Fig. 5: Effects of NtA motif phosphorylation or mutation on structure and activity of CRAF.

a Full-length CRAF^SSYY and MEK1 were co-expressed in Sf9 cells with or without accompanying co-expression of activated Src and PAK1. After purification, the phosphorylation status of the NtA motif of the resulting CRAF^SSYY/MEK1/14-3-3 complexes was assessed by western blotting with phosphospecific antibodies directed against pSer338 (Cell Signaling Technology, #9427) and pTyr341 (Abcam, #ab59223) (upper panels) and against total CRAF as a loading control (lower panels). b Kinase activity of CRAF^SSDD/MEK1/14-3-3 and CRAF^SSYY/MEK1/14-3-3 complexes prepared with or without co-expression of activated Src and PAK1 was assessed in the TR-FRET assay. The FRET ratio at 665/620 nm is plotted for increasing concentrations of each RAF complex. Each data point represents the mean ± SD of triplicate measurements. The decrease in activity at high enzyme concentrations is due to competition from the non-phosphorylatable MEK1^SASA in the enzyme complex, which becomes significant as its concentration approaches that of the WT MEK1 substrate (200 nM). Density maps in the region of the C-helix in cryo-EM structure of the CRAF^SSYY/MEK1/14-3-3 kinase domain open monomer (c) and the crystal structure of CRAF^SSDD and MEK1 kinase domains (PDB ID: 9AY7) (d). In the CRAF^SSYY structure in c, there is clear side chain density for residues in the C-helix and inhibitory turn, and for W496 in the activation loop and W342, the first residue following the NtA motif. By contrast, there is evidence of local disorder in this region in the crystal structure in (d). W342 and W496 are not modeled in the structure and are superimposed from the CRAF^SSYY structure (and colored purple) to indicate their positions. In addition, several large residues in this region of the CRAF^SSDD structure were modeled as alanine owing to a weak or no density for their sidechains (including Q386, F387, R391, and R398). The density map for the cryo-EM structure is shown in c, and the 2Fo − Fc map for the crystal structure in d is contoured at 1.0 σ.