Oncogenic non-V600 mutations evade the regulatory machinery of RAF including the Cdc37/Hsp90 chaperone and the 14-3-3...

The Ser/Thr kinase RAF, particularly BRAF isoform is a dominant target of oncogenic mutations and many mutations have been identified in various cancers. However, how these mutations except V600E evade the regulatory machinery of RAF protein and hence trigger its oncogenicity remains unclear. Methods: In this study, we used mutagenesis, peptide affinity assay, immunoprecipitation, immunoblot, and complementary split luciferase assay as well as mouse xenograft tumour model to investigate how the function of RAF is cooperatively regulated by Cdc37/Hsp90 chaperones and 14-3-3 scaffolds and how this regulatory machinery is evaded by prevalent non-V600 mutations. Results: We found that Cdc37/Hsp90 chaperones engaged with mature BRAF proteins promoted together with 14-3-3 scaffolds a switch of BRAF proteins from active open dimers into inactive close monomers. Most non-V600 mutations were enriched on or around the Cdc37/Hsp90-binding segments of BRAF, which impair association of CDc37/Hsp90 chaperones with BRAF and hence trap BRAF in active open conformation favouring dimerization. These BRAF mutants with high dimer propensity sustained a prolonged ERK signaling, and were effectively targeted by RAF dimer breaker plx8394 in vitro and in vivo. In contrast, CRAF and ARAF existed as immature monomers highly packaged with Cdc37/Hsp90 chaperones, which will be released upon dimerization driven by RAS-GTP binding with their N-terminus as well as 14-3-3 scaffold association with their C-terminus. Mature CRAF and ARAF dimers also sustained a prolonged ERK signaling as non-V600 BRAF mutants by virtue of absence of the C-terminal Cdc37/Hsp90-binding segment. Conclusions: Cdc37/Hsp90 chaperones and 14-3-3 scaffolds cooperatively facilitate the switch of RAF proteins from open active dimers to close inactive monomers. Non-V600 mutations disrupt this regulatory machinery, and trap RAF in dimers, which could be targeted by RAF dimer breakers.