Abstract
p27, an important cell cycle regulator, blocks the G(1)/S transition in cells by binding and inhibiting Cdk2/cyclin A and Cdk2/cyclin E complexes (Cdk2/E). Ubiquitination and subsequent degradation play a critical role in regulating the levels of p27 during cell cycle progression. Here we provide evidence suggesting that both Cdk2/E and phosphorylation of Thr(187) on p27 are essential for the recognition of p27 by the SCF(Skp2/Cks1) complex, the ubiquitin-protein isopeptide ligase (E3). Cdk2/E provides a high affinity binding site, whereas the phosphorylated Thr(187) provides a low affinity binding site for the Skp2/Cks1 complex. Furthermore, binding of phosphorylated p27/Cdk2/E to the E3 complex showed positive cooperativity. Consistently, p27 is also ubiquitinated in a similarly cooperative manner. In the absence of p27, Cdk2/E and Cks1 increase Skp2 phosphorylation. This phosphorylation enhances Skp2 auto-ubiquitination, whereas p27 inhibits both phosphorylation and auto-ubiquitination of Skp2.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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CDC2-CDC28 Kinases
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Carrier Proteins / chemistry*
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Carrier Proteins / metabolism
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Cell-Free System / chemistry
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Cell-Free System / metabolism
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Cyclin A / chemistry
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Cyclin A / metabolism
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Cyclin E / chemistry
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Cyclin E / metabolism
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Cyclin-Dependent Kinase 2 / chemistry
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Cyclin-Dependent Kinase 2 / metabolism
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Cyclin-Dependent Kinase Inhibitor p27 / chemistry
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Cyclin-Dependent Kinase Inhibitor p27 / metabolism
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Cyclin-Dependent Kinases / chemistry*
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Cyclin-Dependent Kinases / metabolism
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G1 Phase / physiology
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Humans
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Multiprotein Complexes / chemistry*
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Multiprotein Complexes / metabolism
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Phosphorylation
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Protein Processing, Post-Translational* / physiology
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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S Phase / physiology
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S-Phase Kinase-Associated Proteins / chemistry*
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S-Phase Kinase-Associated Proteins / metabolism
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Ubiquitin-Protein Ligases / chemistry*
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Ubiquitin-Protein Ligases / metabolism
Substances
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CKS1B protein, human
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Carrier Proteins
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Cyclin A
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Cyclin E
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Multiprotein Complexes
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Recombinant Proteins
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S-Phase Kinase-Associated Proteins
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Cyclin-Dependent Kinase Inhibitor p27
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Ubiquitin-Protein Ligases
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CDC2-CDC28 Kinases
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CDK2 protein, human
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases