title: Method for Assaying the Lipid Kinase Phosphatidylinositol-5-phosphate 4-kinase α in Quantitative High-Throughput Screening (qHTS) Bioluminescent Format.
abstract: Lipid kinases are important regulators of a variety of cellular processes and their dysregulation causes diseases such as cancer and metabolic diseases. Distinct lipid kinases regulate the seven different phosphorylated forms of phosphatidylinositol (PtdIns). Some lipid kinases utilize long-chain lipid substrates that have limited solubility in aqueous solutions, which can lead to difficulties in developing a robust and miniaturizable biochemical assay. The ability to prepare the lipid substrate and develop assays to identify modulators of lipid kinases is important and is the focus of this methods chapter. Herein, we describe a method to prepare a DMSO-based lipid mixture that enables the 1536-well screening of the lipid kinase phosphatidylinositol-5-phosphate 4-kinase α (PI5P4Kα) utilizing the D-myo-di16-PtIns(5)P substrate in quantitative high-throughput screening (qHTS) format using the ADP-Glo™ technology to couple the production of ADP to a bioluminescent readout.
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, 20850, USA.
authors.forename: Mindy I
authors.initials: MI
authors.lastname: Davis
authors.affiliation: Department of Internal Medicine, Division of Hematology Oncology, UC Cancer Institute, College of Medicine, University of Cincinnati, Cincinnati, OH, 45267, USA.|Department of Neurosurgery, Brain Tumor Center, UC Neuroscience Institute, College of Medicine, University of Cincinnati, Cincinnati, OH, 45267, USA.
authors.forename: Atsuo T
authors.initials: AT
authors.lastname: Sasaki
authors.affiliation: Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, 20850, USA. asimeono@mail.nih.gov.
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
mesh_terms: D000255:Adenosine Triphosphate; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D010716:Phosphatidylinositols; D000255:Adenosine Triphosphate; C514871:PIP4K2A protein, human; D017853:Phosphotransferases (Alcohol Group Acceptor)
keywords: ADP-Glo; Bioluminescence; Kinase; Lipid; Luciferase; PI5P4Kα; Phosphorylation; Quantitative high-throughput screening (qHTS)