THE RAS-RELATED PROTEIN R-RAS INTERACTS DIRECTLY WITH RAF-1 IN A GTP-DEPENDENT MANNER

R-ras is a member of the ras family of small GTPases that associates w ith the apoptosis-suppressing proto-oncogene product Bcl-2. Using the yeast two-hybrid system we provide evidence for an interaction between R-ras and the Raf-1 kinase. This interaction requires only the N-term inal regulatory domain (amino acids 1-256) of Raf-1, and is observed w ith both the wild type and a constitutively active R-ras mutant, but n ot with a deletion mutant that lacks the potential effector domain or a mutant of R-ras impaired for GTP binding. Moreover, using an in vitr o binding assay we show a direct GTP-dependent interaction of purified R-ras with a purified Raf-1 fragment corresponding to the proposed 81 -amino-acid H-Ras-binding domain of Raf-1 (amino acids 51-131). Taken together, these data indicate that R-ras may exert its biological effe ct by means of modulating the activity of the Raf-1 kinase as its dire ct downstream effector.