15-Deoxy-Δ12,14-prostaglandin J2 stabilizes, but functionally inactivates p53 by binding to the cysteine 277 residue

Abstract

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a representative cyclopentenone prostaglandin, has many interesting biological effects. In this study, treatment of human breast cancer cells (MCF-7) with 15d-PGJ(2) led to accumulation of p53 protein. However, the p53 DNA binding and its transcriptional activity were significantly reduced. 15d-PGJ(2) directly modified p53 as verified by reacting recombinant p53 with biotinylated 15d-PGJ(2). 9,10-Dihydro-15-deoxy-Delta(12,14)-prostaglandin J(2) lacking the electrophilic alpha,beta-unsaturated functionality failed to inhibit p53 DNA binding as well as to modify p53. Moreover, by conducting an in vitro [(35)S]-labeled p53 translation assay, we identified cysteine 277 as a putative site of p53 modification by 15d-PGJ(2). The DNA-binding ability of a mutant p53 in which cysteine 277 was substituted by alanine was virtually unaffected by 15d-PGJ(2). Likewise, p53 binding activity of biotinylated 15d-PGJ(2) was abolished in mutant cells. In addition, cells expressing wild-type p53 exhibited p53 protein stability to a greater extent than mutant C277A cells. In conclusion, 15d-PGJ(2) can undergo nucleophilic addition to p53, presumably at the cysteine 277 residue, rendering this tumor suppressor less susceptible to proteasomal degradation.