Identification of residues in the cysteine-rich domain of Raf-1 that control Ras binding and Raf-1 activity

We have identified mutations in Raf-l that increase binding to Ras. The mutations were identified making use of three mutant forms of Ras that have reduced Raf-l binding (Winkler, D. G., Johnson, J. C., Cooper, J, A., and Vojtek, A. B. (1997) J. Biol, Chem. 272, 24402-24409). One mutation in Raf-l, N64L, suppresses the Ras mutant R41Q but not other Ras mutants, suggesting that this mutation structurally complements the Ras R41Q mutation. Missense substitutions of residues 143 and 144 in the Raf-l cysteine-rich domain were isolated multiple times. These Raf-l mutants, R143Q, R143W, and K144E, were general suppressors of three different Ras mutants and had increased interaction with non-mutant Ras. Each was slightly activated relative to wild-type Raf-1 in a transformation assay. In addition, two mutants, R143W and K144E, were active when tested for induction of germinal vesicle breakdown in Xenopus oocytes. Interestingly, all three cysteine-rich domain mutations reduced the ability of the Raf-1 N-terminal regulatory region to inhibit Xenopus oocyte germinal vesicle breakdown induced by the C-terminal catalytic region of Raf-l, We propose that a direct or indirect regulatory interaction between the N- and C-terminal regions of Raf-l is reduced by the R143W, R143Q, and K144E mutations, thereby increasing access to the Ras-binding regions of Raf-l and increasing Raf-l activity.