Yoder-Hill J, Golubic M, Stacey D W
Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195, USA.
J Biol Chem. 1995 Nov 17;270(46):27615-21. doi: 10.1074/jbc.270.46.27615.
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.
p120GAP 刺激的 GTP 水解需要 c-Ha-Ras 的效应器结合结构域和开关 II 区域。我们报告了 c-Ha-Ras 的第三个区域，位于α3 螺旋内（氨基酸 101 - 103），高效的 p120GAP 介导的水解也需要该区域，但神经纤维瘤蛋白介导的水解不需要。使用最初由 Willumsen 等人鉴定的插入 - 缺失突变体（Ras-100LIR104）研究了 Ras 蛋白的这个高度保守区域（Willumsen, B. M., Adari, H., Zhang, K., Papageorge, A. G., Stone, J. C., McCormick, F., and Lowy, D. R (1989) 在《鸟嘌呤核苷酸结合蛋白；共同的结构和功能特性》（Bosch, L., Kraal, B., and Parmeggiani, A., 编）第 165 - 178 页，Plenum 出版社，纽约）。100LIR104 替换并未改变该蛋白的内在水解速率。然而，与野生型 Ras 相比，p120GAP 刺激的 Ras-100LIR104 的水解降低了 2 - 3 倍。p120GAP 刺激的水解降低并非由于其无法与 Ras-100LIR104 进行物理结合（通过竞争性结合测定确定）。令人惊讶的是，神经纤维瘤蛋白刺激的 GTP 水解并未因该突变而改变。最后，在 p120GAP 催化结构域或神经纤维瘤蛋白 GRD 加速 Ras-100LIR104 GTP 酶活性的能力方面未观察到差异，表明氨基末端非催化性 GAP 区域对于 p120GAP 刺激的 GTP 水解至关重要。这是关于区分 p120GAP 和神经纤维瘤蛋白活性的 Ras 突变的首次报道。
