title: Binding of a Pleckstrin homology domain protein to phosphoinositide in membranes: a miniaturized FRET-based assay for drug screening.
abstract: Pleckstrin homology (PH) domains are present in key proteins involved in many vital cell processes. For example, the PH domain of Bruton's tyrosine kinase (Btk) binds to phosphatidylinositol triphosphate (PIP(3)) in the plasma membrane after stimulation of the B-cell receptor in B cells. Mutations in the Btk PH domain result in changes in its affinity for PIP(3), with higher binding leading to cell transformation in vitro and lower binding leading to antibody deficiencies in both humans and mice. We describe here a fluorescence resonance energy transfer (FRET)-based biochemical assay that directly monitors the interaction of a PH domain with PIP(3) at a membrane surface. We overexpressed a fusion protein consisting of an enhanced green fluorescent protein (GFP) and the N-terminal 170 amino acids of a Tec family kinase that contains its PH domain (PH170). Homogeneous unilamellar vesicles were made that contained PIP(3) and octadecylrhodamine (OR), a lipophilic FRET acceptor for GFP. After optimization of both protein and vesicle components, we found that binding of the GFP-PH170 protein to PIP3 in vesicles that contain OR results in about a 90% reduction of GFP fluorescence. Using this assay to screen 1440 compounds, we identified three that efficiently inhibited binding of GFP-PH170 to PIP(3) in vesicles. This biochemical assay readily miniaturized to 1.8-microl reaction volumes and was validated in a 3456-well screening format.
authors.affiliation: Aurora Biosciences Corporation, San Diego, CA 92121, USA. hammanb@aurorabio.com
authors.forename: Brian D
authors.initials: BD
authors.lastname: Hamman
authors.forename: Brian A
authors.initials: BA
authors.lastname: Pollok
authors.forename: Todd
authors.initials: T
authors.lastname: Bennett
authors.forename: Janet
authors.initials: J
authors.lastname: Allen
authors.forename: Roger
authors.initials: R
authors.lastname: Heim
mesh_terms: D000077329:Agammaglobulinaemia Tyrosine Kinase; D000818:Animals; D001667:Binding, Competitive; D001798:Blood Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D007700:Kinetics; D050356:Lipid Metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010716:Phosphatidylinositols / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
chemical_list: D001798:Blood Proteins; D008164:Luminescent Proteins; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C039077:platelet protein P47; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; C000625951:Btk protein, mouse