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Updated: Jul 13, 2026
Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay
Published on: May 3, 2018
Spy1 enhances phosphorylation and degradation of the cell cycle inhibitor p27
Christopher W McAndrew1, Randy F Gastwirt, April N Meyer
1Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093-0367, USA.
Abstract:
The cyclin dependent kinase inhibitor (CKI) p27Kip1 binds to cyclin E/CDK2 complexes and prevents premature S-phase entry. During late G1 and throughout S-phase, p27 phosphorylation at T187 leads to its subsequent degradation, which relieves CDK2 inhibition to promote cell cycle progression. However, critical events that trigger CDK2 complexes to phosphorylate p27 remain unclear. Utilizing recombinant proteins, we demonstrate that human Speedy (Spy1) activates CDK2 to phosphorylate p27 at T187 in vitro. Addition of Spy1 or Spy1/CDK2 to a preformed, inhibited cyclin E/CDK2/p27 complex also promoted this phosphorylation. Furthermore, Spy1 protected cyclin E/CDK2 from p27 inhibition toward histone H1, in vitro. Inducible Spy1 expression in U2OS cells reduced levels of endogenous p27 and exogenous p27WT, but not a p27T187A mutant. Additionally, Spy1 expression in synchronized HeLa cells enhanced T187 phosphorylation and degradation of endogenous p27 in late G1 and throughout S-phase. Our studies provide evidence that Spy1 expression enhances CDK2-dependent p27 degradation during late G1 and throughout S-phase.
Insights
The cell cycle regulator Speedy (Spy1) promotes the degradation of p27Kip1 by activating CDK2 phosphorylation. This mechanism enhances cell cycle progression by relieving CDK2 inhibition during S-phase.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- p27Kip1 is a cyclin-dependent kinase inhibitor (CKI) that prevents premature S-phase entry.
- Phosphorylation of p27Kip1 at T187 triggers its degradation, promoting cell cycle progression.
- The specific triggers for CDK2-mediated p27Kip1 phosphorylation remain largely unknown.
Purpose of the Study:
- To investigate the role of human Speedy (Spy1) in the regulation of p27Kip1 phosphorylation and degradation.
- To elucidate the mechanism by which Spy1 influences the cyclin E/CDK2/p27Kip1 complex.
Main Methods:
- In vitro assays using recombinant proteins to assess CDK2 activity and p27Kip1 phosphorylation.
- Cell-based experiments with inducible Spy1 expression in U2OS and HeLa cells.
- Analysis of endogenous and exogenous p27Kip1 levels and phosphorylation status.
Main Results:
- Spy1 directly activates CDK2 to phosphorylate p27Kip1 at T187 in vitro.
- Spy1 protects cyclin E/CDK2 from p27Kip1 inhibition.
- Spy1 expression reduces p27Kip1 levels in cells, an effect dependent on T187 phosphorylation.
- Spy1 enhances p27Kip1 phosphorylation and degradation in synchronized cells during late G1 and S-phase.
Conclusions:
- Spy1 acts as a critical regulator of p27Kip1 degradation.
- Spy1 enhances CDK2-dependent phosphorylation of p27Kip1 at T187, leading to its degradation.
- Spy1 facilitates cell cycle progression by promoting p27Kip1 turnover.
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