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

Nicholas Cope, Christine Candelora, Kenneth Wong, Sujeet Kumar, Haihan Nan, Michael Grasso, Borna Novak, Yana Li, Ronen Marmorstein, Zhihong Wang

Research output: Contribution to journalArticlepeer-review

27 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)1988-1997
Number of pages10
Issue number18
StatePublished - Sep 17 2018
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Organic Chemistry


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