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Src-mediated phosphorylation regulates subcellular distribution and activity of human inducible nitric oxide synthase

Abstract

Inducible nitric oxide synthase (iNOS) expression is regulated at both the transcriptional and post-transcriptional level in epithelial cells. The aim of this study was to characterize the effects of tyrosine phosphorylation on iNOS activity. In a human intestinal epithelial cell line stimulated with cytokines, tyrosine phosphorylation of human iNOS protein was observed after 30 min exposure to pervanadate (PV), an inhibitor of protein tyrosine phosphatases. 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a specific inhibitor of Src tyrosine kinases, abolished the PV-induced iNOS tyrosine phosphorylation. Cotransfection of Src with iNOS cDNA in human embryonic kidney (HEK) 293 cells resulted in a threefold (P<0.001) increase of iNOS protein levels and tyrosine phosphorylation of iNOS. In the presence of Src, 76% of wild-type (wt) iNOS was redistributed to detergent-insoluble domains and iNOS activity was decreased by 28% (P<0.05) despite increased iNOS protein levels. Analysis of iNOS tyrosine mutants revealed decreased Src-induced effects in Y151F iNOS mutant. Using a GST-fusion protein containing a domain encompassing Y151, we show that Y151 is a direct substrate for active Src in vitro. These findings indicate a role for iNOS tyrosine phosphorylation in the regulation of iNOS activity and the implication of Src tyrosine kinases in this pathway.

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Acknowledgements

We thank J Dall'Aglio for his skillful assistance, Drs JR Weidner, W Moolenaar and C Baldari for their generous gift of the anti-human iNOS antiserum and Src or Fyn encoding plasmids, Drs B Hemmings and D Diviani for suggestions and Drs B Thorens, M vanBemmelen and D Firsov for critical reading of the manuscript. This work was supported by the Swiss National Science Foundation (SNSF 3100A0-103928) and EC Grant QLRT2001-02357.

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Correspondence to E Felley-Bosco.

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Supplementary Information accompanies the paper on Oncogene website (http://www.nature.com/onc).

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Hausel, P., Latado, H., Courjault-Gautier, F. et al. Src-mediated phosphorylation regulates subcellular distribution and activity of human inducible nitric oxide synthase. Oncogene 25, 198–206 (2006). https://doi.org/10.1038/sj.onc.1209030

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Keywords

  • inducible nitric oxide synthase
  • Src kinase
  • tyrosine phosphorylation

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