Measuring Insulin-Stimulated Phosphatidyl-Inositol 3-Kinase Activity

Numerous growth factors, hormones, and transforming oncogenes activate phosphatidylinositol 3-kinase (PI3K), an enzyme that phosphorylates the 3′-hydroxyl position of phosphoinositides to generate the intracellular messengers phosphatidylinositol-3-phosphate [PI(3)P], phosphatidylinositol-3,4-bisphos-phate [PI(3,4)P₂ ], and phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P₃ ] (reviewed in ref. 1 ). PI3K commonly participates in signaling pathways leading to increased cell survival, proliferation, and growth, but in some cells and tissues it regulates more specialized biological processes. Notably, PI3K is an obligate intermediate in the signaling pathway linking insulin’s arrival at the cell surface to the regulation of glucose uptake and anabolic metabolism (reviewed in ref. 2 ). This chapter describes a method for quantifying the activation of the class 1A type PI3Ks by insulin.