RESUMO
Cyclin A/cdk2 is active during S and G2 phases of the cell cycle, but its regulation and function during G2 phase is poorly understood. In this study we have examined the regulation of cyclin A/cdk2 activity during normal G2 phase progression and in genotoxin-induced G2 arrest. We show that cyclin A/cdk2 is activated in early G2 phase by a cdc25 activity. In the G2 phase checkpoint arrest initiated in response to various forms of DNA damage, the cdc25-dependent activation of both cyclin A/cdk2 and cyclin B1/cdc2 is blocked. Ectopic expression of cdc25B, but not cdc25C, in G2 phase arrested cells efficiently activated both cyclin A/cdk2 and cyclin B1/cdc2. Finally, we demonstrate that the block in cyclin A/cdk2 activation in the G2 checkpoint arrest is independent of ATM/ATR. We speculate that the ATM/ATR-independent block in G2 phase cyclin A/cdk2 activation may act as a further layer of checkpoint control, and that blocking G2 phase cyclin A/cdk2 activation contributes to the G2 phase checkpoint arrest.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Ciclina A/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Fase G2/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fosfatases cdc25/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia , Cafeína/farmacologia , Quinase 2 Dependente de Ciclina , Dano ao DNA , Proteínas de Ligação a DNA , Ativação Enzimática , Etoposídeo/farmacologia , Humanos , Mutagênicos/farmacologia , Células Tumorais Cultivadas , Proteínas Supressoras de TumorRESUMO
Progression through G2 phase into mitosis is regulated by the activation of the mitotic cyclin/cdk complexes, which are in turn activated cdc25B and cdc25C phosphatases. Here we report that alternate splicing produces at least five variants of cdc25B, although only cdc25B2 and cdc25B3 are detectable as proteins. Analysis of these two variants shows that cdc25B2 is expressed at lower levels relative to cdc25B3 in all cell lines tested, and the expression of both increased markedly during G2 and mitosis. Overexpression of the catalytically inactive version of either cdc25B variant produced a G2 arrest implicating both in regulating G2/M progression.
Assuntos
Proteínas de Ciclo Celular/genética , Fase G2/genética , Mitose/genética , Fosfoproteínas Fosfatases/genética , Isoformas de Proteínas/genética , Splicing de RNA , Fosfatases cdc25 , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiologia , Linhagem Celular , Humanos , Peso Molecular , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/fisiologia , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologiaRESUMO
Cyclin D-Cdk4 complexes have a demonstrated role in G1 phase, regulating the function of the retinoblastoma susceptibility gene product (Rb). Previously, we have shown that following treatment with low doses of UV radiation, cell lines that express wild-type p16 and Cdk4 responded with a G2 phase cell cycle delay. The UV-responsive lines contained elevated levels of p16 post-treatment, and the accumulation of p16 correlated with the G2 delay. Here we report that in UV-irradiated HeLa and A2058 cells, p16 bound Cdk4 and Cdk6 complexes with increased avidity and inhibited a cyclin D3-Cdk4 complex normally activated in late S/early G2 phase. Activation of this complex was correlated with the caffeine-induced release from the UV-induced G2 delay and a decrease in the level of p16 bound to Cdk4. Finally, overexpression of a dominant-negative mutant of Cdk4 blocked cells in G2 phase. These data indicate that the cyclin D3-Cdk4 activity is necessary for cell cycle progression through G2 phase into mitosis and that the increased binding of p16 blocks this activity and G2 phase progression after UV exposure.
