An in vitro BRAF activation assay elucidates molecular mechanisms driving disassembly of the autoinhibited BRAF state

The RAF kinases (ARAF, BRAF, and CRAF) are essential components of the RAS-ERK signaling pathway, which controls vital cellular processes and is frequently dysregulated in human disease. Notably, mutations that alter BRAF function are prominent drivers of human cancer and certain RASopathy disorders, making BRAF an important target for therapeutic intervention. Despite extensive research, several aspects of BRAF regulation remain unclear. In this study, we developed an in vitro BRAF activation assay using purified autoinhibited BRAF:14-3-3<sub>2</sub>:MEK complexes. Our results show that fully processed, active-state KRAS alone can promote dimer-dependent BRAF activation. Moreover, we found that phosphatidylserine (PS)-containing liposomes synergized with KRAS to promote BRAF activation, achieving activity levels comparable to those observed with BRAF proteins that constitutively dimerize. In contrast, the SMP phosphatase complex had only a minimal effect on BRAF catalytic activity in this system but mediated the dephosphorylation of the negative regulatory pS365 14-3-3 binding site in a manner that was accelerated by the presence of KRAS alone or KRAS and 30% PS liposomes. Finally, we show that inhibitors blocking the BRAF RBD:KRAS interaction were able to suppress the in vitro activation of BRAF, underscoring the critical role of RAS binding in initiating the disassembly of the BRAF autoinhibited state. Thus, this assay provides valuable insights into the steps required for BRAF activation and can serve as an effective screening tool for identifying compounds that may inhibit this process and have therapeutic potential.

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Publication Details

Journal
Proceedings of the National Academy of Sciences
Published
2025-09-15
DOI
https://doi.org/10.1073/pnas.2508169122
Citations
2
Primary Topic
Melanoma and MAPK Pathways
Type
article
Field-Weighted Citation Impact
1.61

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article

An in vitro BRAF activation assay elucidates molecular mechanisms driving disassembly of the autoinhibited BRAF state

Daniel A. Ritt, Helgi I. Ingólfsson, Grace Scheidemantle, Felice C. Lightstone et al.
2 citations
Proceedings of the National Academy of Sciences
Melanoma and MAPK Pathways
1.61
article

An in vitro BRAF activation assay elucidates molecular mechanisms driving disassembly of the autoinhibited BRAF state

Daniel A. Ritt, Helgi I. Ingólfsson, Grace Scheidemantle, Felice C. Lightstone, David Durrant, Thomas J. Turbyville, Stuart A. Sandin, Juliana A. Martinez Fiesco, Kelly Snead, Matthew R. Drew, Timothy S. Carpenter, Dwight V. Nissley, Albert S.W. Sohn, Frederick H. Streitz, Yue Yang, Deborah K. Morrison, Rebika Shrestha, Caroline J. DeHart, Andrew G. Stephen, Dominic Esposito, Ping Zhang, Robert A. D’Ippolito, Fikret Aydin, Jeremy O. B. Tempkin, Xiao‐Hua Zhang, Jeffrey S. Hull
article en
2 citations

Abstract

The RAF kinases (ARAF, BRAF, and CRAF) are essential components of the RAS-ERK signaling pathway, which controls vital cellular processes and is frequently dysregulated in human disease. Notably, mutations that alter BRAF function are prominent drivers of human cancer and certain RASopathy disorders, making BRAF an important target for therapeutic intervention. Despite extensive research, several aspects of BRAF regulation remain unclear. In this study, we developed an in vitro BRAF activation assay using purified autoinhibited BRAF:14-3-3<sub>2</sub>:MEK complexes. Our results show that fully processed, active-state KRAS alone can promote dimer-dependent BRAF activation. Moreover, we found that phosphatidylserine (PS)-containing liposomes synergized with KRAS to promote BRAF activation, achieving activity levels comparable to those observed with BRAF proteins that constitutively dimerize. In contrast, the SMP phosphatase complex had only a minimal effect on BRAF catalytic activity in this system but mediated the dephosphorylation of the negative regulatory pS365 14-3-3 binding site in a manner that was accelerated by the presence of KRAS alone or KRAS and 30% PS liposomes. Finally, we show that inhibitors blocking the BRAF RBD:KRAS interaction were able to suppress the in vitro activation of BRAF, underscoring the critical role of RAS binding in initiating the disassembly of the BRAF autoinhibited state. Thus, this assay provides valuable insights into the steps required for BRAF activation and can serve as an effective screening tool for identifying compounds that may inhibit this process and have therapeutic potential.

Proceedings of the National Academy of SciencesVol. 122(38)
Leidos (United States) (US), Lawrence Livermore National Laboratory (US), Cancer Institute (WIA) (IN), Frederick National Laboratory for Cancer Research (US), National Cancer Institute (US), Center for Cancer Research (US), Leidos Biomedical Research Inc. (United States)
U.S. Department of Energy, U.S. Department of Health and Human Services, Frederick National Laboratory for Cancer Research, National Institutes of Health, Office of Science, National Cancer Institute, Advanced Scientific Computing Research, Lawrence Livermore National Laboratory
Openalex Percentile: Top 15%
Melanoma and MAPK Pathways
1.61
DOIOpen Access PDFOpenAlex
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