Fig. 5. KRAS Q61H is resistant to regulation of RAF-RBD binding by tyrosyl phosphorylation.
a Histogram representing the dissociation constant (Kd) for the binding of unmodified, singly or doubly phosphorylated wild-type or Q61H mutant KRAS to BRAF-RBD. The phosphorylated forms were separated using ion-exchange chromatography and binding to immobilized RBD was measured using BLI. The error bars indicate SEM of three independent measurements. b HEK293 cells were transfected with G12V or Q61H HA-KRAS together with increasing amounts of Src. Cells were lysed and activated HA-KRAS was pulled down with RAF1:RBD-conjugated beads and immunoblotted with anti-HA. The whole-cell extract (WCE) was resolved by SDS-PAGE and probed with the indicated antibodies to confirm expression and loading. Numbers indicate normalized ratios of activated KRAS to total KRAS based on densitometry. The immunoblottings are representative of at least three independent experiments. c A line graph that shows root mean square fluctuation (RMSF) of WT-KRAS (top, modified-red, unmodified-blue) and KRAS Q61H (bottom, modified-brown, unmodified-green). The X-axis shows the residue id for the proteins and the Y-axis is for fluctuation distance in Å. d Model summarizing the impact of Q61H mutation and tyrosyl phosphorylation on the KRAS GTPase cycle and RBD binding. Line thicknesses represent relative rates of each step in the GTPase cycle. The upper panels (from left to right) show the impact of phosphorylation on the GTPase cycle of KRAS WT: reduced sensitivity to the activity of GAPs and GEFs, and impaired binding to RAF. The left panels (from top to bottom) show the impact of the Q61H mutation on the GTPase cycle of KRAS: reduced sensitivity to the activity of GAPs and GEFs with little impact on RAF. The lower panels (from left to right) represent how phosphorylation of the KRAS Q61H mutant has little additional impact on its GTPase cycle.