A SINGLE AMINO-ACID CHANGE IN RAF-1 INHIBITS RAS BINDING AND ALTERS RAF-1 FUNCTION

Ras and Raf-1 are key proteins involved in the transmission of develop mental and proliferative signals generated by receptor and nonreceptor tyrosine kinases. Genetic and biochemical studies demonstrate that Ra f-1 functions downstream of Ras in many signaling pathways. Although R af-1 directly associates with GTP-bound Ras, an effect of this interac tion on Raf-1 activity in vivo has not been established. To examine th e biological consequence of the Ras/Raf-1 interaction in vivo, we set out to identify key residues of Raf-1 required for Ras binding. In thi s report, we show that a single amino acid mutation in Raf-1 (Arg(89) to Leu) disrupted the interaction with Ras in vitro and in the yeast t wo-hybrid system. This mutation prevented Ras-mediated but not tyrosin e kinase-mediated enzymatic activation of Raf-1 in the baculovirus/Sf9 expression system. Furthermore, kinase defective Raf-1 proteins conta ining the Arg(89) --> Leu mutation were no longer dominant-inhibitory or capable of blocking Ras-mediated signal transduction in Xenopus lae vis oocytes. These results demonstrate that the association of Raf-1 a nd Ras modulates both the kinase activity and the biological function of Raf-1 and identify Arg(89) as a critical residue involved in this i nteraction. In addition, the finding that tyrosine kinases can stimula te the enzymatic activity of Raf-1 proteins containing a mutation at t he Ras-interaction site suggests that Raf-1 can be activated by Ras-in dependent pathways.