title: Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.
abstract: Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia.
authors.affiliation: Academic Unit of Respiratory Medicine, Department of Infection and Immunity, The University of Sheffield, The Medical School, Sheffield, S10 2RX, United Kingdom.
authors.forename: Lee A
authors.initials: LA
authors.lastname: Borthwick
authors.affiliation: Academic Unit of Respiratory Medicine, Department of Infection and Immunity, The University of Sheffield, The Medical School, Sheffield, S10 2RX, United Kingdom.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Kerbiriou
authors.affiliation: Academic Unit of Child Health, University of Sheffield, Stephenson Wing, Sheffield Children's Hospital, Sheffield, S10 2TH, United Kingdom.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Taylor
authors.affiliation: Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B 35131, Padova, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Cozza
authors.affiliation: University of Bordeaux, France, and Institut de Biochimie et Genetique Cellulaires, Centre Nationale de la Recherche Scientifique UMR 5095, 33077, Bordeaux, France.
authors.forename: Ioan
authors.initials: I
authors.lastname: Lascu
authors.affiliation: Robert Wood Johnson Medical School, UMDNJ, New Brunswick, New Jersey, United States of America.
authors.forename: Edith H
authors.initials: EH
authors.lastname: Postel
authors.affiliation: Medical Research Institute/CVS Diabetes Lung, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, United Kingdom.
authors.forename: Diane
authors.initials: D
authors.lastname: Cassidy
authors.affiliation: Inserm UMR 1078, Génétique, Génomique Fonctionnelle et Biotechnologies, 46 rue Félix Le Dantec, 29218, Brest Cedex2, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Trouvé
authors.affiliation: Medical Research Institute/CVS Diabetes Lung, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, United Kingdom.
authors.forename: Anil
authors.initials: A
authors.lastname: Mehta
authors.affiliation: Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, S10 2TN, United Kingdom.
authors.forename: Louise
authors.initials: L
authors.lastname: Robson
authors.affiliation: Academic Unit of Respiratory Medicine, Department of Infection and Immunity, The University of Sheffield, The Medical School, Sheffield, S10 2RX, United Kingdom.
authors.forename: Richmond
authors.initials: R
authors.lastname: Muimo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D054778:NM23 Nucleoside Diphosphate Kinases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012137:Respiratory System / Q000166:cytology; D055815:Young Adult
chemical_list: D054778:NM23 Nucleoside Diphosphate Kinases; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases