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14-3-3 proteins act as negative regulators of the mitotic inducer Cdc25 in Xenopus egg extracts
A Kumagai1, P S Yakowec, W G Dunphy
1Division of Biology, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
Abstract:
Cdc25, the dual-specificity phosphatase that dephosphorylates the Cdc2-cyclin B complex at mitosis, is highly regulated during the cell cycle. In Xenopus egg extracts, Cdc25 is associated with two isoforms of the 14-3-3 protein. Cdc25 is complexed primarily with 14-3-3epsilon and to a lesser extent with 14-3-3zeta. The association of these 14-3-3 proteins with Cdc25 varies dramatically during the cell cycle: binding is high during interphase but virtually absent at mitosis. Interaction with 14-3-3 is mediated by phosphorylation of Xenopus Cdc25 at Ser-287, which resides in a consensus 14-3-3 binding site. Recombinant Cdc25 with a point mutation at this residue (Cdc25-S287A) is incapable of binding to 14-3-3. Addition of the Cdc25-S287A mutant to Xenopus egg extracts accelerates mitosis and overrides checkpoint-mediated arrests of mitotic entry due to the presence of unreplicated and damaged DNA. These findings indicate that 14-3-3 proteins act as negative regulators of Cdc25 in controlling the G2-M transition.
Insights
14-3-3 proteins bind to Cdc25 (a cell cycle regulator) during interphase, inhibiting its function. This interaction is crucial for controlling the G2-M cell cycle transition.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cdc25 phosphatase is a key regulator of the cell cycle, specifically controlling entry into mitosis by dephosphorylating the Cdc2-cyclin B complex.
- Cdc25 activity is tightly regulated throughout the cell cycle, but the precise mechanisms are not fully understood.
- In Xenopus egg extracts, Cdc25 has been observed to associate with 14-3-3 proteins.
Purpose of the Study:
- To investigate the role of 14-3-3 proteins in the regulation of Cdc25 activity during the cell cycle.
- To identify the specific interactions between Cdc25 and 14-3-3 isoforms.
- To determine the functional consequences of the Cdc25-14-3-3 interaction on cell cycle progression.
Main Methods:
- Utilized Xenopus egg extracts to study cell cycle regulation.
- Investigated protein-protein interactions using co-immunoprecipitation.
- Employed site-directed mutagenesis to create a non-binding Cdc25 mutant (Cdc25-S287A).
- Assessed the impact of the Cdc25-S287A mutant on mitotic entry and checkpoint control.
Main Results:
- Cdc25 primarily associates with 14-3-3epsilon and 14-3-3zeta isoforms in Xenopus egg extracts.
- This association is high during interphase and diminishes significantly at mitosis.
- Phosphorylation of Xenopus Cdc25 at Ser-287 is essential for 14-3-3 binding.
- The Cdc25-S287A mutant failed to bind 14-3-3 proteins.
- Addition of the Cdc25-S287A mutant to egg extracts accelerated mitosis and bypassed G2-M checkpoint arrests.
Conclusions:
- 14-3-3 proteins act as negative regulators of Cdc25 phosphatase.
- The interaction between 14-3-3 and Cdc25, mediated by Ser-287 phosphorylation, is critical for controlling the G2-M cell cycle transition.
- Disruption of this interaction leads to premature entry into mitosis and overrides DNA damage or replication checkpoints.