C-Raf, a member of the Ras-Raf-MEK-ERK signaling pathway, plays key roles in regulating cell proliferation, differentiation and survival. Activating mutations along the pathway are common in many human cancers and are involved in cancer initiation, progression and metastasis. Previous work from our and other groups delineated an intricate regulation of C-Raf kinase by Ras, involving C-Raf dimerization1, protein interactions2 and phosphorylation3-5. The exact mechanism of C-Raf regulation remains, however, incompletely understood, limiting the use of Raf inhibitors in the clinic, as treated patients either show paradoxical activation of the pathway or develop fast resistance to the inhibitors. We obtained recently new data pertaining to C-Raf regulation by phosphorylation and protein-protein interaction allowing a more comprehensive understanding of C-Raf function. Using alanine mutation scanning of all 122 potential C-Raf phosphorylation sites we attained a complete analysis of C-Raf regulation by phosphorylation and identified several novel residues important for C-Raf activation and for substrate binding. Studies on the interaction of C-Raf with MEK revealed a direct effect of MEK on C-Raf through altering the phosphorylation state of C-Raf and allowing C-Raf activation in a Ras-independent manner, defining MEK as a novel Ras-independent C-Raf activator. Interestingly, this activation was not dependent on MEK kinase activity but was rather dependent on the interaction between the two proteins, suggesting that MEK binding confers a C-Raf conformation that is more susceptible to phosphorylation or protected from dephosphorylation. These results also allowed us the development of a Raf-based peptide that specifically inhibits Raf and MEK kinase activities.
References:
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