p53 regulates Cdc2 independently of inhibitory phosphorylation to reinforce radiation-induced G2 arrest in human cells.
We have investigated the influence of p53 on radiation-induced G2 cell cycle arrest using human H1299 cells expressing temperature-sensitive p53. Gamma-irradiated cells lacking p53 arrested transiently in G2 with Cdc2 extensively phosphorylated at the inhibitory sites Thr14 and Tyr15, and with both Cdc2 and cyclin B1 restricted to the cytoplasm. Activation of p53 by temperature shift resulted in a more protracted G2 arrest that could not be overridden by checkpoint-abrogating drugs. Surprisingly, this enhancement of G2 arrest was associated with a marked lack of inhibitory phosphorylation of Cdc2 and with the nuclear localization of both Cdc2 and cyclin B1. While transient expression of an A14F15 mutant form of Cdc2 that is not subject to inhibitory phosphorylation induced mitotic catastrophe in cells lacking p53, the p53-expressing cells were relatively refractory to this effect. Enforced expression of p21WAF1/CJP1 was sufficient to confer nuclear localization on Cdc2 in the p53 null cells, though immunodepletion experiments demonstrated that only a small proportion of Cdc2 was stably associated with p21WAF1/CJP1 in the p53-expressing cells. We conclude that a p53-dependent pathway can operate after exposure of human cells to ionising radiation to promote G2 arrest accompanied by nuclear translocation rather than inhibitory phosphorylation of Cdc2.
Winters
Z E
ZE
Imperial Cancer Research Fund, Molecular Oncology Laboratory, University of Oxford Institute of Molecular Medicine, John Radcliffe Hospital, UK.
Ongkeko
W M
WM
Harris
A L
AL
Norbury
C J
CJ
D001692:Biological Transport; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism*; D002451:Cell Compartmentation; D002467:Cell Nucleus / Q000378:metabolism; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D016195:G2 Phase / Q000502:physiology* / Q000528:radiation effects; D005720:Gamma Rays; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism*; D011485:Protein Binding; D011494:Protein Kinases / Q000032:analysis; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
C534699:CCNB1 protein, human; C493956:CDKN1A protein, human; D019926:Cyclin B; D056744:Cyclin B1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D016159:Tumor Suppressor Protein p53; D011494:Protein Kinases; C102333:histone H1 kinase; D016203:CDC2 Protein Kinase