Peroxynitrite induces tyrosine nitration and modulates tyrosine phosphorylation of synaptic proteins

Peroxynitrite, the product of the radical-radical reaction between nitric o xide and superoxide anion, is a potent oxidant involved in tissue damage in neurodegenerative disorders. We investigated the modifications induced by peroxynitrite in tyrosine residues of proteins from synaptosomes. Peroxynit rite treatment (greater than or equal to 50 mu M) induced tyrosine nitratio n and increased tyrosine phosphorylation, Synaptophysin was identified as o ne of the major nitrated proteins and pp60(src) kinase as one of the major phosphorylated substrates, Further fractionation of synaptosomes revealed n itrated synaptophysin in the synaptic vesicles, whereas phosphorylated pp60 (src) was enriched in the postsynaptic density fraction. Tyrosine phosphory lation was increased by treatment with 50-500 mu M peroxynitrite and decrea sed by higher concentrations, suggesting a possible activation/inactivation of kinases. Immunocomplex kinase assay proved that peroxynitrite treatment of synaptosomes modulated the pp60(src) autophosphorylation activity. The addition of bicarbonate (CO2 1.3 mM) produced a moderate enhancing effect o n some nitrated proteins but significantly protected the activity of pp60(s rc) against peroxynitrite-mediated inhibition so that at 1 mM peroxynitrite , the kinase was still more active than in untreated synaptosomes, The phos photyrosine phosphatase activity of synaptosomes was inhibited by peroxynit rite (greater than or equal to 50 mu M) but significantly protected by CO2. Thus, the increase of phosphorylation cannot be attributed to peroxynitrit e-mediated inhibition of phosphatases. We suggest that peroxynitrite may re gulate the posttranslational modification of tyrosine residues in pre- and postsynaptic proteins. Identification of the major protein targets gives in sight into the pathways possibly involved in neuronal degeneration associat ed with peroxynitrite overproduction.