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Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods
07:49

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

Published on: July 17, 2019

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Role of serine 365 in BRAF V600E sensitivity to RAF inhibition

Michael J Vido1,2, Justin Rock1,2, Andrew E Aplin1,3

  • 1Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA.

Pigment Cell & Melanoma Research
|October 1, 2020
PubMed

Insights

Altering the S365 14-3-3 binding site in BRAF V600E increases BRAF homodimerization and reduces sensitivity to RAF inhibitors, suggesting a role in cancer drug resistance.

Area of Science:

  • Oncology
  • Molecular Biology
  • Biochemistry

Background:

  • BRAF kinase is a key regulator of the MEK-ERK1/2 signaling pathway, frequently mutated in various cancers.
  • 14-3-3 proteins interact with BRAF at N-terminal S365 and C-terminal S729 sites, modulating its activity and dimerization.
  • The role of the N-terminal S365 14-3-3 binding site in full-length BRAF V600E mutants and its impact on drug resistance remain unclear.

Purpose of the Study:

  • To investigate the function of the S365 14-3-3 binding site in full-length BRAF V600E.
  • To determine the effect of S365 site modification on BRAF V600E dimerization and RAF inhibitor sensitivity.

Main Methods:

  • Site-directed mutagenesis was used to create a BRAF V600E mutant with alanine substitution at the S365 site (BRAF V600E S365A).
  • 14-3-3 protein association, BRAF V600E homodimerization, and MEK-ERK1/2 signaling pathway activity were assessed.
  • Cellular responses including cell growth and viability upon RAF inhibitor treatment were evaluated.

Main Results:

  • Mutation of BRAF S365 to alanine (S365A) decreased 14-3-3 binding and enhanced BRAF V600E homodimerization.
  • The BRAF V600E S365A mutant exhibited reduced sensitivity to RAF inhibitors, impacting MEK-ERK1/2 signaling, cell proliferation, and survival.

Conclusions:

  • The S365 14-3-3 binding site plays a crucial role in regulating BRAF V600E dimerization and sensitivity to RAF inhibitors.
  • Alteration or loss of the S365 14-3-3 interaction site may contribute to the development of RAF inhibitor resistance in cancers harboring BRAF V600E mutations.
Keywords:
14-3-3BRAF inhibitorphosphorylationresistancetargeted therapy

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