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Analysis of cyclin D3-cdk4 complexes in fibroblasts expressing and lacking p27(kip1) and p21(cip1)
T K Bagui1, R J Jackson, D Agrawal
1Molecular Oncology Program, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida.
Abstract:
Our studies examined the effects of p27(kip1) and p21(cip1) on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27(kip1)-p21(cip1)-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27(kip1) and/or p21(cip1) removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27(kip1) interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27(kip1)-p21(cip1)-deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27(kip1) or p21(cip1) or were monomeric. We conclude that neither p27(kip1) nor p21(cip1) is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27(kip1) or p21(cip1) are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27(kip1) and p21(cip1).
Insights
Cyclin-dependent kinase inhibitors p27(kip1) and p21(cip1) do not affect cyclin D3-cdk4 complex formation but inactivate them. A small fraction of cyclin D3-cdk4 complexes drives cellular activity.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cyclin D-cyclin-dependent kinase (CDK) complexes regulate cell cycle progression.
- CDK inhibitors p27(kip1) and p21(cip1) are known regulators of CDK activity.
- The precise role of p27(kip1) and p21(cip1) in cyclin D3-CDK4 complex assembly and activity remains to be fully elucidated.
Purpose of the Study:
- To investigate the impact of p27(kip1) and p21(cip1) on cyclin D3-CDK4 complex formation and catalytic activity.
- To characterize the composition of the cyclin D3 protein pool in cells with and without these CDK inhibitors.
- To determine the relationship between p27(kip1)/p21(cip1) binding and cyclin D3-CDK4 complex inactivation.
Main Methods:
- Utilized fibroblasts from p27(kip1)-p21(cip1)-null mice.
- Performed immunodepletion assays on wild-type cell extracts using antibodies against p27(kip1) and p21(cip1).
- Assayed cyclin D3-CDK4 and cyclin D1-CDK4 activities in cell extracts and mixed cell extracts.
Main Results:
- Catalytically active cyclin D3-CDK4 complexes were found in p27(kip1)-p21(cip1)-null cells.
- Immunodepletion of p27(kip1)/p21(cip1) from wild-type cells removed cyclin D3 protein but not associated activity.
- In vitro, p27(kip1) interaction with cyclin D3-CDK4 complexes led to a loss of activity. In p27(kip1)-p21(cip1)-deficient cells, cyclin D3 existed mainly as monomers, while in wild-type cells, it was complexed with CDK4 and/or inhibitors.
Conclusions:
- p27(kip1) and p21(cip1) are not required for cyclin D3-CDK4 complex assembly.
- Complexes of cyclin D3-CDK4 with p27(kip1) or p21(cip1) are catalytically inactive.
- A small fraction of the total cyclin D3 pool is responsible for all cyclin D3-CDK4 activity, irrespective of p27(kip1) and p21(cip1) presence.