Measuring Activity of Phosphoinositide Lipid Kinases Using a Bioluminescent ADP-Detecting Assay.

Abstract

Phosphatidylinositol (PI) and its phosphorylated derivatives, collectively called phosphoinositides, are important second messengers involved in a variety of cellular processes, including cell proliferation, apoptosis, metabolism, and migration. These derivatives are generated by a family of kinases called phosphoinositide lipid kinases (PIKs). Due to the central role of these kinases in signaling pathways, assays for measuring their activity are often used for drug development. Lipid kinase substrates are present in unique membrane environments in vivo and are insoluble in aqueous solutions. Therefore the most important consideration in developing successful lipid kinase assays is the physical state of lipid kinase substrates. Here we describe the preparation of lipid substrates for two major classes of lipid kinases, phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 4-kinases (PI4Ks). Using PI4Ks as an example, we also provide a detailed protocol for small-scale kinase expression and affinity purification from transiently transfected mammalian cells. For measuring lipid kinase activity we apply a universal bioluminescent ADP detection approach. The approach is compatible with diverse lipid substrates and can be used as a single integrated platform for measuring all classes of lipid and protein kinases.