An in vitro BRAF activation assay elucidates molecular mechanisms driving disassembly of the autoinhibited BRAF state
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 2 :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.
Article Details
Journal Info
Proceedings of the National Academy of Sciences
National Academy of Sciences
Authors (26)
Daniel A. Ritt
Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute
David E. Durrant
Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute
Matthew R. Drew
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.
Suzanne I. Sandin
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.
Juliana A. Martinez Fiesco
Center for Structural Biology, Center for Cancer Research, National Cancer Institute
Xiaohua Zhang
Fikret Aydin
Timothy S. Carpenter
Grace M. Scheidemantle
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.
Robert A. D’Ippolito
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.
Alexandria L. Sohn
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.
Caroline J. DeHart
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.
Rebika Shrestha
Kelly Snead
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.
Jenna Hull
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.
Jeremy O. B. Tempkin
Biosciences and Biotechnology Division, Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory
Yue Yang
Felice C. Lightstone
Frederick H. Streitz
Ping Zhang
Thomas J. Turbyville
Andrew G. Stephen
Dominic Esposito
National Cancer Institute RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc.
Helgi I. Ingólfsson
Dwight V. Nissley
Deborah K. Morrison
Laboratory of Cell and Developmental Signaling, Center for Cancer Research, National Cancer Institute