiently than either one alone. These data suggest that efficient cell transformation requires Ras proteins to interact with Cdc42 on the endomembrane and that in order for a given Ras protein to fully transform cells, multiple compartment-specific Ras pathways need to work cooperatively.     

Stimulation of mitogen-activated protein kinase by oncogenic Ras p21 in Xenopus oocytes. Requirement for Ras p21-GTPase-activating protein interaction.

p21ras plays an important role in the control of cell proliferation. The molecular mechanisms implicated are unknown. We report that the injection of oncogenic Lys12 Ras into Xenopus laevis oocytes promoted the activation of mitogen-activated protein kinase (MAP kinase) after a lag of about 90 min. MAP kinase activity was 10-fold higher 4 h after injection of oncogenic Lys12 Ras than after injection of nononcogenic Gly12 Ras. The stimulated MAP kinase activity remained at a plateau for at least 18 h. Maximal stimulation was obtained with 5 ng of Lys12 Ras, which is similar to the amount that elicits germinal vesicle breakdown. DEAE-Sephacel chromatography of extracts from Lys12 Ras-injected oocytes showed one peak of MAP kinase. MAP kinase activation by Lys12 Ras was associated with tyrosine phosphorylation of MAP kinase (p42). As previously shown, the S6-kinase II (likely pp90rsk), which is activated in vitro by MAP kinase, was also activated by oncogenic Lys12 Ras. Lys12 Ras with an additional mutation (Glu38) in the effector region that binds GTPase-activating protein (GAP) did not promote MAP kinase or S6 kinase activations. Thus, GAP may be involved downstream to Ras in these activation processes. Our results indicate that the Ras-GAP complex promotes MAP kinase activation in oocytes. This supports the idea that Ras-GAP controls MAP kinase, a kinase implicated in the action of various stimuli.     

A novel RalGEF-like protein, RGL3, as a candidate effector for rit and Ras

The small GTPase Rit is a close relative of Ras, and constitutively active Rit can induce oncogenic transformation. Although the effector loops of Rit and Ras are highly related, Rit fails to interact with the majority of the known Ras candidate effector proteins, suggesting that novel cellular targets may be responsible for Rit transforming activity. To gain insight into the cellular function of Rit, we searched for Rit-binding proteins by yeast two-hybrid screening. We identified the C-terminal Rit/Ras interaction domain of a protein we have designated RGL3 (Ral GEF-like 3) that shares 35% sequence identity with the known Ral guanine nucleotide exchange factors (RalGEFs). RGL3, through a C-terminal 99-amino acid domain, interacted in a GTP- and effector loop-dependent manner with Rit and Ras. Importantly, RGL3 exhibited guanine nucleotide exchange activity toward the small GTPase Ral that was stimulated in vivo by the expression of either activated Rit or Ras. These data suggest that RGL3 functions as an exchange factor for Ral and may serve as a downstream effector for both Rit and Ras.     

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.     

How does GAP catalyze the GTPase reaction of Ras? A computer simulation study

The formation of a complex between p21(ras) and GAP accelerates the GTPase reaction of p21(ras) and terminates the signal for cell proliferation. The understanding of this rate acceleration is important for the elucidation of the role of Ras mutants in tumor formation. In principle there are two main options for the origin of the effect of GAP. One is a direct electrostatic interaction between the residues of GAP and the transition state of the Ras-GAP complex and the other is a GAP-induced shift of the structure of Ras to a configuration that increases the stabilization of the transition state. This work examines the relative importance of these options by computer simulations of the catalytic effect of Ras. The simulations use the empirical valence bond (EVB) method to study the GTPase reaction along the alternative associative and dissociative paths. This approach reproduces the trend in the overall experimentally observed catalytic effect of GAP: the calculated effect is 7 +/- 3 kcal/mol as compared to the observed effect of approximately 6.6 kcal/mol. Furthermore, the calculated effect of mutating Arg789 to a nonpolar residue is 3-4 kcal/mol as compared to the observed effect of 4.5 kcal/mol for the Arg789Ala mutation. It is concluded, in agreement with previous proposals, that the effect of Arg789 is associated with its direct interaction with the transition state charge distribution. However, calculations that use the coordinates of Ras from the Ras-GAP complex (referred to here as Ras') reproduce a significant catalytic effect relative to the Ras coordinates. This indicates that part of the effect of GAP involves a stabilization of a catalytic configuration of Ras. This configuration increases the positive electrostatic potential on the beta-phosphate (relative to the corresponding situation in the free Ras). In other words, GAP stabilizes the GDP bound configuration of Ras relative to that of the GTP-bound conformation. The elusive oncogenic effect of mutating Gln61 is also explored. The calculated effect of such mutations in the Ras-GAP complex are found to be small, while the observed effect is very large (8.7 kcal/mol). Since the Ras is locked in its Ras-GAP configuration in our simulations, we conclude that the oncogenic effect of mutation of Gln61 is indirect and is associated most probably with the structural changes of Ras upon forming the Ras-GAP complex. In view of these and the results for the Ras' we conclude that GAP activates Ras by both direct electrostatic stabilization of the transition state and an indirect allosteric effect that stabilizes the GDP-bound form. The present study also explored the feasibility of the associative and dissociative mechanism in the GTPase reaction of Ras. It is concluded that the reaction is most likely to involve an associative mechanism.     

Plexin‐B1 is a GTPase activating protein for M‐Ras,remodelling dendrite morphology

Plexins are receptors for axonal guidance molecules known as semaphorins. We recently reported that the semaphorin 4D (Sema4D) receptor, Plexin‐B1, induces axonal growth cone collapse by functioning as an R‐Ras GTPase activating protein (GAP). Here, we report that Plexin‐B1 shows GAP activity for M‐Ras, another member of the Ras family of GTPases. In cortical neurons, the expression of M‐Ras was upregulated during dendritic development. Knockdown of endogenous M‐Ras—but not R‐Ras—reduced dendritic outgrowth and branching, whereas overexpression of constitutively active M‐Ras, M‐Ras(Q71L), enhanced dendritic outgrowth and branching. Sema4D suppressed M‐Ras activity and reduced dendritic outgrowth and branching, but this reduction was blocked by M‐Ras(Q71L). M‐Ras(Q71L) stimulated extracellular signal‐regulated kinase (ERK) activation, inducing dendrite growth, whereas Sema4D suppressed ERK activity and down‐regulation of ERK was required for a Sema4D‐induced reduction of dendrite growth. Thus, we conclude that Plexin‐B1 is a dual functional GAP for R‐Ras and M‐Ras, remodelling axon and dendrite morphology, respectively.     

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.