title: Impaired response to interferon-gamma in activated macrophages due to tyrosine nitration of STAT1 by endogenous nitric oxide.
abstract: 1. Inducible NO synthase (iNOS) expression and activity were measured in the mouse macrophage cell line J774 after exposure to bacterial lipopolysaccharide (LPS) with or without interferon-gamma (IFN-gamma). 2. Inhibition of NOS activity by concomitant N(G)-monomethyl-L-arginine (L-NMMA) treatment further increased iNOS protein levels, with a substantial increase in iNOS half-life. 3. Western blotting and ELISA demonstrated that several cell proteins were tyrosine-nitrated when iNOS activity was high. 4. Rapid IFN-gamma-induced phosphorylation of STAT1 was reduced by about 40% when cells were pretreated to induce iNOS, unless L-NMMA was present during the pretreatment period. 2D gel electrophoresis demonstrated the presence of nitrotyrosine in STAT1 after iNOS induction, and confirmed the reduction in phospho-STAT1 on subsequent stimulation with IFN-gamma for 15 min and its partial restoration when L-NMMA was present during the pretreatment period. 5. We did not detect tyrosine nitration of the upstream kinase JAK2 in LPS+IFN-gamma pretreated cells, but JAK2 activity was also impaired, and was partially restored by concomitant L-NMMA pretreatment. 6. We conclude that endogenous production of NO induces feedback inhibition of signalling pathways activated by IFN-gamma, at least in part by nitrating tyrosine residues in STAT1 which prevents phosphorylation.
authors.affiliation: Centre for Cardiovascular Biology & Medicine, School of Biomedical Sciences, King's College London, Guy's Campus, London SE1 1UL.
authors.forename: M
authors.initials: M
authors.lastname: Llovera
authors.forename: J D
authors.initials: JD
authors.lastname: Pearson
authors.forename: C
authors.initials: C
authors.lastname: Moreno
authors.forename: V
authors.initials: V
authors.lastname: Riveros-Moreno
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003720:Densitometry; D015180:Electrophoresis, Gel, Two-Dimensional; D007371:Interferon-gamma / Q000494:pharmacology*; D053613:Janus Kinase 1; D053614:Janus Kinase 2; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008715:Methionine / Q000378:metabolism; D051379:Mice; D009566:Nitrates / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011518:Proto-Oncogene Proteins; D050794:STAT1 Transcription Factor; D015534:Trans-Activators / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D008070:Lipopolysaccharides; D009566:Nitrates; D011518:Proto-Oncogene Proteins; D050794:STAT1 Transcription Factor; C494080:Stat1 protein, mouse; D015534:Trans-Activators; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D007371:Interferon-gamma; D008715:Methionine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D011505:Protein-Tyrosine Kinases; C507905:Jak1 protein, mouse; C507926:Jak2 protein, mouse; D053613:Janus Kinase 1; D053614:Janus Kinase 2