ANALYSIS OF PHYSIOLOGICAL FUNCTION OF LIPID SIGNALLING SYSTEM

脂质激酶磷脂酰肌醇4-磷酸5-激酶（PIP 5 K）使磷脂酰肌醇4-磷酸磷酸化以产生磷脂酰肌醇4，5-二磷酸（PIP 2），其充当脂质信号传导分子。哺乳动物PIP 5 K的三种同工酶α β和γ，以及PIP 5 K γ型的三种剪接变体γ635、γ661和γ687已被鉴定。我们一直在研究每个PIP 5 K同工酶的激活机制和生理功能，以了解PIP 5 K的分子多样性。在这个项目的2年中，我们获得了如下所述的结果。PIP 5 K同工酶和剪接变体的PIP 5 K γ 661的特异性伴侣蛋白被发现与小鼠海马神经元中的衔接子复合物AP-2特异性相互作用并被其激活。此外，在小鼠海马神经元中，发现这些分子的相互作用是去极化诱导的网格蛋白依赖性突触囊泡内吞所必需的. PIP 5 K β的特异性伴侣蛋白PIP 5 K β同工酶与KIF 2A特异性相互作用。这种相互作用刺激了PIP 5 K β激酶活性和KIF 2A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的轴突生长具有负调控作用. PIP 5 K激活剂ARF 6的结合位点小G蛋白ARF 6激活PIP 5 K。我们鉴定了ARF 6与PIP 5 K的激酶核心结构域结合，该结构域在PIP 5 K同工酶中高度保守。PIP 5 K α基因敲除小鼠的制备与分析我们制备了PIP 5 K α基因敲除小鼠，这是迄今为止尚未报道的。这些小鼠明显健康，未观察到任何缺陷。

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.