Related Experiment Videos
Cell cycle-dependent phosphorylation of p27 cyclin-dependent kinase (Cdk) inhibitor by cyclin E/Cdk2
H Morisaki1, A Fujimoto, A Ando
1Department of Geriatric Research, National Institute for Longevity Sciences, Aichi, Japan.
Abstract:
The cyclin-dependent kinase (Cdk) inhibitor p27 interrupts progression of the cell cycle by inhibiting various cyclin/Cdk activities. Since the protein level of p27 does not correlate with its mRNA level or protein synthesis rate in most cases, it is suggested that degradation of the protein may be regulated via an unidentified mechanism(s) involving a post-translational modification(s). We present evidence here that p27 phosphorylation is cell cycle-dependent and peaks in the late G1 phase and that the level of p27 protein is inversely correlated with its phosphorylation. Although both cyclin D1- and cyclin-E-dependent kinases are active in the late G1 phase in human fibroblasts, cyclin E/Cdk2 specifically phosphorylates p27 on threonine-187 in vitro. Interestingly, ectopic expression of T187A revealed that it was far more stable in vivo than wild type p27. Thus, phosphorylation of p27 by cyclin E/ Cdk2 may affect the stability of its protein and play a role in how the protein functions.
Insights
The cell cycle inhibitor p27's protein levels are regulated by phosphorylation, specifically by cyclin E/Cdk2 at threonine-187. This post-translational modification impacts p27 protein stability and function.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
Background:
- The cyclin-dependent kinase (Cdk) inhibitor p27 regulates cell cycle progression.
- p27 protein levels often do not correlate with mRNA or synthesis rates, suggesting post-translational regulation.
Purpose of the Study:
- To investigate the role of post-translational modifications, specifically phosphorylation, in regulating p27 protein stability and function.
- To identify the specific kinases involved in p27 phosphorylation and the sites of modification.
Main Methods:
- Cell cycle analysis
- Western blotting to assess protein levels and phosphorylation
- In vitro kinase assays
- Site-directed mutagenesis (T187A) and ectopic expression studies
Main Results:
- p27 phosphorylation is cell cycle-dependent, peaking in late G1 phase.
- p27 protein levels are inversely correlated with its phosphorylation status.
- Cyclin E/Cdk2 specifically phosphorylates p27 at threonine-187 in vitro.
- A T187A mutant of p27 exhibits increased stability in vivo compared to wild-type p27.
Conclusions:
- Phosphorylation of p27 by cyclin E/Cdk2 at threonine-187 is a key mechanism regulating p27 protein stability.
- This phosphorylation event likely influences p27's role in cell cycle control.