DISCRIMINATION OF AMINO-ACIDS MEDIATING RAS BINDING FROM NONINTERACTING RESIDUES AFFECTING RAF ACTIVATION BY DOUBLE MUTANT ANALYSIS

The contribution of residues outside the Has binding domain of Raf (Ra fRBD) to Ras-Raf interaction and Ras-dependent Raf activation has rema ined unresolved. Here, we utilize a double mutant approach to identify complementary interacting amino acids that are involved in Ras-Raf in teraction and activation. Biochemical analysis demonstrates that Raf-A rg(59) and Raf-Arg(67) from RafRBD are interacting residues complement ary to Ras-Glu(37) located in the Ras effector region. Raf-Arg(59) and Raf-Arg(67) also mediate interaction with Ras-Glu(37) in Has-dependen t Raf activation, The characteristics observed here can be used as cri teria for a role of residues from other regions of Raf in Ras-Raf inte raction and activation. We developed a quantitative two-hybrid system as a tool to investigate the effect of point mutations on protein-prot ein interactions that elude biochemical analysis of bacterially expres sed proteins. This assay shows that Raf-Ser(257) in the RafCR2 domain does not contribute to Ras-Raf interaction and that the Raf-S257L muta tion does not restore Raf binding to Ras-E37G, Yet, Raf-S257L displays high constitutive kinase activity and further activation by Ras-G12V/ E37G is still impaired as compared with activation by Ras-G12V, This s trongly suggests that the RafCR2 domain is an independent domain invol ved in the control of Raf activity and a common mechanism for constitu tively activating mutants may be the interference with the inactive gr ound state of the kinase.