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Formation of p27-CDK complexes during the human mitotic cell cycle
T J Soos1, H Kiyokawa, J S Yan
1Program in Molecular Biology, Memorial Sloan-Kettering Cancer Research Center, New York, New York 10021, USA.
Cell Growth & Differentiation : the Molecular Biology Journal of the American Association for Cancer Research
|February 1, 1996
Summary
The protein p27 binds to cyclin-dependent kinases (CDKs) CDK2, CDK4, and CDK6 in MANCA cells. Cyclin D3 appears to sequester p27
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- Cyclin-dependent kinases (CDKs) control cell cycle progression into S-phase.
- The protein p27 is known to inhibit cyclin/CDK complexes in vitro and in nonproliferating cells.
- The precise interactions of p27 with cyclin/CDK complexes and their role in proliferating cells remain incompletely understood.
Purpose of the Study:
- To investigate the cell cycle-dependent interactions of p27 with CDK2, CDK4, and CDK6 in proliferating MANCA cells.
- To characterize the kinase activity associated with p27/CDK complexes.
- To elucidate the functional consequences of p27 binding to different CDK complexes during the cell cycle.
Main Methods:
- Cell cycle analysis of p27 protein levels in MANCA cells.
- Immunoprecipitation assays to detect p27 association with CDK2, CDK4, and CDK6.
- Assays to measure Rb-kinase activity in p27-immunoprecipitates.
- Depletion experiments using CDK6-specific antibodies.
Main Results:
- p27 protein levels remain constant throughout the cell cycle in MANCA cells.
- p27 associates with CDK2, CDK4, and CDK6, with CDK2 association varying across the cell cycle (lowest in early G1, increasing to mid-G1).
- p27-immunoprecipitates exhibit Rb-kinase activity, partially mediated by CDK6, while p27/CDK2 complexes do not phosphorylate histone H1.
Conclusions:
- Cyclin D3 levels are higher than p27 levels in MANCA cells.
- The cell cycle-dependent association of p27 with CDK2 suggests a regulatory role.
- A model is proposed where cyclin D/CDK complexes sequester the inhibitory activity of p27 towards CDK2.