Abstract
cAMP-dependent protein kinase A (PKA) can modulate synaptic transmission by acting directly on unknown targets in the neurotransmitter secretory machinery. Here we identify Snapin, a protein of relative molecular mass 15,000 that is implicated in neurotransmission by binding to SNAP-25, as a possible target. Deletion mutation and site-directed mutagenetic experiments pinpoint the phosphorylation site to serine 50. PKA-phosphorylation of Snapin significantly increases its binding to synaptosomal-associated protein-25 (SNAP-25). Mutation of Snapin serine 50 to aspartic acid (S50D) mimics this effect of PKA phosphorylation and enhances the association of synaptotagmin with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Furthermore, treatment of rat hippocampal slices with nonhydrolysable cAMP analogue induces in vivo phosphorylation of Snapin and enhances the interaction of both Snapin and synaptotagmin with the SNARE complex. In adrenal chromaffin cells, overexpression of the Snapin S50D mutant leads to an increase in the number of release-competent vesicles. Our results indicate that Snapin may be a PKA target for modulating transmitter release through the cAMP-dependent signal-transduction pathway.
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Acknowledgements
We thank Q. Su for constructs of pSFV1-Snapin mutants, C. M. Gerwin, A. Bührmann and F. Friedlein for technical assistance, X. Zheng for synaptsome preparations, J. S. Diamond for hippocampal slices, M. Takahashi for antibodies, and J. W. Nagle for DNA sequencing. We also thank H. Gainer, C. T. Yokoyama, S. Das and J. Coulombe for comments on the manuscript, and E. Neher for continuous support. This work was supported by the intramural research program of NINDS, NIH (to Z-H. S.), the Deutsche Forschungsgemeinschaft (to J.R.), an EU Network Grant (to U.A.), and the HHMI-NIH Research Scholars Program (to M.G.C.).
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Chheda, M., Ashery, U., Thakur, P. et al. Phosphorylation of Snapin by PKA modulates its interaction with the SNARE complex. Nat Cell Biol 3, 331–338 (2001). https://doi.org/10.1038/35070000
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DOI: https://doi.org/10.1038/35070000
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