The importance of two conserved arginine residues for catalysis by the ras GTPase-activating protein, neurofibromin.

Ras proteins are guanine-nucleotide binding proteins that have a low intrinsic GTPase activity that is enhanced 10(5)-fold by the GTPase-activating proteins (GAPs) p120-GAP and neurofibromin. Comparison of the primary sequences of RasGAPs shows two invariant arginine residues (Arg1276 and Arg1391 of neurofibromin). In this study, site-directed mutagenesis was used to change each of these residues in the catalytic domain of neurofibromin (NF1-334) to alanine. The ability of the mutant proteins to bind to Ras.GTP and to stimulate their intrinsic GTPase rate was then determined by kinetic methods under single turnover conditions using a fluorescent analogue of GTP. The separate contributions of each of these residues to catalysis and binding affinity to Ras were measured. Both the R1276A and the R1391A mutant NF1-334 proteins were 1000-fold less active than wild-type NF1-334 in activating the GTPase when measured at saturating concentrations. In contrast, there was only a minor effect of either mutation on NF1-334 affinity for wild-type Ha-Ras. These data are consistent with both arginines being required for efficient catalysis. Neither arginine is absolutely essential, because the mutant NF1-334 proteins increase the intrinsic Ras.GTPase by at least 100-fold. The roles of Arg1276 and Arg1391 in neurofibromin are consistent with proposals based on the recently published x-ray structure of p120-GAP complexed with Ras.

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.