Mapping of phosphomonoester and apparent phosphodiester bonds of the oncogene product p53 from simian virus 40-transformed 3T3 cells.

Abstract

The oncogene product p53, isolated from SV3T3 cells where it forms a complex with simian virus 40 large tumor antigen (T antigen) in the nucleus, has been found to be phosphorylated at at least four distinct sites on the 390 amino acid protein. Separation of tryptic phosphopeptides has permitted identification of two sites as Ser-312 and Ser-389, and permitted analysis of the types of phosphate bonds. The peptide containing Ser-312 separates electrophoretically into three charged forms; two are resistant to dephosphorylation by both alkaline phosphatase and alkaline hydrolysis, suggesting a phosphodiester. The carboxyl-terminal phosphopeptide containing Ser-389 was alkaline phosphatase-resistant and liberated four ribonucleoside monophosphates upon base or RNase hydrolysis, suggesting that Ser-389 may be covalently linked to RNA. Phosphorylation of Ser-389 decreased markedly at the nonpermissive temperature in simian virus 40 tsA58-transformed cells, indicating a dependence on native T antigen function and a possible role in transformation by T antigen. Two additional phosphorylation sites, one involving serine and one involving threonine, probably reside in the amino-terminal segment of p53 and appear to be peptide-phosphate monoesters.

Nuclear protein phosphorylation and growth control

“…Phosphorylation of p53 is also modulated by the transforming proteins of DNA tumour viruses, which suggests that it may play a central role in controlling the growth of the cell. There are two C-terminal phosphorylation sites in p53, Ser-309 and -386 (in mouse p53) and a cluster ofphosphorylation sites at the N-terminus, including Ser-4, 6, 15 and 34 (Samad et al, 1986;Meek & Eckhart, 1988). Different cell lines or species show similar but not identical phosphorylation patterns and additional, as yet unidentified, sites of phosphorylation may be present.…”

“…Interestingly, a DNA repairdefective mutation (hrr25-1) in Saccharomyces cerevisiae lies within a gene encoding a protein kinase with striking similarity to CKI (Hoekstra et al, 1991 (Sturzbecher et al, 1987). These changes may reflect increased phosphorylation only at a specific site or sites (Samad et al, 1986;Meek & Eckhart, 1988). In this respect, a recent study by Scheidtmann's group is particularly interesting.…”

Serine 312 phosphorylation is dispensable for wild-type p53 functions in vivo

“…17 S312 has also been shown to be phosphorylated on transformation in murine cell-based studies. 18,19 To confirm this, we transfected p53 À/À mouse embryonic fibroblasts (MEFs) with wild-type (WT) or S312A mouse p53 cDNAs and treated them with the ER-stress inducer thapsigargin (TG) 12 and analyzed the phosphorylation status. We observed increased phosphsorylation of WT p53 over time, whilst this was not the case in the S312A-transfected cells (Supplementary Figure 1B), indicating that mouse S312 can indeed be phosphorylated in vivo.…”

“…18 Figure 3 Stability and degradation of S312A p53. (a, b) Unstressed (a) or g-irradiated (10 Gy, 1 h) (b) p53 þ / þ and p53 S312A/S312A MEFs were treated with 50 mg/ml cycloheximide and harvested for p53 analysis.…”

p53 regulation upon genotoxic stress: intricacies and complexities

“…Phosphorylation was the first functionally relevant post-transcriptional modification of p53 to be discovered 35 and since then 23 phosphorylation sites on p53 have been uncovered. Several serines and threonines of p53 have been shown to be differentially phosphorylated by kinases, some under genotoxic stress (S6, S9, S15, S20, S46, S215, S366, S376, T388, S392) and others under basal conditions (T55, S376).…”