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Cell cycle dependent subcellular distribution of Cdc25B subtypes
1Department of Pharmacology, University of Pittsburgh Cancer Institute, University of Pittsburgh, Pennsylvania 15261, USA.
Abstract:
The dual specificity phosphatase and oncogene Cdc25B has been implicated in the G2/M cell cycle checkpoint, but the mode by which it is regulated remains poorly understood. Regional subcellular redistribution of proteins represents a unique potential regulatory mechanism. Thus, we examined in live cells the subcellular localization characteristics of Cdc25B2 and Cdc25B3 fused to green fluorescent protein. Cdc25B2 partitioned primarily in the cytoplasm during G1 and progressively migrated to the nucleus as cells transited from S to G2/M phase. In contrast, Cdc25B3 maintained a homogeneously staining diffuse phenotype irrespective of cell cycle phase. Treatment of the Cdc25B2-green fluorescent protein stable transfectants with vanadate inhibited the cell cycle dependency of intracellular distribution, while okadaic acid had little effect except in G1, suggesting regulation by at least one phosphorylation-dependent pathway. The DNA topoisomerase II poison and DNA damaging agent, etoposide, inhibited nuclear localization of Cdc25B2 in S phase, possibly by invoking a sequestration cascade. Thus, differences in the spatial distribution of Cdc25B subtypes exist within cells and the 41 amino acid insert in the N-terminus of the Cdc25B3 splice variant encodes an important inhibitory determinant for such regulation. The subcellular redistribution of Cdc25B2 could be functionally important for G2/M checkpoint regulation.
Insights
Cdc25B protein subtypes exhibit distinct subcellular localizations, influencing cell cycle regulation. Cdc25B2 moves to the nucleus during cell division, a process regulated by phosphorylation and DNA damage.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The dual specificity phosphatase and oncogene Cdc25B is crucial for the G2/M cell cycle checkpoint.
- Mechanisms regulating Cdc25B activity and function, particularly its subcellular localization, are not fully understood.
Purpose of the Study:
- To investigate the subcellular localization of Cdc25B2 and Cdc25B3 splice variants in live cells.
- To explore the regulatory mechanisms, including phosphorylation and DNA damage, influencing Cdc25B2 localization and its role in cell cycle control.
Main Methods:
- Utilized live-cell imaging of green fluorescent protein (GFP)-tagged Cdc25B2 and Cdc25B3.
- Applied chemical treatments (vanadate, okadaic acid, etoposide) to assess regulatory pathway involvement.
- Observed protein redistribution in response to cell cycle progression and DNA damage.
Main Results:
- Cdc25B2 exhibited cell cycle-dependent nuclear translocation from G1 to G2/M phase, while Cdc25B3 remained diffusely localized.
- Vanadate treatment disrupted the cell cycle-dependent localization of Cdc25B2, suggesting phosphorylation-dependent regulation.
- Etoposide inhibited Cdc25B2 nuclear import during S phase, indicating a DNA damage-induced sequestration mechanism.
- A 41-amino acid N-terminal insert in Cdc25B3 acts as an inhibitory determinant for subcellular redistribution.
Conclusions:
- Distinct spatial distribution patterns of Cdc25B subtypes exist within cells.
- Subcellular redistribution of Cdc25B2 is a regulated process potentially critical for G2/M checkpoint function.
- The N-terminal insert in Cdc25B3 plays a key role in regulating its localization and function.