Abstract
Entry into S phase is dependent on the coordinated activation of CDK4,6 and CDK2 kinases. Once a cell commits to S phase, there must be a mechanism to ensure the irreversibility of this decision. The activity of these kinases is inhibited by their association with p27. In many cells, p27 plays a major role in the withdrawal from the cell cycle in response to environmental cues. Thus, it is likely that p27 is a target of the machinery required to ensure the irreversibility of S-phase entry. We have been interested in understanding the mechanisms regulating p27 at the G1/S transition. In this report, we define a cell-free degradation system which faithfully recapitulates the cell cycle phase-specific degradation of p27. We show that this reaction is dependent on active CDK2 activity, suggesting that CDK2 activity is directly required for p27 degradation. In addition to CDK2, other S-phase-specific factors are required for p27 degradation. At least some of these factors are ubiquitin and proteasome dependent. We discuss the relationships between CDK2 activity, ubiquitin-dependent, and possibly ubiquitin-independent proteasomal activities in S-phase extracts as related to p27.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Base Sequence
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CDC2-CDC28 Kinases*
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Cell Cycle
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Cell Cycle Proteins*
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Cell-Free System
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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 / metabolism*
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Cyclins / metabolism
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Cysteine Endopeptidases / metabolism*
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Enzyme Inhibitors / pharmacology
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G1 Phase
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HeLa Cells
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Humans
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In Vitro Techniques
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Microtubule-Associated Proteins / genetics
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Microtubule-Associated Proteins / metabolism*
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Multienzyme Complexes / metabolism*
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Oligonucleotide Probes / genetics
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Proteasome Endopeptidase Complex
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Protein Serine-Threonine Kinases / antagonists & inhibitors
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Protein Serine-Threonine Kinases / metabolism*
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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S Phase
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Tumor Suppressor Proteins*
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Ubiquitins / metabolism
Substances
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Cell Cycle Proteins
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Cyclins
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Enzyme Inhibitors
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Microtubule-Associated Proteins
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Multienzyme Complexes
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Oligonucleotide Probes
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Recombinant Proteins
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Tumor Suppressor Proteins
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Ubiquitins
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Cyclin-Dependent Kinase Inhibitor p27
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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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Cyclin-Dependent Kinase 2
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Cyclin-Dependent Kinases
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex