Inhibition of Ras/Raf interaction by anti-oncogenic mutants of neurofibromin, the neurofibromatosis type 1 (NF1) gene product, in cell-free systems.
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 mammalian 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) (GTP gamma S) 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.GTP gamma S 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.
D016475:3T3 Cells; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002474:Cell-Free System; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology; D051379:Mice; D009154:Mutation; D025542:Neurofibromin 1; D009857:Oncogenes; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf; D051381:Rats; D018631:ras Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*
D025542:Neurofibromin 1; D011506:Proteins; D011518:Proto-Oncogene Proteins; D006160:Guanosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins