Figure 1

SETD6 methylates PAK4 at lysine 473. (A) A multiple alignment of lysine 473 residue of PAK4 in different organisms. Multiple alignment was performed using COBALT tool⁵⁵ for Homo sapiens, Mus musculus, Danio rerio and Drosophila melanogaster PAK4 protein sequences. (B) In-vitro methylation assay. Recombinant His-Sumo-PAK4 wild-type (wt) or the His-Sumo-PAK4 K473R mutant were incubated with or without His-SETD6 in the presence of ³H-labeled SAM. Proteins were then subjected to SDS-PAGE followed by exposure to autoradiogram to detect ³H-labeled proteins or Coomassie staining to detect all proteins. (C) Methylation assay in cells. MDA-MB-231 wild-type cells were transfected with Flag PAK4 wild-type or Flag PAK4 K473R, and both with HA SETD6 plasmids. Cell lysates were immunoprecipitated (IP) with FLAG-M2 beads, and proteins in IP and input samples were detected by Western blot with indicated antibodies. Methylation was detected with pan-methyl antibody. Uncropped gels are shown in Supplementary Fig. S9.