1. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A
- Author
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Borthwick, L.A., Kerbiriou, M., Taylor, C.J., Cozza, G., Lascu, I., Postel, E.H., Cassidy, D., Trouvé, P., Mehta, A., Robson, L., and Muimo, R.
- Subjects
Models, Molecular ,Genetics and Molecular Biology (all) ,Cell Membranes ,Respiratory System ,Cystic Fibrosis Transmembrane Conductance Regulator ,lcsh:Medicine ,Biochemistry ,Cytosol ,Cyclic AMP ,Post-Translational Modification ,Phosphorylation ,lcsh:Science ,Medicine (all) ,Cell Polarity ,NM23 Nucleoside Diphosphate Kinases ,Precipitation Techniques ,Chemistry ,Protein Transport ,Physical Sciences ,Cellular Structures and Organelles ,Research Article ,Protein Binding ,Immunoblotting ,Molecular Sequence Data ,Molecular Probe Techniques ,Research and Analysis Methods ,Cell Line ,Young Adult ,Chlorides ,Immunoprecipitation ,Animals ,Humans ,Amino Acid Sequence ,Agricultural and Biological Sciences (all) ,Biochemistry, Genetics and Molecular Biology (all) ,Protein Interactions ,Molecular Biology Techniques ,Molecular Biology ,Binding Sites ,Cell Membrane ,lcsh:R ,Chemical Compounds ,Biology and Life Sciences ,Membrane Proteins ,Proteins ,Epithelial Cells ,Cell Biology ,Cyclic AMP-Dependent Protein Kinases ,Protein Structure, Tertiary ,lcsh:Q - 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.
- Published
- 2016