Chapter Six - Methods to Measure the Enzymatic Activity of PI3Ks

Abstract

Phosphoinositide-3-kinase (PI3K) signaling has been implicated in a panoply of cellular responses including survival, proliferation, protein synthesis, migration, and vesicular trafficking. In addition, alterations in the enzymatic activity of PI3Ks have been involved in the pathogenesis of multiple diseases, ranging from cancer to chronic inflammation. The emerging interest in PI3K as a pharmacological target has prompted the development of several molecules with inhibitory activity. In this context, the quantification of the second messenger generated by PI3Ks, phosphoinositide-3-phosphate, offers an opportunity to directly test variations in the lipid kinase activity of PI3K in physiological as well as pathological conditions. Here, we will describe common methods to measure the lipid kinase activity of PI3K in vitro and new techniques to follow the production of phosphoinositide-3-phosphate in vivo. These methods are relevant to study the alterations of the PI3K systems at the interface between signaling and oncometabolism.