P53 IS PHOSPHORYLATED IN-VITRO AND IN-VIVO BY THE DELTA-ISOFORM AND EPSILON-ISOFORM OF CASEIN-KINASE-1 AND ENHANCES THE LEVEL OF CASEIN-KINASE-1-DELTA IN RESPONSE TO TOPOISOMERASE-DIRECTED DRUGS

The p53 tumour suppressor protein plays a key role in the integration of stress signals, Multi-site phosphorylation of p53 may play an integ ral part in the transmission of these signals and is catalysed by many different protein kinases including an unidentified p53-N-terminus-ta rgeted protein kinase (p53NK) which phosphorylates a group of sites at the N-terminus of the protein. In this paper, we present evidence tha t the delta and epsilon isoforms of casein kinase 1 (CK1 delta and CK1 epsilon) show identical features to p53NK and can phosphorylate p53 b oth in vitro and in vivo. Recombinant, purified glutathione S-transfer ase (GST)-CK1 delta and GST-CK1 epsilon fusion proteins each phosphory late p53 in vitro at serines 4, 6 and 9, the sites recognised by p53NK . Furthermore, p53NK (i) co-purifies with CK1 delta/epsilon, (ii) shar es identical kinetic properties to CK1 delta/epsilon, and (iii) is inh ibited by a CK1 delta/epsilon-specific inhibitor (IC261). In addition, CK1 delta is also present in purified preparations of p53NK as judged by immunoanalysis using a CK1 delta-specific monoclonal antibody. Tre atment of murine SV3T3 cells with IC261 specifically blocked phosphory lation in vivo of the CK1 delta/epsilon phosphorylation sites in p53, indicating that p53 interacts physiologically with CK1 delta and/or CK 1 epsilon. Similarly, over-expression of a green fluorescent protein ( GFP)-CK1 delta fusion protein led to hyper-phosphorylation of p53 at i ts N-terminus. Treatment of MethAp53ts cells with the topoisomerase-di rected drugs etoposide or camptothecin led to increases in both CK1 de lta-mRNA and -protein levels in a manner dependent on the integrity of p53, These data suggest that p53 is phosphorylated by CK1 delta and C K1 epsilon and additionally that there may be a regulatory feedback lo op involving p53 and CK1 delta.