Phosphomimetic mutation of Ser-187 of SNAP-25 increases both syntaxin binding and highly Ca2+-sensitive exocytosis.
Phosphomimetic mutation of Ser-187 of SNAP-25 increases both syntaxin binding and highly Ca2+-sensitive exocytosis.
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DOI：
10.1085/jgp.200609685
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发表时间：
2007-03
期刊：
影响因子：
--
通讯作者：
Gillis KD
中科院分区：
文献类型：
--
作者：
Yang Y;Craig TJ;Chen X;Ciufo LF;Takahashi M;Morgan A;Gillis KD
介导PKC增强胞吐作用的磷酸化靶点尚不清楚。PKC磷酸化SNARE蛋白SNAP-25的Ser-187。我们使用塞姆利基森林病毒系统在牛肾上腺嗜铬细胞中表达SNAP-25的突变体，然后使用笼状Ca 2+的光释放与膜片钳电容测量一起直接测量胞吐的Ca 2+依赖性。用紫外线闪光将[Ca 2 +]i升高至几μM并释放高度Ca 2+敏感的囊泡池（HCSP），然后用一系列去极化脉冲引发Ca 2+敏感性较低的囊泡易释放池（RRP）的胞吐作用。碳纤维安培法证实，从单个颗粒释放的儿茶酚胺的量和动力学是相似的两个阶段的胞吐。用S187 E SNAP-25突变体的表达模拟PKC磷酸化导致HCSP增加约3倍，而对去极化的反应仅增加1.5倍。磷酸化模拟S187 D突变导致HCSP增加1.5倍，但对去极化的反应小30%。进行了重组SNARE蛋白的体外结合试验，以检查可能促进高度Ca 2+敏感状态的蛋白质-蛋白质结合的变化。S187 E突变体表现出与突触融合蛋白结合增加，但与突触结合蛋白I的钙非依赖性结合减少。模拟具有T138 E突变的SNAP-25的推定PKA磷酸化位点的磷酸化降低了体外与突触融合蛋白和突触结合蛋白I的结合。在嗜铬细胞中表达T138 E/S187 E双突变体表明，HCSP的大小增加与SNAP-25体外与突触融合蛋白结合的增加相关，但与SNAP-25与突触结合蛋白I的钙非依赖性结合无关。我们的研究结果支持这一假设，胞吐触发较低的Ca 2+浓度（从HCSP）发生不同的分子机制比胞吐触发较高的Ca 2+水平。
The phosphorylation targets that mediate the enhancement of exocytosis by PKC are unknown. PKC phosporylates the SNARE protein SNAP-25 at Ser-187. We expressed mutants of SNAP-25 using the Semliki Forest Virus system in bovine adrenal chromaffin cells and then directly measured the Ca2+ dependence of exocytosis using photorelease of caged Ca2+ together with patch-clamp capacitance measurements. A flash of UV light used to elevate [Ca2+]i to several μM and release the highly Ca2+-sensitive pool (HCSP) of vesicles was followed by a train of depolarizing pulses to elicit exocytosis from the less Ca2+-sensitive readily releasable pool (RRP) of vesicles. Carbon fiber amperometry confirmed that the amount and kinetics of catecholamine release from individual granules were similar for the two phases of exocytosis. Mimicking PKC phosphorylation with expression of the S187E SNAP-25 mutant resulted in an approximately threefold increase in the HCSP, whereas the response to depolarization increased only 1.5-fold. The phosphomimetic S187D mutation resulted in an ∼1.5-fold increase in the HCSP but a 30% smaller response to depolarization. In vitro binding assays with recombinant SNARE proteins were performed to examine shifts in protein–protein binding that may promote the highly Ca2+-sensitive state. The S187E mutant exhibited increased binding to syntaxin but decreased Ca2+-independent binding to synaptotagmin I. Mimicking phosphorylation of the putative PKA phosphorylation site of SNAP-25 with the T138E mutation decreased binding to both syntaxin and synaptotagmin I in vitro. Expressing the T138E/ S187E double mutant in chromaffin cells demonstrated that enhancing the size of the HCSP correlates with an increase in SNAP-25 binding to syntaxin in vitro, but not with Ca2+-independent binding of SNAP-25 to synaptotagmin I. Our results support the hypothesis that exocytosis triggered by lower Ca2+ concentrations (from the HCSP) occurs by different molecular mechanisms than exocytosis triggered by higher Ca2+ levels.
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影响因子：
16.2
作者：
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通讯作者：
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影响因子：
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