Related Experiment Video

Updated: Sep 9, 2025

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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An In Vitro BRAF Activation Assay Elucidates Molecular Mechanisms Driving Disassembly of the Autoinhibited BRAF State

Daniel A Ritt1, David E Durrant1, Matthew R Drew2

  • 1Laboratory of Cell and Developmental Signaling, Center for Cancer Research (CCR), National Cancer Institute (NCI), Frederick, MD 21702.

Biorxiv : the Preprint Server for Biology
|September 2, 2025
PubMed

Insights

Active-state KRAS alone or with liposomes activates BRAF kinase. This new in vitro assay reveals KRAS binding is critical for BRAF activation and can screen for therapeutic inhibitors.

Area of Science:

  • Molecular Biology
  • Cell Signaling
  • Biochemistry

Background:

  • The RAS-ERK signaling pathway, including RAF kinases (ARAF, BRAF, CRAF), is crucial for cellular processes and often dysregulated in diseases like cancer.
  • BRAF mutations are key drivers in human cancers and RASopathies, making it a significant therapeutic target.
  • Despite extensive research, the precise mechanisms regulating BRAF activation remain incompletely understood.

Purpose of the Study:

  • To develop an in vitro BRAF activation assay to investigate the molecular steps governing BRAF regulation.
  • To elucidate the roles of KRAS, phosphatidylserine (PS), and phosphatase complexes in BRAF activation.
  • To establish a screening platform for identifying BRAF inhibitors with therapeutic potential.

Main Methods:

  • Development of an in vitro BRAF activation assay using purified autoinhibited BRAF:14-3-3:MEK complexes.
  • Assessment of BRAF activation by active-state KRAS, with and without PS-containing liposomes.
  • Evaluation of the SMP phosphatase complex's effect on BRAF activity and phosphorylation.
  • Testing the efficacy of inhibitors targeting the BRAF RBD:KRAS interaction.

Main Results:

  • Active-state KRAS alone promotes dimer-dependent BRAF activation.
  • PS-containing liposomes synergize with KRAS to significantly enhance BRAF activation.
  • The SMP phosphatase complex minimally affects catalytic activity but dephosphorylates a negative regulatory site, accelerated by KRAS.
  • Inhibitors of the BRAF RBD:KRAS interaction suppress in vitro BRAF activation, confirming RAS binding's critical role.

Conclusions:

  • RAS binding initiates BRAF autoinhibited monomer disassembly, facilitating membrane recruitment, dimerization, and activation.
  • The developed in vitro assay provides critical insights into BRAF activation mechanisms.
  • This assay serves as a valuable platform for discovering novel BRAF-targeting therapeutic compounds.
Keywords:
BRAFKRASRAF kinasesRAS GTPaseSignal transduction

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