Abstract
The biochemical interactions between the Cdk2/Cyclin E kinase and its inhibitor p27, were investigated using purified, recombinant p27 and CAK-phosphorylated Cdk2/Cyclin E. From kcat/Km determinations using either histone H1 or pRb as substrates, we found that Cdk2/Cyclin E has 60-fold higher specificity for pRb than for histone H1. The IC50 value of p27 increased with increasing Cdk2/Cyclin E concentrations while it remained constant at various ATP and histone H1 concentrations, suggesting that p27 acts as a tight binding inhibitor of Cdk2/Cyclin E. We also found that p27 could be phosphorylated by Cdk2/Cyclin E only at high enzyme concentrations, and that p27 forms a stable interaction with Cdk2/Cyclin E regardless of its phosphorylation state. Our results further indicate that the Cdk2/Cyclin E/p27 ternary complex is kinetically inactive as an enzyme; instead it serves as a substrate for Cdk2/Cyclin E. These results suggest that if phosphorylation of p27 by Cdk2/Cyclin E is involved in its ubiquitin-dependent degradation, as previously suggested, then the target for such event is the phosphorylated p27 bound to Cdk2/Cyclin E and not free p27.
MeSH terms
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Adenosine Triphosphate / metabolism
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Adenosine Triphosphate / physiology
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Amino Acid Sequence
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Animals
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Binding Sites
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CDC2-CDC28 Kinases*
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Cell Cycle Proteins*
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Cyclin E / antagonists & inhibitors*
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Cyclin E / genetics
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Cyclin E / metabolism
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinase Inhibitor p27
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Cyclin-Dependent Kinases / antagonists & inhibitors*
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Cyclin-Dependent Kinases / genetics
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Cyclin-Dependent Kinases / metabolism
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Dose-Response Relationship, Drug
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Enzyme Inhibitors / metabolism*
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Humans
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Kinetics
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Macromolecular Substances
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Mice
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Microtubule-Associated Proteins / biosynthesis
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Microtubule-Associated Proteins / genetics
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Microtubule-Associated Proteins / metabolism*
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Molecular Sequence Data
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Protein Binding
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Protein Serine-Threonine Kinases / antagonists & inhibitors*
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism
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Recombinant Proteins / biosynthesis
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Recombinant Proteins / isolation & purification
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Substrate Specificity
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Tumor Suppressor Proteins*
Substances
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Cdkn1b protein, mouse
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Cell Cycle Proteins
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Cyclin E
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Enzyme Inhibitors
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Macromolecular Substances
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Microtubule-Associated Proteins
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Recombinant Proteins
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Tumor Suppressor Proteins
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Cyclin-Dependent Kinase Inhibitor p27
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Adenosine Triphosphate
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Protein Serine-Threonine Kinases
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CDC2-CDC28 Kinases
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CDK2 protein, human
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Cdk2 protein, mouse
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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases