Tian Jin-Hua, Das Sunit, Sheng Zu-Hang
Synaptic Function Unit, NINDS, National Institutes of Health, Bethesda, Maryland 20892-4154, USA.
J Biol Chem. 2003 Jul 11;278(28):26265-74. doi: 10.1074/jbc.M300492200. Epub 2003 May 2.
Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that binds with VAMP/synaptobrevin and SNAP-25 to form the SNARE complex. Modulation of syntaxin binding properties by protein kinases could be critical to control of neurotransmitter release. Using yeast two-hybrid selection with syntaxin-1A as bait, we have isolated a cDNA encoding the C-terminal domain of death-associated protein (DAP) kinase, a calcium/calmodulin-dependent serine/threonine protein kinase. Expression of DAP kinase in adult rat brain is restricted to particular neuronal subpopulations, including the hippocampus and cerebral cortex. Biochemical studies demonstrate that DAP kinase binds to and phosphorylates syntaxin-1 at serine 188. This phosphorylation event occurs both in vitro and in vivo in a Ca2+-dependent manner. Syntaxin-1A phosphorylation by DAP kinase or its S188D mutant, which mimics a state of complete phosphorylation, significantly decreases syntaxin binding to Munc18-1, a syntaxin-binding protein that regulates SNARE complex formation and is required for synaptic vesicle docking. Our results suggest that syntaxin is a DAP kinase substrate and provide a novel signal transduction pathway by which syntaxin function could be regulated in response to intracellular [Ca2+] and synaptic activity.
Syntaxin-1是突触小泡对接/融合机制的关键组成部分，它与VAMP/突触囊泡蛋白和SNAP-25结合形成SNARE复合体。蛋白激酶对Syntaxin结合特性的调节可能对神经递质释放的控制至关重要。以Syntaxin-1A为诱饵进行酵母双杂交筛选，我们分离出了一个编码死亡相关蛋白（DAP）激酶C末端结构域的cDNA，DAP激酶是一种钙/钙调蛋白依赖性丝氨酸/苏氨酸蛋白激酶。DAP激酶在成年大鼠脑中的表达仅限于特定的神经元亚群，包括海马体和大脑皮层。生化研究表明，DAP激酶与Syntaxin-1结合并使其丝氨酸188位点磷酸化。这种磷酸化事件在体外和体内均以钙依赖性方式发生。DAP激酶或其模拟完全磷酸化状态的S188D突变体对Syntaxin-1A的磷酸化显著降低了Syntaxin与Munc18-1的结合，Munc18-1是一种Syntaxin结合蛋白，调节SNARE复合体的形成，是突触小泡对接所必需的。我们的结果表明Syntaxin是DAP激酶的底物，并提供了一种新的信号转导途径，通过该途径可以根据细胞内[Ca2+]和突触活动来调节Syntaxin的功能。
