Mechanism of BRAF Activation through Biochemical Characterization of the Recombinant Full‐Length Protein

Abstract

BRAF kinase plays an important role in mitogen-activated protein kinase (MAPK) signaling and harbors activating mutations in about half of melanomas and in a smaller percentage in many other cancers. Despite its importance, few in vitro studies have been performed to characterize the biochemical properties of full-length BRAF. Herein, a strategy to generate an active, intact form of BRAF protein suitable for in vitro enzyme kinetics is described. It is shown that purified, intact BRAF protein autophosphorylates the kinase activation loop and this can be enhanced by binding the MEK protein substrate through an allosteric mechanism. These studies provide in vitro evidence that BRAF selectively binds to active RAS and that the BRAF/CRAF heterodimer is the most active form, relative to their respective homodimers. Full-length BRAF analysis with small-molecule BRAF inhibitors shows that two drugs, dabrafenib and vemurafenib, can modestly enhance kinase activity of BRAF at low concentration. Taken together, this characterization of intact BRAF contributes to a framework for understanding its role in cell signaling.

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

“…We realized that BRAF V600E co‐purified with a trace amount of HSP70 (Figure B). Our previous results have demonstrated that the presence of HSP70 has no impact on enzyme kinetics . In addition, smaller bands corresponding to ≈50 kDa were identified by mass spectrometry to be fragments of BRAF, and no kinase activity was detected for those fragments.…”

“…Despite the extensive interest in developing inhibitors targeting BRAF V600E , the potential role of V600E mutation in oncogenic activation and drug sensitivity is not fully understood. Most in vitro BRAF studies reported to date used the catalytic domain, which we have shown to be less active and a poor physiological replicate compared to full‐length BRAF . The biochemical characterization of purified full‐length oncogenic BRAF reported here complements current knowledge obtained from truncated kinase domain and cell‐based studies.…”

“…Steady‐state kinetics analysis of FL‐BRAF V600E was performed by using previously developed radioactive kinase assays, in which the consumption of ATP was monitored as the readout of BRAF activity, and kinase‐dead FL‐MEK1 purified from Escherichia coli was used as the phosphorylated substrate . As shown in Figure D and E, the reaction rate is linear versus reaction time and enzyme concentration, thus supporting the idea that the reactions catalyzed by BRAF under the conditions of our assay meet the prerequisite for steady‐state kinetics.…”

“…We measured the IC 50 values of three types of BRAF inhibitor against the V600E mutant in an ELISA assay in which phosphorylated MEK1 was quantified by using phospho‐specific antibody. As shown in Table and Figure S3, unlike wild‐type BRAF, the V600E mutant is potently inhibited by all three categories of RAF inhibitors, regardless the position of the αC‐helix and DFG motif. The biochemical selectivity towards V600E revealed here has been demonstrated to translate to cellular selectivity in V600E mutant tumors .…”

“…The biochemical selectivity towards V600E revealed here has been demonstrated to translate to cellular selectivity in V600E mutant tumors . Previously, we observed that dabrafenib and vemurafenib paradoxically activate purified FL‐wild‐type BRAF . Consistent with cellular and in vivo data, purified FL‐BRAF V600E did not display paradoxical activation toward dabrafenib and vemurafenib; this suggests that the potential structural perturbation introduced by the V600E mutation plays a role in evading paradoxical activation.…”