Src-mediated phosphorylation regulates subcellular distribution and activity of human inducible nitric oxide synthase.
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.
Hausel
P
P
Department of Pharmacology and Toxicology, Rue du Bugnon 27, Lausanne 1005, Switzerland.
Latado
H
H
Courjault-Gautier
F
F
Felley-Bosco
E
E
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D007413:Intestinal Mucosa / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D004791:Enzyme Inhibitors; C076715:pervanadate; D009569:Nitric Oxide; D014638:Vanadates; D014443:Tyrosine; D052247:Nitric Oxide Synthase Type II; D016392:Proto-Oncogene Proteins pp60(c-src); D017027:Protein Tyrosine Phosphatases