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 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.
Synaptic Function Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 36 Convent Drive, Bethesda, Maryland 20892-4154, USA.
M G
MG
Chheda
U
U
Ashery
P
P
Thakur
J
J
Rettig
Z H
ZH
Sheng
D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002874:Chromosome Mapping; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; C494210:Snap25 protein, rat; C498452:Snapin protein, rat; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D017868:Cyclic AMP-Dependent Protein Kinases