Regulation of RAF family kinases: new insights from recent structural and biochemical studies

Abstract

The RAF kinases are required for signal transduction through the RAS-RAF-MEK-ERK pathway, and their activity is frequently up-regulated in human cancer and the RASopathy developmental syndromes. Due to their complex activation process, developing drugs that effectively target RAF function has been a challenging endeavor, highlighting the need for a more detailed understanding of RAF regulation. This review will focus on recent structural and biochemical studies that have provided 'snapshots' into the RAF regulatory cycle, revealing structures of the autoinhibited BRAF monomer, active BRAF and CRAF homodimers, as well as HSP90/CDC37 chaperone complexes containing CRAF or BRAFV600E. In addition, we will describe the insights obtained regarding how BRAF transitions between its regulatory states and examine the roles that various BRAF domains and 14-3-3 dimers play in both maintaining BRAF as an autoinhibited monomer and in facilitating its transition to an active dimer. We will also address the function of the HSP90/CDC37 chaperone complex in stabilizing the protein levels of CRAF and certain oncogenic BRAF mutants, and in serving as a platform for RAF dephosphorylation mediated by the PP5 protein phosphatase. Finally, we will discuss the regulatory differences observed between BRAF and CRAF and how these differences impact the function of BRAF and CRAF as drivers of human disease.

Keywords: HSP90/CDC37 chaperone complexes; RAF kinases; RAS pathway; RASopathies; cancer.

Figure 1.. Recent cryo-EM structures of RAF regulatory complexes.

(A) Domain structure of the RAF kinases. Key regions of the RAF regulatory and catalytic domains are indicated as are the N′ and C′ phosphoserine (PS) docking sites for 14-3-3 dimers. (B) Structure of an active BRAF dimer complex, with the BRAF kinase domains (KDs) forming back-to-back dimers and a single 14-3-3 dimer binding the C′ phosphosites (shown in dark gray) on each protomer. Shown is PDB: 6UAN and the PDB numbers of other RAF dimer structures are listed. (C) Structure of an autoinhibited BRAF monomer in which the RBD, CRD and KD are resolved. A 14-3-3 dimer is bound to the N′ and C′ phosphosites (shown in light gray and dark gray, respectively) and the active sites of the MEK and BRAF kinase domains interact in a face-to-face manner. Shown is PDB: 7MFD and the PDB numbers of other RAF monomer structures are listed. (D) Structure of CRAF:HSP90₂:CDC37 (RHC complex) bound to protein phosphatase PP5. Shown is PDB: 8GAE and the PDB numbers of other RHC complexes are listed.

Figure 2.. Summary of RAF kinase regulation.

(A) BRAF monomer to dimer transition. The autoinhibited BRAF monomer is recruited from the cytosol to the plasma membrane by GTP-loaded RAS. Binding of RAS to the BRAF RBD generates a steric clash and electrostatic repulsion between RAS and the C′-bound 14-3-3 protomer, which releases the RBD-CRD from the autoinhibition complex and in turn promotes the dissociation of 14-3-3 from the N′ site, thus exposing the BRAF dimer interface. The exposed N′ 14-3-3 binding site can now be dephosphorylated by SHOC2:MRAS:PP1C and the BRAF kinase domains can form dimers, bridged by a 14-3-3 dimer binding to the C′ phosphosite on each BRAF protomer. (B) Functions of the RAF:HSP90₂:CDC37 (RHC) complex. CDC37 and dimeric HSP90 interact directly with the RAF kinase domain to form the RHC complex, which is required for the folding and stability of the RAF kinases. In response to signaling cues, the RAF kinases become phosphorylated on activating sites and are also the targets of inhibitory feedback phosphorylation events. Post-signaling hyperphosphorylated RAF proteins may be recycled to a pre-signaling state through widespread dephosphorylation mediated by RHC complexes containing the PP5 protein phosphatase. Phosphorylation sites are depicted as black balls contain the letter P. (C) Listed are the known differences between the BRAF and CRAF kinases.