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 (RafRBD) to Ras-Raf interaction and Ras-dependent Raf activation has remained unresolved. Here, we utilize a double mutant approach to identify complementary interacting amino acids that are involved in Ras-Raf interaction and activation. Biochemical analysis demonstrates that Raf-Arg(59) and Raf-Arg(67) from RafRBD are interacting residues complementary 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-dependent Raf activation, The characteristics observed here can be used as criteria for a role of residues from other regions of Raf in Ras-Raf interaction and activation. We developed a quantitative two-hybrid system as a tool to investigate the effect of point mutations on protein-protein interactions that elude biochemical analysis of bacterially expressed proteins. This assay shows that Raf-Ser(257) in the RafCR2 domain does not contribute to Ras-Raf interaction and that the Raf-S257L mutation 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 strongly suggests that the RafCR2 domain is an independent domain involved in the control of Raf activity and a common mechanism for constitutively activating mutants may be the interference with the inactive ground state of the kinase.