KOIDE Hiroshi Tokyo Institute of Technology, Fac.Biosci.Biotech., Assistant, 生命理工学部, 寄附講座教員 (70260536)
SATO Takaya Tokyo Institute of Technology, Fac.Biosci.Biotech., Assistant, 生命理工学部, 寄附講座教員 (20251655)
|Budget Amount *help
¥5,200,000 (Direct Cost : ¥5,200,000)
Fiscal Year 1996 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1995 : ¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1994 : ¥2,200,000 (Direct Cost : ¥2,200,000)
The neurofibromatosis type 1 (NF1) gene encodes a protein, neurofibromin, containing GTPase-activating protein-related domain (GRD) that stimulates intrinsic GTPase activity of Ras protein. By screening a randomly mutagenized NF1-GRD library in Saccharomyces cerevisiae, we isolated two NF1-GRD mutants (NF201 and NF204) with single amino acid substitutions, which suppress the heat shock-sensitive phenotype of the RAS2 (G19V) mutant. The NF1-GRD mutants also suppress the oncogenic Ras-induced transformation of NIH 3T3 mouse fibroblasts (Nakafuku, M., Nagamine, M., Ohtoshi, A., Tanaka, K., Toh-e, A., and Kaziro, Y.(1993) Proc.Natl.Acad.Sci.U.S.A.90,6706-6710). In this paper, we investigated the molecular mechanism of inhibition of the transforming Ras-specific function by the NF1-GRD mutants in mammalia cells. In human embryonic kidney (HEK) 293 cells, the mutant NF1-GRDs attenuated the stimulation of mitogen-activated protein kinase by Ras (G12V), but not by platelet-derived growth factor. In cell-free systems, purified recombinant NF1-GRD mutants showed an inhibitory effect on the association of Ras・guanosine 5'-O- (3-thiotriphosphate) (GTPgammaS) with Raf at several times lower concentrations than the wild type. Furthermore, it was revealed that the binding affinity of the mutant NF1-GRDs toward Ras-GTPgammaS is approximately 5-10 times higher than the wild type.These results suggest that the mutant NF1-GRDs tightly bind to an oncogenic Ras in its GTP-bound active conformation and block the interaction between Ras and its effector, Raf.