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首页> 外文期刊>The Journal of Neuroscience: The Official Journal of the Society for Neuroscience >Phosphorylation of SNAP-25 at Ser187 mediates enhancement of exocytosis by a phorbol ester in INS-1 cells.
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Phosphorylation of SNAP-25 at Ser187 mediates enhancement of exocytosis by a phorbol ester in INS-1 cells.

机译：SNAP-25在Ser187上的磷酸化介导佛波酯在INS-1细胞中增强胞吐作用。

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摘要

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.
机译：用佛波酯酯激活二酰基甘油（DAG）信号传导途径可显着增强Ca2 +触发的内分泌细胞和神经元胞吐作用，但DAG的相关靶标尚存争议。该信号传导途径的可能效应子机制是PKC使Ser187上的SNAP-25（25 kDa突触体相关蛋白）磷酸化。在这里，我们研究了Ser187在佛波酯PMA（佛波12-肉豆蔻酸酯13-乙酸酯）增强胞吐作用中的作用。我们使用膜电容的膜片钳测量以及笼状Ca2 +的光释放和膜去极化来研究胞吐作用。不可表达的S187C SNAP-25突变体的表达不会减弱PMA在牛嗜铬细胞或INS-1胰岛素分泌细胞系中对胞吐作用的增强作用。为了测试内源性SNAP-25被禁用的条件下Ser187突变的影响，我们表达了肉毒杆菌毒素血清型E以在INS-1细胞中裂解SNAP-25。共表达抗毒素突变体（TR），但不是野生型SNAP-25，能够挽救PMA调节的胞吐作用。毒素与TR-S187C SNAP-25突变体的共表达能够完全阻止PMA响应[Ca2 +] i的光升高至低microM水平或去极化序列而增强胞吐作用。磷酸化S187E突变增强了Ca2 +分解引起的小而快速的胞吐爆发，但与PMA一样，对去极化链引起的胞吐作用较小。这项工作支持以下假设，即PKC对SNAP-25的Ser187的磷酸化是DAG增强胞吐作用的关键步骤。

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