p53 N-terminus-targeted protein kinase activity is stimulated in response to wild type p53 and DNA damage.
The p53 tumour suppressor protein plays a central role in the cellular defence against agents which cause genetic damage. The activity of p53 is regulated at different levels and is subject to multi-site phosphorylation by a variety of different protein kinases. In this paper we have characterised p53 N-terminus-targeted protein kinase (p53NK) activities, present in a range of cell lines, following fractionation of cellular lysates by ion exchange chromatography on HiTrap Q and Mono Q resins. Three peaks of p53NK activity were observed following fractionation of HeLa cell lysates; these activities were each able to catalyse phosphorylation of up to three residues (serines 4, 6 and 9 in murine p53) within the N-terminus of the p53 protein. Similarly, multiple p53NK activities were detected in the MethAp53(ts) cell line (which expresses the valine 135 temperature-sensitive p53 protein). Strikingly, when these cells were shifted from 38 degrees C (the non-permissive temperature) to 28 degrees C, at which the p53 adopts a wild type conformation, a fivefold stimulation of kinase activity was detected. Moreover, when the DNA damage-inducing drugs etoposide or camptothecin were added to the cells, a further stimulation of kinase activity was observed following growth at 28 degrees C, but not 38 degrees C. These data are consistent with a regulatory model in which p53 is sensitive to stress or DNA damage through phosphorylation at its N-terminus.
Knippschild
U
U
Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, UK.
Milne
D
D
Campbell
L
L
Meek
D
D
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D004249:DNA Damage; D005978:Glutathione / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D002352:Carrier Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D011494:Protein Kinases; D005978:Glutathione