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A membrane capture assay for lipid kinase activity

Abstract

Phosphoinositide kinases such as PI3-kinase synthesize lipid second messengers that control diverse cellular processes. Recently, these enzymes have emerged as an important class of drug targets, and there is significant interest in discovering new lipid kinase inhibitors. We describe here a procedure for the high-throughput determination of lipid kinase inhibitor IC50 values. This assay exploits the fact that phosphoinositides, but not nucleotides such as ATP, bind irreversibly to nitrocellulose membranes. As a result, the radiolabeled lipids from a kinase assay can be isolated by spotting the crude reaction on a nitrocellulose membrane and then washing. We show that diverse phosphoinositide kinases can be assayed using this approach and outline how to perform the assay in 96-well plates. We also describe a MATLAB script that automates the data analysis. The complete procedure requires 3–4 h.

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Figure 1: Routes for the synthesis of phosphoinositides in mammalian cells.
Figure 2
Figure 3: Substrate specificity of lipid kinases.
Figure 4: The membrane capture assay of PI(5)P4-KIIβ is linear with respect to time and enzyme concentration.
Figure 5: Determination of IC50 values for p110α with the PI3-kinase inhibitors PIK-108 and PIK-93 using the membrane capture assay.
Figure 6: 'Spot' image analysis tool.

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Acknowledgements

Z.A.K. is a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation. M.E.F. is an ARCS Foundation Fellow. We acknowledge funding from NIH training grant GM08284. K.M.S. received funding from the Howard Hughes Medical Institute. The research of T.B. and A.B. was supported by the Intramural Research Program of the National Institute of Child Health and Human Development of the National Institutes of Health. We thank James Hurley for the generous gift of PI(5)P4-KIIβ.

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Correspondence to Kevan M Shokat.

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Knight, Z., Feldman, M., Balla, A. et al. A membrane capture assay for lipid kinase activity. Nat Protoc 2, 2459–2466 (2007). https://doi.org/10.1038/nprot.2007.361

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