The tumor suppressor and transcriptional factor p53 is a phosphorylated protein. There are multiple potential phosphorylation sites on the amino- and carboxyl-terminal regions of p53, but the role of phosphorylation at each site in the regulation of the functions of p53 is not well understood. The functions of p53 include transcriptional activation of genes involved in cell cycle arrest (WAF1/Cip1) and apoptosis (Bax anf Fas/APO-1). The preliminary studies showed that radiation exposure caused the wild-type p53-containing leukemia cell line BV173 to apoptosis rapidly and that treatment with arabinosyl cytosine (ara-C) caused a relatively slow apoptosis. Analyses of gene expression in the treated cells revealed that radiation activated both Bax and Fas/AP0-1 expression, whereas ara-C activated Bax but not Fas/APO-1 expression. Two-dimensional gel electrophoresis of immunoprecipitated, 35S-methionine-labeled p53 and the treated cells showed that p53 existed as different isoforms with distinct isoelectric points in radiated and ara-C-treated cells. The previous study characterized 143Ala as a temperature-sensitive (ts) mutant that activates endogenous WAF1/Cip1 and Fas/APO-1 genes at permissive temperatures. Two-dimensional gel analyses indicated that 143 Ala has diffferent isoelectric points at permissive and non-permissive temperatures, whereas the isoelectric points of the non-temperature-sensitive mutant 248Trp did not change with temperature switch. Phosphorylation is a key form of posttranslational modification that alters the isoelectric points of proteins. Therefore, these results strongly suggest that phosphorylation may play an important role in regulating the transcriptional activation functions of p53 in response to radiation and ara-C and in conferring the functional switch in the 143Ala ts mutant. The investigators hypothesize that differential phosphorylation of p53 accounts for its different transcriptional activities in irradiated and ara-C-treated BV173 cells. In this study they hope to identify important phosphorylation sites on p53 and shed light on the role of phosphorylation in the regulation of the p53 functions.
