Measuring Activity of Phosphoinositide Lipid Kinases Using a Bioluminescent ADP-Detecting Assay.
Phosphatidylinositol (PI) and its phosphorylated derivatives, collectively called phosphoinositides, are important second messengers involved in a variety of cellular processes, including cell proliferation, apoptosis, metabolism, and migration. These derivatives are generated by a family of kinases called phosphoinositide lipid kinases (PIKs). Due to the central role of these kinases in signaling pathways, assays for measuring their activity are often used for drug development. Lipid kinase substrates are present in unique membrane environments in vivo and are insoluble in aqueous solutions. Therefore the most important consideration in developing successful lipid kinase assays is the physical state of lipid kinase substrates. Here we describe the preparation of lipid substrates for two major classes of lipid kinases, phosphatidylinositol 3-kinases (PI3Ks) and phosphatidylinositol 4-kinases (PI4Ks). Using PI4Ks as an example, we also provide a detailed protocol for small-scale kinase expression and affinity purification from transiently transfected mammalian cells. For measuring lipid kinase activity we apply a universal bioluminescent ADP detection approach. The approach is compatible with diverse lipid substrates and can be used as a single integrated platform for measuring all classes of lipid and protein kinases.
University of Michigan, 6520 MSRB I SPC 5682, 1150 W Medical Center Dr, Ann Arbor, MI, 48109-5682, USA. andrewwt@med.umich.edu.
Andrew W
AW
Tai
Promega Corporation, Research and Development Department, 2800 Woods Hollow Road, Madison, WI, 53711, USA. jvidugir@promega.com.
Jolanta
J
Vidugiriene
D019870:1-Phosphatidylinositol 4-Kinase / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D000244:Adenosine Diphosphate / Q000032:analysis*; D057809:HEK293 Cells; D006801:Humans; D007202:Indicators and Reagents; D049409:Luciferases, Firefly / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008823:Micelles; D019869:Phosphatidylinositol 3-Kinases / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D010716:Phosphatidylinositols / Q000378:metabolism; D011934:Reagent Strips; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013379:Substrate Specificity; D014162:Transfection
D007202:Indicators and Reagents; D008823:Micelles; D010716:Phosphatidylinositols; D011934:Reagent Strips; D011993:Recombinant Fusion Proteins; D000244:Adenosine Diphosphate; D049409:Luciferases, Firefly; D019869:Phosphatidylinositol 3-Kinases; D019870:1-Phosphatidylinositol 4-Kinase
ADP assay; Lipid kinase; Phosphatidylinositol 3-kinases; Phosphatidylinositol 4-kinases; Phosphatidylinositol phosphate; Phosphoinositides