This work studies the coordination of the action of GTPase-activating protein (GAP) and guanine nucleotide exchange factor (GEF) on activated human c-Ha-Ras p21. Purified human p120-GAP was obtained with a new efficient procedure. To distinguish the GTPase-activating effect of p120-GAP from other effects dependent on the interaction with activated Ha-Ras, the nonhydrolyzable GTP analogue guanosine 5-O-(thiotriphosphate) (GTP␥S) was used. The results showed that the GTP␥S/GTP␥S exchange enhanced

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... by the C-terminal catalytic domain of the yeast GEF Sdc25p (C-Sdc25p) is prevented by p120-GAP. This effect is strictly specific for the activated form of Ha-Ras, the target of GAP; no effect on Ha-Ras⅐GDP was detectable. The GAP catalytic domain also inhibited C-Sdc25p but to a lower extent. The interfering effect by p120-GAP was also evident in a homologous mammalian system, using full-length mouse RasGEF, its C-terminal half-molecule, or C-terminal catalytic domain. As a consequence of this inhibition, presence of p120-GAP enhanced the regeneration of Ha-Ras⅐GTP␥S by GEF at a GDP:GTP␥S ratio mimicking the in vivo GDP:GTP ratio. Our work describes a novel function of p120-GAP and suggests a mechanism by which GAP protects Ha-Ras⅐GTP in vivo against unproductive exchanges. This constrain is likely involved in the regulation of the physiological GDP/GTP cycle of Ras and in the action of p120-GAP as downstream effector of Ras. Helix ␣3 is proposed as a Ras element playing a key-role in the interference between GAP and GEF on Ras. * This work was supported by the EC contract BIOTECH BIO4-CT96 -1110, Ligue Nationale Franc ¸aise Contre le Cancer, Association pour la Recherche sur le Cancer (grant no. 6377) and Fe ´de ´ration Nationale des Centres de Lutte Contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Recipient of a fellowship of the Ministe 're des Affaires Etrange 'res. § Recipient of fellowships of the Association pour la Recherche sur le Cancer and the Fondation pour la Recherche Me ´dicale. ¶ Recipient of a fellowship of the Association pour la Recherche sur le Cancer.