2 TYPES OF RAS MUTANTS THAT DOMINANTLY INTERFERE WITH ACTIVATORS OF RAS

In the fission yeast Schizosaccharomyces pombe, ras1 regulates both se xual development (conjugation and sporulation) and cellular morphology . Two types of dominant interfering mutants were isolated in a genetic screen for ras1 mutants that blocked sexual development. The first ty pe of mutation, at Ser-22, analogous to the H-ras(Asn-17) mutant (L. A . Feig and G. M. Cooper, Mol. Cell. Biol. 8:3235-3243, 1988), blocked only conjugation, whereas a second type of mutation, at Asp-62, interf ered with conjugation, sporulation, and cellular morphology. Analogous mutations at position 64 of Saccharomyces cerevisiae RAS2 or position 57 of human H-ras also resulted in dominant interfering mutants that interfered specifically and more profoundly than mutants of the first type with RAS-associated pathways in both S. pombe or S. cerevisiae. G enetic evidence indicating that both types of interfering mutants func tion upstream of RAS is provided. Biochemical evidence showing that th e mutants are altered in their interaction with the CDC25 class of exc hange factors is presented. We show that both H-ras(Asn-17) and H-ras( Tyr-57), compared with wild-type H-ras, are defective in their guanine nucleotide-dependent release from human cdc25 and that this defect is more severe for the H-ras(Tyr-57) mutant. Such a defect would allow t he interfering mutants to remain bound to, thereby sequestering RAS ex change factors. The more severe interference phenotype of this novel i nterfering mutant suggests that it functions by titrating out other po sitive regulators of RAS besides those encoded by ste6 and CDC25.