The Eyes Absent gene family (EYA1-4) encodes a group of developmentally essential transcriptional coactivators with tyrosine phosphatase activity. EYA protein levels are very low in adult tissues but are often increased during tumorigenesis where EYA tyrosine phosphatase activity has been shown to promote cancer cell phenotypes such as proliferation, migration and invasion. Additionally, while most protein phosphatases are difficult to chemically inhibit with specificity because they share a common cysteine-based reaction mechanism, EYA protein phosphatases use a unique aspartate-catalysed reaction. This suggests that EYA proteins may be good drug targets.
To determine the biological implications and chemotherapeutic potential of EYA inhibition, we aimed to determine the substrates of EYA phosphatase activity. First, we elucidated EYA4 interacting proteins using a BioID proximity proteomics approach, wherein fusion of EYA4 with a biotin ligase results in biotinylation of interacting proteins which are detected with LC-MS/MS. Next, using LC-MS/MS again, we detected proteins with enhanced tyrosine phosphorylation in EYA4-depleted cancer cells. By comparing these two datasets, we identified polo-like kinase 1 (PLK1), a master regulator of mitosis. We further confirmed that PLK1 is a substrate of EYA4 using overexpression of EYA4 or a phosphatase defective mutant of EYA4. Next, we examined PLK1 tyrosine phosphorylation through the cell cycle and found that EYA4 dephosphorylates PLK1 immediately prior to mitotic entry. Furthermore, preventing PLK1 tyrosine dephosphorylation by depletion of EYA4 or following treatment with benzbromarone, a recently identified EYA phosphatase inhibitor, strongly enhanced both mitotic duration and levels of mitotic cell death.
Overall, our results indicate PLK1 as an EYA4 substrate and suggest mitotic cell death as a potential chemotherapeutic mechanism of targeting EYA4 tyrosine phosphatase activity.