Nitric oxide induces phosphorylation of p53 and impairs nuclear export

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Abstract
The tumor suppressor p53 accumulates under diverse stress conditions and affects cell cycle progression and/or apoptosis. This has been exemplified for endogenously produced or exogenously supplied nitric oxide (NO) and thus accounts at least in part for pathophysiological signaling of that bioactive molecule, although detailed mechanisms remain to be elucidated. By using luciferase reporter assays, we show that NO stabilized a transcriptionally active p53 protein. Considering that p53 is targeted by murine double minute (Mdm2) for ubiquitination and subsequent proteasomal degradation and knowing that this interaction is impaired by, for example, UV-treatment with concomitant stabilization of p53 we questioned the p53/Mdm2 interaction in the presence of NO. Although p53 became phosphorylated at serine 15 under the impact of NO, coimmunoprecipitation with Mdm2 and ubiquitination remained intact, thus excluding any interference of NO with this pathway. The importance of N-terminal p53 phosphorylation was verified with p53 mutants where the first six serine residues have been converted to alanine, and which do not accumulate in response to NO. Regulation of p53 stability can be also achieved by affecting nuclear–cytoplasmic shuttling and it was presented that leptomycin B, an inhibitor of nuclear export, caused p53 accumulation. Cell fractionation and immunofluorescence staining following NO-treatment revealed predominant nuclear accumulation of p53 in close association with serine 15-phosphorylation, which suggests impaired nuclear–cytoplasmic shuttling. This was verified by heterokaryon analysis. We conclude that attenuated nuclear export contributes to stabilization and activation of p53 under the influence of NO.