Abstract PR02: Factors modulating RAF dimerization downstream of RAS – A mechanistic overview

The RAS-RAF pathway is one of the most commonly dysregulated in human cancers. The activation of this pathway is tightly regulated with appropriate spatial and temporal signaling cues. The central step in activation of this pathway is the dimerization of RAF kinases. Recent advances in biochemical, enzymatic and structural characterization of various multiprotein complexes in this pathway provide insight into various modes of RAF activity modulation. Kinase RAF and its substrate MEK exist as a pre-formed complex prior to pathway activation. Multiple factors prevent MEK phosphorylation by RAF in this pre-formed complex, including phosphorylation of a Serine residue N-terminal to the kinase domain, which enables 14-3-3 to trap RAF as a monomer. One of our intriguing findings is that cellular ATP exerts a negative regulatory effect on RAF kinase and stabilizes the inactive conformation of RAF monomer. We characterize this negative regulatory effect of ATP of RAF kinase structurally by solving a RAF-ATP complex and enzymology. Upon membrane recruitment of RAF by RAS-GTP, dephosphorylation of the above-mentioned serine residue in RAF occurs by a multiprotein complex composed of phosphatase PP1C, a scaffolding protein SHOC2 and RAS-GTP. We solved the structure of this RAS-SHOC2-PP1C complex and performed a thorough enzymatic analysis of this complex for RAF dephosphorylation. Our results show that RAF specificity is determined by SHOC2 and RAS GTP is responsible for spatial localization both inactive RAF and SHOC2/PP1C to the membrane. This dephosphorylation results in RAF being stabilized as a dimer by 14-3-3, which now binds to phospho-serine residues C-terminal to the kinase domain (S729 in BRAF) in 2 RAF molecules. We solve a structure of the RAF-14-3-3 complex. 14-3-3 induced RAF dimerization increases activity of RAF ~500 fold. Further, oncogenic mutations in RAF and MEK occur basally in tumors, and additional mutations are induced upon pathway inhibitor treatment in the clinic, especially for recent KRAS-G12C treatments. These mutations include RAF mutations that either activate or inactivate the kinase activity of the mutated RAF, but nevertheless result in pathway activation. Our data suggests that these mutations appear to tilt the RAF monomer-dimer equilibrium towards RAF dimer. The molecular mechanism of how the MEK mutations active the pathway in poorly understood. Biochemical and structural characterization of these mutations occurring in MEK kinas