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Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216
C Y Peng1, P R Graves, R S Thoma
1Department of Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.
Abstract:
Human Cdc25C is a dual-specificity protein phosphatase that controls entry into mitosis by dephosphorylating the protein kinase Cdc2. Throughout interphase, but not in mitosis, Cdc25C was phosphorylated on serine-216 and bound to members of the highly conserved and ubiquitously expressed family of 14-3-3 proteins. A mutation preventing phosphorylation of serine-216 abrogated 14-3-3 binding. Conditional overexpression of this mutant perturbed mitotic timing and allowed cells to escape the G2 checkpoint arrest induced by either unreplicated DNA or radiation-induced damage. Chk1, a fission yeast kinase involved in the DNA damage checkpoint response, phosphorylated Cdc25C in vitro on serine-216. These results indicate that serine-216 phosphorylation and 14-3-3 binding negatively regulate Cdc25C and identify Cdc25C as a potential target of checkpoint control in human cells.
Insights
Human Cdc25C protein phosphorylation at serine-216 and 14-3-3 protein binding negatively regulate its activity. This identifies Cdc25C as a key target in human cell checkpoint control.
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- Human Cdc25C is a dual-specificity protein phosphatase crucial for mitotic entry.
- Cdc25C dephosphorylates the protein kinase Cdc2 to regulate cell cycle progression.
- During interphase, Cdc25C is phosphorylated on serine-216 and binds to 14-3-3 proteins.
Purpose of the Study:
- To investigate the role of serine-216 phosphorylation and 14-3-3 binding in Cdc25C regulation.
- To determine if Cdc25C is a target of DNA damage checkpoint pathways.
Main Methods:
- Site-directed mutagenesis to prevent serine-216 phosphorylation.
- Conditional overexpression of wild-type and mutant Cdc25C.
- Assessing mitotic timing and G2 checkpoint integrity.
- In vitro phosphorylation assays using Chk1 kinase.
Main Results:
- A mutation preventing serine-216 phosphorylation abolished 14-3-3 binding.
- Overexpression of the non-phosphorylatable mutant disrupted mitotic timing.
- Cells expressing the mutant escaped G2 checkpoint arrest induced by DNA damage or replication stress.
- Human Chk1 kinase phosphorylated Cdc25C on serine-216 in vitro.
Conclusions:
- Serine-216 phosphorylation and subsequent 14-3-3 binding negatively regulate human Cdc25C activity.
- Cdc25C is a critical target for checkpoint control mechanisms in human cells.
- This regulation is essential for maintaining genomic integrity during the cell cycle.