P53 N-terminal binding and stabilisation by PIAS3 inhibits MDM2-induced p53 ubiquitination and regulates cell growth

Abstract

Mouse double minute 2 homolog (MDM2) binds to p53 through the 1-52 amino acid region of p53, in order to ubiquitinate p53. MDM2 thus destabilises p53 and inhibits the effect of p53 on cell cycle arrest and apoptosis. In the present study, the 1-52 amino acid region of the wild-type (wt) p53 protein was stably expressed in H1299 cells. The lysate of the transfected cells was then analysed using co-immunoprecipitation. A specific fraction of the precipitate was subjected to mass spectrometry analysis, which revealed that p53 bound to protein inhibitor of activated STAT3 (PIAS3). To the best of our knowledge, the present study is the first to report that the interaction of PIAS3 with p53 occurs through the 1-52 amino acid region of p53. Overexpression of PIAS3 in the A549 wt p53-expressing cell line was found to significantly increase the half‑life of p53 in the presence of cycloheximide, an inhibitor of protein synthesis. However, PIAS3 overexpression did not affect p53 mRNA levels. Furthermore, PIAS3 overexpression was observed to decrease p53 ubiquitination. Protein-protein interaction analysis revealed that PIAS3 binds to the 1-52 amino acid region of p53, thus disrupts the formation of the p53‑MDM2 complex. In addition, overexpression of PIAS3 in A549 cells was found to enhance the transcription of the p53‑transactivated target p21/waf1, due to p53 accumulation, which led to an increase in p53 binding to the p21 gene promoter. These findings indicate that this newly identified p53‑PIAS3 interaction through the 1-52 amino acid region of p53, reduces p53‑MDM2 complex formation, which not only increases the half-life of p53, but also its transactivation of target genes.