Species-specific phosphorylation of mouse and rat p53 in simian virus 40-transformed cells

Abstract

We have analyzed in detail the phosphorylation of p53 from normal (3T3) and simian virus 40 (SV40)-transformed (SV3T3) BALB/c mouse cells and from normal (F111) and SV40-transformed [FR(wt648)] rat cells by two-dimensional tryptic peptide mapping and phosphoamino acid analyses. To accommodate the different half-lives of p53 in normal (half-life, 15 min) and transformed (half-life, 20 h) cells and possible differences in the rates of turnover of phosphate at specific sites, cells were labeled for 2 h (short-term labeling) or 18 h (long-term labeling). Depending on the labeling conditions, either close similarities or marked differences were observed in the phosphorylation patterns of p53 from normal and transformed cells. After the 2-h labeling, the phosphorylation patterns of p53 from normal and transformed mouse cells were quite similar. In contrast, p53 from normal and transformed rat cells exhibited dramatic quantitative and qualitative differences under these labeling conditions. The reverse was found after an 18-h label leading to steady-state phosphorylation of p53 in transformed cells: while p53 in transformed mouse cells revealed a marked quantitative increase in phosphorylation compared with p53 from normal cells, the corresponding patterns of p53 from normal and transformed rat cells were similar. Our data thus indicate species-specific differences in the phosphorylation of mouse and rat p53 in SV40-transformed cells, reflected by (i) different turnover rates at specific sites in mouse and rat p53 and (ii) phosphorylation of nonhomologous serine and threonine residues in rat p53, as revealed by indirect assignment of phosphorylation sites to the phosphopeptides of rat p53. Analyses of p53 from the SV40 tsA58 mutant-transformed F111 cell lines FR(tsA58)A (N type) and FR(tsA58)57 (A type) yielded no conclusive evidence for a direct correlation between phosphorylation of p53, the metabolic stabilization of p53, and expression of the transformed phenotype.