The N-terminal domains of cyclin-dependent kinase inhibitory proteins block the phosphorylation of cdk2/Cyclin E by the CDK-activating kinase

Biochem Biophys Res Commun. 2000 May 10;271(2):469-73. doi: 10.1006/bbrc.2000.2648.

Abstract

It has been suggested that binding of p27 and p21 kinase inhibitory proteins (KIPs) to cyclin-dependent kinases (cdks) render them inaccessible to cdk-activating kinase (CAK), presumably by steric hindrance by the C-terminal residues. However, this common mechanism of inhibition is inconsistent with the known structural divergence in the p27 and p21 C-terminal domains. Therefore, we studied the direct binding of N-terminal minimal domain of p27 (amino acids 28-81) to cdk2/cyclin E. An unlabeled p27 minimal domain, mutated in the N-terminal LFG motif, was unable to compete with a labeled minimal domain for binding to cdk2/cyclin E. The p27 and its minimal domain inhibited CAK-mediated phosphorylation of cdk2/cyclin E. This inhibitory effect was significantly diminished with p27 minimal domain mutated in the LFG motif. A synthetic peptide, ACRRLFGPVDSE, from the N-terminal residues 17-28 of p21, was also a potent inhibitor of CAK-mediated cdk2/cyclin E phosphorylation. Taken together, these results show that anchoring of p27 or p21 KIPs to cyclin E via the N-terminal LFG-containing motif can block CAK access to its cdk2/cyclin E substrate.

MeSH terms

  • Animals
  • CDC2-CDC28 Kinases*
  • Cell Cycle Proteins*
  • Cell Line
  • Chromatography
  • Cyclin E / metabolism*
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinase Inhibitor p27
  • Cyclin-Dependent Kinases / metabolism*
  • Humans
  • Insecta
  • Microtubule-Associated Proteins / antagonists & inhibitors
  • Microtubule-Associated Proteins / chemistry*
  • Microtubule-Associated Proteins / metabolism
  • Peptides / metabolism
  • Phosphorylation
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Scintillation Counting
  • Streptavidin / metabolism
  • Tumor Suppressor Proteins*

Substances

  • Cell Cycle Proteins
  • Cyclin E
  • Microtubule-Associated Proteins
  • Peptides
  • Recombinant Proteins
  • Tumor Suppressor Proteins
  • Cyclin-Dependent Kinase Inhibitor p27
  • Streptavidin
  • Protein-Serine-Threonine Kinases
  • CDC2-CDC28 Kinases
  • CDK2 protein, human
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases
  • cyclin-dependent kinase-activating kinase