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

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. Home of the BRAF: A strategy to obtain full‐length BRAF that is significantly more active than its truncated counterpart is reported. To gain a better understanding of BRAF regulation, a detailed kinetic analysis of purified full‐length BRAF is conducted to investigate the impact of BRAF inhibitors, MEK binding, RAS binding, and heterodimerization with CRAF on kinase activity in vitro.