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p12(DOC-1) is a novel cyclin-dependent kinase 2-associated protein
S Shintani1, H Ohyama, X Zhang
1Laboratory of Molecular Pathology, Medical School, Harvard University, Boston, Massachusetts 02115, USA.
Abstract:
Regulated cyclin-dependent kinase (CDK) levels and activities are critical for the proper progression of the cell division cycle. p12(DOC-1) is a growth suppressor isolated from normal keratinocytes. We report that p12(DOC-1) associates with CDK2. More specifically, p12(DOC-1) associates with the monomeric nonphosphorylated form of CDK2 (p33CDK2). Ectopic expression of p12(DOC-1) resulted in decreased cellular CDK2 and reduced CDK2-associated kinase activities and was accompanied by a shift in the cell cycle positions of p12(DOC-1) transfectants ( upward arrow G(1) and downward arrow S). The p12(DOC-1)-mediated decrease of CDK2 was prevented if the p12(DOC-1) transfectants were grown in the presence of the proteosome inhibitor clasto-lactacystin beta-lactone, suggesting that p12(DOC-1) may target CDK2 for proteolysis. A CDK2 binding mutant was created and was found to revert p12(DOC-1)-mediated, CDK2-associated cell cycle phenotypes. These data support p12(DOC-1) as a specific CDK2-associated protein that negatively regulates CDK2 activities by sequestering the monomeric pool of CDK2 and/or targets CDK2 for proteolysis, reducing the active pool of CDK2.
Insights
p12(DOC-1) protein binds to cyclin-dependent kinase 2 (CDK2), reducing its activity and promoting cell cycle arrest. This suggests p12(DOC-1) acts as a growth suppressor by targeting CDK2 for degradation.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- Cyclin-dependent kinase (CDK) regulation is essential for cell cycle progression.
- p12(DOC-1) is a known growth suppressor identified in keratinocytes.
Purpose of the Study:
- To investigate the interaction between p12(DOC-1) and CDK2.
- To elucidate the mechanism by which p12(DOC-1) influences cell cycle progression.
Main Methods:
- Co-immunoprecipitation to assess protein association.
- Cell cycle analysis via flow cytometry.
- Proteasome inhibition studies.
- Site-directed mutagenesis to create CDK2 binding mutants.
Main Results:
- p12(DOC-1) specifically associates with the monomeric, nonphosphorylated form of CDK2.
- Ectopic p12(DOC-1) expression decreased CDK2 levels and kinase activity, leading to G1 cell cycle arrest.
- Proteasome inhibition blocked p12(DOC-1)-induced CDK2 degradation.
- A CDK2 binding mutant abrogated the p12(DOC-1)-mediated cell cycle effects.
Conclusions:
- p12(DOC-1) negatively regulates CDK2 activity.
- p12(DOC-1) may sequester monomeric CDK2 or target it for proteasomal degradation.
- These actions contribute to p12(DOC-1)'s role as a growth suppressor.