A Novel PI 3-Kinase Assay Utilising HTRF Technology on the RUBYstar
Christine J. Rossant, Jason P. Brown, Marie L. Marshall.
BMG LABTECH, Germany. Upstate Ltd., Cambridge, UK
Inositol phospholipids provide a wide range of cellular signals that play crucial roles in normal cell function. These lipids are interconverted by families of kinases and phosphatases, generating at least seven different lipid species that differ with respect to the number and distribution of phosphate groups around the inositol ring. Type I PI 3-Kinases (p110 a, ß, ? and d) convert phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol 3,4,5-trisphosphate (PIP3) upon activation by growth factors, insulin and G-protein coupled receptors. PIP3 is a ubiquitous second messenger that binds to protein effector molecules such as protein Ser/Thr kinases (PKB, PDK1), Tec family protein tyrosine kinases, and regulators of monomeric G-proteins (e.g. GRP-1). These binding interactions modulate cellular processes such as growth, proliferation, motility, apoptosis and mediation of the metabolic actions of insulin. PI 3-Kinases have traditionally been assayed using labour intensive radiometric methods. In this application note we describe a novel assay format for measuring PI 3-Kinase activity (data is presented for human PI 3-Kinase ?) that exploits the specifi c protein/lipid interactions described above. This assay has been confi gured into a robust homogeneous format utilising Homogeneous Time Resolved Fluorescence (HTRF®) technology.