Fig. 4

Experimental validation of functional impact of mutations in 3D clusters in MAP2K1 and RAC1. a Seven residues in a 3D cluster in MAP2K1, in the context of the domain structure of the protein. Notation as in Fig. 1: each circle is an occurrence in a sample; connecting lines (bottom) indicate cluster membership, i.e., statistically significant proximity in 3D in the protein structure. b The same cluster of mutated residues in the 3D structure of MAP2K1. The purple helix is known to negatively regulate the kinase activity of MAP2K1/MEK1. c Functional characterization of MAP2K1/MEK1 mutants in HEK-293H cells. Expression of G128D and Y130C (as well as the previously characterized F53L, Q56P, and K57N) mutants each resulted in increased expression of phosphorylated ERK compared to wild-type MAP2K1 — but not the cluster member A52V. d ERK phosphorylation was inhibited by trametinib in cells expressing the Q56P or Y130C MAP2K1 mutations in HEK-293H cells. e The four residues (two single-residue hotspots: P29 and A159, and two rarely mutated residues: G15 and C18) in the identified 3D cluster in RAC1 in the linear domain structure of the protein. f The same cluster in the 3D structure of RAC1. g Western blot analysis of RAC1 activation (GTP-bound RAC1 levels) by PAK1 pulldown (left) and of total RAC1 levels (right) in HEK-293 T cells. The RAC1 3D cluster mutations G15S and C18Y, as well as the previously characterized P29S and A159V, were associated with significant RAC1 activation, as compared to wild-type RAC1