A new function of p120-GTPase-activating protein - Prevention of the guanine nucleotide exchange factor-stimulated nucleotide exchange on the active form of Ha-Ras p21

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-Has, the nonhydrolyzable GTP analogue guanosine 5'-O-(thiotriphosphate) (GTP gamma S) was used, The results showed that the GTP gamma S/GTP gamma S exchange enhanced 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 Ras GDP 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 cat alytic domain, As a consequence of this inhibition, presence of p120-GAP enhanced the regeneration of Ha-Ras.GTP gamma S by GEF at a GDP:GTP gamma S ratio mimicking the in vice GDP:GTP ratio, Our work describes a novel function of p120-GAP and suggests a mechanism by which GAP protects Ha-Ras.GTP 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 alpha 3 is proposed as a Ras element playing a key-role in the interference between GAP and GEF on Ras.