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Role of the cdc25C phosphatase in G2 arrest induced by nitrogen mustard
P M O'Connor1, D K Ferris, I Hoffmann
1Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
Abstract:
G2 arrest induced by nitrogen mustard in human lymphoma CA46 cells is associated with a failure to activate hyperphosphorylated cdc2/cyclin B1 complexes. We investigated the possibility that this might be due to a suppression of cdc25C phosphatase activity. cdc25C from interphase cells migrated as a 54- to 57-kDa doublet in SDS gels and exhibited basal phosphatase activity. cdc25C from mitotic cells migrated as a 66-kDa hyperphosphorylated species and exhibited elevated phosphatase activity. cdc25C hyperphosphorylation and activation were mediated by cdc2, supporting the view of a cdc2-cdc25C autocatalytic feedback loop. Immunofluorescence and cell fractionation studies suggested cdc2-cdc25C interaction occurred within the cytoplasm. Cells arrested in G2 phase following nitrogen mustard treatment or cells arrested in S phase with aphidicolin failed to dephosphorylate and activate cdc2, and this correlated with failure to convert cdc25C into the most active hyperphosphorylated species. Our findings suggest that checkpoints guarding against mitotic entry in the presence of unreplicated or damaged DNA suppress formation of the cdc2-cdc25C autocatalytic feedback loop that normally brings about rapid activation of cdc2.
Insights
Cell cycle checkpoints prevent cdc2-cdc25C feedback loop activation, inhibiting cdc2/cyclin B1 complex formation and G2 arrest in response to DNA damage. This study reveals a key mechanism in DNA damage response.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The G2 cell cycle checkpoint prevents entry into mitosis when DNA is damaged or unreplicated.
- Activation of cyclin-dependent kinases (CDKs), particularly cdc2/cyclin B1, is crucial for mitotic entry.
- cdc25C phosphatase plays a key role in activating cdc2/cyclin B1 complexes.
Purpose of the Study:
- To investigate the role of cdc25C phosphatase activity in G2 arrest induced by DNA damage.
- To determine if suppression of cdc25C activity contributes to the failure of cdc2/cyclin B1 complex activation.
Main Methods:
- Utilized human lymphoma CA46 cells treated with nitrogen mustard (DNA damaging agent) or aphidicolin (S-phase arrest agent).
- Analyzed cdc25C phosphatase activity and phosphorylation status using SDS-PAGE and Western blotting.
- Employed immunofluorescence and cell fractionation to study protein-protein interactions (cdc2-cdc25C).
Main Results:
- Nitrogen mustard treatment induced G2 arrest without activating hyperphosphorylated cdc2/cyclin B1 complexes.
- cdc25C phosphatase activity was suppressed in nitrogen mustard- or aphidicolin-treated cells.
- cdc25C failed to convert into its highly active hyperphosphorylated form, suggesting impaired activation.
- cdc2-cdc25C interaction, crucial for the autocatalytic feedback loop, was inhibited under DNA-damaging conditions.
Conclusions:
- Cell cycle checkpoints suppress the cdc2-cdc25C autocatalytic feedback loop in response to DNA damage.
- This suppression prevents the activation of cdc2/cyclin B1 complexes, leading to G2 arrest.
- The findings elucidate a critical mechanism by which cells maintain genomic integrity.