Turbyville, T. J. | bioRxiv Science

bioRxiv2025

eIF2B Selectively Anchors and Activates Mutant KRAS

Much is known about how RAS oncoproteins regulate mRNA translation factors, but the reverse relationship, how translation factors influence RAS activity, has remained largely unexplored. At the plasma membrane (PM), Son of Sevenless (SOS) acts as the canonical guanine nucleotide exchange factor (GEF) for RAS proteins, yet mechanisms governing its specificity for individual RAS isoforms remain unknown. Here, we show that the translation initiation factor eIF2B, best known for its GEF function in translation initiation, forms a distinct complex with SOS and mutant KRAS at the PM, but not with other oncogenic RAS variants. Mechanistically, eIF2B acts as an allosteric regulator of SOS, selectively enhancing GDP-GTP exchange on mutant KRAS. This specificity arises from the translational activity of eIF2B, which upregulates glycosphingolipid (GSL) biosynthesis to remodel PM lipids and preferentially anchor mutant KRAS. Together, our results uncover an unexpected moonlighting function of eIF2B: acting both as a direct activator of SOS and as a regulator of GSL pathway that shapes the membrane landscape, both required for mutant KRAS activation. These insights redefine our understanding of eIF2B and mutant KRAS functions in cancer and have profound implications for KRAS-driven oncogenesis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/686860v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1f50934org.highwire.dtl.DTLVardef@2f55b9org.highwire.dtl.DTLVardef@1a51e39org.highwire.dtl.DTLVardef@1634aac_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIeIF2B interacts with mutant KRAS and SOS at the plasma membrane (PM). C_LIO_LIThe eIF2B:SOS complex promotes the GTP-bound active state of mutant KRAS. C_LIO_LIeIF2B enhances the translation of B4GALT5 mRNA, encoding a key enzyme of glycosphingolipid (GSL) biosynthesis. C_LIO_LIUpregulation of the GSL metabolites, ganglioside GM3 and sulfatide SM4, remodels PM lipid composition to facilitate eIF2B:SOS:KRAS complex formation and mutant KRAS nanoclustering. C_LIO_LIThrough its interaction with SOS and stimulation of GSL synthesis, eIF2B selectively activates mutant KRAS at the PM among RAS isoforms. C_LIO_LIeIF2B is required for the development of mKRAS-driven lung adenocarcinoma in mice. C_LIO_LIeIF2B is a marker of poor prognosis in mutant KRAS-driven cancers. C_LI

cancer biology↗

bioRxiv2025

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.

biochemistry↗