Nocodazole-Induced Expression and Phosphorylation of Anillin and Other Mitotic Proteins Are Decreased in DNA-Dependent Protein Kinase Catalytic Subunit-Deficient Cells and Rescued by Inhibition of the Anaphase-Promoting Complex/Cyclosome with proTAME but Not Apcin

Mol Cell Biol. 2020 Jun 15;40(13):e00191-19. doi: 10.1128/MCB.00191-19. Print 2020 Jun 15.

Abstract

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has well-established roles in DNA double-strand break repair, and recently, nonrepair functions have also been reported. To better understand its cellular functions, we deleted DNA-PKcs from HeLa and A549 cells using CRISPR/Cas9. The resulting cells were radiation sensitive, had reduced expression of ataxia-telangiectasia mutated (ATM), and exhibited multiple mitotic defects. Mechanistically, nocodazole-induced upregulation of cyclin B1, anillin, and securin was decreased in DNA-PKcs-deficient cells, as were phosphorylation of Aurora A on threonine 288, phosphorylation of Polo-like kinase 1 (PLK1) on threonine 210, and phosphorylation of targeting protein for Xenopus Klp2 (TPX2) on serine 121. Moreover, reduced nocodazole-induced expression of anillin, securin, and cyclin B1 and phosphorylation of PLK1, Aurora A, and TPX2 were rescued by inhibition of the anaphase-promoting complex/cyclosome (APC/C) by proTAME, which prevents binding of the APC/C-activating proteins Cdc20 and Cdh1 to the APC/C. Altogether, our studies suggest that loss of DNA-PKcs prevents inactivation of the APC/C in nocodazole-treated cells.

Keywords: APC/C; Aurora A; DNA-PK; PLK1; TPX2; anillin; mitosis; nocodazole; protein kinase.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • A549 Cells
  • Anaphase-Promoting Complex-Cyclosome / antagonists & inhibitors*
  • Anaphase-Promoting Complex-Cyclosome / metabolism
  • Animals
  • Antineoplastic Agents / pharmacology*
  • Aurora Kinase A / metabolism
  • CRISPR-Cas Systems
  • Carbamates / pharmacology
  • Cell Cycle Proteins / metabolism
  • Contractile Proteins / genetics*
  • Contractile Proteins / metabolism
  • DNA-Activated Protein Kinase / genetics*
  • DNA-Activated Protein Kinase / metabolism
  • Diamines / pharmacology
  • Down-Regulation / drug effects
  • Enzyme Inhibitors / pharmacology*
  • HeLa Cells
  • Humans
  • Nocodazole / pharmacology*
  • Phosphorylation / drug effects
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / metabolism
  • Up-Regulation / drug effects
  • Xenopus

Substances

  • Antineoplastic Agents
  • Carbamates
  • Cell Cycle Proteins
  • Contractile Proteins
  • Diamines
  • Enzyme Inhibitors
  • Proto-Oncogene Proteins
  • anillin
  • apcin
  • Anaphase-Promoting Complex-Cyclosome
  • AURKA protein, human
  • Aurora Kinase A
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • Protein-Serine-Threonine Kinases
  • polo-like kinase 1
  • Nocodazole