TYROSINE PHOSPHORYLATION OF INDUCIBLE NITRIC-OXIDE SYNTHASE - IMPLICATIONS FOR POTENTIAL POSTTRANSLATIONAL REGULATION

The activation of cultured Raw 264.7 murine macrophages with interfero n 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 ce lls was characterized for possible post-translational protein modifica tion by endogenous tyrosine protein kinases. Western-blot analysis usi ng phosphotyrosine antibodies revealed that i-NOS was phosphorylated o n tyrosine residues and that this was an early event coinciding with t he appearance of newly synthesized i-NOS. A brief exposure of activate d cells to vanadate, a tyrosine phosphatase inhibitor, significantly i ncreased the level of i-NOS tyrosine phosphorylation, suggesting that tyrosine phosphatases are dynamically involved in the regulation of th is process. Vanadate treatment of activated cells also resulted in a r apid increase in enzyme activity, occurring within 5 min of exposure. Taken together, these results demonstrate that tyrosine kinases and ph osphatases are involved in the post-translational modification of i-NO S and may potentially play a role in modulating the functional activit y of the enzyme in macrophages.