title: Role of binding and nucleoside diphosphate kinase A in the regulation of the cystic fibrosis transmembrane conductance regulator by AMP-activated protein kinase.
abstract: Cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel mutations cause cystic fibrosis lung disease. A better understanding of CFTR regulatory mechanisms could suggest new therapeutic strategies. AMP-activated protein kinase (AMPK) binds to and phosphorylates CFTR, attenuating PKA-activated CFTR gating. However, the requirement for AMPK binding to CFTR and the potential role of other proteins in this regulation are unclear. We report that nucleoside diphosphate kinase A (NDPK-A) interacts with both AMPK and CFTR in overlay blots of airway epithelial cell lysates. Binding studies in Xenopus oocytes and transfected HEK-293 cells revealed that a CFTR peptide fragment that binds AMPK (CFTR-1420-57) disrupted the AMPK-CFTR interaction. Introduction of CFTR-1420-57 into human bronchial Calu-3 cells enhanced forskolin-stimulated whole cell conductance in patch clamp measurements. Similarly, injection of CFTR-1420-57 into Xenopus oocytes blocked the inhibition of cAMP-stimulated CFTR conductance by AMPK in two-electrode voltage clamp studies. AMPK also inhibited CFTR conductance with co-expression of WT NDPK-A in two-electrode voltage clamp studies, but co-expression of a catalytically inactive H118F mutant or various Ser-120 NDPK-A mutants prevented this inhibition. In vitro phosphorylation of WT NDPK-A was enhanced by purified active AMPK, but phosphorylation was prevented in H118F and phosphomimic Ser-120 NDPK-A mutants. AMPK does not appear to phosphorylate NDPK-A directly but rather promotes an NDPK-A autophosphorylation event that involves His-118 and Ser-120. Taken together, these results suggest that NDPK-A exists in a functional cellular complex with AMPK and CFTR in airway epithelia, and NDPK-A catalytic function is required for the AMPK-dependent regulation of CFTR.
authors.affiliation: Department of Medicine, Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
authors.forename: J Darwin
authors.initials: JD
authors.lastname: King
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Lee
authors.forename: Claudia E
authors.initials: CE
authors.lastname: Riemen
authors.forename: Dietbert
authors.initials: D
authors.lastname: Neumann
authors.forename: Sheng
authors.initials: S
authors.lastname: Xiong
authors.forename: J Kevin
authors.initials: JK
authors.lastname: Foskett
authors.forename: Anil
authors.initials: A
authors.lastname: Mehta
authors.forename: Richmond
authors.initials: R
authors.lastname: Muimo
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Hallows
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000378:metabolism*; D000818:Animals; D001980:Bronchi / Q000166:cytology; D002384:Catalysis; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007477:Ions / Q000737:chemistry; D008954:Models, Biological; D009154:Mutation; D054778:NM23 Nucleoside Diphosphate Kinases / Q000502:physiology*; D009865:Oocytes / Q000166:cytology; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D011485:Protein Binding; D014982:Xenopus laevis
chemical_list: C505032:CFTR protein, human; D007477:Ions; D054778:NM23 Nucleoside Diphosphate Kinases; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D055372:AMP-Activated Protein Kinases