Phosphorylation of Snapin by PKA modulates its interaction with the SNARE complex

cAMP-dependent protein kinase A (PKA) can modulate synaptic transmission by acting directly on unknown targets in the neurotransmitter secretory machi nery. Here we identify Snapin, a protein of relative molecular mass 15,000 that is implicated in neurotransmission by binding to SNAP-25, as a possibl e target. Deletion mutation and site-directed mutagenetic experiments pinpo int the phosphorylation site to serine 50. PKA-phosphorylation of Snapin si gnificantly increases its binding to synaptosomal-associated protein-25 (SN AP-25). Mutation of Snapin serine 50 to aspartic acid (S50D) mimics this ef fect of PKA phosphorylation and enhances the association of synaptotagmin w ith the soluble N-ethylmaleimide-sensitive factor attachment protein recept or (SNARE) complex. Furthermore, treatment of rat hippocampal slices with n onhydrolysable cAMP analogue induces in vivo phosphorylation of Snapin and enhances the interaction of both Snapin and synaptotagmin with the SNARE co mplex. In adrenal chromaffin cells, overexpression of the Snapin S50D mutan t leads to an increase in the number of release-competent vesicles. Our res ults indicate that Snapin may be a PKA target for modulating transmitter re lease through the cAMP-dependent signal-transduction pathway.