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Differential contribution of inhibitory phosphorylation of CDC2 and CDK2 for unperturbed cell cycle control and DNA

Jeremy P H Chow1, Wai Yi Siu, Horace T B Ho

  • 1Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.

The Journal of Biological Chemistry
|August 13, 2003
PubMed

Insights

Inhibitory phosphorylation of cyclin-dependent kinases (CDKs) critically regulates cell cycle progression. This study reveals CDK2 plays a minor role in normal cell cycles, while CDC2 is heavily regulated by inhibitory phosphorylation, impacting DNA damage checkpoints.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Cyclin-dependent kinases (CDKs) control cell cycle progression.
  • Inhibitory phosphorylation at Thr14/Tyr15 is crucial for cell cycle checkpoints.
  • Differential regulation of CDK complexes impacts cellular functions.

Purpose of the Study:

  • Compare the roles of inhibitory phosphorylation in cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes.
  • Investigate the impact of non-phosphorylatable CDK mutants on cell cycle progression and DNA damage response.
  • Elucidate the roles of specific regulators in Thr14/Tyr15 phosphorylation during normal and stressed cell cycles.

Main Methods:

  • Utilized HeLa cells for cell cycle analysis.
  • Employed non-phosphorylation mutants of CDC2 and CDK2.
  • Induced DNA damage using radiomimetic drugs and replication blocks (hydroxyurea).
  • Assessed histone H3 phosphorylation, DNA replication, and cell division.
  • Investigated the effects of CDC25A, CDC25B, CHK1, CDC25C, and WEE1 manipulations.

Main Results:

  • Inhibitory phosphorylation significantly regulates CDC2 but minimally affects CDK2 in unperturbed HeLa cell cycles.
  • Non-phosphorylatable CDC2 and CDK2 mutants induced unscheduled histone H3 phosphorylation and cytotoxicity.
  • DNA damage and replication blocks increased inhibitory phosphorylation of CDC2, inhibiting cyclin-CDK pairs.
  • Ectopic expression of non-phosphorylated CDC2 promoted cell division post-DNA damage.
  • A non-phosphorylatable CDK2 mutant, but not CDK4, disrupted the G2 DNA damage checkpoint.

Conclusions:

  • Differential regulation of CDK2 and CDC2 by inhibitory phosphorylation reflects their distinct cellular roles.
  • Non-phosphorylatable CDK mutants can override cell cycle checkpoints, leading to cytotoxicity.
  • Specific regulators like CDC25A, CDC25B, and CHK1 are involved in regulating histone H3 phosphorylation after DNA damage.

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