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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.

We used clone 6 cells (rat embryo fibroblasts transformed by the temperature sensitive mutant p53val135 and an activated H-ras-gene (Michalovitz et al., 1990)), growth arrested at 32 degrees C, as a model to analyse whether and how transformed cells, growth-arrested by an overexpressed wild-type p53, might overcome p53-mediated growth inhibition. When clone 6 cells were kept at 32 degrees C for about 2 weeks, foci of cells appeared which grew temperature-independent. Analysis of individual clones of such cell demonstrated that the ectopically expressed tsp53-gene had not been altered by an additional mutation, but that the tsp53 in these cells at 32 degrees C had lost its ability to upregulate expression of the p53 target genes waf1 and mdm2. This loss of p53-specific transactivation correlated with nuclear exclusion of the tsp53 at 32 degrees C, which was most likely mediated by cytoplasmic retention of the tsp53 protein via short-lived anchor proteins. Cytoplasmic retention of the tsp53 at 32 degrees C was also observed in PC12 pheochromocytoma cells ectopically expressing tsp53val135, there occurring without specific selection. Also in these cells nuclear exclusion of the tsp53 correlated with loss of p53 mediated growth inhibition. Nuclear exclusion of p53 thus might serve as an epigenetic mechanism to eliminate the growth-inhibitory function of p53.

The temperature-sensitive mutant p53 tsp53val135 (tsp53) displays a mutant phenotype at 38 degrees C, but assumes properties of a wild-type (wt) p53 at 32 degrees C. We analysed the cellular responses of two cell lines which ectopically overexpress tsp53, and dramatically differ in their responses to tsp53 expressed at 32 degrees C. Clone 6 (cl6) cells [precrisis rat embryo fibroblasts transformed by tsp53val135 and an activated ras oncogene at 38 degrees C (Michalovitz et al., 1990. Cell 62, 671-680) stop to grow and arrest mainly in the G1 phase of the cell cycle, whereas MethAp53ts cells [BALB/c mouse MethA tumor cells, transfected with the same tsp53 encoding vector as cl6 cells (Otto and Deppert, 1993. Oncogene 8, 2591-2603)] do not growth arrest at 32 degrees C. Both cell lines expressed similar amounts of tsp53, which was mainly cytoplasmic at 38 degrees C and mainly nuclear at 32 degrees C. At 32 degrees C, both cell lines contained similar amounts of waf1/cip1 mRNA. However, the amount of mdm2 mRNA in MethAp53ts cells was considerably higher compared to that in cl6 cells. The different transcriptional regulation of the mdm2-gene in cl6 and MethAp53ts cells at 32 degrees C indicated that the tsp53 proteins in these cells were functionally different. This assumption was supported by our finding that at 32 degrees C phosphorylation of the tsp53 in these cells was markedly different. We conclude that the cellular environment is an important determinant of p53 function.

The Rev2 cell line is a cellular revertant of the SV40 wild-type transformed rat cell line SV-52 [Bauer, M., Guhl, E., Graessmann, M. & Graessmann, A. (1987). J. Virol., 61, 1821-1827]. To characterize the level of cellular interference with the SV40 large T antigen (large T)-induced transformation pathway in Rev2 cells, we analysed the biological and biochemical properties of large T expressed in Rev2 cells. We found that Rev2 cells encoded an authentic wild-type large T, with regard to its sequence and its transforming functions. No differences were found in the metabolic stability of large T, or in complex formation with the cellular p53 protein, or in p53 metabolic stabilization. In contrast to SV-52 cells, Rev2 cells showed no association of large T with the chromatin fraction of isolated nuclei. This difference correlated with a reduced affinity of the Rev2 large T to SV40 DNA in vitro. The T proteins from both cell lines were phosphorylated at the same multiple sites. However, in Rev2 cells the phosphorylation of large T at specific serine -residues was significantly reduced. Thus the revertant phenotype of Rev2 cells may be due to an altered phosphorylation state of its large T protein, leading to altered nuclear localization and reduced transforming activity. The alterations of Rev2 large T properties and phosphorylation were very similar to the changes observed with mutant large T in FR(tsA58)A cells, an SV40 tsA58 N-type transformant, when the cells had reverted to the normal phenotype at the non-permissive growth temperature. Thus altered phosphorylation might provide a common structural basis for the biological inactivation of the large T proteins in these cells.