Phosphorylation of p53 in primary, immortalised and transformed Balb/c mouse cells.

To address the question whether phosphorylation of p53 might be functionally involved in its metabolic stabilisation and in cellular transformation processes, we have analysed the phosphorylation of the cellular protein p53 in normal and transformed cells of Balb/c mouse origin. Two-dimensional tryptic peptide maps of metabolically unstable p53 from normal Balb/c 3T3 cells and of metabolically stable p53 from 3T3 cells transformed by Simian virus 40 (SV3T3 cells) revealed no qualitative differences between their phosphorylation sites. Except for the unique lack of one or possibly two sites, the phosphopeptide map of p53 from cells transformed by the chemical carcinogen methylcholanthrene (MethA cells), expressing two different, metabolically stable mutant forms of p53, was identical to that of wild-type p53 from normal and SV40-transformed 3T3 cells. These results suggest that no direct relationship exists between phosphorylation of p53, its metabolic stabilisation, and cellular transformation processes. We have included in our analyses p53 from primary Balb/c mouse embryo fibroblast (MEF) cells and the immortalised MEFP27 cell line, established from primary cells after 27 passages. The phosphorylation sites of p53 from these cells were found to be identical to that of p53 from 3T3 cells. In addition, primary and established (immortalised) Balb/c mouse cells revealed no major differences in abundance of p53 mRNA and p53 protein, as well as stability of p53. These results indicate that immortalisation of normal cells involves neither gross alterations of p53 expression nor changes in specific phosphorylation of p53.