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Negative regulation of Wee1 expression and Cdc2 phosphorylation during p53-mediated growth arrest and apoptosis
S D Leach1, C D Scatena, C J Keefer
1Department of Surgery, The Vanderbilt Cancer Center, Nashville Veterans Affairs Medical Center, Tennessee 37232, USA.
Abstract:
The G2 cell cycle checkpoint protects cells from potentially lethal mitotic entry after DNA damage. This checkpoint involves inhibitory phosphorylation of Cdc2 at the tyrosine-15 (Y15) position, mediated in part by the Wee1 protein kinase. Recent evidence suggests that p53 may accelerate mitotic entry after DNA damage and that the override of the G2 checkpoint may play a role in the induction of apoptosis by p53. To determine the biochemical mechanism by which p53 inactivates the G2 checkpoint, the effects of p53 activation on Wee1 expression, Cdc2-Y15 phosphorylation, and cyclin B1-associated Cdc2 kinase activity were examined. Under conditions of either growth arrest or apoptosis, p53 activation resulted in the down-regulation of Wee1 expression and dephosphorylation of Cdc2. A parallel increase in cyclin B1/Cdc2 kinase activity was observed during p53-mediated apoptosis. Negative regulation of the Wee1 expression and Cdc2 phosphorylation by p53 was also evident in thymus tissue from p53+/+ mice but not from p53-/- mice. Inactivation of the G2 checkpoint may contribute to the tumor suppressor activity of p53.
Insights
The tumor suppressor p53 protein inactivates the G2 cell cycle checkpoint by downregulating Wee1 kinase. This mechanism accelerates mitotic entry and promotes apoptosis following DNA damage.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- The G2 cell cycle checkpoint prevents entry into mitosis after DNA damage.
- Wee1 kinase phosphorylates Cdc2 at tyrosine-15 (Y15), inhibiting its activity and maintaining the G2 arrest.
- p53 is a tumor suppressor protein involved in DNA damage response and apoptosis.
Purpose of the Study:
- To elucidate the biochemical mechanism by which p53 inactivates the G2 checkpoint.
- To investigate the role of p53 in regulating Wee1 expression and Cdc2 phosphorylation.
- To determine if p53-mediated G2 checkpoint override contributes to apoptosis.
Main Methods:
- Western blotting to assess Wee1 expression and Cdc2-Y15 phosphorylation.
- Kinase assays to measure cyclin B1-associated Cdc2 activity.
- Analysis of thymus tissue from p53 wild-type and knockout mice.
Main Results:
- p53 activation led to decreased Wee1 expression and Cdc2-Y15 dephosphorylation.
- Cyclin B1/Cdc2 kinase activity increased during p53-mediated apoptosis.
- These effects were observed in p53+/+ mice but not in p53-/- mice.
Conclusions:
- p53 inactivates the G2 checkpoint by downregulating Wee1, leading to Cdc2 activation.
- This inactivation promotes mitotic entry and may contribute to p53's tumor suppressor function.
- The findings reveal a novel mechanism for p53 in DNA damage response and cell fate determination.