Phosphorylation of SNAP-25 at Ser187 mediates enhancement of exocytosis by a phorbol ester in INS-1 cells.
Activation of diacylglycerol (DAG) signaling pathways with phorbol esters dramatically enhances Ca2+-triggered exocytosis from both endocrine cells and neurons, however the relevant targets of DAG are controversial. A possible effector mechanism for this signaling pathway is phosphorylation of SNAP-25 (25 kDa synaptosome-associated protein) at Ser187 by PKC. Here, we investigated the role of Ser187 in the enhancement of exocytosis by the phorbol ester PMA (phorbol 12-myristate 13-acetate). We used patch-clamp measurements of membrane capacitance together with photorelease of caged-Ca2+ and membrane depolarization to study exocytosis. Expression of the nonphosphorylatable S187C SNAP-25 mutant did not attenuate the enhancement of exocytosis by PMA in either bovine chromaffin cells or the INS-1 insulin-secreting cell line. To test the effects of Ser187 mutations under conditions in which the endogenous SNAP-25 is disabled, we expressed botulinum toxin serotype E to cleave SNAP-25 in INS-1 cells. Coexpression of a toxin-resistant mutant (TR), but not wild-type SNAP-25, was able to rescue PMA-modulated exocytosis. Coexpression of the toxin with the TR-S187C SNAP-25 mutant was able to completely block the enhancement of exocytosis by PMA in response to photoelevation of [Ca2+]i to low microM levels or to a depolarizing train. The phospho-mimetic S187E mutation enhanced the small, fast burst of exocytosis evoked by photelevation of Ca2+, but, like PMA, had smaller effects on exocytosis evoked by a depolarizing train. This work supports the hypothesis that phosphorylation of Ser187 of SNAP-25 by PKC is a key step in the enhancement of exocytosis by DAG.
Shu
Yilong
Y
Interdisciplinary Neuroscience Program, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
Liu
Xin
X
Yang
Yan
Y
Takahashi
Masami
M
Gillis
Kevin D
KD
D000818:Animals; D019274:Botulinum Toxins, Type A / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells; D004307:Dose-Response Relationship, Radiation; D005089:Exocytosis / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007340:Insulinoma / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D009154:Mutation / Q000502:physiology; D018408:Patch-Clamp Techniques; D010703:Phorbol Esters / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D012694:Serine / Q000235:genetics / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D014162:Transfection / Q000379:methods
D010703:Phorbol Esters; D050825:Synaptosomal-Associated Protein 25; D049452:Green Fluorescent Proteins; D012694:Serine; C020922:4-O-methyl-12-O-tetradecanoylphorbol 13-acetate; D019274:Botulinum Toxins, Type A; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium