Role of binding and nucleoside diphosphate kinase A in the regulation of the cystic fibrosis transmembrane conductance regulator by AMP-activated protein kinase.

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.

NDPK-A (but not NDPK-B) and AMPK alpha1 (but not AMPK alpha2) bind the cystic fibrosis transmembrane conductance regulator in epithelial cell membranes.

Cystic fibrosis (CF) results from mutations within the cystic fibrosis transmembrane-conductance regulator (CFTR) protein. The AMP-activated protein kinase (AMPK) is a heterotrimer composed of different isoforms of the alphabetagamma subunits, where the alpha1 catalytic subunit binds CFTR. Nucleoside diphosphate kinase (NDPK, NM23/awd) converts nucleoside diphosphates to nucleoside triphosphates but also acts as a protein kinase. We recently showed that AMPK alpha1 binds NDPK-A in lung epithelial cytosol. Here we report that in the plasma membrane of human airway epithelial cells, NDPK-A and AMPK alpha1 associate with the plasma membrane via CFTR. We show that the regulatory domain of CFTR binds NDPK-A whereas AMPK gamma1 or gamma2 bind the first nucleotide binding domain (NBD1) and AMPK alpha1 binds the second (NBD2) of CFTR. We also show that NDPK-A specifically binds AMPK alpha1 and AMPK gamma2 subunits, thereby specifying the isozyme of AMPK heterotrimer that associates with CFTR at the membrane. Thus, the combined data provide novel insight into the subunit composition of the epithelial CFTR/AMPK/NDPK complex, such that: CFTR interacts specifically with AMPK alpha1, gamma2 and NDPK-A and not NDPK-B or AMPK gamma1.

A novel physical and functional association between nucleoside diphosphate kinase A and AMP-activated protein kinase alpha1 in liver and lung.

Nucleoside diphosphate kinase (NDPK, NM23/awd) belongs to a multifunctional family of highly conserved proteins (approximately 16-20 kDa) containing two well-characterized isoforms (NM23-H1 and -H2; also known as NDPK A and B). NDPK catalyses the conversion of nucleoside diphosphates into nucleoside triphosphates, regulates a diverse array of cellular events and can act as a protein histidine kinase. AMPK (AMP-activated protein kinase) is a heterotrimeric protein complex that responds to cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting. AMPK was first discovered as an activity that inhibited preparations of ACC1 (acetyl-CoA carboxylase), a regulator of cellular fatty acid synthesis. We report that NM23-H1/NDPK A and AMPK alpha1 are associated in cytosol from two different tissue sources: rat liver and a human lung cell line (Calu-3). Co-immunoprecipitation and binding assay data from both cell types show that the H1/A (but not H2/B) isoform of NDPK is associated with AMPK complexes containing the alpha1 (but not alpha2) catalytic subunit. Manipulation of NM23-H1/NDPK A nucleotide transphosphorylation activity to generate ATP (but not GTP) enhances the activity of AMPK towards its specific peptide substrate in vitro and also regulates the phosphorylation of ACC1, an in vivo target for AMPK. Thus novel NM23-H1/NDPK A-dependent regulation of AMPK alpha1-mediated phosphorylation is present in mammalian cells.