An In Vitro BRAF Activation Assay Elucidates Molecular Mechanisms Driving the 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:14-3-32: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. Significance StatementBRAF is a central intermediate in RAS pathway signaling, and its activity is often elevated in human cancers and RASopathy disorders. Due to the complexity of BRAF activation, identifying compounds that sustainably inhibit BRAF function has proven difficult, emphasizing the need for a more comprehensive understanding of BRAF regulation. Here, we have developed an in vitro BRAF activation assay that elucidates key steps in this process. Our findings demonstrate that RAS binding not only recruits BRAF to the plasma membrane but initiates the disassembly of the autoinhibited monomer, which in the context of the membrane, facilitates BRAF dimerization and activation. This assay advances our understanding of BRAF regulation and provides a novel platform for drug discovery efforts targeting BRAF.