Mechanism of inhibition by arachidonic acid of the catalytic activity of Ras GTPase-activating proteins.

Ras is a guanine nucleotide-binding protein that acts as a molecular switch controlling cell growth. The Ras GTPase-activating proteins (GAPs) p120-GAP and neurofibromin are candidates as Ras effectors. The GTPase-activating activity of both proteins is inhibited by mitogenic lipids, such as arachidonic acid and phosphatidic acid, and differential inhibition of the two GAPs led to the hypothesis that both were effectors in a Ras-controlled mitogenic pathway (Bollag, G., and McCormick, F. (1991) Nature 351, 576-579). We have studied the mechanism of inhibition by arachidonic acid in three ways: first, by measurements of catalytic activity under multiple turnover conditions; second, using p-((6-phenyl)-1,3,5-hexatrienyl)benzoic acid as a fluorescent probe for ligands binding to GAPs; and third, by using a scintillation proximity assay to measure direct binding of Ras to neurofibromin. We found no significant differential inhibition between p120-GAP and neurofibromin by arachidonic acid. The inhibition by arachidonic acid included a major component that is competitive with Ras GTP. These data suggest that insomuch as the mitogenic effects of lipids are mediated via inhibition of GAPs, GAPs are not Ras effector proteins. Additionally, lipids can exert a non-competitive type effect, consistent with a protein denaturing activity, making difficult extrapolations from in vitro data to the situation within cells, and possibly explaining the variability of literature data on inhibition by lipids.