title: Uncovering Aberrant Mutant PKA Function with Flow Cytometric FRET.
abstract: Biology has been revolutionized by tools that allow the detection and characterization of protein-protein interactions (PPIs). Förster resonance energy transfer (FRET)-based methods have become particularly attractive as they allow quantitative studies of PPIs within the convenient and relevant context of living cells. We describe here an approach that allows the rapid construction of live-cell FRET-based binding curves using a commercially available flow cytometer. We illustrate a simple method for absolutely calibrating the cytometer, validating our binding assay against the gold standard isothermal calorimetry (ITC), and using flow cytometric FRET to uncover the structural and functional effects of the Cushing-syndrome-causing mutation (L206R) on PKA's catalytic subunit. We discover that this mutation not only differentially affects PKAcat's binding to its multiple partners but also impacts its rate of catalysis. These findings improve our mechanistic understanding of this disease-causing mutation, while illustrating the simplicity, general applicability, and power of flow cytometric FRET.
authors.affiliation: Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205, USA. Electronic address: slee222@jhmi.edu.
authors.forename: Shin-Rong
authors.initials: SR
authors.lastname: Lee
authors.affiliation: Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205, USA.
authors.forename: Lingjie
authors.initials: L
authors.lastname: Sang
authors.affiliation: Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205, USA; Department of Neuroscience, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205, USA; Center for Cell Dynamics, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205, USA.
authors.forename: David T
authors.initials: DT
authors.lastname: Yue
mesh_terms: D002151:Calorimetry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D060066:Protein Interaction Maps
chemical_list: D017868:Cyclic AMP-Dependent Protein Kinases