Activation of cyclin E/CDK2 is coupled to site-specific autophosphorylation and ubiquitin-dependent degradation of cyclin E.
A yeast screen was developed to identify mutations in human cyclin E that lead to stabilization of the protein in order to identify determinants important for cyclin E turnover. Both C-terminal truncations and missense mutations near the C-terminus of cyclin E conferred hyperstability in vivo, suggesting that sequences in this region were critical for turnover. The following observations indicate that autophosphorylation of CDK2/cyclin E on Thr380 of the cyclin regulates cyclin E destruction: (i) mutation of Thr380 to Ala stabilizes cyclin E in yeast and mammalian cells; (ii) cyclin E/CDK2 autophosphorylates on cyclin E in vitro and cyclin E is a phosphoprotein in vivo in mammalian cells; (iii) the T380A mutation eliminates phosphorylation on the same site in mammalian cells and in vitro; (iv) inhibiting CDK2 activity in vivo stabilizes cyclin E; (v) the T380A mutation prevents ubiquitination of cyclin E. These results suggest a model where activation of cyclinE/CDK2 is coupled to cyclin E turnover via site-specific phosphorylation, which acts as a signal for ubiquitination and proteasome processing.
Won
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Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Reed
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D000818:Animals; D042846:CDC2-CDC28 Kinases; D017489:CDC28 Protein Kinase, S cerevisiae / Q000172:deficiency / Q000235:genetics; D002478:Cells, Cultured; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics; D016193:G1 Phase / Q000502:physiology; D015723:Gene Library; D005816:Genetic Complementation Test; D006801:Humans; D016296:Mutagenesis; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014162:Transfection; D014452:Ubiquitins / Q000502:physiology*
D016213:Cyclins; D018076:DNA, Complementary; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D010769:Phosphothreonine; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D017489:CDC28 Protein Kinase, S cerevisiae; C495894:CDK2 protein, human; C495892:Cdk2 protein, rat; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases