title: PLD Protein-Protein Interactions With Signaling Molecules and Modulation by PA.
abstract: We describe methods for studying phospholipase D (PLD) interactions with signaling proteins and modulation of these interactions by the PLD reaction product, phosphatidic acid (PA). PLD is fundamental to the physiological maintenance of cellular/intracellular membranes, protein trafficking, cytoskeletal dynamics, membrane remodeling, cell proliferation, meiotic division and sporulation. PA is an acidic phospholipid involved in the biosynthesis of many other lipids that affects the enzymatic activities of many different signaling proteins via protein-lipid interactions or as a substrate. The involvement of PLD as an effector of protein-protein interactions and downstream signaling via PA-mediated processes has led to the investigation of PA-binding domains in target protein partners. We present here data and protocols detailing the interaction between PLD2-Rac2 interaction and modulation of this interaction by PA. We describe biochemical techniques to measure interactions between PLD, PA, and the small GTPase Rac2, which are associated in the cell. We found two maxima concentrations of PA that contributed to association or dissociation of Rac2 with PLD2, as well as the PLD2 lipase and guanine nucleotide exchange factor (GEF) activities. Fluctuations in the Rac2-PLD2 protein-protein binding interaction facilitate shuttling of Rac2 and/or PLD2 within the cell dependent on local cellular PA concentration. Fluorescence resonance emission transfer stoichiometry for PLD2 and Rac2 binding yielded a 3:1 ratio of Rac2:PLD2. Detection of PA in mammalian cells with a new biosensor showed colocalization in and around the nucleus. We also described methods for quantitation of PA in biological materials by HPLC electrospray ionization tandem mass spectrometry.
authors.affiliation: Wright State University, Boonshoft School of Medicine, Dayton, OH, United States. Electronic address: julian.cambronero@wright.edu.
authors.forename: J
authors.initials: J
authors.lastname: Gomez-Cambronero
authors.affiliation: The Gill Heart Institute, College of Medicine, Lexington Veterans Affairs Medical Center, University of Kentucky, Lexington, KY, United States.
authors.forename: A J
authors.initials: AJ
authors.lastname: Morris
authors.affiliation: Wright State University, Boonshoft School of Medicine, Dayton, OH, United States.
authors.forename: K M
authors.initials: KM
authors.lastname: Henkels
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D019556:COS Cells; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer; D015870:Gene Expression; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047468:Immunoprecipitation; D010712:Phosphatidic Acids / Q000378:metabolism* / Q000494:pharmacology; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D015398:Signal Transduction; D021241:Spectrometry, Mass, Electrospray Ionization; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000097766:RAC2 GTP-Binding Protein
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D010712:Phosphatidic Acids; D010739:Phospholipase D; D020744:rac GTP-Binding Proteins; D000097766:RAC2 GTP-Binding Protein
keywords: GEF; PABD; Phosphatidic acid; Phospholipase D; Rac2