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Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complex formation.

The cellular abundance of the cyclin-dependent kinase (Cdk) inhibitor p27 is regulated by the ubiquitin-proteasome system. Activation of p27 degradation is seen in proliferating cells and in many types of aggressive human carcinomas. p27 can be phosphorylated on threonine 187 by Cdks, and cyclin E/Cdk2 overexpression can stimulate the degradation of wild-type p27, but not of a threonine 187-to-alanine p27 mutant [p27(T187A)]. However, whether threonine 187 phosphorylation stimulates p27 degradation through the ubiquitin-proteasome system or an alternative pathway is still not known. Here, we demonstrate that p27 ubiquitination (as assayed in vivo and in an in vitro reconstituted system) is cell-cycle regulated and that Cdk activity is required for the in vitro ubiquitination of p27. Furthermore, ubiquitination of wild-type p27, but not of p27(T187A), can occur in G1-enriched extracts only upon addition of cyclin E/Cdk2 or cyclin A/Cdk2. Using a phosphothreonine 187 site-specific antibody for p27, we show that threonine 187 phosphorylation of p27 is also cell-cycle dependent, being present in proliferating cells but undetectable in G1 cells. Finally, we show that in addition to threonine 187 phosphorylation, efficient p27 ubiquitination requires formation of a trimeric complex with the cyclin and Cdk subunits. In fact, cyclin B/Cdk1 which can phosphorylate p27 efficiently, but cannot form a stable complex with it, is unable to stimulate p27 ubiquitination by G1 extracts. Furthermore, another p27 mutant [p27(CK-)] that can be phosphorylated by cyclin E/Cdk2 but cannot bind this kinase complex, is refractory to ubiquitination. Thus throughout the cell cycle, both phosphorylation and trimeric complex formation act as signals for the ubiquitination of a Cdk inhibitor.

Pubmed ID: 10323868

Authors

  • Montagnoli A
  • Fiore F
  • Eytan E
  • Carrano AC
  • Draetta GF
  • Hershko A
  • Pagano M

Journal

Genes & development

Publication Data

May 1, 1999

Associated Grants

  • Agency: NCI NIH HHS, Id: R01 CA76584
  • Agency: NIGMS NIH HHS, Id: R01 GM57587

Mesh Terms

  • Cell Cycle
  • Cell Cycle Proteins
  • Cell Division
  • Cell Line
  • Cyclin-Dependent Kinase Inhibitor p27
  • Cyclin-Dependent Kinases
  • Cyclins
  • G1 Phase
  • HeLa Cells
  • Humans
  • Microtubule-Associated Proteins
  • Phosphorylation
  • Protein Conformation
  • Signal Transduction
  • Tumor Suppressor Proteins
  • Ubiquitins