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Nitric oxide induces phosphorylation of p53 and impairs nuclear export
Nicole Schneiderhan1, Andreja Budde, Yanping Zhang
1Department of Cell Biology, Faculty of Biology, University of Kaiserslautern, Erwin-Schrödinger-Strasse, 67663 Kaiserslautern, Germany.
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.
Insights
Nitric oxide (NO) stabilizes the tumor suppressor p53 by promoting its nuclear accumulation, independent of the Mdm2 degradation pathway. This NO-induced p53 stabilization is linked to serine 15 phosphorylation and impaired nuclear export.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The tumor suppressor p53 is a critical regulator of cell cycle progression and apoptosis, accumulating under various stress conditions.
- Nitric oxide (NO) is a bioactive molecule involved in cellular signaling, with its effects on p53 stabilization partially understood.
- p53 degradation is primarily mediated by Mdm2 through ubiquitination and proteasomal pathways, a process that can be inhibited by certain stresses like UV treatment.
Purpose of the Study:
- To elucidate the detailed mechanisms by which nitric oxide (NO) influences the stability and activity of the tumor suppressor p53.
- To investigate whether NO affects the interaction between p53 and its negative regulator, Mdm2.
- To determine the role of p53 phosphorylation and nuclear-cytoplasmic shuttling in NO-mediated p53 stabilization.
Main Methods:
- Luciferase reporter assays to assess p53 transcriptional activity.
- Co-immunoprecipitation and ubiquitination assays to analyze the p53/Mdm2 interaction.
- Site-directed mutagenesis to create p53 mutants lacking N-terminal phosphorylation sites.
- Cell fractionation, immunofluorescence staining, and heterokaryon analysis to study p53 localization and nuclear export.
Main Results:
- Nitric oxide (NO) was found to stabilize a transcriptionally active p53 protein.
- Despite p53 phosphorylation at serine 15 by NO, the interaction with Mdm2 and subsequent ubiquitination remained intact, ruling out interference with this degradation pathway.
- p53 mutants lacking N-terminal serine phosphorylation failed to accumulate in response to NO, highlighting the importance of these sites.
- NO treatment led to predominant nuclear accumulation of p53, associated with serine 15 phosphorylation, suggesting impaired nuclear export.
- Heterokaryon analysis confirmed that NO treatment attenuates the nuclear export of p53.
Conclusions:
- Nitric oxide (NO) stabilizes and activates the tumor suppressor p53.
- p53 stabilization by NO is mediated by impaired nuclear export and requires N-terminal phosphorylation at sites like serine 15.
- The Mdm2-dependent degradation pathway is not directly affected by NO in the context of p53 stabilization.