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

Abstract

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 p110α catalytic domain with an N-terminal His-tag and the p85α regulatory domain in Sf9 insect cells. The complex consisting of p110α and p85α was purified by nickel affinity chromatography. The authors established an adenosine triphosphate (ATP) depletion assay to measure the activity of p110α/p85α. The assay was optimized by testing different lipids as substrates, as well as various kinase and lipid concentrations. Furthermore, they analyzed autophosphorylation of p110α/p85α 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 μM. p110α/p85α underwent autophosphorylation most prominently at the p85α 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)

Keywords

phosphoinositide 3-kinase PI3K PI3Kα ATP depletion assay AlphaScreen

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