Fig. 4: Assembling a model for RAS-driven activation of RAF.

In the quiescent state, BRAF is maintained in an autoinhibited complex with MEK and a 14-3-3 dimer in the cytosol. As a first step in activation (1), this complex is recruited to the membrane by GTP-bound RAS to form a recruitment complex, as visualized in this study. The interaction with farnesylated RAS in a membrane context induces release of autoinhibitory interactions in the BRAF/MEK1/14-3-3 complex, resulting in formation of an “open” monomer complex (2). This second step in activation may result from extraction of the CRD domain from its relatively buried site in the autoinhibited complex, due to preferential binding to RAS and the plasma membrane. Opening exposes pS365 for dephosphorylation by the SHOC2 phosphatase complex, and also allows the 14-3-3 dimer to rearrange to bind the C-terminal pS729 site of two BRAF molecules, driving and stabilizing the active BRAF dimer (3). Because the pS365 site is buried and not accessible for dephosphorylation in the recruitment complex, and because S365 plays no known role in the formation of the active dimer, we hypothesize that the SHOC2 phosphatase complex acts on the open monomer state and contributes to activation by preventing its “reclosure”. Once pS365 is dephosphorylated, BRAF cannot reassume the closed, autoinhibited state observed in the recruitment complex, and the resulting accumulation of open monomers favors rearrangement into active dimers. Protein Data Bank accession codes for structures supporting distinct states and components of this model include: 6NYB and 7MFD (autoinhibited complex); 8DGS and 8DGT (recruitment complex, this study); 6PP9 and 6U2G (BRAF/MEK kinase domain portion of open monomer); 6XHB and 7JHP (RAS complexes with RBD/CRD fragments of RAF, relevant to the open monomer and active dimer states); 6Q0J, 6Q0K, 6UAN, 6XAG, 7MFF (14-3-3-bound BRAF dimers, with or without MEK); and 7SD0, 7UPI, 7TXH, and 7TVF (ternary SHOC2, MRAS, PP1C phosphatase complex).