Chk2-dependent phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) regulates centrosome maturation

Cell Cycle. 2019 Oct;18(20):2651-2659. doi: 10.1080/15384101.2019.1654795. Epub 2019 Aug 15.

Abstract

Checkpoint kinase 2 (Chk2) is a pivotal effector kinase in the DNA damage response, with an emerging role in mitotic chromosome segregation. In this study, we show that Chk2 interacts with myosin phosphatase targeting subunit 1 (MYPT1), the targeting subunit of protein phosphatase 1cβ (PP1cβ). Previous studies have shown that MYPT1 is phosphorylated by CDK1 at S473 during mitosis, and subsequently docks to the polo-binding domain of PLK1 and dephosphorylates PLK1. Herein we present data that Chk2 phosphorylates MYPT1 at S507 in vitro and in vivo, which antagonizes pS473. Chk2 inhibition results in failure of γ-tubulin recruitment to the centrosomes, phenocopying Plk1 inhibition defects. These aberrancies were also observed in the MYPT1-S507A stable transfectants, suggesting that Chk2 exerts its effect on centrosomes via MYPT1. Collectively, we have identified a Chk2-MYPT1-PLK1 axis in regulating centrosome maturation. Abbreviations: Chk2: checkpoint kinase 2; MYPT1: myosin phosphatase targeting subunit 1; PP1cβ: protein phosphatase 1c β; Noc: nocodazole; IP: immunoprecipitation; IB: immunoblotting; LC-MS/MS: liquid chromatography-tandem mass spectrometry; Chk2: checkpoint kinase 2; KD: kinase domain; WT: wild type; Ub: ubiquitin; DAPI: 4',6-diamidino-2-phenylindole; IF: Immunofluorescence; IR: ionizing radiation; siCHK2: siRNA targeting CHK2.

Keywords: Chk2; MYPT1; Phosphoprotein phosphatase 1β (PP1β); Plk1; centrosome.

MeSH terms

  • Cell Cycle Proteins / metabolism
  • Centrosome / metabolism*
  • Checkpoint Kinase 2 / genetics
  • Checkpoint Kinase 2 / metabolism*
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Mitosis / genetics*
  • Myosin-Light-Chain Phosphatase / genetics
  • Myosin-Light-Chain Phosphatase / metabolism*
  • Phosphorylation / genetics
  • Plasmids / genetics
  • Polo-Like Kinase 1
  • Protein Phosphatase 1 / metabolism
  • Protein Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / metabolism
  • Transfection
  • Tubulin / metabolism

Substances

  • Cell Cycle Proteins
  • Proto-Oncogene Proteins
  • Tubulin
  • Checkpoint Kinase 2
  • CHEK2 protein, human
  • Protein Serine-Threonine Kinases
  • PPP1CB protein, human
  • Protein Phosphatase 1
  • Myosin-Light-Chain Phosphatase
  • PPP1R12A protein, human

Grants and funding

This work was supported by the National Natural Science Foundation of China (NSFC) fund (31872720) and Capacity Building for Sci-Tech Innovation - Fundamental Scientific Research Funds (19530050137) to J. L. X.X. was supported by the National Natural Science Foundation of China (NSFC) grants 31761133012 and 31530016, the 973 projects 2017YFA0503900 and 2015CB910601, the Shenzhen Science and Technology Innovation Commission grants JCYJ20170412113009742 and JCYJ20180507182213033.