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Inhibition of cyclin-dependent kinases by p21
J W Harper1, S J Elledge, K Keyomarsi
1Verna & Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas 77030, USA.
Abstract:
p21Cip1 is a cyclin-dependent kinase (Cdk) inhibitor that is transcriptionally activated by p53 in response to DNA damage. We have explored the interaction of p21 with the currently known Cdks. p21 effectively inhibits Cdk2, Cdk3, Cdk4, and Cdk6 kinases (Ki 0.5-15 nM) but is much less effective toward Cdc2/cyclin B (Ki approximately 400 nM) and Cdk5/p35 (Ki > 2 microM), and does not associate with Cdk7/cyclin H. Overexpression of P21 arrests cells in G1. Thus, p21 is not a universal inhibitor of Cdks but displays selectivity for G1/S Cdk/cyclin complexes. Association of p21 with Cdks is greatly enhanced by cyclin binding. This property is shared by the structurally related inhibitor p27, suggesting a common biochemical mechanism for inhibition. With respect to Cdk2 and Cdk4 complexes, p27 shares the inhibitory potency of p21 but has slightly different kinase specificities. In normal diploid fibroblasts, the vast majority of active Cdk2 is associated with p21, but this active kinase can be fully inhibited by addition of exogenous p21. Reconstruction experiments using purified components indicate that multiple molecules of p21 can associate with Cdk/cyclin complexes and inactive complexes contain more than one molecule of p21. Together, these data suggest a model whereby p21 functions as an inhibitory buffer whose levels determine the threshold kinase activity required for cell cycle progression.
Insights
p21Cip1 is a selective cyclin-dependent kinase (Cdk) inhibitor that halts cell cycle progression. Its interaction with Cdks, particularly G1/S complexes, is enhanced by cyclin binding, acting as a buffer for cell cycle control.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
Background:
- p21Cip1 is a Cdk inhibitor activated by p53 during DNA damage.
- Cdks regulate cell cycle progression, and their dysregulation is linked to diseases like cancer.
Purpose of the Study:
- To investigate the interaction specificity of p21Cip1 with various cyclin-dependent kinases (Cdks).
- To elucidate the role of p21Cip1 in cell cycle arrest and its mechanism of Cdk inhibition.
Main Methods:
- In vitro kinase assays to determine inhibition constants (Ki) of p21Cip1 against different Cdk/cyclin complexes.
- Cell-based assays involving p21 overexpression to observe cell cycle effects.
- Reconstruction experiments with purified proteins to study p21-Cdk complex formation.
Main Results:
- p21Cip1 potently inhibits G1/S phase Cdks (Cdk2, Cdk3, Cdk4, Cdk6) but shows weaker inhibition of Cdc2/cyclin B and Cdk5/p35.
- p21Cip1 does not associate with Cdk7/cyclin H.
- Cyclin binding significantly enhances p21Cip1 association with Cdks, a mechanism shared with p27.
- Overexpression of p21Cip1 leads to G1 cell cycle arrest.
- Inactive Cdk/cyclin complexes can bind multiple p21Cip1 molecules.
Conclusions:
- p21Cip1 is a selective inhibitor of G1/S phase Cdks, not a universal Cdk inhibitor.
- p21Cip1 acts as an inhibitory buffer, with its levels dictating the threshold kinase activity required for cell cycle progression.
- The findings suggest a common mechanism of Cdk inhibition involving cyclin binding for p21Cip1 and p27.