ras and GAP—Who's controlling whom?

Cell, Vol. 61, 921-923,

June

15, 1990, Copyright

ras and GAPWho’s Controlling

0 1990 by Cell Press

Minireview Whom?

Alan Hall Chester Beatty Laboratories Institute of Cancer Research London SW3 6JB England

The discovery of the GTPase activating protein, GAP (Trahey and McCormick, 1987), came as a welcome relief to those of us struggling to understand the biochemical function of mammalian ras. The importance of ras as a regulator of intracellular signaling pathways, particularly those controlling growth, had long been apparent, but its biochemical mechanism of action remained obscure. Here at last was a protein with which it interacted. Since ras proteins are guanine nucleotide binding proteins that can switch from an inactive GDP-bound form to an active GTPbound form, it is essential to identify both upstream activating factors (that catalyze nucleotide exchange) and downstream signals elicited by rasGTP to understand its regulatory function. The excitement generated by the discovery of GAP, therefore, was tempered only by the likelihood that its role was to down-regulate ras.GTP The situation soon changed when mutational analysis of the GAP-ras interaction suggested that GAP might also be the biological target for regulation by ras (Cal& et al., 1988; Adari et al., 1988). Currently, the role of GAP in rasregulated signal transduction is a major focus of attention -does GAP control fas (upstream model) or does ras control GAP (downstream model)? A variety of signal transduction pathways can be blocked by microinjecting antibodies that neutralize ras into cells, including the stimulation of quiescent fibroblasts by serum, the neuronal differentiation of PC12 cells induced by nerve growth factor, and the insulin-dependent maturation of Xenopus oocytes. It has been argued from these experiments that ras is a key regulatory molecule linking growth factor receptors to their known signal transduction pathways (Figure la) or perhaps to a novel uncharacterized signal essential for biological responses (Figure lb). However, also shown is an alternative possibility where ras is activated by other upstream signals and then either contributes to the known growth factor-induced pathways (Figure lc) or activates a novel signal that acts synergistically with growth factors (Figure Id). To try to resolve these possibilities and define rasregulated events, a number of groups have turned to yeast genetics. As a result we know that in Saccharomyces cerevisiae, the two RAS proteins are key regulatory molecules in a major signaling pathway linking nutritional status to intracellular CAMP levels and growth (best described by Figure la). The control of RAS by nutritional factors is mediated by the product of the CDC25 gene, which is thought to catalyze guanine nucleotide exchange. The single ras protein in Schizosaccharomyces pombe is also controlled by nutritional factors acting through an upstream factor, sfe6, which has some homology to CDC25

(Hughes et al., 1990). However, S. pombe ras does not affect CAMP levels or cell growth but is essential for conjugation and meiosis. It appears that ras-induced intracellular signals act synergistically with a mating factor signal transduction pathway to promote conjugation (best described by Figure Id). Clearly the function of ras in these two yeasts has diverged, and it is not known which, if either, most closely resembles mammalian ras function. Candidate upstream factors that catalyze guanine nucleotide exchange on mammalian p21ras have been identified (West et al., 1990; Wolfman and Macara, 1990); their characterization should reveal if the assumption that the upstream control of ras by nutrients in yeast has been replaced by growth factors in mammalian cells is justified. The major role of the RAS proteins in S. cerevisiae is to control the activity of adenylate cyclase, and the lethality induced by RAS deletions is overcome by introducing mammalian ras genes into yeast. Despite this, ras does not control adenylate cyclase in mammalian cells. These results could simply be explained if the effector protein in the two organisms had diverged while maintaining a common recognition sequence within the ras protein (believed to be amino acids 32-40 in mammalian ras). However, an alternative possibility has recently been raised. Cyclase in S. cerevisiae exists as a tight complex with a 70 kd protein, CAP (cyclase-associated protein), and in membranes isolated from CAP- cells, adenylate cyclase no longer responds to RAS. It is possible, therefore, that CAP is the RAS effector protein. Since CAP- cells also exhibit additional phenotypes not related to the RASlcyclase pathway, CAP might link RASwith more than one signaling pathway. The gene for CAP has now been cloned by two groups (Field et al., 1990; Fedor-Chaiken et al., 1990) and the obvious next step is to see if mammalian or S. pombe homologs exist.

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Figure 1. The Role of ras in Growth tion Pathways

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The requirement for upstream activation can be overcome by single amino acid substitutions in ras that block its GTPase activity (for example, at codon 12). The introduction of such activated (oncogenic) proteins into cells can have dramatic effects: malignant transformation and growth factor-independent proliferation in 3T3 fibroblasts, factor-independent neuronal differentiation in the PC12 cell line, and maturation in Xenopus oocytes. These effects would be consistent with the simplest model shown in Figure la, where ras is a regulatory molecule controlling the known signal transduction pathways of these growth factor receptors. The difficulty is that when these signaling pathways are looked at a little more closely, it is very hard to see where ras fits in. Addition of plateletderived growth factor (PDGF) to fibroblasts, for example, leads to the stimulation of three known enzymatic activities, phospholipase C-r (PLC-y), c-raf (a cytoplasmic serine/threonine kinase) and 3’ phosphatidylinositol kinase (PI-3 kinase); each of these proteins complexes directly with the PDGF receptor. How ras relates to these growth factor-induced responses is not known, but several papers published recently suggest that GAP could be a link. The 120 kd cytoplasmic GAP protein interacts with ras.GTP and stimulates its intrinsic GTPase activity dramatically, suggesting that the role of GAP is to downregulate rasGTl? In that case, signaling by ras would first require inactivation of GAP to allow ra.sGTP to interact with its real effector; a possible mechanism for this has been proposed (Tsai et al., 1989). Certain phospholipids and their breakdown products (e.g., arachidonic acid) that are known to be produced in response to growth factor stimulation of cells efficiently block the ability of GAP to stimulate ras GTPase activity in vitro. Perhaps this is the link between growth factors and ras. A closer look at GAP, however, reveals a picture that is not so clear-cut. First, the protein has at least two domains, with the carboxy-terminal third alone able to stimulate ras GTPase activity. Second, GAP is more abundant in cells than ras-an unexpected result if GAP is acting enzymatically to remove ras.GTP And third, mutational analysis of ras reveals that amino acids essential for GTPase stimulation by GAP (residues 32-40) are also essential for biological activity. This mutational analysis has suggested that in addition to downregulating ras.GTl? GAP might also be the target. Consistent with this, GAP interacts with activated (oncogenic) ras proteins, though because it has no effect on their intrinsic GTPase activity, they are constitutively and predominantly in the GTPbound state in vivo. But sequence analysis of GAP (Trahey et al., 1988; Vogel et al., 1988) has not revealed any clues as to a possible effector function for this protein. The only significant homology with other proteins is a region in the amino-terminal noncatalytic domain, which contains two SH2 (src homology) domains related to putative regulatory sequences of some nonreceptor tyrosine kinases and PLCy. Does yeast help us define the function of GAP? GAP activity has not been identified biochemically either in S. cerevisiae or S. pombe cells, and normal mammalian ras proteins introduced into yeast remain constitutively in the

GTP-bound form. Nevertheless, normal yeast RAS is found predominantly in the GDP-bound state, implying that yeast must contain a GAP-like activity. Two genes have been identified whose products appear to fulfill this role, IRA7 and IRA2 (Tanakaet al., 1990). Deletion of either gene results in a phenotype similar to activating mutations in RAS and leads to high constitutive levels of RASGTt? IRA7 and IRA2 are clearly therefore not targets for RAS but instead down-regulate its activity by stimulating GTPase rates. Sequence analysis of IRA1 and IRA2 has revealed some homology with the carboxy-terminal catalytic domain of GAP; furthermore, mammalian GAP can rescue the normal phenotype in iral-/ira2strains. This has been taken to indicate that the function of mammalian GAP must also be to down-regulate ras, but the experiments really only show that mammalian GAP can stimulate the GTPase activity of yeast RAS. The strongest evidence yet that GAP is a biological target for mammalian ras comes from an unexpected system. Yatani et al. (1990) were examining the control of K+ channels in isolated membranes by the G protein Gk after activation of muscarinic receptors. They found that addition of recombinant GAP or ras rapidly blocks the current through these activated channels; the effect of adding recombinant GAP is dependent on endogenous ras in the membranes (i.e., it could be blocked by anti-ras antibodies), and the effect of recombinant ras is through interaction with endogenous GAP The authors conclude that a ras.GTP-GAP complex must be responsible for blocking the channels. Since this block could be overcome by binding GTPyS to Gk, it seems likely that ras-GAP either acts directly by inhibiting the coupling of Gk to its upstream receptor, or indirectly by inhibiting the association of Gka with its 8~ subunits. Whether this is through direct protein-protein interaction or intermediary signals induced byras-GAP is not clear. This represents the first biochemical assay for GAP as a downstream signaling molecule, and it has already been used to demonstrate that the carboxy-terminal GTPase activating domain of GAP is not sufficient for channel blocking. Furthermore, since 8~ subunits are often shared by G proteins, the experiment may bear on why growth factor-induced phosphoinositide turnover is down-regulated in ras-transformed cells. Perhaps ras can also interfere with this and other G protein-controlled processes. A further twist to the GAP story has emerged from three laboratories that have shown GAP is phosphorylated on tyrosine in response to PDGF or EGF (but not insulin or basic fibroblast growth factor) and the tyrosine kinase oncogene products v-src or v-fps (Molloy et al., 1989; Ellis et al., 1990; Kaplan et al., 1990)ln addition, after stimulation of cells with PDGF, phosphorylated GAP is found tightly associated with the PDGF receptor. This receptor is now known to bind at least four proteins in response to stimulation (Figure 2): PI-3 kinase, c-raf, PLC-y, and GAP The consequences of GAP phosphorylation and its association with growth factor receptors are not yet clear. Less than 10% of cellular GAP appears to be phosphorylated and complexed to receptors; using recombinant proteins, GAP has been shown to bind to the PDGF receptor in the

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absence of fas. An 83 amino acid deletion in the PDGF receptor (in the kinase insert domain shown in black in Figure 2) that blocks PDGF-induced mitogenesis (in some cells) also blocks binding of PI-3 kinase, but not PLC-r or c-raf. This deletion also blocks GAP binding, implying that GAP and PI-3 kinase are essential components of the mitogenic response. The picture is frustratingly close to completing a link between ras and growth factor receptors. Somewhat of a surprise is that the link seems to be through GAP and not nucleotide exchange, as had been expected. The simple upstream model for mammalian GAP looks increasingly less likely, in view of the experiments of Yatani et al. showing that the MS-GAP complex has a biochemical activity. However, until an assay is available to show that the complex can generate a proliferative signal, it cannot be ruled out that GAP is a downregulatory molecule both for ras and (as ras-GAP) for other G protein-controlled signaling pathways. In this event the real downstream target of ras awaits identification. The downstream model for GAP looks more promising, but a simple linear relationship between receptors, ras, and GAP as shown in Figure la is also unlikely. GAP does not seem to be required for binding of PLC-y, PI-3 kinase, or c-raf to the PDGF receptor, and GAP itself can bind in the absence of ras. It would seem, therefore, that the target for ras is phosphorylated GAP, perhaps complexed with receptor (Figure 2). In ras-transformed cells, GAP is not associated with the PDGF receptor, which might suggest that the phosphorylated GAP-receptor complex is not important per se if fas is locked in the GTP form. Perhaps the effect of GAP complexing with the receptor is to attenuate its GTPase stimulatory effect. In any case, the available information points to the distinct possibility that ras is controlling a novel signal. Two proteins (~62 and p190) that associate with GAP are phosphorylated on tyrosine in response to growth factor stimulation (Ellis et al., 1990). Their characterization is eagerly awaited and may help define a function for GAP The rasrelated proteins, rho and rapl, have been shown to each have their own GTPase activating protein. rap1 can also affect proliferative responses, and in addition to interacting with a rap.GAP, it also interacts with ras.GAP Clearly the control of mammalian cell growth involves a complex network of signal transduction pathways; working out how they interact with one another may take some time yet. But, to return to the original question, does ras control GAP, or does GAP control ras? The answer seems to be: yes. References Adari, H., Lowy, D. IX, Willumsen, F. (1988). Science 240, 518-521. Cal& C., Hancock, 332, 548-551.

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