Phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase C - Its implication in regulating the interaction of Munc-18/n-Sec1/rbSec1 with syntaxin
Phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase C - Its implication in regulating the interaction of Munc-18/n-Sec1/rbSec1 with syntaxin
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DOI：
10.1074/jbc.271.13.7265
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发表时间：
1996-03-29
影响因子：
4.8
通讯作者：
Takai, Y
中科院分区：
文献类型：
--
作者：
Fujita, Y;Sasaki, T;Takai, Y
Munc-18/n-Sec 1/rbSec 1与突触融合蛋白相互作用，这种相互作用抑制囊泡相关膜蛋白（VAMP）/小突触泡蛋白和25 kDa突触小体相关蛋白（SNAP-25）与突触融合蛋白的结合。突触融合蛋白、VAMP和SNAP-25作为可溶性N-乙基马来酰亚胺敏感性融合蛋白附着蛋白（SNAP）受体，对于突触囊泡与突触前质膜的对接和/或融合是必需的。酵母、秀丽隐杆线虫和果蝇中的遗传分析表明，Munc-18对囊泡运输至关重要。另一方面，蛋白激酶C（PKC）刺激各种类型的分泌细胞中的Ca 2+依赖性胞吐作用。然而，Munc-18和PKC在囊泡运输中的作用方式尚未阐明。在这里，我们表明，重组Munc-18是磷酸化的常规PKC在钙和磷脂依赖性的方式在无细胞系统。约Imol磷酸盐最大掺入Imol Munc-18中。主要的磷酸化位点是Ser（306）和Ser（313）。与突触融合蛋白复合的Munc-18不被磷酸化。PKC催化的Munc-18磷酸化抑制其与syntaxin的相互作用。这些结果表明，PKC催化的Munc-18磷酸化在调节Munc-18与突触融合蛋白的相互作用，从而突触囊泡与突触前质膜的对接和/或融合中起着重要作用。
Munc-18/n-Sec1/rbSec1 interacts with syntaxin and this interaction inhibits the association of vesicle-associated membrane protein (VAMP)/synaptobrevin and synaptosomal-associated protein of 25 kDa (SNAP-25) with syntaxin. Syntaxin, VAMP, and SNAP-25 serve as soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptors essential for docking and/or fusion of synaptic vesicles with the presynaptic plasma membrane. Genetic analyses in yeast, Caenorhabditis elegans, and Drosophila suggest that Munc-18 is essential for vesicle transport. On the other hand, protein kinase C (PKC) stimulates Ca2+-dependent exocytosis in various types of secretory cells. However, the modes of action of Munc-18 and PKC in vesicle transport have not been clarified. Here, we show that recombinant Munc-18 is phosphorylated by conventional PKC in a Ca2+- and phospholipid-dependent manner in a cell-free system. About 1 mol of phosphate is maximally incorporated into 1 mol of Munc-18. The major phosphorylation sites are Ser(306) and Ser(313). The Munc-18 complexed with syntaxin is not phosphorylated. The PKC-catalyzed phosphorylation of Munc-18 inhibits its interaction with syntaxin. These results suggest that the PKC-catalyzed phosphorylation of Munc-18 plays an important role in regulating the interaction of Munc-18 with syntaxin and thereby the docking and/or the fusion of synaptic vesicles with the presynaptic plasma membrane.