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Nitric oxide induces phosphorylation of p53 and impairs nuclear export

Oncogene volume 22, pages 2857–2868 (2003)Cite this article

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.

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Abbreviations

NO:

nitric oxide

GSNO:

S-nitrosoglutathione

SpNO:

spermine-NONOate

DAPI:

4′,6-diamino-2-phenylindole

CRM1:

chromosome region maintenance 1

Mdm2:

murine double minute

NES:

nuclear export sequence

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Acknowledgements

We thank M Decker and B Rogge for technical assistance. Thanks go to B Vogelstein for providing p53 reporter plasmids, D Bohmann for providing the His-ubiquitin expression plasmid, C Jost for providing the HA-p53 construct and K. Vousden as well as J Chen for providing the Mdm2 construct and M Scheffner for providing p53 Δ62–96 and p53 Δ62–96 S>A plasmids. We also thank M Yoshida for his generous gift of LMB. This work was supported by the Deutsche Forschungsgemeinschaft (BR999), Wilhelm Sander-Foundation and Boehringer Ingelheim Fonds for a travel award to NS.

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  1. Department of Cell Biology, Faculty of Biology, University of Kaiserslautern, Erwin-Schrödinger-Strasse, Kaiserslautern, 67663, Germany

    Nicole Schneiderhan, Andreja Budde & Bernhard Brüne

  2. Department of Molecular and Cellular Oncology, University of Texas, MD Anderson Cancer Center, Houston, 77030, TX, USA

    Yanping Zhang

  3. Department of Medicine IV-Experimental Division, Faculty of Medicine, University of Erlangen-Nürnberg, Erlangen, 91054, Germany

    Nicole Schneiderhan & Andreja Budde

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Correspondence to Bernhard Brüne.

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Schneiderhan, N., Budde, A., Zhang, Y. et al. Nitric oxide induces phosphorylation of p53 and impairs nuclear export. Oncogene 22, 2857–2868 (2003). https://doi.org/10.1038/sj.onc.1206431

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