Activation of cyclin E/CDK2 is coupled to site-specific autophosphorylation and ubiquitin-dependent degradation of cyclin E

EMBO J. 1996 Aug 15;15(16):4182-93.

Abstract

A yeast screen was developed to identify mutations in human cyclin E that lead to stabilization of the protein in order to identify determinants important for cyclin E turnover. Both C-terminal truncations and missense mutations near the C-terminus of cyclin E conferred hyperstability in vivo, suggesting that sequences in this region were critical for turnover. The following observations indicate that autophosphorylation of CDK2/cyclin E on Thr380 of the cyclin regulates cyclin E destruction: (i) mutation of Thr380 to Ala stabilizes cyclin E in yeast and mammalian cells; (ii) cyclin E/CDK2 autophosphorylates on cyclin E in vitro and cyclin E is a phosphoprotein in vivo in mammalian cells; (iii) the T380A mutation eliminates phosphorylation on the same site in mammalian cells and in vitro; (iv) inhibiting CDK2 activity in vivo stabilizes cyclin E; (v) the T380A mutation prevents ubiquitination of cyclin E. These results suggest a model where activation of cyclinE/CDK2 is coupled to cyclin E turnover via site-specific phosphorylation, which acts as a signal for ubiquitination and proteasome processing.

Publication types

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

MeSH terms

  • Animals
  • CDC2-CDC28 Kinases*
  • CDC28 Protein Kinase, S cerevisiae / deficiency
  • CDC28 Protein Kinase, S cerevisiae / genetics
  • Cells, Cultured
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases / genetics
  • Cyclin-Dependent Kinases / metabolism*
  • Cyclins / genetics
  • Cyclins / metabolism*
  • DNA, Complementary / genetics
  • Fibroblasts / metabolism
  • Fungal Proteins / genetics
  • G1 Phase / physiology
  • Gene Library
  • Genetic Complementation Test
  • Humans
  • Mutagenesis
  • Phosphorylation
  • Phosphothreonine / metabolism
  • Protein Processing, Post-Translational*
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism*
  • Rats
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / metabolism
  • Transfection
  • Ubiquitins / physiology*

Substances

  • Cyclins
  • DNA, Complementary
  • Fungal Proteins
  • Recombinant Fusion Proteins
  • Ubiquitins
  • Phosphothreonine
  • Protein-Serine-Threonine Kinases
  • CDC2-CDC28 Kinases
  • CDC28 Protein Kinase, S cerevisiae
  • CDK2 protein, human
  • Cdk2 protein, rat
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases