Article: article from journal or magazin.
Src-mediated phosphorylation regulates subcellular distribution and activity of human inducible nitric oxide synthase.
Publication types: Journal Article ; Research Support, Non-U.S. Gov't Publication Status: ppublish
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.
Amino Acid Sequence, Cells, Cultured, Enzyme Inhibitors/pharmacology, Epithelial Cells/cytology, Epithelial Cells/drug effects, Humans, Immunoblotting, Immunoprecipitation, Intestinal Mucosa/cytology, Intestinal Mucosa/drug effects, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation/genetics, Nitric Oxide/metabolism, Nitric Oxide Synthase Type II/genetics, Nitric Oxide Synthase Type II/metabolism, Phosphorylation/drug effects, Protein Tyrosine Phosphatases/antagonists & inhibitors, Proto-Oncogene Proteins pp60(c-src)/pharmacology, Sequence Homology, Amino Acid, Subcellular Fractions/enzymology, Transfection, Tyrosine/metabolism, Vanadates/pharmacology
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