Related Experiment Videos
p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest
K Polyak1, J Y Kato, M J Solomon
1Cell Biology and Genetics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Abstract:
Cell-cell contact and TGF-beta can arrest the cell cycle in G1. Mv1Lu mink epithelial cells arrested by either mechanism are incapable of assembling active complexes containing the G1 cyclin, cyclin E, and its catalytic subunit, Cdk2. These growth inhibitory signals block Cdk2 activation by raising the threshold level of cyclin E necessary to activate Cdk2. In arrested cells the threshold is set higher than physiological cyclin E levels and is determined by an inhibitor that binds to cyclin E-Cdk2 complexes. A 27-kD protein that binds to and prevents the activation of cyclin E-Cdk2 complexes can be purified from arrested cells but not from proliferating cells, using cyclin E-Cdk2 affinity chromatography. p27 is present in proliferating cells, but it is sequestered and unavailable to interact with cyclin E-Cdk2 complexes. Cyclin D2-Cdk4 complexes bind competitively to and down-regulate the activity of p27 and may thereby act in a pathway that reverses Cdk2 inhibition and enables G1 progression.
Insights
Cell cycle arrest in G1 is mediated by an inhibitor protein (p27) that blocks cyclin E-Cdk2 complex activation. Cyclin D2-Cdk4 complexes may reverse this inhibition, allowing cell cycle progression.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cell-cell contact and TGF-beta induce G1 cell cycle arrest.
- During arrest, cyclin E-Cdk2 complexes are inactive.
- This inactivity is linked to elevated thresholds for cyclin E-Cdk2 activation.
Purpose of the Study:
- To identify the molecular mechanisms underlying G1 cell cycle arrest.
- To investigate the role of specific inhibitors in regulating cyclin E-Cdk2 activity.
- To elucidate the pathway that reverses cell cycle arrest.
Main Methods:
- Cell cycle synchronization and arrest.
- Biochemical assays to assess cyclin E-Cdk2 complex activity.
- Affinity chromatography using cyclin E-Cdk2 to purify interacting proteins.
- Western blotting to detect protein levels and interactions.
Main Results:
- A 27-kD inhibitor protein (p27) was purified from arrested cells but not proliferating cells.
- p27 binds to and inhibits cyclin E-Cdk2 complex activation.
- p27 is present in proliferating cells but sequestered.
- Cyclin D2-Cdk4 complexes down-regulate p27 activity.
Conclusions:
- p27 is a key inhibitor of cyclin E-Cdk2 activation during G1 arrest.
- The sequestration of p27 in proliferating cells allows cell cycle progression.
- Cyclin D2-Cdk4 complexes play a role in reversing p27-mediated inhibition and promoting G1 progression.