title: The C-terminal regulatory domain of p53 contains a functional docking site for cyclin A.
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.
authors.affiliation: Department of Molecular and Cellular Pathology, Cancer Research Campaign Laboratories, University of Dundee Medical School, Scotland.
authors.forename: M G
authors.initials: MG
authors.lastname: Luciani
authors.forename: J R
authors.initials: JR
authors.lastname: Hutchins
authors.forename: D
authors.initials: D
authors.lastname: Zheleva
authors.forename: T R
authors.initials: TR
authors.lastname: Hupp
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D042846:CDC2-CDC28 Kinases; D019925:Cyclin A / Q000378:metabolism*; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000906:Antibodies; C534699:CCNB1 protein, human; D019925:Cyclin A; D019926:Cyclin B; D056744:Cyclin B1; D006657:Histones; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D010768:Phosphoserine; D011494:Protein Kinases; C102333:histone H1 kinase; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases