Assaying Phosphoinositide Phosphatases

The roles of phosphoinositide second messengers as signaling molecules in a vast array of cellular processes including cell growth, metabolism, vesicular transport, programmed cell death, and responses to extracellular signals are only beginning to be understood. The recent identification of novel phosphoinositide signaling molecules underscores the need for methodology with which to characterize the enzymes responsible for regulating cellular phosphoinositide levels. One of the ways in which cells control these lipids is through dephosphorylation by phosphoinositide phosphatases, which oppose and regulate the actions of phosphoinositide kinases. We describe herein two rapid and simple assays for characterizing phosphoinositide phosphatases that can be used to provide a basis for understanding the activity and specificity of these enzymes.