Kanaho Yasunori, Nakayama Kazuhisa, Frohman Michael A, Yokozeki Takeaki
Graduate School of Comprehensive Human Sciences, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
Methods Enzymol. 2007;434:155-69. doi: 10.1016/S0076-6879(07)34009-3.
The remarkably versatile phospholipid, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], plays crucial roles in signal transduction, actin cytoskeleton reorganization, clathrin-dependent endocytosis, and regulation of membrane morphology. In mammalian cells, PI(4,5)P(2) is synthesized predominantly by phosphatidylinositol 4-phosphate [PI(4)P] 5-kinase (PIP5K) through phosphorylation of PI(4)P at the D-5 position of the inositol ring. PIP5K is composed of three isoforms, PIP5Kalpha, beta, and gamma, and three splicing variants of the gamma isozyme. Although the PIP5Kgamma splicing variant PIP5Kgamma661 appears to be very specifically activated by talin, which plays a crucial role in focal adhesion formation, and the adaptor complex AP-2, the regulation of activities of other PIP5K isozymes is not fully understood at present. To understand the activation mechanism and the physiological function specific to each PIP5K isozyme, it is required to identify a specific activator of each PIP5K isozyme. This chapter describes common assays used to measure interaction and activation of PIP5K isozymes by activators thus far identified. In addition, procedures for preparation of PIP5K isozymes and activators are described.
功能极其多样的磷脂酰肌醇4,5-二磷酸[PI(4,5)P₂]在信号转导、肌动蛋白细胞骨架重组、网格蛋白介导的内吞作用以及膜形态调控中发挥着关键作用。在哺乳动物细胞中，PI(4,5)P₂主要由磷脂酰肌醇4-磷酸[PI(4)P] 5-激酶（PIP5K）通过将PI(4)P的肌醇环D-5位磷酸化而合成。PIP5K由三种同工型组成，即PIP5Kα、β和γ，以及γ同工酶的三种剪接变体。尽管PIP5Kγ剪接变体PIP5Kγ661似乎非常特异性地被在粘着斑形成中起关键作用的踝蛋白和衔接蛋白复合物AP-2激活，但目前对其他PIP5K同工酶活性的调节尚未完全了解。为了了解每种PIP5K同工酶的激活机制和特定的生理功能，需要鉴定每种PIP5K同工酶的特异性激活剂。本章描述了用于测量迄今为止已鉴定的激活剂与PIP5K同工酶的相互作用和激活的常用测定方法。此外，还描述了PIP5K同工酶和激活剂的制备程序。
