J Biol Chem, Vol. 274, Issue 15, 9915-9917, April 9, 1999

COMMUNICATION
Rapid Inactivation of NOS-I by Lipopolysaccharide Plus Interferon--induced Tyrosine Phosphorylation

Marco Colasanti, Tiziana Persichini, Elisabetta Cavalieri^§, Cinzia Fabrizi, Sofia Mariotto^§, Marta Menegazzi^§, Giuliana M. Lauro, and Hisanori Suzuki^§

From the  Department of Biology, University of Rome, ROMA TRE, Viale Marconi 446, I-00146 Rome, Italy and the ^§ Department of Neuroscience and Vision, Laboratory of Biological Chemistry, University of Verona, Strada Le Grazie 8, I-37134 Verona, Italy

Human astrocytoma T67 cells constitutively express a neuronal NO synthase (NOS-I) and, following administration of lipopolysaccharide (LPS) plus interferon- (IFN), an inducible NOS isoform (NOS-II). Previous results indicated that a treatment of T67 cells with the combination of LPS plus IFN, by affecting NOS-I activity, also inhibited NO production in a very short time. Here, we report that under basal conditions, a NOS-I protein of about 150 kDa was weakly and partially tyrosine-phosphorylated, as verified by immunoprecipitation and Western blotting. Furthermore, LPS plus IFN increased the tyrosine phosphorylation of NOS-I, with a concomitant inhibition of its enzyme activity. The same effect was observed in the presence of vanadate, an inhibitor of phosphotyrosine-specific phosphatases. On the contrary, genistein, an inhibitor of protein-tyrosine kinases, reduced tyrosine phosphorylation of NOS-I, enhancing its enzyme activity. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a suboptimal induction of NOS-II mRNA expression in T67 cells was enhanced by vanadate (or L-NAME) and inhibited by genistein. Because exogenous NO has been found to suppress NOS-II expression, the decrease of NO production that we have obtained from the inactivation of NOS-I by LPS/IFN-induced tyrosine phosphorylation provides the best conditions for NOS-II expression in human astrocytoma T67 cells.