Shu Yilong, Liu Xin, Yang Yan, Takahashi Masami, Gillis Kevin D
Interdisciplinary Neuroscience Program, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, Missouri 65211, USA.
J Neurosci. 2008 Jan 2;28(1):21-30. doi: 10.1523/JNEUROSCI.2352-07.2008.
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.
佛波酯激活二酰基甘油（DAG）信号通路可显著增强内分泌细胞和神经元中Ca2+触发的胞吐作用，然而DAG的相关靶点仍存在争议。该信号通路的一种可能效应机制是蛋白激酶C（PKC）使SNAP-25（25 kDa突触体相关蛋白）的Ser187位点发生磷酸化。在此，我们研究了Ser187在佛波酯PMA（佛波醇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技术，结合笼锁Ca2+的光释放和膜去极化来研究胞吐作用。在牛嗜铬细胞或INS-1胰岛素分泌细胞系中，非磷酸化的S187C SNAP-25突变体的表达并未减弱PMA对胞吐作用的增强。为了在内源性SNAP-25失活的条件下测试Ser187突变的影响，我们在INS-1细胞中表达了肉毒杆菌毒素E型以切割SNAP-25。共表达毒素抗性突变体（TR）而非野生型SNAP-25能够挽救PMA调节的胞吐作用。毒素与TR-S187C SNAP-255突变体共表达能够完全阻断PMA在[Ca2+]i光升高至低 microM水平或去极化串刺激时对胞吐作用的增强。磷酸化模拟物S187E突变增强了Ca2+光升高诱发的小而快速的胞吐爆发，但与PMA一样，对去极化串刺激诱发的胞吐作用影响较小。这项工作支持了PKC使SNAP-25的Serl87位点磷酸化是DAG增强胞吐作用的关键步骤这一假说。
