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Degradation of p57Kip2 mediated by SCFSkp2-dependent ubiquitylation
Takumi Kamura1, Taichi Hara, Shuhei Kotoshiba
1Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan.
Abstract:
The abundance of the cyclin-dependent kinase (CDK) inhibitor p57Kip2, an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCFSkp2 has now been shown to be responsible for regulating the cellular level of p57Kip2 by targeting it for ubiquitylation and proteolysis. The elimination of p57Kip2 was impaired in Skp2-/- cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with p57Kip2 in vivo. Overexpression of WT Skp2 promoted degradation of p57Kip2, whereas expression of a dominant negative mutant of Skp2 prolonged the half-life of p57Kip2. Mutation of the threonine residue (Thr-310) of human p57Kip2 that is conserved between the COOH-terminal QT domains of p57Kip2 and p27Kip1 prevented the effect of Skp2 on the stability of p57Kip2, suggesting that phosphorylation at this site is required for SCFSkp2-mediated ubiquitylation. Finally, the purified recombinant SCFSkp2 complex mediated p57Kip2 ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCFSkp2 complex plays an important role in cell-cycle progression by determining the abundance of p57Kip2 and that of the related CDK inhibitor p27Kip1.
Insights
The SCFSkp2 complex targets the cell cycle regulator p57Kip2 for degradation, controlling its abundance. This regulation is crucial for normal cell cycle progression and involves phosphorylation at Thr-310.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The cyclin-dependent kinase (CDK) inhibitor p57Kip2 regulates cell cycle progression.
- Its cellular abundance is primarily controlled by the ubiquitin-proteasome pathway.
Purpose of the Study:
- To identify the E3 ubiquitin ligase responsible for p57Kip2 degradation.
- To elucidate the mechanism by which p57Kip2 levels are regulated.
Main Methods:
- Coimmunoprecipitation assays to detect protein interactions.
- Analysis of p57Kip2 stability in Skp2 knockout and overexpression systems.
- In vitro ubiquitylation assays using purified SCFSkp2 complex.
Main Results:
- SCFSkp2 directly targets p57Kip2 for ubiquitylation and proteasomal degradation.
- Skp2 interacts with p57Kip2 in vivo, and its activity is modulated by phosphorylation at Thr-310.
- SCFSkp2-mediated degradation of p57Kip2 is dependent on the cyclin E-CDK2 complex.
Conclusions:
- The SCFSkp2 complex is a key regulator of p57Kip2 abundance.
- This regulation is essential for proper cell cycle progression.
- SCFSkp2 also regulates the related CDK inhibitor p27Kip1.