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 necessary for p120GAP-stimulated GTP hydrolysis. We report a third region of c-Ha-Ras located within the alpha 3 helix (amino acids 101-103) which is also required for efficient p120GAP, but not neurofibromin-mediated hydrolysis. This highly conserved region of the Ras protein was investigated using an insertion-deletion mutant (Ras-100LIR104) originally 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 Structural and Functional Properties (Bosch, L., Kraal, B., and Parmeggiani, A., eds) pp. 165-178, Plenum Press, New York). The 100LIR104 substitution did not alter the intrinsic hydrolytic rate of the protein. The p120GAP-stimulated hydrolysis of Ras-100LIR104, however, was decreased by 2-3-fold compared to wild type Ras. This decrease in p120GAP-stimulated hydrolysis was not due to its inability to physically associate with Ras-100LIR104. GTP (as determined by competitive binding assays). Surprisingly, neurofibromin-stimulated GTP hydrolysis was unaltered by the mutation. Finally, no differences were observed in the ability of either the p120GAP catalytic domain or the neurofibromin GRD to accelerate Ras-100LIR104 GTPase activity, indicating that the amino-terminal noncatalytic GAP region is critical for p120GAP-stimulated GTP hydrolysis. This is the first report of a Ras mutation which differentiates between p120GAP and neurofibromin activity.
D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D017124:Conserved Sequence; D003600:Cytosol / Q000378:metabolism; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D025542:Neurofibromin 1; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017384:Sequence Deletion; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D025542:Neurofibromin 1; D011506:Proteins; D011994:Recombinant Proteins; D020703:ras GTPase-Activating Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D016283:Proto-Oncogene Proteins p21(ras)