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The Role of Cdc2 Feedback Loop Control in the DNA Damage Checkpoint in Mammalian Cells¹

Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong [R. Y. C. P., M. S. C.]; Department of Microbiology, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1, Takara-machi, Kanazawa 920, Japan [K. Y.]; and The Salk Institute for Biological Studies, La Jolla, California 92037 [T. H.]

DNA damage inactivates cyclin-dependent kinases (CDKs) and arrests the cell cycle. Following DNA damage, the G₁-S CDKs are inhibited by a mechanism involving p53-dependent induction of p21^Cip1/Waf1; but how the Cdc2 is inhibited is less apparent. We found that the signal generated by the DNA damage checkpoint in G₂ was dominant over that from the spindle microtubule-assembly checkpoint, because the high Cdc2 activity present in nocodazole or Taxol-arrested cells was reduced by DNA damage. Phosphorylation of the inhibitory residues in Cdc2, Thr¹⁴, and Tyr¹⁵ coincided with the inactivation of Cdc2 after DNA damage. Interpretation of this result, however, was not straightforward due to the regulation of Thr¹⁴/Tyr¹⁵ phosphorylation by feedback loops; hence, their phosphorylation can in principle result merely from the inhibition of Cdc2 activity. Consistent with this, Thr¹⁴/Tyr¹⁵ phosphorylation was induced when Cdc2 kinase activity was inhibited with butyrolactone-I. Given these complications, we undertook a more critical analysis of the mechanisms that regulate Cdc2 after DNA damage. Caffeine reversed the DNA damage-induced inhibition of Cdc2 by causing dephosphorylation of Cdc2, and this dephosphorylation still occurred even when the Cdc2 feedback loops were blocked with butyrolactone-I. These data suggest that the DNA damage checkpoint in part acts through Thr¹⁴/Tyr¹⁵ phosphorylation by a mechanism independent of Cdc2 activity, and this phosphorylation can be accentuated by the Cdc2 feedback loops involving Thr¹⁴/Tyr¹⁵ protein kinases and phosphatases. The kinase activity of the Wee1Hu Tyr¹⁵ protein kinase was unaltered after DNA damage, but the phosphatase activity of Cdc25C was reduced. Thus, the decrease in Cdc25C activity may in part account for the DNA damage-induced increase in Thr¹⁴/Tyr¹⁵ phosphorylation.

¹ This work was supported in part by the USPHS Grants CA14195 and CA39780 (to T. H.), the Research Grants Council Grant DAG96/97-SC26 (to R. Y. C. P.), and the Dan Charitable Trust Fund International Fellowship (to R. Y. C. P. and K. Y.). T. H. is an American Cancer Society Research Professor.

² To whom requests for reprints should be addressed, at Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. Phone: 852-2358-8703; Fax: 852-2358-1552; E-mail: bcrandy@usthk.ust.hk.

Received 6/12/97. Accepted 9/19/97.

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