DNA-PKcs associates with PLK1 and is involved in proper chromosome segregation and cytokinesis

J Cell Biochem. 2014 Jun;115(6):1077-88. doi: 10.1002/jcb.24703.

Abstract

Accurate mitotic regulation is as important as intrinsic DNA repair for maintaining genomic stability. It is believed that these two cellular mechanisms are interconnected with DNA damage. DNA-PKcs is a critical component of the non-homologous end-joining pathway of DNA double-stranded break repair, and it was recently discovered to be involved in mitotic processing. However, the underlying mechanism of DNA-PKcs action in mitotic control is unknown. Here, we demonstrated that depletion of DNA-PKcs led to the dysregulation of mitotic progression in response to DNA damage, which eventually resulted in multiple failures, including failure to segregate sister chromatids and failure to complete cytokinesis, with daughter cells becoming fused again. The depletion of DNA-PKcs resulted in a notable failure of cytokinesis, with a high incidence of multinucleated cells. There were also cytoplasmic bridges containing DNA that continuously connected the daughter cells after DNA damage was induced. Phosphorylated DNA-PKcs (T2609) colocalizes with PLK1 throughout mitosis, including at the centrosomes from prophase to anaphase and at the kinetochores from prometaphase to metaphase, with accumulation at the midbody during cytokinesis. Importantly, DNA-PKcs was found to associate with PLK1 in the mitotic phase, and the depletion of DNA-PKcs resulted in the overexpression of PLK1 due to increased protein stability. However, deficiency in DNA-PKcs attenuated the recruitment of phosphorylated PLK1 to the midbody but not to the kinetochores and centrosomes. Our results demonstrate the functional association of DNA-PKcs with PLK1, especially in chromosomal segregation and cytokinesis control.

Keywords: CYTOKINESIS; DNA DAMAGE RESPONSE; DNA-PKcs; GENOMIC STABILITY; MITOTIC PROGRESSION; PLK1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Catalytic Domain
  • Cell Cycle
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Centrosome / metabolism
  • Chromosome Segregation*
  • Cytokinesis*
  • DNA Damage
  • DNA Repair
  • DNA-Activated Protein Kinase / genetics
  • DNA-Activated Protein Kinase / metabolism*
  • Flow Cytometry
  • HeLa Cells
  • Humans
  • Immunoblotting
  • Kinetochores / metabolism
  • Microscopy, Confocal
  • Mitosis
  • Phosphorylation
  • Protein Binding
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • RNA Interference
  • Time-Lapse Imaging / methods

Substances

  • Cell Cycle Proteins
  • Proto-Oncogene Proteins
  • DNA-Activated Protein Kinase
  • Protein-Serine-Threonine Kinases
  • polo-like kinase 1