Abstract
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.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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CDC2-CDC28 Kinases*
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CDC28 Protein Kinase, S cerevisiae / deficiency
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CDC28 Protein Kinase, S cerevisiae / genetics
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Cells, Cultured
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases / genetics
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Cyclin-Dependent Kinases / metabolism*
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Cyclins / genetics
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Cyclins / metabolism*
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DNA, Complementary / genetics
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Fibroblasts / metabolism
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Fungal Proteins / genetics
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G1 Phase / physiology
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Gene Library
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Genetic Complementation Test
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Humans
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Mutagenesis
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Phosphorylation
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Phosphothreonine / metabolism
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Protein Processing, Post-Translational*
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Rats
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Recombinant Fusion Proteins / metabolism
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Saccharomyces cerevisiae / metabolism
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Transfection
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Ubiquitins / physiology*
Substances
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Cyclins
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DNA, Complementary
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Fungal Proteins
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Recombinant Fusion Proteins
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Ubiquitins
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Phosphothreonine
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Protein Serine-Threonine Kinases
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CDC2-CDC28 Kinases
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CDC28 Protein Kinase, S cerevisiae
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CDK2 protein, human
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Cdk2 protein, rat
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases