Expression and Purification of PI3 Kinase α and Development of an ATP Depletion and an AlphaScreen PI3 Kinase Activity Assay

Phosphoinositide-3-kinases are important targets for drug development because many proteins in the PI3 kinase signaling pathway are mutated, hyperactivated, or overexpressed in human cancers. Here, the authors coexpressed the human class Ia PI3 kinase p110 alpha catalytic domain with an N-terminal His-tag and the p85 alpha regulatory domain in Sf9 insect cells. The complex consisting of p110a and p85a was purified by nickel affinity chromatography. The authors established an adenosine triphosphate (ATP) depletion assay to measure the activity of p110 alpha/p85 alpha. The assay was optimized by testing different lipids as substrates, as well as various kinase and lipid concentrations. Furthermore, they analyzed autophosphorylation of p110 alpha/p85 alpha and determined the IC50 for wortmannin, a known PI3 kinase inhibitor. The IC50 for wortmannin was determined to be 7 nM. From a selection of substrates, phosphatidylinositol-4,5-biphosphate turned out to be the best substrate at a concentration of 50 mu M. p110 alpha/ p85a underwent autophosphorylation most prominently at the p85 alpha subunit. However, in the presence of lipid substrate, the autophosphorylation was negligible. In parallel, a second assay format using the AlphaScreen technology was optimized to measure PI3 kinase activity. Both assay formats used should be suitable for high-throughput screening for the identification of PI3 kinase inhibitors. ( Journal of Biomolecular Screening 2008: 1035-1040)