Poster Presentation 44th Lorne Genome Conference 2023

Eyes absent family members EYA4 and EYA1 promote the kinase activity and mitotic function of PLK1 through dephosphorylation of pY445 (#126)

Christopher B Nelson 1 , Samuel Rogers 1 , Kaushik Roychoudhury 2 , Yaw Sing Tan 3 , Caroline J Atkinson 4 , Alexander P Sobinoff 1 , Christopher G Tomlinson 1 , Anton Hsu 1 , Robert Lu 1 , Eloise Dray 5 , Michelle Haber 4 , Jamie I Fletcher 4 , Anthony J Cesare 1 , Rashmi S Hegde 2 , Hilda A Pickett 1
  1. Children's Medical Research Institute, Westmead, NSW, Australia
  2. Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA
  3. Bioinformatics Institute, Agency for Science, Technology and Research (A*STAR), Singapore
  4. Children's Cancer Institute, Lowy Cancer Research Centre, UNSW, Sydney, NSW, Australia
  5. Health Science Center, University of Texas at San Antonio, San Antonio, TX, USA

Introduction

The Eyes Absent family (EYA1-4) is a uniquely druggable group of protein tyrosine phosphatases with established oncogenic activity across a range of tumour types.  Several small molecules have been identified that can specifically disrupt the phosphatase activity of EYA proteins; however, fundamental knowledge of the phosphatase substrates and downstream signalling pathways by which the EYAs promote tumour biology is currently lacking. This is particularly true of EYA4, which has no known substrates, but is tumour promoting in cancers of neurological origin, including glioblastoma and malignant peripheral nerve sheath tumours.

Methods/Results

Using a combination of proximity proteomics, phosphoproteomics, in-vitro phosphatase assays, and phosphosite mutants we have identified the master mitotic regulator, and notable oncogene, Polo-like kinase 1 (PLK1) as a direct interactor and phosphatase substrate of both EYA4 and EYA1, with pY445 on PLK1 being the primary target site. EYA-mediated dephosphorylation of pY445 promotes the interaction between PLK1 and PLK1-activating proteins BORA and CEP192, increases PLK1 activity, and enhances PLK1 functions in centrosome maturation and mitosis. These results are supported by molecular dynamics simulations that predict an inhibitory effect of pY445 on PLK1-substrate interactions. Finally, depletion of EYA4 or EYA1, or inhibition of EYA phosphatase activity, dramatically reduce PLK1 activation, resulting in mitotic defects and cell death.

Conclusions

Overall, this work characterizes a novel phosphotyrosine signalling network important for the regulation of mitosis in cells expressing EYA4 and/or EYA1. Additionally, it provides mechanistic rationale for the use of EYA phosphatase inhibitors as chemotherapeutic agents.