Effects of protein phosphatase inhibition and phosphatase gene disruption on p53 biochemistry and function

The protein phosphatase inhibitor okadaic acid (OA) previously has been shown to induce hyperphosphorylation of p53 protein both grossly and at specific tryptic peptide sites. However, the consequences of OA induced phosphorylation (and phosphorylation in general) on p53 function in vivo remain unclear. The focus of this study was to determine if hyperphosphorylation wrought by OA or expression of human p53 in protein phosphatase-deficient yeast strains could indeed regulate the interaction between p53 and a physiological downstream target, the cdk inhibitor, p21waf1. In S. pombe, one strain containing a mutant p53 (arg->his 175) and a type 1 protein phosphatase gene knockout was unable to grow whereas both parental strains were both able to thrive, indicating a possible gain of function related to p53 phosphorylation. Rat embryonic fibroblasts harboring a highly expressed mouse p53 transgene and a p53 null control cell line were treated with 50nM doses of OA. This treatment resulted in: (1) the formation and retention of acidic p53 protein isoforms, and, more specifically, phosphorylation of tryptic peptide sites in the transactivation domain, (2) an increase in p53 affinity for a p21waf1 promotor oligonucleotide, (3) an increase in cellular steady state levels of p21waf1 message, (4) an increase in p53-dependent transcriptional activity from a waf1 reporter construct, and (5) a G2/M cell cycle blockage that is associated with intact p53. These results demonstrate for the first time that hyperphosphorylation of p53 induced by OA may regulate a critical downstream affector of cell growth suppression in an intact cellular environment