Expression of wild-type CFTR suppresses NF-kappaB-driven inflammatory signalling.

Background: Mutation of the cystic fibrosis transmembrane-conductance regulator (CFTR) causes cystic fibrosis (CF) but not all CF aspects can easily be explained by deficient ion transport. CF-inflammation provides one example but its pathogenesis remains controversial. Here, we tested the simple but fundamental hypothesis that wild-type CFTR is needed to suppress NF-kappaB activity.

Cystic fibrosis transmembrane regulator fragments with the Phe508 deletion exert a dual allosteric control over the master kinase CK2.

Cystic fibrosis mostly follows a single Phe508 deletion in CFTR (cystic fibrosis transmembrane regulator) (CFTRDeltaF508), thereby causing premature fragmentation of the nascent protein with concomitant alterations of diverse cellular functions. We show that CK2, the most pleiotropic protein kinase, undergoes allosteric control of its different cellular forms in the presence of short CFTR peptides encompassing the Phe508 deletion: these CFTRDeltaF508 peptides drastically inhibit the isolated catalytic subunit (alpha) of the kinase and yet up-regulate the holoenzyme, composed of two catalytic and two non-catalytic (beta) subunits. Remarkable agreement between in silico docking and our biochemical data point to different sites for the CFTRDeltaF508 peptide binding on isolated CK2alpha and on CK2beta assembled into the holoenzyme, suggesting that CK2 targeting may be perturbed in cells expressing CFTRDeltaF508; this could shed light on some pleiotropic aspects of cystic fibrosis disease.

Inhibition of protein kinase CK2 closes the CFTR Cl channel, but has no effect on the cystic fibrosis mutant deltaF508-CFTR.

Background: Deletion of phenylalanine-508 (DeltaF508) from the first nucleotide-binding domain (NBD1) in the wild-type cystic fibrosis (CF) transmembrane-conductance regulator (wtCFTR) causes CF. However, the mechanistic relationship between DeltaF508-CFTR and the diversity of CF disease is unexplained. The surface location of F508 on NBD1 creates the potential for protein-protein interactions and nearby, lies a consensus sequence (SYDE) reported to control the pleiotropic protein kinase CK2.

Transglutaminase 2 and nucleoside diphosphate kinase activity are correlated in epithelial membranes and are abnormal in cystic fibrosis.

Tissue transglutaminase (tgase2) is a multifunctional enzyme that crosslinks proteins but also acts as a G-protein, differential functions regulated by calcium and GTP. In the epithelial cell membrane, we show that manipulation of tgase2 function by monodansylcadaverine or retinoic acid (RA) alters the activity of a membrane-bound protein kinase, nucleoside diphosphate kinase (NDPK, nm23-H1/H2) that is known to control G-protein function. We find that NDPK function is abnormally low in cystic fibrosis but can be restored by RA treatment in vitro.

Epithelial IgG and its relationship to the loss of F508 in the common mutant form of the cystic fibrosis transmembrane conductance regulator.

The most debilitating feature of cystic fibrosis (CF) disease is uncontrolled inflammation of respiratory epithelium. The relationship between the commonest mutated form of CFTR (F508del or DeltaF508) and inflammation has not yet been elucidated. Here, we present a new paradigm suggesting that CFTR can interact with intra-epithelial IgG, establishing a direct link between normal CFTR and the immune system.

The phosphorylation status of membrane-bound nucleoside diphosphate kinase in epithelia and the role of AMP.

Nucleoside diphosphate kinase (NDPK) has many roles and is present in different locations in the cell. Membrane-bound NDPK is present in epithelial fractions enriched for the apical membrane. Here, we show in human, mouse and sheep airway membranes, that the phosphorylation state of membrane-bound NDPK on histidine and serine residues differs dependent on many regulatory factors.

Small interfering peptide (siP) for in vivo examination of the developing lung interactonome.

To understand the role of reactive oxygen species in mechanosensory control of lung development a new approach to interfere with protein-protein interactions by means of a short interacting peptide was developed. This technology was used in the developing rodent lung to examine the role of NADPH oxidase (NOX), casein kinase 2 (CK2), and the cystic fibrosis transmembrane conductance regulator (CFTR) in stretch-induced differentiation. Interactions between these molecules was targeted in an in utero system with recombinant adeno-associated virus (rAAV) containing inserted DNA sequences that express a control peptide or small interfering peptides (siPs) specific for subunit interaction or phosphorylation predicted to be necessary for multimeric enzyme formation.

Mechanistic insight into control of CFTR by AMPK.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and protein kinase A (PKA)-regulated Cl(-) channel in the apical membrane of epithelial cells. The metabolically regulated and adenosine monophosphate-stimulated kinase (AMPK) is colocalized with CFTR and attenuates its function. However, the sites for CFTR phosphorylation and the precise mechanism of inhibition of CFTR by AMPK remain obscure.

Modulation of protein kinase CK2 activity by fragments of CFTR encompassing F508 may reflect functional links with cystic fibrosis pathogenesis.

Deletion of F508 in the first nucleotide binding domain (NBD1) of cystic fibrosis transmembrane conductance regulator protein (CFTR) is the commonest cause of cystic fibrosis (CF). Functional interactions between CFTR and CK2, a highly pleiotropic protein kinase, have been recently described which are perturbed by the F508 deletion. Here we show that both NBD1 wild type and NBD1 DeltaF508 are phosphorylated in vitro by CK2 catalytic alpha-subunit but not by CK2 holoenzyme unless polylysine is added.

Protein kinase CK2, cystic fibrosis transmembrane conductance regulator, and the deltaF508 mutation: F508 deletion disrupts a kinase-binding site.

Deletion of phenylalanine 508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common mutation in cystic fibrosis. The F508 region lies within a surface-exposed loop that has not been assigned any interaction with associated proteins. Here we demonstrate that the pleiotropic protein kinase CK2 that controls protein trafficking, cell proliferation, and development binds wild-type CFTR near F508 and phosphorylates NBD1 at Ser-511 in vivo and that mutation of Ser-511 disrupts CFTR channel gating.

Protein kinase CK2 acts as a signal molecule switching between the NDPK-A/AMPK alpha1 complex and NDPK-B.

Previously we elucidated the molecular interaction between the nucleoside diphosphate kinase A (NDPK-A)/AMP-activated protein kinase (AMPK) alpha1 complex, discovering a process we termed "substrate channeling." Here, we investigate the protein-protein interaction of the substrate channeling complex with the pleiotropic protein kinase, CK2 (formerly casein kinase 2). We show that CK2 is part of the NDPK-A/AMPK alpha1 complex under basal (background AMPK activity) conditions, binding directly to each of the complex components independently.

Understanding the molecular basis of the interaction between NDPK-A and AMPK alpha 1.

Nucleoside diphosphate kinase (NDPK) (nm23/awd) belongs to a multifunctional family of highly conserved proteins (approximately 16 to 20 kDa) including two well-characterized isoforms (NDPK-A and -B). NDPK catalyzes the conversion of nucleoside diphosphates to nucleoside triphosphates, regulates a diverse array of cellular events, and can act as a protein histidine kinase. AMP-activated protein kinase (AMPK) is a heterotrimeric protein complex that responds to the cellular energy status by switching off ATP-consuming pathways and switching on ATP-generating pathways when ATP is limiting.

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.

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.

4alpha-Phorbol negates the inhibitory effects of phorbol-12-myristate-13-acetate on human cilia and alters the phosphorylation of PKC.

In medium 199, ciliary beat frequency (CBF) in human nasal epithelium declines to 60% of baseline by 2 h and 1 nM phorbol-12-myristate-13-acetate (PMA) doubles the rate of decline by activating protein kinase C (PKC). We find that a reported negative control for PMA, 4alpha-phorbol (1 pM-1 nM)+/-1 nM PMA, not only maintains CBF at baseline, but arrests a pre-existing PMA-induced decline in CBF and alters the profile of multiple phosphorylated PKC species. Thus, 4alpha-phorbol not only potently prevents PMA from inhibiting CBF but also has potent effects on the phosphorylation of PKC.