Elucidating the Regulatory Hotspots of Full-Length BRAF through Enzymological Analyses

• Main Author: Cope, Nicholas J
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01.01.2020
• Subjects: Biochemistry
• ISBN: 1658426789; 9781658426787

BRAF kinase plays an important role in MAPK signaling and harbors activating mutations in about half of melanomas and in a smaller percentage in other cancers. Despite its importance, few in vitro studies have been performed to characterize the biochemical properties of full-length (FL) BRAF. Herein, a strategy to generate an active, intact form of wild-type and mutated BRAF protein suitable for in vitro enzyme kinetics is described. FL-BRAF analysis with small-molecule BRAF inhibitors shows that two drugs, dabrafenib and vemurafenib, can modestly enhance kinase activity of BRAF at low concentration. Second we examine the most prevalent BRAF mutation, V600E. Our biochemical characterization of intact BRAFV600E together with molecular dynamics (MD) simulations of BRAF kinase domain and cell-based assays demonstrate that BRAFV600E has several unique features that contribute to its tumorigenesis. First, steady-state kinetic analyses reveal that purified BRAFV600E is more active than fully-activated wild-type BRAF, consistent with the notion that elevated signaling output is necessary for malignant transformation. Second, BRAFV600E 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. Third, BRAFV600E bypasses inhibitory P-loop phosphorylation to enforce necessary elevated signaling output for tumorigenesis. Together, we provide new insight into the biochemical function of BRAFV600E, which complement the understanding of BRAF regulation under normal and disease conditions. Lastly, we inspected class 3 BRAF mutations, kinase-impaired or kinase-dead, which have the highest mutation frequency in BRAF in lung adenocarcinoma. Herein we established that BRAFD594G, a representative of class 3 mutants, has an increased dimerization potential as compared to wild-type BRAF. MD simulations revealed that the D594G mutation changes the position of the αC-helix and extends the activation loop, shifting the equilibrium towards the active, dimeric conformation, thus priming BRAFD594G as an effective allosteric activator of CRAF. Additionally, we show that BRAFD594G:CRAF heterodimers bypass autoinhibitory P-loop phosphorylation, which might lead to higher duration of MAPK pathway signaling in cancer cells. Lastly, we propose that the dimer interface of BRAFD594G:CRAF heterodimer may serve as a promising target for the design of novel cancer therapeutics.