Peroxynitrite Induces Tyrosine Nitration and Modulates Tyrosine Phosphorylation of Synaptic Proteins

Abstract : Peroxynitrite, the product of the radical‐radical reaction between nitric oxide 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. Peroxynitrite treatment (≥50 μ M ) induced tyrosine nitration and increased tyrosine phosphorylation. Synaptophysin was identified as one of the major nitrated proteins and pp60 src kinase as one of the major phosphorylated substrates. Further fractionation of synaptosomes revealed nitrated synaptophysin in the synaptic vesicles, whereas phosphorylated pp60 src was enriched in the postsynaptic density fraction. Tyrosine phosphorylation was increased by treatment with 50‐500 μ M peroxynitrite and decreased 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 (CO 2 1.3 m M ) produced a moderate enhancing effect on some nitrate proteins but significantly protected the activity of pp60 src against peroxynitrite‐mediated inhibition so that at 1 m M peroxynitrite, the kinase was still more active than in untreated synaptosomes. The phosphotyrosine phosphatase activity of synaptosomes was inhibited by peroxynitrite (≥50 μ M ) but significantly protected by CO 2 . Thus, the increase of phosphorylation cannot be attributed to peroxynitrite‐mediated inhibition of phosphatases. We suggest that peroxynitrite may regulate the posttranslational modification of tyrosine residues in pre‐ and postsynaptic proteins. Identification of the major protein targets gives insight into the pathways possibly involved in neuronal degeneration associated with peroxynitrite overproduction.