title: The role of Cdc2 feedback loop control in the DNA damage checkpoint in mammalian cells.
abstract: 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 p21Cip1/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.
authors.affiliation: Department of Biochemistry, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong. bcrandy@usthk.ust.hk
authors.forename: R Y
authors.initials: RY
authors.lastname: Poon
authors.forename: M S
authors.initials: MS
authors.lastname: Chau
authors.forename: K
authors.initials: K
authors.lastname: Yamashita
authors.forename: T
authors.initials: T
authors.lastname: Hunter
mesh_terms: D016475:3T3 Cells / Q000187:drug effects / Q000528:radiation effects; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002110:Caffeine / Q000494:pharmacology; D019925:Cyclin A / Q000378:metabolism*; D019926:Cyclin B / Q000378:metabolism*; D056744:Cyclin B1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004249:DNA Damage; D004317:Doxorubicin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D016193:G1 Phase / Q000187:drug effects / Q000528:radiation effects; D016195:G2 Phase / Q000187:drug effects* / Q000528:radiation effects*; D006801:Humans; D051379:Mice; D008938:Mitosis / Q000187:drug effects; D015739:Nocodazole / Q000494:pharmacology; D017239:Paclitaxel / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D013912:Threonine / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D000970:Antineoplastic Agents; C534699:CCNB1 protein, human; C493956:CDKN1A protein, human; C534700:Ccnb1 protein, mouse; C493957:Cdkn1a protein, mouse; D019925:Cyclin A; D019926:Cyclin B; D056744:Cyclin B1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D013912:Threonine; D002110:Caffeine; D014443:Tyrosine; D004317:Doxorubicin; D011494:Protein Kinases; C102333:histone H1 kinase; D016203:CDC2 Protein Kinase; D017239:Paclitaxel; D015739:Nocodazole