RAS INTERACTION WITH 2 DISTINCT BINDING DOMAINS IN RAF-1 MAY BE REQUIRED FOR RAS TRANSFORMATION

Although Raf-1 is a critical has effector target, how Ras mediates Raf -1 activation remains unresolved, Raf-1 residues 55-131 define a Ras-b inding domain essential for Raf-1 activation. Therefore, our identific ation of a second Ras-binding site in the Raf-1 cysteine-rich domain ( residues 139-184) was unexpected and suggested a more complex role for Ras in Raf-1 activation, Both Ras recognition domains preferentially associate with Ras-GTP, Therefore, mutations that impair Ras activity by perturbing regions that distinguish Ras-GDP from Ras-GTP (switch I and II) may disrupt interactions with either Raf-1 binding domain. We observed that mutations of Ras that impaired Ras transformation by per turbing its switch I (T35A and E37G) or switch II (G60A and Y64W) doma in preferentially diminished binding to Raf-1-(55-131) or the Raf-1 cy steine-rich domain, respectively. Thus, these Ras binding domains reco gnize distinct Ras-GTP determinants, and both may be essential for Ras transforming activity. Finally, since Ha-Ras T35A and E37G mutations prevent Ras interaction with full-length Raf-1, we suggest that Raf-Cy s is a cryptic binding site that is unmasked upon Ras interaction with Raf-1-(55-131).