Supplementary Figure 5: Substitutions of Lys241 and Lys245 in Ezh2 disrupt interactions of Ezh2 with Vav1.
(a) Ezh2–Vav1 interaction domains, Ezh2 (Ezh2201-252) and Vav1 (Vav11-172) with C-terminal His-tag or N-terminal GST-tag, respectively, were purified from E. coli. Mutations of crucial residues (through computational prediction) for this interaction were introduced into Ezh2201-252 and the interactions of these mutated recombinant proteins with Vav1 were tested in His pull down experiments. Ezh2201-252K241/245A mutant was referred to as Ezh2VavMT in the main text. The enzymatic activity of Ezh2 is not affected by this mutation (Venkatesan et al., unpublished data). (b) Methylation of endogenous talin1 from cells expressing wild-type Ezh2 (Ezh2), hyperactive Ezh2 (Ezh2Y641F) or Vav interaction mutant Ezh2 (Ezh2VavMT) were determined as described in Fig. 5g. Data shown in this figure are representative from one of 3 independent experiments. (c) The effects of Vav interaction mutant Ezh2 on its co-localization with focal adhesions were determined by transfecting indicated Ezh2 and mCherry-paxillin constructs into MCF10a cells and the fluorescence signals were measured by confocal microscopy. White boxes indicate the regions enlarged in the following panels (box 1 middle panel, box 2 lower panel). Scar bars, 20 µm.
