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Biochemical Characterization of Full‐Length Oncogenic BRAF V600E together with Molecular Dynamics Simulations Provide Insight into the Activation and Inhibition Mechanisms of RAF Kinases
Author(s) -
Cope Nicholas,
Novak Borna,
Candelora Christine,
Wong Kenneth,
Cavallo Maria,
Gunderwala Amber,
Liu Zhiwei,
Li Yana,
Wang Zhihong
Publication year - 2019
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201900266
Subject(s) - v600e , carcinogenesis , kinase , melanoma , cancer research , protein kinase domain , mutation , biology , phosphorylation , allosteric regulation , biochemistry , microbiology and biotechnology , chemistry , cancer , enzyme , genetics , gene , mutant
The most prevalent BRAF mutation, V600E, occurs frequently in melanoma and other cancers. Although extensive progress has been made toward understanding the biology of RAF kinases, little in vitro characterization of full‐length BRAF V600E is available. Herein, we show the successful purification of active, full‐length BRAF V600E from mammalian cells for in vitro experiments. Our biochemical characterization of intact BRAF V600E together with molecular dynamics (MD) simulations of the BRAF kinase domain and cell‐based assays demonstrate that BRAF V600E has several unique features that contribute to its tumorigenesis. Firstly, steady‐state kinetic analyses reveal that purified BRAF V600E is more active than fully activated wild‐type BRAF; this is consistent with the notion that elevated signaling output is necessary for transformation. Secondly, BRAF V600E has a higher potential to form oligomers, despite the fact that the V600E substitution confers constitutive kinase activation independent of an intact side‐to‐side dimer interface. Thirdly, BRAF V600E bypasses inhibitory P‐loop phosphorylation to enforce the necessary elevated signaling output for tumorigenesis. Together, these results provide new insight into the biochemical properties of BRAF V600E , complementing the understanding of BRAF regulation under normal and disease conditions.