Fig. 1: EYA4 interacts with and dephosphorylates PLK1 in G2.

A Volcano plot of EYA4-Myc-BioID interactome. Grey coloured box represents 156 high confidence EYA4 interactors with -log p-value > 1.3 and Log2 fold difference > 2. EYA4, PLK1 and AURKA are indicated on the plot (n = 4, two-sided Student’s t-test). B EYA4 interactors with known localization to mitotic structures including PLK1. Proteins have been colour coded by Log2 fold difference. C PLK1 tyrosine phosphorylation was assessed by western blots of eluates from immunoprecipitation (IP) of tyrosine phosphorylated proteins using an anti-phosphotyrosine antibody in 293 T or HeLa protein lysates following EYA4 depletion (n = 3 biological repeats). Asterisk represents non-specific band on the EYA4 blot. D Immunopurified endogenous PLK1 from control or EYA4 depleted 293 T cells that had been arrested in G2 with RO3306 or released into mitosis for 15 or 30 min were probed using an anti-phosphotyrosine antibody (pY, n = 1). E Schematic representation of EYA4 protein with N-terminal transactivation domain and C-terminal tyrosine phosphatase domain as well as putative PDS (AA 123- 129) and known phosphosite (pS128). F Co-immunopurification reactions between immunopurified endogenous PLK1 and an EYA4-Myc construct or EYA4-Myc mutants. Cells have been arrested in G2 with RO3306 or released into early M phase (n = 2 biological repeats). Densitometry of the relative EYA4-Myc or EYA4-Myc mutant in the IP eluate to that in the lysate is shown below the blot. G Immunoflourescence of endogenous PLK1 and EYA4 in G2 arrested HeLa cells treated with control siRNA or siRNA targeting EYA4. Enlarged images show closeups of PLK1 centrosomal foci demonstrating colocalization with EYA4 in the siCon treatment. Source data are provided as a Source data file.