Mechanism of Cdk2/Cyclin E inhibition by p27 and p27 phosphorylation.
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.
Xu
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Mitotix Inc., One Kendall Square, Cambridge, Massachusetts 02139, USA.
Nakano
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T
Wick
S
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Dubay
M
M
Brizuela
L
L
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases; D018797:Cell Cycle Proteins; D019927:Cyclin E / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013379:Substrate Specificity; D025521:Tumor Suppressor Proteins
C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D019927:Cyclin E; D004791:Enzyme Inhibitors; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases