Tyrosine phosphorylation of inducible nitric oxide synthase: implications for potential post-translational regulation.
The activation of cultured Raw 264.7 murine macrophages with interferon gamma and lipopolysaccharide results in the expression of inducible nitric oxide synthase (i_NOS) and the subsequent production of nitric oxide. In the present study, the i-NOS expressed in these activated cells was characterized for possible post-translational protein modification by endogenous tyrosine protein kinases. Western-blot analysis using phosphotyrosine antibodies revealed that i-NOS was phosphorylated on tyrosine residues and that this was an early event coinciding with the appearance of newly synthesized i-NOS. A brief exposure of activated cells to vanadate, a tyrosine phosphatase inhibitor, significantly increased the level of i-NOS tyrosine phosphorylation, suggesting that tyrosine phosphatases are dynamically involved in the regulation of this process. Vanadate treatment of activated cells also resulted in a rapid increase in enzyme activity, occurring within 5 min of exposure. Taken together, these results demonstrate that tyrosine kinases and phosphatases are involved in the post-translational modification of i-NOS and may potentially play a role in modulating the functional activity of the enzyme in macrophages.
Pan
J
J
Neuroscience Therapeutic Domain, Hoechst-Roussel Pharmaceuticals Inc., Somerville, NJ 08876, USA.
Burgher
K L
KL
Szczepanik
A M
AM
Ringheim
G E
GE
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000494:pharmacology; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D017931:DNA Primers; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D007371:Interferon-gamma / Q000494:pharmacology; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects; D008264:Macrophages; D051379:Mice; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase / Q000096:biosynthesis / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational; D013997:Time Factors; D019323:omega-N-Methylarginine
D017931:DNA Primers; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008070:Lipopolysaccharides; D019000:Phosphotyrosine; D019323:omega-N-Methylarginine; D007371:Interferon-gamma; D001120:Arginine; D019001:Nitric Oxide Synthase; D002118:Calcium