a, Screening scheme used to identify MEK1 mutants rescuing the interaction with KSR1 W831R. Screening was conducted in two rounds. In round 1, MEK1 was randomly mutagenized by error-prone-PCR, which identified 16 mutants corresponding exclusively to M219V and N221Y substitutions. In round 2, saturation mutagenesis was used to test all possible residue combinations in the region encoding amino acids 219–221. This identified 70 mutants that either complemented the Y2H interaction on SD-TLH or on both SD-TLH and SD-TLA. Sequence diversity of the recovered mutants is summarized by sequence logos shown on the right (see Supplementary Table 2 for details of mutant sequences). After further phenotypic screening, the fittest mutant on SD-TLA corresponded to MEK1 M219W–A220L. b, Position of MEK1 mutations that rescue the interaction with KSR1 W831R. Mutations systematically mapped to the MEK1 activation segment between Ser218 and Ser222, which are target phosphorylation sites for RAF proteins. Multiple sequence alignments of MAP2K1-7 activation segments illustrate that residues 219–221 vary within the MAP2K family, suggesting that substitutions of this sequence do not drastically affect enzyme function. The M219V substitution recovered in our screen is also found at the homologous position in MAP2K3 and MAP2K6. c, MEK1 activation segment mutations MEK1 M219V, MEK1 N221Y, MEK1 M219V/N221Y, and MEK1 M219W/A220L stimulate MEK1 binding to endogenous KSR1, BRAF, and CRAF. Co-IP of Flag-tagged MEK1 variants was performed in HEK293T cells. d, MEK1 M219W–A220L but not WT MEK1 stimulates KSR1 W831R–BRAF dimerization in BRET assays. mCherry-tagged MEK1 WT or MEK1 M219W–A220L were titrated in cells expressing the BRAF–KSR1 W831R BRET biosensors. BRET log 2(fold-changes) were reported as a function of log 10-transformed mCherry relative fluorescence units. e, Strategy to distinguish activator and substrate MEK in BRAF transactivation assays. Eight repeats of a G 4S flexible linker (symbolized by an octagon) were added onto WT MEK1 to separate it by size from co-transfected MEK variants lacking the linker. Experimental conditions corresponding to lanes 7 and 8 of Fig. 2d, respectively, are depicted at the top and bottom of the diagram. Experiment in e was repeated at least three times. For gel source data, see Supplementary Fig. 1.