Fig. 1: Domain organization, expression, and characterization of CRAF/MEK1/14-3-3 complexes.
a, Schematic showing the domain organization of CRAF and MEK1. Residue numbers for domain boundaries are shown below and selected regulatory phosphorylation sites for ARAF, BRAF, and CRAF are indicated above the schematics. b Rationale for the engineered salt-bridge in CRAF. The autoinhibited BRAF/MEK1/14-3-3 complex (PDB ID: 6NYB) is superimposed on an AlphaFold3 model of an autoinhibited CRAF/MEK1/14-3-3 complex. The BRAF kinase domain and CRD are shown in dark blue and dark red, respectively, and the corresponding domains of CRAF in light blue and pink. The Q156R/D587E mutation in CRAF introduces a salt-bridge between the CRD and kinase domain analogous to the R252 to E695 salt-bridge observed in autoinhibited BRAF. c Coomassie-stained SDS-PAGE analysis of purified CRAFSSYY/MEK1/14-3-3 and CRAFSSDD/MEK1/14-3-3 complexes. The Q156R and D587E mutations (QRDE) are incorporated in both CRAF constructs. Staining is consistent with a stoichiometric complex of CRAF, MEK1, and a 14-3-3 dimer. Sf9 cells express two 14-3-3 isoforms, ε and ζ. The experiments were independently repeated twice with similar results. d Kinase activity of purified RAF complexes measured in a time-resolved (TR)-FRET-based biochemical assay (see Methods for details). The FRET ratio at 665/620 nm is plotted for increasing concentrations of each RAF protein. RAF dimers were generated using N-terminally truncated (ΔN) constructs lacking the RBD and CRD, while RAF monomers were obtained with full-length constructs. Data are presented as mean values ± standard deviation (SD); experiments were conducted three times independently, each time in triplicate, with similar results. Source data are provided as a Source Data file. NtA N-terminal acidic motif, RBD RAS-binding domain, CRD cysteine-rich domain, KD kinase domain, CR1-CR3 conserved regions 1-3.
