Phosphorylation of syntaphilin by cAMP-dependent protein kinase modulates its interaction with syntaxin-1 and annuls its inhibitory effect on vesicle exocytosis
Phosphorylation of syntaphilin by cAMP-dependent protein kinase modulates its interaction with syntaxin-1 and annuls its inhibitory effect on vesicle exocytosis
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DOI：
10.1074/jbc.m400496200
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发表时间：
2004-04-30
影响因子：
4.8
通讯作者：
Sheng, ZH
中科院分区：
文献类型：
--
作者：
Boczan, J;Leenders, AGM;Sheng, ZH
环腺苷酸（cAMP）依赖性蛋白激酶（PKA）可通过直接作用于神经递质分泌机制来调节突触传递。在此，我们确定了一个可能的靶点：突触结合蛋白（syntaphilin），它被认定为一种分子夹，可控制游离的突触融合蛋白 -1（syntaxin -1）和发动蛋白 -1（dynamin -1）的可利用性，从而调节突触小泡的胞吐和胞吞作用。缺失突变和定点诱变实验确定了主要的PKA磷酸化位点为丝氨酸43和56。在体外，突触结合蛋白的PKA磷酸化显著降低了它与突触融合蛋白 -1A的结合。将丝氨酸43突变为天冬氨酸（S43D）的突触结合蛋白突变体在结合方面显示出类似的效果。为了表征体内磷酸化事件，我们制备了针对含有磷酸化丝氨酸43的突触结合蛋白肽段的抗血清。用cAMP类似物BIMPS处理大鼠脑突触体或转染了突触结合蛋白的人胚肾293（HEK 293）细胞，可诱导突触结合蛋白在体内磷酸化，并抑制其在神经元中与突触融合蛋白 -1的相互作用。为了确定突触结合蛋白的PKA磷酸化是否参与Ca²⁺依赖性胞吐的调节，我们研究了突触结合蛋白及其S43D突变体的过表达对PC12细胞中人生长激素调节性分泌的影响。尽管在PC12细胞中野生型突触结合蛋白的表达在高钾诱导的人生长激素释放方面表现出显著降低，但S43D突变体未能抑制胞吐作用。我们的数据预测，突触结合蛋白可能是一种受到高度调节的分子，并且PKA磷酸化可作为突触结合蛋白的“关闭”开关，从而通过环腺苷酸依赖性信号转导途径阻断其抑制功能。
cAMP-dependent protein kinase (PKA) can modulate synaptic transmission by acting directly on the neurotransmitter secretory machinery. Here, we identify one possible target: syntaphilin, which was identified as a molecular clamp that controls free syntaxin-1 and dynamin-1 availability and thereby regulates synaptic vesicle exocytosis and endocytosis. Deletion mutation and site-directed mutagenesis experiments pinpoint dominant PKA phosphorylation sites to serines 43 and 56. PKA phosphorylation of syntaphilin significantly decreases its binding to syntaxin-1A in vitro. A syntaphilin mutation of serine 43 to aspartic acid (S43D) shows similar effects on binding. To characterize in vivo phosphorylation events, we generated antisera against a peptide of syntaphilin containing a phosphorylated serine 43. Treatment of rat brain synaptosomes or syntaphilin-transfected HEK 293 cells with the cAMP analogue BIMPS induces in vivo phosphorylation of syntaphilin and inhibits its interaction with syntaxin-1 in neurons. To determine whether PKA phosphorylation of syntaphilin is involved in the regulation of Ca2+-dependent exocytosis, we investigated the effect of overexpression of syntaphilin and its S43D mutant on the regulated secretion of human growth hormone from PC12 cells. Although expression of wild type syntaphilin in PC12 cells exhibits significant reduction in high K+-induced human growth hormone release, the S43D mutant fails to inhibit exocytosis. Our data predict that syntaphilin could be a highly regulated molecule and that PKA phosphorylation could act as an "off" switch for syntaphilin, thus blocking its inhibitory function via the cAMP-dependent signal transduction pathway.