Phosphorylation of RAF Kinase Dimers Drives Conformational Changes that Facilitate Transactivation

Abstract

RAF kinases are key players in the MAPK signaling pathway and are important targets for personalized cancer therapy. RAF dimerization is part of the physiological activation mechanism, together with phosphorylation, and is known to convey resistance to RAF inhibitors. Herein, molecular dynamics simulations are used to show that phosphorylation of a key N‐terminal acidic (NtA) motif facilitates RAF dimerization by introducing several interprotomer salt bridges between the αC‐helix and charged residues upstream of the NtA motif. Additionally, we show that the R‐spine of RAF interacts with a conserved Trp residue in the vicinity of the NtA motif, connecting the active sites of two protomers and thereby modulating the cooperative interactions in the RAF dimer. Our findings provide a first structure‐based mechanism for the auto‐transactivation of RAF and could be generally applicable to other kinases, opening new pathways for overcoming dimerization‐related drug resistance.

Targeting MAPK Signaling in Cancer: Mechanisms of Drug Resistance and Sensitivity

“…Interestingly, dimers are drug resistant and actually can cause "paradoxical" activation of the ERK pathway as one drug bound protomer can activate its drug free binding partner through allosteric transactivation [28,52]. It is counterintuitive that a drug in large stoichiometric excess of RAF should only be able to bind to one RAF molecule in a dimer, but it was experimentally observed [53] and is explainable on thermodynamic grounds [54], which include a slight structural asymmetry of RAF protomers in the dimer [55] (Figure 4). This type of resistance can be broken by RAF inhibitors that prevent dimerization [56], or by a combination of two structurally different RAF inhibitors that target the two different conformations of RAF proteins in a dimer.…”

Targeting Aberrant RAS/RAF/MEK/ERK Signaling for Cancer Therapy

“…Once RAF achieves active conformation, its dimer interface becomes further stabilized by the hydrophobic R-spine residue in the αChelix (L505 for B-Raf, L397 for C-Raf, and L358 for A-Raf) located adjacent to the conserved RKTR motif, which is allosterically connected αC-helix and dimer interface [163]. Upon the relocation of R509 to the center of the dimer interface, αC-helix interacts with the NTA motif of the trans RAF molecule and adopts the "IN" conformation [80,164] The dimerization of RAF proteins can be promoted by shortening their β3-αC loop; and in-frame deletions of β3-αC loop activate ARAF by enforcing homodimer formation, showing that ARAF can function as a "true" kinase to induce ERK phosphorylation [84], even though it is the weakest kinase in the family. The corresponding deletions in BRAF also ramp up its kinase activity through enhancing its homodimer formation.…”

Dynamic, structural and thermodynamic basis of insulin-like growth factor 1 kinase allostery mediated by activation loop phosphorylation

“…25–29 On the other hand, relatively few studies have been carried out to understand the allosteric regulation of A-loop phosphorylation on kinase conformational dynamics 22,30–32 and catalytic activities. 33 Thus, detailed mechanisms of kinase allostery mediated by A-loop phosphorylation remain poorly understood.…”