Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF

Over 30 mutations of the B-Raf gene associated with human cancers have been identified, the majority of which are located within the kinase domain. Here we show that of 22 B-Raf mutants analyzed, 18 have elevated kinase activity and signal to ERK in vivo. Surprisingly, three mutants have reduced kinase activity towards MEK in vitro but, by activating C-Raf in vivo, signal to ERK in cells. The structures of wild type and oncogenic V599EB-RAF kinase domains in complex with the Raf Inhibitor BAY43-9006 show that the activation segment is held in an inactive conformation by association with the P loop. The clustering of most mutations to these two regions suggests that disruption of this interaction converts B-Raf into its active conformation. The high activity mutants signal to ERK by directly phosphorylating MEK, whereas the impaired activity mutants stimulate MEK by activating endogenous C-Raf, possibly via an allosteric or transphosphorylation mechanism.