The C-terminal regulatory domain of p53 contains a functional docking site for cyclin A

J Mol Biol. 2000 Jul 14;300(3):503-18. doi: 10.1006/jmbi.2000.3830.

Abstract

Radiation injury to cells enhances C-terminal phosphorylation of p53 at both Ser315 and Ser392 in vivo, suggesting the existence of two cooperating DNA damage-responsive pathways that play a role in stimulating p53-dependent gene expression. Our previous data has shown that cyclin A-cdk2 is the major enzyme responsible for modifying p53 at Ser315 in vivo after irradiation damage and in this report we dissect the mechanism of cyclinA-cdk2 binding to and phosphorylation of p53. Although cyclin B(1)-dependent protein kinases can phosphorylate small peptides containing the Ser315 site, cyclin A-cdk2 does not phosphorylate such small peptides suggesting that additional determinants are required for cyclin A-cdk2 interaction with p53. Peptide competition studies have localized a cyclin A interaction site to a Lys381Lys382Leu383Met384Phe385 sequence within C-terminal negative regulatory domain of human p53. An alanine mutation at any one of four key positions abrogates the efficacy of a synthetic peptide containing this motif as an inhibitor of cyclin A-cdk2 phosphorylation of p53 protein. Single amino acid mutations of full-length p53 protein at Lys382, Leu383, or Phe385 decreases cyclin A-cdk2 dependent phosphorylation at Ser315. Cyclin B(1)-cdk2 complexes are not inhibited by KKLMF motif-containing peptides nor is p53 phosphorylation by cyclin B-cdk2 reduced by mutation of the cyclin A interaction site. These data identifying a KKLMF cyclin A docking site on p53 protein highlight a common cyclin A interaction motif that is shared between the tumour suppressor proteins pRb and p53.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Amino Acid Substitution / genetics
  • Antibodies / immunology
  • Binding Sites
  • CDC2-CDC28 Kinases*
  • Cyclin A / metabolism*
  • Cyclin B / metabolism
  • Cyclin B1
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases / antagonists & inhibitors
  • Cyclin-Dependent Kinases / metabolism
  • Histones / metabolism
  • Humans
  • Molecular Sequence Data
  • Mutation / genetics
  • Peptide Fragments / chemical synthesis
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism
  • Peptide Fragments / pharmacology
  • Phosphorylation / drug effects
  • Phosphoserine / immunology
  • Phosphoserine / metabolism
  • Protein Binding
  • Protein Kinase Inhibitors
  • Protein Kinases / metabolism
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / antagonists & inhibitors
  • Protein-Serine-Threonine Kinases / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Fusion Proteins / pharmacology
  • Sequence Alignment
  • Tumor Cells, Cultured
  • Tumor Suppressor Protein p53 / chemistry*
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism*
  • Tumor Suppressor Protein p53 / pharmacology

Substances

  • Antibodies
  • CCNB1 protein, human
  • Cyclin A
  • Cyclin B
  • Cyclin B1
  • Histones
  • Peptide Fragments
  • Protein Kinase Inhibitors
  • Recombinant Fusion Proteins
  • Tumor Suppressor Protein p53
  • Phosphoserine
  • Protein Kinases
  • histone H1 kinase
  • Protein-Serine-Threonine Kinases
  • CDC2-CDC28 Kinases
  • CDK2 protein, human
  • Cyclin-Dependent Kinase 2
  • Cyclin-Dependent Kinases