Kate J. Treharne

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. We demonstrate that CFTR normally remains closed at baseline, but nevertheless, opens after inhibition of AMPK. AMPK phosphorylates CFTR in vitro at two essential serines (Ser737 and Ser768) in the R domain, formerly identified as “inhibitory” PKA sites. Replacement of both serines by alanines (i) reduced phosphorylation of the R domain, with Ser768 having dramatically greater impact, (ii) produced CFTR channels that were partially open in the absence of any stimulation, (iii) significantly augmented their activation by IBMX/forskolin, and (iv) eliminated CFTR inhibition post AMPK activation. Attenuation of CFTR by AMPK activation was detectable in the absence of cAMP-dependent stimulation but disappeared in maximally stimulated oocytes. Our data also suggest that AMP is produced by local phosphodiesterases in close proximity to CFTR. Thus we propose that CFTR channels are kept closed in nonstimulated epithelia with high baseline AMPK activity but CFTR may be basally active in tissues with lowered endogenous AMPK activity.

Expression of Wild-Type CFTR Suppresses NF-κB-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-κB activity. Methodology/Principal Findings In lung epithelial (H441) and engineered (H57) cell lines; we report that inflammatory markers are significantly suppressed by wild-type CFTR. Transient-transfection of wild-type CFTR into CFTR-naïve H441 cells, dose-dependently down-regulates both basal and Tumour Necrosis Factor-α evoked NF-κB activity when compared to transfection with empty vector alone (p<0.01, n>5). This effect was also observed in CFTR-naïve H57-HeLa cells which stably express a reporter of NF-κB activity, confirming that the CFTR-mediated repression of inflammation was not due to variable reporter gene transfection efficiency. In contrast, H57 cells transfected with a control cyano-fluorescent protein show a significantly elevated basal level of NF-κB activity above control. Initial cell seeding density may be a critical factor in mediating the suppressive effects of CFTR on inflammation as only at a certain density (1×105 cells/well) did we observe the reduction in NF-κB activity. CFTR channel activity may be necessary for this suppression because the CFTR specific inhibitor CFTRinh172 significantly stimulates NF-κB activity by ∼30% in CFTR expressing 16HBE14o− cells whereas pharmacological elevation of cyclic-AMP depresses activity by ∼25% below baseline. Conclusions/Significance These data indicate that CFTR has inherent anti-inflammatory properties. We propose that the hyper-inflammation found in CF may arise as a consequence of disrupted repression of NF-κB signalling which is normally mediated by CFTR. Our data therefore concur with in vivo and in vitro data from Vij and colleagues which highlights CFTR as a suppressor of basal inflammation acting through NF-κB, a central hub in inflammatory signalling.

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 ΔF508 are phosphorylated in vitro by CK2 catalytic α-subunit but not by CK2 holoenzyme unless polylysine is added. MS analysis reveals that, in both NBD1 wild type and ΔF508, the phosphorylated residues are S422 and S670, while phosphorylation of S511 could not be detected. Accordingly, peptides encompassing the 500−518 sequence of CFTR are not phosphorylated by CK2; rather they inhibit CK2α catalytic activity in a manner which is not competitive with respect to the specific CK2 peptide substrate. In contrast, 500−518 peptides promote the phosphorylation of NBD1 by CK2 holoenzyme overcoming inhibition by the β-subunit. Such a stimulatory efficacy of the CFTR 500−518 peptide is dramatically enhanced by deletion of F508 and is abolished by deletion of the II507 doublet. Kinetics of NBD1 phosphorylation by CK2 holoenzyme, but not by CK2α, display a sigmoid shape denoting a positive cooperativity which is dramatically enhanced by the addition of the ΔF508 CFTR peptide. SPR analysis shows that NBD1 ΔF508 interacts more tightly than NBD1 wt with the α-subunit of CK2 and that CFTR peptides which are able to trigger NBD1 phosphorylation by CK2 holoenzyme also perturb the interaction between the α- and the β-subunits of CK2.

Inhibition of Protein Kinase CK2 Closes the CFTR Cl − Channel, but has no Effect on the Cystic Fibrosis Mutant ΔF508-CFTR

Background: Deletion of phenylalanine-508 (ΔF508) 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 ΔF508-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. Methods: Electrophysiology, immunofluorescence and biochemistry applied to CFTR-expressing cells, Xenopus oocytes, pancreatic ducts and patient biopsies. Results: Irrespective of PKA activation, CK2 inhibition (ducts, oocytes, cells) attenuates CFTR-dependent Cl- transport, closing wtCFTR in cell-attached membrane patches. CK2 and wtCFTR co-precipitate and CK2 co-localized with wtCFTR (but not ΔF508-CFTR) in apical membranes of human airway biopsies. Comparing wild-type and ΔF508CFTR expressing oocytes, only ΔF508-CFTR Cl- currents were insensitive to two CK2 inhibitors. Furthermore, wtCFTR was inhibited by injecting a peptide mimicking the F508 region, whereas the ΔF508-equivalent peptide had no effect. Conclusions: CK2 controls wtCFTR, but not ΔF508-CFTR. Others find that peptides from the F508 region of NBD1 allosterically control CK2, acting through F508. Hence, disruption of CK2-CFTR interaction by ΔF508-CFTR might disrupt multiple, membrane-associated, CK2-dependent pathways, creating a new molecular disease paradigm for deleted F508 in CFTR.

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.

CFTR, chloride concentration and cell volume: could mammalian protein histidine phosphorylation play a latent role?

A considerable body of evidence indicates that the intracellular chloride concentration ([Cl−]i) is an important regulatory signal in epithelial ion transport. [Cl−]i regulates the open channel probability of sodium and chloride channels, the rate of chloride channel recycling to the apical membrane, cell volume homeostasis, the activity of sodium‐coupled chloride entry pathways and G‐protein activity. Cell volume goes awry in epithelial cells bearing mutant forms of the cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR); however, the pathways that mediate this [Cl−]i effect at the apical membrane of polarized epithelia are unknown. Recently, we proposed a mechanism for the transduction of in vitro chloride concentration into a phosphorylation signal to proteins within the apical membrane of respiratory epithelia. Our studies show that an apically enriched plasma membrane fraction from a variety of species, including sheep, human and mouse airway, contains at least two membrane‐bound protein kinases which exhibit a number of novel properties. Firstly, the phosphate is located on histidine residues within different families of proteins; one kinase(s) utilizes GTP rather than ATP as a phosphate donor and each kinase has its own unique profile of membrane protein phosphorylation (which itself varies with anion species). Secondly, both kinases mediate Cl−‐dependent phosphorylation of an apical membrane protein around the established physiological values for [Cl−]i in airway epithelial cells (∼40 mm); associated phosphatases also alter the net phosphoprotein profile of the apical membrane. These findings are reviewed and their potential roles explored in relation to the pathogenesis of CF using the control of cell volume as a model for disrupted cellular function in CF‐affected epithelia.

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. GTP (but not ATP) promotes serine phosphorylation (pSer) of NDPK. Further we find that rising [AMP] promotes pSer (only with GTP) but inhibits histidine phosphorylation (pHis) of NDPK from both donors. We find that NDPK co-immunoprecipitates reciprocally with AMP-activated kinase and that these two proteins can co-localise in human airways. AMP concentrations rise rapidly when ATP is depleted or during hypoxia. We find that, in human airway cells exposed to hypoxia (3% oxygen), membrane-bound NDPK is inhibited. Although histidine phosphorylation should in principle be independent of the nucleotide triphosphates used, we speculate that this membrane pool of NDPK may be able to switch function dependent on nucleotide species.

Understanding the molecular basis of the interaction between NDPK-A and AMPK α1

ABSTRACT Nucleoside diphosphate kinase (NDPK) (nm23/awd) belongs to a multifunctional family of highly conserved proteins (∼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. AMPK was first discovered as an activity that inhibited preparations of acetyl coenzyme A carboxylase 1 (ACC1), a regulator of cellular fatty acid synthesis. We recently reported that NDPK-A (but not NDPK-B) selectively regulates the α1 isoform of AMPK independently of the AMP concentration such that the manipulation of NDPK-A nucleotide trans-phosphorylation activity to generate ATP enhanced the activity of AMPK. This regulation occurred irrespective of the surrounding ATP concentration, suggesting that “substrate channeling” was occurring with the shielding of NDPK-generated ATP from the surrounding medium. We speculated that AMPK α1 phosphorylated NDPK-A during their interaction, and here, we identify two residues on NDPK-A targeted by AMPK α1 in vivo. We find that NDPK-A S122 and S144 are phosphorylated by AMPK α1 and that the phosphorylation status of S122, but not S144, determines whether substrate channeling can occur. We report the cellular effects of the S122 mutation on ACC1 phosphorylation and demonstrate that the presence of E124 (absent in NDPK-B) is necessary and sufficient to permit both AMPK α1 binding and substrate channeling.

Protein Kinase CK2, Cystic Fibrosis Transmembrane Conductance Regulator, and the ΔF508 Mutation F508 DELETION DISRUPTS A KINASE-BINDING SITE

Deletion of phenylalanine 508 (ΔF508) 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. Importantly, the interaction of CK2 with NBD1 is selectively abrogated by the ΔF508 mutation without disrupting four established CFTR-associated kinases and two phosphatases. Loss of CK2 association is functionally corroborated by the insensitivity of ΔF508-CFTR to CK2 inhibition, the absence of CK2 activity in ΔF508 CFTR-expressing cell membranes, and inhibition of CFTR channel activity by a peptide that mimics the F508 region of CFTR (but not the equivalent ΔF508 peptide). Disruption of this CK2-CFTR association is the first described ΔF508-dependent protein-protein interaction that provides a new molecular paradigm in the most frequent form of cystic fibrosis.

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

MINT‐7219905, MINT‐7219896: tgase2 (uniprotkb:P21980) physically interacts (MI:0914) with NDPK (uniprotkb:P15531) by anti bait coimmunoprecipitation (MI:0006)