␣-Synuclein (␣-Syn) is implicated in the pathogenesis of Parkinson's Disease, genetically through missense mutations linked to early onset disease and pathologically through its presence in Lewy bodies. ␣-Syn is phosphorylated on serine residues; however, tyrosine phosphorylation of ␣-Syn has not been established (1, 2). A comparison of the protein sequence between Synuclein family members revealed that all four tyrosine residues of ␣-Syn are conserved in all orthologs and ␤-Syn paralogs described to date, suggesting that these residues may be of functional importance (3). For this reason, experiments were performed to determine whether ␣-Syn could be phosphorylated on tyrosine residue(s) in human cells. Indeed, ␣-Syn is phosphorylated within 2 min of pervanadate treatment in ␣-Syn-transfected cells. Tyrosine phosphorylation occurs primarily on tyrosine 125 and was inhibited by PP2, a selective inhibitor of Src protein-tyrosine kinase (PTK) family members at concentrations consistent with inhibition of Src function (4). Finally, we demonstrate that ␣-Syn can be phosphorylated directly both in cotransfection experiments using c-Src and Fyn expression vectors and in in vitro kinase assays with purified kinases. These data suggest that ␣-Syn can be a target for phosphorylation by the Src family of PTKs.

The 38-amino-acid isoform of pituitary adenylate cyclase-activating polypeptide (PACAP38) elicits a robust outgrowth of neurites in cultured PC12 cells. Initiation of neurite outgrowth occurs within 4-8 hr after the addition of PACAP38. Treatment with PACAP38 does not elicit collateral activation of p140(trk) nerve growth factor receptor tyrosine kinase activity, nor is it associated with tyrosine phosphorylation of suc1-associated neurotrophic factor target, a selective target of neurotrophin tyrosine kinase receptors. Coadministration of epidermal growth factor with PACAP38 elicits an enhanced response. Induction of neurites is also observed on the addition of PACAP38 to dominant negative Src and Ras PC12 cell variants. PACAP38 stimulates extracellular signal-regulated kinase (Erk) activity >10-fold within 5 min, and the effect is augmented by cotreatment with epidermal growth factor. Pretreatment with the cAMP-dependent protein kinase-selective inhibitor, H-89, is ineffective as an antagonist of PACAP38-induced neurite outgrowth, whereas down-regulation of protein kinase C (PKC) by phorbol ester or incubation with PKC-selective inhibitors GF109203X and calphostin C effectively blocks PACAP38-stimulated neurite formation. Stimulation of Erk activity is inhibited by incubation with PD90859, a pharmacological antagonist of the threonine/tyrosine dual-specificity Erk. Inhibition of ligand-stimulated Erk activation prevents PACAP38-induced neurite outgrowth. Collectively, these findings indicate that PACAP38-stimulated neuritogenesis requires PKC and Erk activation but is independent of cAMP-dependent protein kinase, nerve growth factor receptor tyrosine kinase, p21(ras) G protein, and pp60(c-src) cytoplasmic tyrosine kinase.