title: Qualitative and Quantitative In Vitro Analysis of Phosphatidylinositol Phosphatase Substrate Specificity.
abstract: Phosphoinositides compromise a family of eight membrane lipids which play important roles in many cellular signaling pathways. Signaling through phosphoinositides has been shown in a variety of cellular functions such cell proliferation, cell growth, apoptosis, and vesicle trafficking. Phospholipid phosphatases regulate cell signaling by modifying the concentration of phosphoinositides and their dephosphorylated products. To understand the role of individual lipid phosphatases in phosphoinositide turnover and functional signaling, it is crucial to determine the substrate specificity of the lipid phosphatase of interest. In this chapter we describe how the substrate specificity of an individual lipid phosphatase can be qualitatively and quantitatively measured in an in vitro radiometric assay. In addition, we specify the different expression systems and purification methods required to produce the necessary yield and functionality in order to further characterize these enzymes. The outstanding versatility and sensitivity of this assay system are yet unmatched and are therefore currently considered the standard of the field.
authors.affiliation: Research Department of Cancer Biology, UCL Cancer Institute, Paul O'Gorman Building, University College London, 72 Huntley Street, London, WC1E 6BT, UK.
authors.forename: Laura Ren Huey
authors.initials: LR
authors.lastname: Ip
authors.affiliation: Translational Innovation Group, UCL-Eisai Collaborative, University College London, 1st Floor Maple House, 149 Tottenham Court Road, London, W1T 7DN, UK. c.gewinner@ucl.ac.uk.
authors.forename: Christina Anja
authors.initials: CA
authors.lastname: Gewinner
mesh_terms: D000818:Animals; D002460:Cell Line; D057075:Enzyme Assays / Q000379:methods*; D015870:Gene Expression; D066298:In Vitro Techniques; D008055:Lipids / Q000737:chemistry; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013194:Staining and Labeling; D013379:Substrate Specificity
chemical_list: D008055:Lipids; D010716:Phosphatidylinositols; D011993:Recombinant Fusion Proteins; D019869:Phosphatidylinositol 3-Kinases; D010744:Phosphoric Monoester Hydrolases
keywords: Cell signaling; Enzyme assay; GST-pull-down assay; Inositol-polyphosphate-4-phosphatase type II; Insect cell culture; Lipid phosphatase assay; Mammalian overexpression system; PI-3 kinase; PTEN; Phosphatidylinositol; Phosphoinositide; Phosphorylation; Thin-layer chromatography