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Cdc25B activity is regulated by 14-3-3
1Joint Oncology Program, Department of Pathology, University of Queensland School of Medicine, Herston Road, Brisbane, Queensland 4006, Australia.
Abstract:
In the G2 phase cell cycle checkpoint arrest, the cdc25-dependent activation of cyclin B/cdc2, a critical step in regulating entry into mitosis, is blocked. Studies in yeast have demonstrated that the inhibition of cdc25 function involves 14-3-3 binding to cdc25. In humans, two cdc25 isoforms have roles in G2/M progression, cdc25B and cdc25C, both bind 14-3-3. Abrogating 14-3-3 binding to cdc25C attenuates the G2 checkpoint arrest, but the contribution of 14-3-3 binding to the regulation of cdc25B function is unknown. Here we demonstrate that high level over-expression of cdc25B in G2 checkpoint arrested cells can activate cyclin B/cdc2 and overcome the checkpoint arrest. Mutation of the major 14-3-3 binding site, S323, or removal of the N-terminal regulatory domain are strong activating mutations, increasing the efficiency with which the mutant forms of cdc25B not only overcome the arrest, but also initiate aberrant mitosis. We also demonstrate that 14-3-3 binding to the S323 site on cdc25B blocks access of the substrate cyclin/cdks to the catalytic site of the enzyme, thereby directly inhibiting the activity of cdc25B. This provides direct mechanistic evidence that 14-3-3 binding to cdc25B can regulate its activity, thereby controlling progression into mitosis.
Insights
14-3-3 proteins inhibit cell cycle progression by binding to cdc25B, a key regulator of mitosis. Disrupting this interaction overcomes G2 arrest, revealing a direct mechanism for controlling entry into mitosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The G2 cell cycle checkpoint prevents entry into mitosis until DNA is repaired.
- cdc25 phosphatases (cdc25B and cdc25C in humans) are crucial for activating cyclin B/cdc2 kinase, which drives mitotic entry.
- 14-3-3 proteins bind to cdc25C, inhibiting its function and contributing to the G2 arrest.
Purpose of the Study:
- To investigate the role of 14-3-3 binding in regulating the function of the cdc25B phosphatase.
- To determine if cdc25B activity can overcome G2 checkpoint arrest.
- To elucidate the mechanism by which 14-3-3 binding affects cdc25B activity.
Main Methods:
- Over-expression of wild-type and mutant cdc25B in G2-arrested human cells.
- Site-directed mutagenesis of the major 14-3-3 binding site (S323) on cdc25B.
- Analysis of cyclin B/cdc2 activation and mitotic entry.
- Biochemical assays to assess cdc25B catalytic activity and substrate access.
Main Results:
- High-level expression of cdc25B in G2-arrested cells activated cyclin B/cdc2 and overcame the G2 arrest.
- Mutations at the S323 14-3-3 binding site or deletion of the N-terminal regulatory domain strongly activated cdc25B, promoting aberrant mitosis.
- 14-3-3 binding to S323 directly inhibited cdc25B activity by blocking substrate access to the catalytic site.
Conclusions:
- 14-3-3 binding to cdc25B is a key regulatory mechanism that inhibits its phosphatase activity.
- Disruption of 14-3-3 binding to cdc25B can release the G2 checkpoint and promote mitotic entry.
- This study provides direct mechanistic insight into how cdc25B activity is controlled to regulate cell cycle progression into mitosis.