Cell-free degradation of p27(kip1), a G1 cyclin-dependent kinase inhibitor, is dependent on CDK2 activity and the proteasome

Mol Cell Biol. 1999 Feb;19(2):1190-201. doi: 10.1128/MCB.19.2.1190.

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

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • CDC2-CDC28 Kinases*
  • Cell Cycle
  • Cell Cycle Proteins*
  • Cell-Free System
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinase Inhibitor p27
  • Cyclin-Dependent Kinases / antagonists & inhibitors*
  • Cyclin-Dependent Kinases / metabolism*
  • Cyclins / metabolism
  • Cysteine Endopeptidases / metabolism*
  • Enzyme Inhibitors / pharmacology
  • G1 Phase
  • HeLa Cells
  • Humans
  • In Vitro Techniques
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Multienzyme Complexes / metabolism*
  • Oligonucleotide Probes / genetics
  • Proteasome Endopeptidase Complex
  • Protein Serine-Threonine Kinases / antagonists & inhibitors
  • Protein Serine-Threonine Kinases / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • S Phase
  • Tumor Suppressor Proteins*
  • Ubiquitins / metabolism

Substances

  • Cell Cycle Proteins
  • Cyclins
  • Enzyme Inhibitors
  • Microtubule-Associated Proteins
  • Multienzyme Complexes
  • Oligonucleotide Probes
  • Recombinant Proteins
  • Tumor Suppressor Proteins
  • Ubiquitins
  • Cyclin-Dependent Kinase Inhibitor p27
  • Protein Serine-Threonine Kinases
  • CDC2-CDC28 Kinases
  • CDK2 protein, human
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex