Method for Assaying the Lipid Kinase Phosphatidylinositol-5-phosphate 4-kinase α in Quantitative High-Throughput Screening (qHTS) Bioluminescent Format

Mindy I Davis; Atsuo T Sasaki; Anton Simeonov

doi:10.1007/978-1-4939-3170-5_1

Method for Assaying the Lipid Kinase Phosphatidylinositol-5-phosphate 4-kinase α in Quantitative High-Throughput Screening (qHTS) Bioluminescent Format

Methods Mol Biol. 2016:1376:1-9. doi: 10.1007/978-1-4939-3170-5_1.

Authors

Mindy I Davis¹, Atsuo T Sasaki^{2

3}, Anton Simeonov⁴

Affiliations

¹ Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, 20850, USA.
² Department of Internal Medicine, Division of Hematology Oncology, UC Cancer Institute, College of Medicine, University of Cincinnati, Cincinnati, OH, 45267, USA.
³ Department of Neurosurgery, Brain Tumor Center, UC Neuroscience Institute, College of Medicine, University of Cincinnati, Cincinnati, OH, 45267, USA.
⁴ Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, 20850, USA. asimeono@mail.nih.gov.

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.

Keywords: ADP-Glo; Bioluminescence; Kinase; Lipid; Luciferase; PI5P4Kα; Phosphorylation; Quantitative high-throughput screening (qHTS).

Publication types

Research Support, N.I.H., Extramural

MeSH terms

Adenosine Triphosphate
High-Throughput Screening Assays / methods*
Humans
Luminescent Measurements / methods*
Phosphatidylinositols / metabolism
Phosphorylation
Phosphotransferases (Alcohol Group Acceptor) / metabolism*
Substrate Specificity

Substances

Phosphatidylinositols
Adenosine Triphosphate
PIP4K2A protein, human
Phosphotransferases (Alcohol Group Acceptor)

Grants and funding

Z01 HG200319-05/Intramural NIH HHS/United States