Extended Data Figure 4: MEK1 binding to KSR1 is required for the transactivation of BRAF by side-to-side dimerization.

a, BRAF activation by KSR1 depends on an intact side-to-side dimerization surface in each protomer. Co-transfection of WT BRAF and KSR1 induces a strong pMEK signal while KSR1^R665H or BRAF^R509H mutants do not. b, Human KSR1 and KSR2 can both transactivate BRAF. C-terminal GFP10 fusions of KSR1 and KSR2 were co-transfected with BRAF and the resulting pMEK signal was monitored by western immunoblotting. c, Disruption of KSR1–MEK1 interaction with the W831R mutation perturbs BRAF transactivation by KSR1. The BRAF I666R mutation also prevents MEK phosphorylation. Similarly, MEK1^F311S is not phosphorylated when co-transfected with WT BRAF and KSR1. d, Transactivation of Drosophila RAF (dRAF) by Drosophila KSR (dKSR) requires MEK binding (W896R mutation is homologous to W831R in human KSR1) and dRAF–dKSR dimerization (R732H mutation is homologous to R665H in human KSR1). e, The transactivation potential of FRB–dKSR^KD towards FKBP–dRAF^KD was assessed by monitoring the levels of phosphorylated MEK in the presence or absence of rapamycin in S2 cells (1 μM). Blocking MEK binding to dKSR (W896R mutation) and dRAF–dKSR dimerization (R732H mutation) abrogated RAF transactivation and MEK phosphorylation as judged by western immunoblotting. f, MEK phosphorylation induced by co-transfection of BRAF and KSR1 depends on the catalytic activity of BRAF and not on the integrity of the active site region of KSR1. g, KSR1 A637F (AF) and A637F/YLQE_602-604>DDEE_602-604 (AFDDEE) gain-of-function mutants are dependent on binding to endogenous MEK1 or MEK2 to dimerize with endogenous BRAF and to activate the cellular pool of RAF as judged by the pMEK1 or pMEK2 signal. The W831R substitution in KSR1 was used to abrogate KSR1^AF and KSR1^AFDDEE mutant binding to MEK. Experiments were repeated at least three times. For gel source data, see Supplementary Fig. 1.