GAP as ras Effector or Negative Regulator? Frank McCormick

CETUS Corporation, Emeryville, California

INTRODUCTION

GTPase-activating protein (GAP) was identified on the basis of its ability to downregulate normal rds p21 biolog- ical activity without affecting oncogenic ras p21 mutants [l]. Existence of GAP explained why normal ras p21 pro- teins are orders of magnitude less active than oncogenic mutants in biological assays (promotion of oocyte matu- ration, for example); these differences could not be ex- plained on the basis of the intrinsic properties of ras p21 proteins alone. At the biochemical level, GAP acts as an enzyme that catalyzes conversion of p21 .GTP (the active form the ras p21 protein) to p21 .GDP (the inactive form). Since GAP fails to catalyze conversion of oncogenic p21 .GTP mutants(positi0n 12, 13, 61 mutants) to their GDP-bound states, they remain constitutively in their active state. Onco- genic mutants can then be seen as mutants that escape this aspect of GAP action, and, in these terms, GAP clearly acts as a negative regulator of normal ras p21 function.

EVIDENCE IN FAVOR OF GAP AS AN EFFECTOR

The first suspicion that GAP may have an additional role came from analysis of the region of ras p21 to which GAP appears to bind. These early studies [2,31 examined the abil- ity of GAP to stimulate GTPase activity of various ras mu- tants. It was found that point mutations in a region defined as the ”effector binding region” [4,51 and that destroyed biological activity of v-Ha-ras oncogenes, prevented GAP- mediated stimulation of normal ras p21. For example, the mutation D38E in v-Ha-rasfails to form foci; the same muta- tion in c-Ha-ras renders it insensitive to GAP-mediated GTPase activation. These results led to the hypothesis that GAP binding is essential for ra5 p21 action. According to this hypothesis, “effector” mutants would fail to bind GAP, whereas oncogenic mutants would bind GAP but fail to undergo GTP hydrolysis. Competition binding studies ful- filled these predictions: G12Vand Q61 Loncogenic mutants were shown to form complexes with GAP, in the latter case with an affinity about 50 times higher than normal ras p21. On the other hand, an ”effector” mutant (D38A) failed to show detectable binding to GAP [6].

The ability of Q61 L to bind GAP tightly was used in ele- gant experiments initiated by the late Irving Sigal [7]; he made a double mutant of Ha-ras with both Q61L and C186S mutations. The latter mutation prevents membrane localization and thus inactivates the ra5 p21 protein. Injection of this double mutant into Xenopus oocytes inter- fered with the biological potency of co-injected G12V ras p21. This experiment was interpreted as evidence that the double mutant traps in the cytosol a factor that is necessary

for G12V ras action. Injection of purified GAP into these oocytes partially restored G 12V function, suggesting that the cytosolic factor trapped by the double mutant is actu- ally GAP

A similar mechanism has been proposed to explain how the product of the k-rev1 gene [8], the raplA protein, reverts transformation by activated ras genes. GAP binds to raplA with high affinity (Kd = 50 nM; Frech et al., in press), but it fails to stimulate raplA GTPase activity. Over- expression of rap1 A protein could therefore interfere with ras function by sequestering GAP One difficulty with this model is the necessary prediction that the complex formed between raplA and GAP is biologically inactive (as mea- sured by transformation assays), whereas the complexes formed between oncogenic ras p21 proteins and GAP are fully active. This difficulty can be resolved by assuming that biological activity requires other interactions (binding to other proteins, for example) that occur efficiently with ras:GAP but not with rap1A:GAP complexes. Alternatively, GAP binding to ras p21 may result in conformational changes that are necessary for signaling, whereas GAP binding to raplA may not lead to such changes.

Recently, it has been shown that GAP binds to activated platelet-derived growth factor (PDGFI-receptors 19,101, and so forms part of a putative signaling complex that also con- tains phospholipase C-y, type I phosphatidylinositol kinase, and the c-rafproto-oncogene [l 1 I. Analysis of GAP binding to mutant PDGF receptors indicated that binding may be necessary for receptor function. Furthermore, ras p21 may be necessary for PDGF-receptor function, since antibodies against p21 block mitogenic responses 1121. How are ras p21 and GAPconnected in thissignaling system? First, GAP and ras p21 could function independently. This seems unlikely but remains a formal possibility. Second, GAP‘s function in PDGF receptor signaling could be ras p21 depen- dent. In this case, GAP would clearly be an effector of ras action. Binding of GAP to PDGF receptor is not ras p21 dependent, since GAP and PDGF receptor bind in vitro [9]. Activation of some other, unknown function of GAP could still depend on ras, however. Third, interaction of GAP with PDGF receptor could inhibit GAP’s GTPase-accelerating effect ras p21, which could then accumulate in the active, GTP- bound state. An immediate objection to this hypothesis is that the amount of GAP in the cell far exceeds the amount PDGF-receptor, making it appear unlikely that GAP could be effectively sequestered by these activated receptors. However, it is conceivable that activation of the receptor could lead to indirect effects on GAP that prevent its asso- ciation with ras p21. In support of this, Ellis and cowork-

0 1990 WILEY-LISS, INC.

MOLECULAR CARCINOGENESIS 3: 185- 187 (1990)

186 MCCOR MlCK

ers [I31 found that in cells stimulated by tyrosine kinase oncogenes or growth factor receptors, GAP becomes asso- ciated with other cellular proteins(P190, P62); these com- plexes could perhaps, prevent GAP-mediated GTP hydrolysis by ras p2 1 and thus activate ras indirectly. The biochemical properties of these complexes are under investigation.

Direct evidence that ras p21 and GAP work together to produce a biochemical effect has come recently from an unexpected source: the study of K + channels in iso- lated patches of atrial cell membranes [14]. In this system, a G protein referred to as Gk (probably synonymous with Gi-3) couples a muscarinic receptor to a K + channel, so that when the receptor is activated channel opening begins. Recombinant GAP at subnanomolar concentra- tions prevents coupling of the receptor to the G protein. Recombinant ras p2 1, at nanomolar concentrations, does the same thing. Furthermore, the effect of ras proteins depends on GAP and vice versa, indicating that the effect is a result of the concerted action of a ras:GAP complex. The biochemical nature of the effect is not clear, but it is hoped that this system will help to define a role for GAP that is clearly distinct from its role in stimulating ras p21 GTPase activity,

EVIDENCE NOT SUPPORTING GAP AS AN EFFECTOR OF RAS ACTION

The discovery that an "effector" mutant of ras p21 (D38A) is unable to bind to GAP [61 supported the notion that GAP binding is necessary for ra5 action. However, recent data from Wittinghofer and coworkers indicates that another effector mutant, D38E, retains its ability to bind GAP [ 151. No stimulation of GTPase occurs on binding, how- ever. Biochemically, this mutant appears to resemble onco- genic ras mutants that also bind GAP but fail to undergo GTPase stimulation. Biologically, however, they have very different activities. The simplest interpretation is that GAP binding has nothing to do with transformation. Along the same lines, R-ras is sensitive to GAP-mediated GTPase stimulation [ 161, but is unable to transform cells, and, as discussed above, raplA binds GAP tightly but fails to trans- form. Clearly GAP binding is not enough to generate a biological signal. A role for GAP cannot be ruled out by these kinds of analyses, unless a mutant of ras that retains biological activity but fails to bind GAP is discovered. Pres- ently, we do not know of such a mutant.

If GAP is not the effector of ras action, it may seem sur- prising that it binds to r ~ s p21 at a site that is supposed to be the effector binding site. This could be a coincidence, but more likely, it could reflect the fact that GAP binding, like the binding of a putative effector, is GTP dependent. For GAP, recognition of the GTP state is a signal for downregu- lation; for the effector it is for signal transmission. The region of ra5 p21 that undergoes GTP/GDP conformational changes is rather limited (the "effector" loop and a helical region close to the Y13-259 binding site), and it may not be sur- prising that GAP and effector share the same site. In a model in which the effector of ras action is distinct from GAP, we have to assume that ras p21, in its GTP-bound state, is ca-

pable of binding to its true effector and activating it before encountering GAP and being converted to the GDP-bound state. This appears to be the situation in 5. cerevkiae: RAS proteins bound to GTP activate adenylyl cyclase and are sub- sequently converted to their GDP-bound states by the GAP- related proteins referred to as IRA1 and IRA2 [ 171. These IRA proteins are not essential for cell growth under any known conditions, and do not appear to have any effector role in 5. cerevisiae growth. Indeed, the function of IRA proteins can be replaced with mammalian GAP [17]. These observations do not argue against an effector role for GAP in mammalian cells, but they certainly establish that an ef- fector function can be totally distinct from GTPase activation.

FUTURE PROSPECTS

The role of GAP in ras function may well be resolved by one of the following experimental results:

identification of a ras mutant that is fully functional but fails to interact with GAP (conclusion: GAP is not an effector), inactivation of GAP by neutralizing antibodies, gene knock-out or dominant interfering mutants have no effect on rasaction (conclusion: GAP is not an effector), the above manipulations prevent rasfunction (conclu- sion: GAP IS necessary for ras function; it may be an effector), a biochemical function IS ascribed to GAP that is consis- tent with GAP as effector (e.g., ras p21 .GTP-dependent production of a mitogenic second messenger), the true effector is identified and has properties con- sistent with rasfunction (see above), or human tumors are identified that are homozygous for inactive GAP

Until one of these results is documented, the role of GAP in rasfunction will be unclear, and the signal transduction pathways with which ras is involved will remain elusive.

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