Elucidation of binding determinants and functional consequences of Ras/Raf-cysteine-rich domain interactions

Raf-1 is a critical downstream target of Ras and contains two distinct doma ins that bind Ras. The first Ras-binding site (RBS1) in Raf-1 has been show n to be essential for Ras-mediated translocation of Raf-1 to the plasma mem brane, whereas the second site, in the Raf-1 cysteine rich domain (Raf-CRD) , has been implicated in regulating Raf kinase activity. While recognition elements that promote Ras RBS1 complex formation have been characterized, r elatively little is known about Ras/Raf-CRD interactions. In this study, we have characterized interactions important for Ras binding to the Raf-CRB. Reconciling conflicting reports, we found that these interactions are essen tially independent of the guanine nucleotide bound state, but instead, are enhanced by post-translational modification of Ras. Specifically, our findi ngs indicate that Res farnesylation is sufficient for stable association of Ras with the Raf-CRD. Furthermore, we have also identified a Raf-CRD varia nt that is impaired specifically in its interactions with Ras. MMR data als o suggests that residues proximal to this mutation site on the Raf-CRD form contacts with Res. This Raf-CRD mutant impairs the ability of Ras to activ ate Raf kinase, thereby providing additional support that Ras interactions with the Raf-CRD are important for Ras-mediated activation of Raf-1.