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

Oncogene. 2006 Jan 12;25(2):198-206. doi: 10.1038/sj.onc.1209030.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cells, Cultured
  • Enzyme Inhibitors / pharmacology
  • Epithelial Cells / cytology
  • Epithelial Cells / drug effects*
  • Epithelial Cells / enzymology
  • Humans
  • Immunoblotting
  • Immunoprecipitation
  • Intestinal Mucosa / cytology
  • Intestinal Mucosa / drug effects*
  • Intestinal Mucosa / enzymology
  • 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

Substances

  • Enzyme Inhibitors
  • pervanadate
  • Nitric Oxide
  • Vanadates
  • Tyrosine
  • Nitric Oxide Synthase Type II
  • Proto-Oncogene Proteins pp60(c-src)
  • Protein Tyrosine Phosphatases