title: Cell-free degradation of p27(kip1), a G1 cyclin-dependent kinase inhibitor, is dependent on CDK2 activity and the proteasome.
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.
authors.affiliation: Program in Molecular Biology and Cell Biology and Genetics, Cornell University Graduate School of Medical Sciences, New York, New York 10021, USA.
authors.forename: H
authors.initials: H
authors.lastname: Nguyen
authors.forename: D M
authors.initials: DM
authors.lastname: Gitig
authors.forename: A
authors.initials: A
authors.lastname: Koff
mesh_terms: D001483:Base Sequence; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D002474:Cell-Free System; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016193:G1 Phase; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism*; D015345:Oligonucleotide Probes / Q000235:genetics; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016196:S Phase; D025521:Tumor Suppressor Proteins; D014452:Ubiquitins / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D016213:Cyclins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D009097:Multienzyme Complexes; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D014452:Ubiquitins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex