A CONSERVED REGION OF C-HA-RAS IS REQUIRED FOR EFFICIENT GTPASE STIMULATION BY GTPASE-ACTIVATING PROTEIN BUT NOT NEUROFIBROMIN

The effector binding domain and the switch II region of c-Ha-Ras are n ecessary for p120(GAP)-stimulated GTP hydrolysis. We report a third re gion of c-Ha-Ras located within the alpha3 helix (amino acids 101-103) which is also required for efficient p120(GAP), but not neurofibromin -mediated hydrolysis. This highly conserved region of the Ras protein was investigated using an insertion-deletion mutant (Ras-100LIR104) or iginally characterized by Willumsen et al. (Willumsen, B.M., Adari, H. , Zhang, K., Papageorge, A.G., Stone, J.C., McCormick, F., and Lowy, D .R. (1989) in The Guanine Nucleotide Binding Proteins; Common Structur al and Functional Properties (Bosch, L., Kraal, B., and Parmeggiane, A ., eds) pp. 165-178, Plenum Press, New York). The 100LIR substitution did not alter the intrinsic hydrolytic rate of the protein. The p120(G AP)-stimulated hydrolysis of Ras-100LIR104, however, was decreased by 2-3-fold compared to wild type Ras. This decrease in p120(GAP)-stimula ted hydrolysis was not due to its inability to physically associate wi th Ras-100LIR104-GTP (as determined by competitive binding assays). Su rprisingly, neurofibromin-stimulated GTP hydrolysis was unaltered by t he mutation. Finally, no differences were observed in the ability of e ither the p120(GAP) catalytic domain or the neurofibromin GRD to accel erate Ras-100LIR104 GTPase activity, indicating that the amino-termina l noncatalytic GAP region is critical for p120(GAP)-stimulated GTP hyd rolysis. This is the first report of a Ras mutation which differentiat es between p120(GAP) and neurofibromin activity.