Signal Transduction

Fig 4.19  Some of the many influences and interactions of Ras.

RasGAPs

In cell-free preparations, the half-life of the GTP bound to Ras is between 1 and 5 h. With a rate constant for hydrolysis of about 4.6 10 4 s 1, wild-

type Ras hardly functions as a GTPase and one might expect it to be almost permanently activated. It would also be hard to understand why the differences in the rates of GTP hydrolysis by the wild-type and the oncogenic mutants should be so crucial. However, when the rate of GTP hydrolysis in cells is measured, things are very different.118 RasGTP injected into Xenopus oocytes is converted to its inactive GDP form within 5 min. Cells contain a protein or proteins that accelerate the rate of GTP hydrolysis enormously. By contrast, GTP bound to GTPase-defective transforming mutants of Ras remains intact. These observations led to the discovery and isolation of the GTPase activating protein RasGAP (also known as p120GAP). The GTPase activating proteins (GAPs) were the first proteins found to interact directly with Ras.

RasGAP

This GAP protein interacts specifically with wild-type Ras, accelerating the GTPase activity by up to five orders of magnitude. When over-expressed,119,120 or injected into fibroblasts at a concentration in excess of the normal level,121 RasGAP inhibits the mitogenic action of growth factors. In this respect it acts, as one might expect, as a negative regulator of Ras. However, in T- and B- lymphocytes and in adipocytes a mitogenic signal is mediated by a decrease in the activity of RasGAP.122–124

In addition to its (Ras-interacting) catalytic function, RasGAP contains a number of identifiable domains125,126 (Figure 4.20). These include a PH

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GTP-Binding Proteins and Signal Transduction

Fig 4.20  The domain organization of RasGAP and related GTPase activating proteins.

domain,127 a C2 domain,128 two SH2 domains, and one SH3 domain129 (these and other domain structures are described in Chapter 24). This implies the potential for a multiplicity of interactions with other signalling proteins, scaffolds, adaptors and other molecules. The Ca2 -sensitivity of RasGAP is discussed in Chapter 8.

GAPs and effectors

Does RasGAP do more than regulate GTPase activity? Is it, like the effectors of heterotrimeric G proteins, also an effector of downstream processes?130 The fact that Ras-GAP interacts only with RasGTP and not with RasGDP is certainly consistent with the idea of an effector function. Furthermore, the interaction is mediated through its contact with the effector domain of Ras. Some (though not all) mutations in this domain prevent the interaction with GAP. Against this is the finding that RasGAP suppresses the transformation of

fibroblasts induced by over-expression of normal wild-type Ras. Here, the GAP appears to play the role of a negative regulator, simply accelerating the rate of GTP hydrolysis.

An alternative possibility would be that RasGAP might be the mediator of just a subset of Ras functions, the rest of which are linked to other effector proteins. For instance, it can stimulate transcription of the c-fos promoter,131 but here its catalytic domain plays no part since deletion mutants comprising just the SH2 and SH3 domains are equally effective. However, Ras still appears to play a part since the induction of c-fos by the GAP deletion mutant is prevented by N17Ras, a dominant inhibitory mutant. It would appear that the GAP deletion mutant cooperates with another signal emanating from activated Ras. Also, as

a result of complex formation with Rho-GAP (specific for the Ras related GTPase Rho), RasGAP acts to inhibit the formation of stress fibres and focal adhesions, both of which are Rho-dependent processes132 (see figure 16.11 page 499). The assignment of GAP functions will only be resolved when we can identify the relevant protein–protein interactions in normal cells.

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Signal Transduction

Mechanism of GTPase activation

In trying to understand the mechanism of activation of the intrinsic GTPase activity of Ras by GAP proteins, two main ideas have been examined.133 Firstly, there is the possibility that the GAP acts simply by driving the Ras protein into a conformation active for GTP hydrolysis, without itself forming a part of the active site. In this case, the action of GAP on Ras would be catalytic and hence non-stoichiometric. Now, however, it is thought that the GAP interacts with the Ras stoichiometrically, contributing cationic residues (arginines)

that stabilize the transition state of the reacting GTP (Figure 4.21). This allows access of Q61 which directs an incoming water molecule for the hydrolysis of GTP. Mutations of this residue, frequently found in human tumours, prevent the enhancement of GTPase activity by GAP and lock the protein in its active conformation. The analogous interaction of Rho with Rho-GAP defines the catalytic centre of the Rho GTPase. For both Rho and Ras, the interaction with their respective GAPs is transient. They form active heterodimeric enzymes and then separate as the GTPases take up the conformations determined by the presence of GDP in the nucleotide binding pocket.

Fig 4.21  Organization of the active site of Ras and its interaction with RasGAP.The point of contact between the catalytic GTPase site of Ras and its GTPase activating protein (RasGAP). A primary arginine finger extends across the cleft between the two proteins and neutralizes the negative charge (pink) that develops in the transition state of the reaction. A second arginine residue (R2) stabilizes the primary arginine finger. The departing phosphate is depicted as a pentacoordinate intermediate. The Ras Q61 also contributes to the transition state.Adapted from Scheffzek et al.133

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