Differential Contribution of Inhibitory Phosphorylation of CDC2 and CDK2 for Unperturbed Cell Cycle Control and DNA Integrity Checkpoints

doi:10.1074/jbc.M306683200

Differential Contribution of Inhibitory Phosphorylation of CDC2 and CDK2 for Unperturbed Cell Cycle Control and DNA Integrity Checkpoints*

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

¶ Croucher Foundation Senior Fellow. To whom correspondence should be addressed: Dept. of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong. Tel.: 852-23588718; Fax: 852-23581552; E-mail: bcrandy{at}ust.hk.

Abstract

Inhibition of cyclin-dependent kinases (CDKs) by Thr¹⁴/Tyr¹⁵ phosphorylation is critical for normal cell cycle progression and is a converging event for several cell cycle checkpoints. In this study, we compared the relative contribution of inhibitory phosphorylation for cyclin A/B1-CDC2 and cyclin A/E-CDK2 complexes. We found that inhibitory phosphorylation plays a major role in the regulation of CDC2 but only a minor role for CDK2 during the unperturbed cell cycle of HeLa cells. The relative importance of inhibitory phosphorylation of CDC2 and CDK2 may reflect their distinct cellular functions. Despite this, expression of nonphosphorylation mutants of both CDC2 and CDK2 triggered unscheduled histone H3 phosphorylation early in the cell cycle and was cytotoxic. DNA damage by a radiomimetic drug or replication block by hydroxyurea stimulated a buildup of cyclin B1 but was accompanied by an increase of inhibitory phosphorylation of CDC2. After DNA damage and replication block, all cyclin-CDK pairs that control S phase and mitosis were to different degrees inhibited by phosphorylation. Ectopic expression of nonphosphorylated CDC2 stimulated DNA replication, histone H3 phosphorylation, and cell division even after DNA damage. Similarly, a nonphosphorylation mutant of CDK2, but not CDK4, disrupted the G₂ DNA damage checkpoint. Finally, CDC25A, CDC25B, a dominantnegative CHK1, but not CDC25C or a dominant-negative WEE1, stimulated histone H3 phosphorylation after DNA damage. These data suggest differential contributions for the various regulators of Thr¹⁴/Tyr¹⁵ phosphorylation in normal cell cycle and during the DNA damage checkpoint.

Footnotes

↵1 The abbreviations used are: CDK, cyclin-dependent kinase; ADR, adriamycin; CMP, camptothecin; Dox, doxycycline; HU, hydroxyurea; GST, glutathione S-transferase; HA, hemagglutinin; PBS, phosphate-buffered saline; GFP, green fluorescent protein; BrdUrd, bromode-oxyuridine; TRITC, tetramethylrhodamine isothiocyanate.
↵2 W. Y. Siu and R. Y. C. Poon, manuscript in preparation.
↵3 J. P. H. Chow, W. Y. Siu, H. T. B. Ho, K. H. T. Ma, C. C. Ho, and R. Y. C. Poon, unpublished results.
↵* This work was supported in part by the Croucher Foundation, the Philip Morris External Research Program, and Research Grants Council Grant HKUST6129/02M (to R. Y. C. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Croucher Foundation Scholar.
↵§ Recipient of a grant from the Hongkong Bank Foundation Local Bursary and Scholarship Program.
- Received June 24, 2003.
The American Society for Biochemistry and Molecular Biology, Inc.