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Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells
P Jin1, Y Gu, D O Morgan
1Department of Physiology, University of California, San Francisco 94143-0444, USA.
Abstract:
The activity of the mitosis-promoting kinase CDC2-cyclin B is normally suppressed in S phase and G2 by inhibitory phosphorylation at Thr14 and Tyr15. This work explores the possibility that these phosphorylations are responsible for the G2 arrest that occurs in human cells after DNA damage. HeLa cell lines were established in which CDC2AF, a mutant that cannot be phosphorylated at Thr14 and Tyr15, was expressed from a tetracycline-repressible promoter. Expression of CDC2AF did not induce mitotic events in cells arrested at the beginning of S phase with DNA synthesis inhibitors, but induced low levels of premature chromatin condensation in cells progressing through S phase and G2. Expression of CDC2AF greatly reduced the G2 delay that resulted when cells were X-irradiated in S phase. However, a significant G2 delay was still observed and was accompanied by high CDC2-associated kinase activity. Expression of wild-type CDC2, or the related kinase CDK2AF, had no effect on the radiation-induced delay. Thus, inhibitory phosphorylation of CDC2, as well as additional undefined mechanisms, delay mitosis after DNA damage.
Insights
Inhibitory phosphorylation of CDC2 (cell division cycle 2) suppresses its activity, preventing premature mitosis. This study shows this phosphorylation is crucial for G2 arrest after DNA damage, but other mechanisms also contribute to the delay.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- CDC2-cyclin B activity is regulated by inhibitory phosphorylation at Thr14 and Tyr15.
- DNA damage induces a G2 cell cycle arrest, preventing entry into mitosis.
Purpose of the Study:
- To investigate the role of CDC2 inhibitory phosphorylation in the G2 arrest following DNA damage in human cells.
- To determine if preventing Thr14 and Tyr15 phosphorylation affects the G2 delay induced by DNA damage.
Main Methods:
- Established HeLa cell lines expressing a non-phosphorylatable CDC2 mutant (CDC2AF) under a tetracycline-repressible promoter.
- Assessed the effect of CDC2AF expression on mitotic progression and G2 delay after DNA damage (X-irradiation) or S-phase arrest.
Main Results:
- Expression of CDC2AF partially reduced the G2 delay after X-irradiation but did not eliminate it.
- CDC2AF induced low levels of premature chromatin condensation in cells progressing through S and G2 phases.
- High CDC2-associated kinase activity was observed during the remaining G2 delay, indicating active suppression mechanisms.
- Expression of wild-type CDC2 or CDK2AF did not affect the radiation-induced G2 delay.
Conclusions:
- Inhibitory phosphorylation of CDC2 is a significant but not the sole mechanism responsible for delaying mitosis after DNA damage.
- Additional, undefined pathways also contribute to the G2 arrest response to DNA damage.