Phosphorylation of syntaphilin by cAMP-dependent protein kinase modulates its interaction with syntaxin-1 and annuls its inhibitory effect on vesicle exocytosis.
cAMP-dependent protein kinase (PKA) can modulate synaptic transmission by acting directly on the neurotransmitter secretory machinery. Here, we identify one possible target: syntaphilin, which was identified as a molecular clamp that controls free syntaxin-1 and dynamin-1 availability and thereby regulates synaptic vesicle exocytosis and endocytosis. Deletion mutation and site-directed mutagenesis experiments pinpoint dominant PKA phosphorylation sites to serines 43 and 56. PKA phosphorylation of syntaphilin significantly decreases its binding to syntaxin-1A in vitro. A syntaphilin mutation of serine 43 to aspartic acid (S43D) shows similar effects on binding. To characterize in vivo phosphorylation events, we generated antisera against a peptide of syntaphilin containing a phosphorylated serine 43. Treatment of rat brain synaptosomes or syntaphilin-transfected HEK 293 cells with the cAMP analogue BIMPS induces in vivo phosphorylation of syntaphilin and inhibits its interaction with syntaxin-1 in neurons. To determine whether PKA phosphorylation of syntaphilin is involved in the regulation of Ca(2+)-dependent exocytosis, we investigated the effect of overexpression of syntaphilin and its S43D mutant on the regulated secretion of human growth hormone from PC12 cells. Although expression of wild type syntaphilin in PC12 cells exhibits significant reduction in high K(+)-induced human growth hormone release, the S43D mutant fails to inhibit exocytosis. Our data predict that syntaphilin could be a highly regulated molecule and that PKA phosphorylation could act as an "off" switch for syntaphilin, thus blocking its inhibitory function via the cAMP-dependent signal transduction pathway.
Synaptic Function Unit, National Institute of Neurological Dideases and Stroke, National Institutes of Health, Bethesda, Maryland 20892-4154, USA.
Judit
J
Boczan
A G Miriam
AG
Leenders
Zu-Hang
ZH
Sheng
D019943:Amino Acid Substitution; D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001921:Brain; D002352:Carrier Proteins / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism / Q000502:physiology*; D005089:Exocytosis; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D051381:Rats; D015398:Signal Transduction; D013574:Synaptosomes / Q000737:chemistry / Q000378:metabolism; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D002352:Carrier Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C405513:SNPH protein, human; C494212:STX1A protein, human; C494213:Stx1a protein, rat; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D017868:Cyclic AMP-Dependent Protein Kinases