ANALYSIS OF PHYSIOLOGICAL FUNCTION OF LIPID SIGNALLING SYSTEM

磷脂酰肌醇4-磷酸5激酶（PIP5K）磷酸化磷脂酰肌醇4-磷酸，生成磷脂酰肌醇4,5-二磷酸（PIP2），它是一种脂质信号分子。已经鉴定出哺乳动物PIP5K、α β和γ的3个同工酶，以及PIP5K γ型的3个剪接变体γ635、γ661和γ687。我们一直在研究PIP5K各同工酶的激活机制和生理功能，以了解PIP5K的分子多样性。在2年的项目中，我们获得了如下结果：1。PIP5K同工酶和剪接变异体PIP5K γ661PIP5K γ661的特异性伴侣蛋白在小鼠海马神经元中被发现与接头复合体AP-2特异性相互作用并被其激活。此外，我们还发现，这些分子的相互作用对于小鼠海马神经元去极化诱导的网格蛋白依赖性突触囊泡内吞是绝对必需的。PIP5K同工酶的PIP5Kβ特异性伴侣蛋白与KIF2A相互作用。这种相互作用刺激了PIP5Kβ激酶活性和KIF2A的微管解聚活性。最后，发现这两种分子的相互作用对小鼠海马神经元轴突生长具有负向调节作用。PIP5K激活剂ARF6的结合位点小G蛋白ARF6激活PIP5K。我们发现ARF6与PIP5K的激酶核心结构域结合，在PIP5K同工酶中高度保守。PIP5K α敲除小鼠的制备和分析我们制备了PIP5K α敲除小鼠，尚未有报道。这些小鼠显然是健康的，没有观察到任何缺陷。

The lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) phosphorylates phosphatidylinositol 4-phosphate to produce phosphatidylinositol 4,5-bisphosphate (PIP2), which functions as a lipid signaling molecule. Three isozymes for mammalian PIP5K, α β and γ, and three splicing variants of γ type of PIP5K, γ635, γ661 and γ687, have been identified. We have been investigating activation mechanisms and physiological functions of each PIP5K isozyme to understand the molecular multiplicity of PIP5K. During 2 years of this project, we obtained the results described below.1. Specific partner protein of PIP5K γ661PIP5K γ661 of PIP5K isozymes and splicing variants, was found to specifically interact with and is activated by the adaptor complex AP-2 in mouse hippocampal neurons. Furthermore, it was found that the interaction of these molecules is absolutely required for the clathrin-dependent synaptic vesicle endocytosis induced by depolarization, in mouse hippocampal neurons.2. Specific partner protein of PIP5KβPIP5Kβ of PIP5K isozymes specifically interact with KIF2A. The interaction stimulated PIP5Kβ kinase activity and microtubule depolymerizing activity of KIF2A. Finally, the interaction of these two molecules was found to negatively regulate axonal outgrowth of mouse hippocampal neurons.3. PIP5K binding site for its activator ARF6The small G protein ARF6 activates PIP5K. We identified that ARF6 binds to the kinase core domain of PIP5K, which is highly conserved among PIP5K isozymes.4. Preparation and analysis of PIP5K α knockout miceWe prepared PIP5K α knockout mice, which have not yet been reported. These mice were apparently healthy and any defects were not observe.