title: Dual-color click beetle luciferase heteroprotein fragment complementation assays.
abstract: Understanding the functional complexity of protein interactions requires mapping biomolecular complexes within the cellular environment over biologically relevant time scales. Herein, we describe a set of reversible multicolored heteroprotein complementation fragments based on various firefly and click beetle luciferases that utilize the same substrate, D-luciferin. Luciferase heteroprotein fragment complementation systems enabled dual-color quantification of two discrete pairs of interacting proteins simultaneously or two distinct proteins interacting with a third shared protein in live cells. Using real-time analysis of click beetle green and click beetle red luciferase heteroprotein fragment complementation applied to β-TrCP, an E3-ligase common to the regulation of both β-catenin and IκBα, GSK3β was identified as a candidate kinase regulating IκBα processing. These dual-color protein interaction switches may enable directed dynamic analysis of a variety of protein interactions in living cells.
authors.affiliation: Molecular Imaging Center, Mallinckrodt Institute of Radiology, Department of Developmental Biology, Washington University, St. Louis, MO 63110, USA.
authors.forename: Victor
authors.initials: V
authors.lastname: Villalobos
authors.forename: Snehal
authors.initials: S
authors.lastname: Naik
authors.forename: Monique
authors.initials: M
authors.lastname: Bruinsma
authors.forename: Robin S
authors.initials: RS
authors.lastname: Dothager
authors.forename: Mei-Hsiu
authors.initials: MH
authors.lastname: Pan
authors.forename: Mustapha
authors.initials: M
authors.lastname: Samrakandi
authors.forename: Britney
authors.initials: B
authors.lastname: Moss
authors.forename: Adnan
authors.initials: A
authors.lastname: Elhammali
authors.forename: David
authors.initials: D
authors.lastname: Piwnica-Worms
mesh_terms: D000818:Animals; D052160:Benzothiazoles / Q000737:chemistry; D002460:Cell Line; D001517:Coleoptera / Q000201:enzymology*; D003116:Color; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D025941:Protein Interaction Mapping; D020123:Sirolimus / Q000494:pharmacology; D051176:beta Catenin / Q000378:metabolism; D044844:beta-Transducin Repeat-Containing Proteins / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; C532924:D-luciferin; D007166:Immunosuppressive Agents; D051176:beta Catenin; D044844:beta-Transducin Repeat-Containing Proteins; D008156:Luciferases; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D020123:Sirolimus