The dimer-dependent catalytic activity of RAF family kinases is revealed through characterizing their oncogenic mutants

Abstract

Although extensively studied for three decades, the molecular mechanisms that regulate the RAF/MEK/ERK kinase cascade remain ambiguous. Recent studies identified the dimerization of RAF as a key event in the activation of this cascade. Here, we show that in-frame deletions in the β3-αC loop activate ARAF as well as BRAF and other oncogenic kinases by enforcing homodimerization. By characterizing these RAF mutants, we find that ARAF has less allosteric and catalytic activity than the other two RAF isoforms, which arises from its non-canonical APE motif. Further, these RAF mutants exhibit a strong oncogenic potential, and a differential inhibitor resistance that correlates with their dimer affinity. Using these unique mutants, we demonstrate that active RAFs, including the BRAF(V600E) mutant, phosphorylate MEK in a dimer-dependent manner. This study characterizes a special category of oncogenic kinase mutations, and elucidates the molecular basis that underlies the differential ability of RAF isoforms to stimulate MEK-ERK pathway. Further, this study reveals a unique catalytic feature of RAF family kinases that can be exploited to control their activities for cancer therapies.

Biochemical Characterization of Full‐Length Oncogenic BRAFV600E together with Molecular Dynamics Simulations Provide Insight into the Activation and Inhibition Mechanisms of RAF Kinases

“…Although an increased dimerization potential observed in vitro does not necessarily reflect the actual oligomeric status of BRAF in cells, multiple research groups have provided solid cellular evidence supporting the idea that BRAF is indeed oligomeric in living cells . In addition, BRAF V600E was shown to phosphorylate MEK in a dimer‐dependent manner …”

AMPKi overcomes the paradoxical activation of CRAF driven by RAF inhibitors through blocking the 14-3-3 binding to its carboxyl-terminus

“…The dimerization of RAF kinase is required for both activation and catalytic activity (11,31). Our recent study has shown that even constitutively active RAF mutants function as a dimer to phosphorylate MEK (11).…”

“…The Ser/Thr protein kinase, RAF, is a core component of this signaling cascade, which includes three isoforms: BRAF, CRAF, and ARAF (5)(6)(7). All RAF isoforms have similar molecular structures albeit distinct traits that result in their differential ability to activate their downstream effector, MEK (8)(9)(10)(11). Recent studies have identified RAF dimerization as a key event in the activation and regulation of this signaling cascade (12)(13)(14)(15)(16)(17).…”

“…Similarly, RAF inhibitors promote RAF dimerization through altering the conformation of its kinase domain (4). The dimerization-favored conformation of RAF molecule could be also achieved by catalytic spine mutations or β3-αC loop deletions that exist in cancer genomes (11,21). Furthermore, the differential molecular traits among RAF isoforms result in their distinct propensities in dimerization-driven transactivation.…”

“…Furthermore, the differential molecular traits among RAF isoforms result in their distinct propensities in dimerization-driven transactivation. ARAF has a non-canonic APE motif that weakens its dimerization ability and accounts for its less activity toward MEK (11). The unique N-terminal acidic motif (NtA motif) of BRAF enables this isoform strongly transactivate the other two isoforms through dimerization, whereas the transactivation ability of CRAF and ARAF is regulated by their NtA motif phosphorylation (8).…”