Regulation of Phosphatidylinositol 4-Phosphate 5-Kinase Activity by Partner Proteins

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, PIP5Kα, β, and γ, and three splicing variants of the γ isozyme. Although the PIP5Kγ splicing variant PIP5Kγ661 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.