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Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15
Y Gu1, J Rosenblatt, D O Morgan
1Department of Physiology, University of California, San Francisco 94143-0444.
Abstract:
We have examined the role of phosphorylation in the regulation of human cyclin-dependent kinase-2 (CDK2), a protein closely related to the cell cycle regulatory kinase CDC2. We find that CDK2 from HeLa cells contains three major tryptic phosphopeptides. Analysis of site-directed mutant proteins, expressed by transient transfection of COS cells, demonstrates that the two major phosphorylation sites are Tyr15 (Y15) and Thr160 (T160). Additional phosphorylation probably occurs on Thr14 (T14). Replacement of T160 with alanine abolishes the kinase activity of CDK2, indicating that phosphorylation at this site (as in CDC2) is required for kinase activity. Mutation of Y15 and T14 stimulates kinase activity, demonstrating that phosphorylation at these sites (as in CDC2) is inhibitory. Similarly, CDK2 is activated in vitro by dephosphorylation of Y15 and T14 by the phosphatase CDC25. Analysis of HeLa cells synchronized at various cell cycle stages indicates that CDK2 phosphorylation on T160 increases during S phase and G2, when CDK2 is most active. Phosphorylation on the inhibitory sites T14 and Y15 is also maximal during S phase and G2. Thus, the activity of a subpopulation of CDK2 molecules is inhibited at a time in the cell cycle when overall CDK2 activity is increased.
Insights
Phosphorylation regulates human cyclin-dependent kinase-2 (CDK2) activity. Activating phosphorylation at Thr160 is essential, while inhibitory phosphorylation at Tyr15 and Thr14 is regulated during the cell cycle.
Area of Science:
- Molecular Biology
- Cell Cycle Regulation
Background:
- Cyclin-dependent kinase-2 (CDK2) is crucial for cell cycle progression.
- Phosphorylation is a key regulatory mechanism for CDK2 activity.
Purpose of the Study:
- To investigate the specific phosphorylation sites regulating human CDK2 activity.
- To understand the role of phosphorylation in controlling CDK2 function during the cell cycle.
Main Methods:
- Site-directed mutagenesis of CDK2 phosphorylation sites.
- Expression of mutant CDK2 proteins in COS cells.
- In vitro kinase assays and phosphatase treatments.
- Analysis of CDK2 phosphorylation in synchronized HeLa cells.
Main Results:
- CDK2 contains three major phosphopeptides, with Tyr15 (Y15) and Thr160 (T160) identified as key sites.
- Phosphorylation at T160 is required for CDK2 kinase activity, while phosphorylation at Y15 and Thr14 (T14) is inhibitory.
- CDK2 activity is enhanced by dephosphorylation of Y15 and T14 via CDC25 phosphatase.
- T160 phosphorylation increases during S and G2 phases, coinciding with maximal CDK2 activity.
- Inhibitory phosphorylation at T14 and Y15 also peaks during S and G2 phases.
Conclusions:
- Phosphorylation at T160 is essential for CDK2 activation, mirroring CDC2 regulation.
- Phosphorylation at Y15 and T14 acts as an inhibitory mechanism for CDK2.
- CDK2 activity is finely tuned by a balance of activating and inhibitory phosphorylation during the cell cycle.