1. Role of Binding and Nucleoside Diphosphate Kinase A in the Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator by AMP-activated Protein Kinase
- Author
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Jeffrey K. Lee, Richmond Muimo, Claudia E. Riemen, Anil Mehta, J. Kevin Foskett, J Darwin King, Dietbert Neumann, Sheng Xiong, Kenneth R. Hallows, Moleculaire Genetica, RS: CARIM School for Cardiovascular Diseases, and Genetica & Celbiologie
- Subjects
AMP-activated Kinase (AMPK) ,Oocyte ,Patch-Clamp Techniques ,Cystic Fibrosis Transmembrane Conductance Regulator ,AMP-Activated Protein Kinases ,Biochemistry ,Ion Channels ,Xenopus laevis ,0302 clinical medicine ,AMP-activated protein kinase ,CFTR ,Phosphorylation ,0303 health sciences ,education.field_of_study ,biology ,Autophosphorylation ,NM23 Nucleoside Diphosphate Kinases ,respiratory system ,Nucleoside-diphosphate kinase ,Cystic fibrosis transmembrane conductance regulator ,3. Good health ,Cell biology ,030220 oncology & carcinogenesis ,Protein Binding ,Nucleoside diphosphate kinase A ,congenital, hereditary, and neonatal diseases and abnormalities ,Bronchi ,Models, Biological ,Catalysis ,Cell Line ,Nucleoside Diphosphate Kinase ,03 medical and health sciences ,Membrane Biology ,Animals ,Humans ,Protein kinase A ,education ,Molecular Biology ,Nm23 ,030304 developmental biology ,Ions ,AMPK ,Chloride Transport ,Epithelial Cells ,Cell Biology ,Molecular biology ,digestive system diseases ,Calu-3 Cells ,respiratory tract diseases ,Patch Clamp Electrophysiology ,HEK293 Cells ,Gene Expression Regulation ,Mutation ,Oocytes ,biology.protein - Abstract
Background: AMPK phosphorylates CFTR and inhibits PKA-stimulated CFTR channel gating by unclear mechanisms. Results: NDPK-A, AMPK, and CFTR exist in a membrane-associated complex. AMPK-CFTR binding and NDPK-A catalytic function are required for CFTR inhibition by AMPK. Conclusion: NDPK-A plays an integral role in the regulation of CFTR by AMPK. Significance: Targeting the AMPK-CFTR interaction and NDPK-A function could yield new therapeutic strategies for CF., 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.
- Published
- 2012