title: p53 is phosphorylated in vitro and in vivo by the delta and epsilon isoforms of casein kinase 1 and enhances the level of casein kinase 1 delta in response to topoisomerase-directed drugs.
abstract: The p53 tumour suppressor protein plays a key role in the integration of stress signals. Multi-site phosphorylation of p53 may play an integral part in the transmission of these signals and is catalysed by many different protein kinases including an unidentified p53-N-terminus-targeted protein kinase (p53NK) which phosphorylates a group of sites at the N-terminus of the protein. In this paper, we present evidence that the delta and epsilon isoforms of casein kinase 1 (CK1delta and CK1epsilon) show identical features to p53NK and can phosphorylate p53 both in vitro and in vivo. Recombinant, purified glutathione S-transferase (GST)-CK1delta and GST-CK1epsilon fusion proteins each phosphorylate p53 in vitro at serines 4, 6 and 9, the sites recognised by p53NK. Furthermore, p53NK (i) co-purifies with CK1delta/epsilon, (ii) shares identical kinetic properties to CK1delta/epsilon, and (iii) is inhibited by a CK1delta/epsilon-specific inhibitor (IC261). In addition, CK1delta is also present in purified preparations of p53NK as judged by immunoanalysis using a CK1delta-specific monoclonal antibody. Treatment of murine SV3T3 cells with IC261 specifically blocked phosphorylation in vivo of the CK1delta/epsilon phosphorylation sites in p53, indicating that p53 interacts physiologically with CK1delta and/or CK1epsilon. Similarly, over-expression of a green fluorescent protein (GFP)-CK1delta fusion protein led to hyper-phosphorylation of p53 at its N-terminus. Treatment of MethAp53ts cells with the topoisomerase-directed drugs etoposide or camptothecin led to increases in both CK1delta-mRNA and -protein levels in a manner dependent on the integrity of p53. These data suggest that p53 is phosphorylated by CK1delta and CK1epsilon and additionally that there may be a regulatory feedback loop involving p53 and CK1delta.
authors.affiliation: Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, UK.
authors.forename: U
authors.initials: U
authors.lastname: Knippschild
authors.forename: D M
authors.initials: DM
authors.lastname: Milne
authors.forename: L E
authors.initials: LE
authors.lastname: Campbell
authors.forename: A J
authors.initials: AJ
authors.lastname: DeMaggio
authors.forename: E
authors.initials: E
authors.lastname: Christenson
authors.forename: M F
authors.initials: MF
authors.lastname: Hoekstra
authors.forename: D W
authors.initials: DW
authors.lastname: Meek
mesh_terms: D000818:Animals; D019556:COS Cells; D002166:Camptothecin / Q000494:pharmacology; D047388:Casein Kinases; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005047:Etoposide / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism*; D051379:Mice; D010748:Phosphopeptides / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D059005:Topoisomerase II Inhibitors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010748:Phosphopeptides; D012333:RNA, Messenger; D059005:Topoisomerase II Inhibitors; D016159:Tumor Suppressor Protein p53; D005047:Etoposide; D011494:Protein Kinases; D047388:Casein Kinases; D002166:Camptothecin