Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA

CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (P(o)) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted P(o)-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.     

Phosphorylation of the cystic fibrosis transmembrane conductance regulator.

Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP-dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.     

The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATP-driven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA). The R domain has multiple excitatory phosphorylation sites, but Ser(737) and Ser(768) are inhibitory. The underlying mechanism is unclear. Here, sulfhydryl-specific cross-linking strategy was employed to demonstrate that Ser(768) or Ser(737) could interact with outwardly facing hydrophilic residues of cytoplasmic loop 3 regulating channel gating. Furthermore, mutation of these residues to alanines promoted channel opening by curcumin in an ATP-dependent manner even in the absence of PKA. However, mutation of Ser(768) and His(950) with different hydrogen bond donors or acceptors clearly changed ATP- and PKA-dependent channel activity no matter whether curcumin was present or not. More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Finally, in vitro and in vivo single channel recordings suggest that Ser(768) may form a putative hydrogen bond with His(950) of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating.     

Identification of protein kinase A phosphorylation sites on NBD1 and R domains of CFTR using electrospray mass spectrometry with selective phosphate ion monitoring.

HPLC-electrospray mass spectrometry was used to identify the phosphorylated sites on a bacterially expressed cystic fibrosis transmembrane conductance regulator (CFTR) fragment containing the first nucleotide binding domain (NBD1) and the regulatory domain (R). Tryptic digests of NBD1-R (CFTR residues 404-830) were analyzed after protein kinase A (PKA) treatment for all possible peptides and phosphopeptides (a total of 118 species) containing Ser residues within "high-probability" PKA consensus sequences: R-R/K-X-S/T, R-X-X-S/T, and R-X-S/T. Three criteria were used to assign phosphorylated sites: (1) an 80-Da increase in the predicted average molecular weight of the tryptic peptides; (2) co-elution with the PO3- ion induced by stepped energy collision; and (3) the relative elution positions of the phosphorylated and unmodified peptides. Ser residues within the eight dibasic sites in the NBD1 and R domains (positions 422, 660, 700, 712, 737, 768, 795, and 813) were phosphorylated, a pattern similar to that observed for full-length CFTR. The serine at position 753, which in CFTR is phosphorylated in vivo, was not phosphorylated. The remaining potential PKA sites, Ser489, Ser519, Ser557, Ser670, and Thr788, were not phosphorylated. The "low-probability" PKA sites (those not containing an Arg residue) were not phosphorylated. The results suggest that isolated domains of CFTR developed useful models for investigating the biochemical and structural effects of phosphorylation within CFTR. The mass spectrometry approach in this study should prove useful for defining phosphorylation sites of CFTR in vitro and in vivo.     

Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry.

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes an apical membrane Cl- channel regulated by protein phosphorylation. To identify cAMP-dependent protein kinase (PKA)-phosphorylated residues in full-length CFTR, immobilized metal-ion affinity chromatography (IMAC) was used to selectively purify phosphopeptides. The greater specificity of iron-loaded (Fe3+) nitrilotriacetic (NTA). Sepharose compared to iminodiacetic acid (IDA) metal-chelating matrices was demonstrated using a PKA-phosphorylated recombinant NBD1-R protein from CFTR. Fe(3+)-loaded NTA Sepharose preferentially bound phosphopeptides, whereas acidic and poly-His-containing peptides were co-purified using the conventional IDA matrices. IMAC using NTA Sepharose enabled the selective recovery of phosphopeptides and identification of phosphorylated residues from a complex proteolytic digest. Phosphopeptides from PKA-phosphorylated full-length CFTR, generated in Hi5 insect cells using a baculovirus expression system, were purified using NTA Sepharose. Phosphopeptides were identified using matrix-assisted laser desorption mass spectrometry (MALDI/MS) with post-source decay (PSD) analysis and collision-induced dissociation (CID) experiments. Phosphorylated peptides were identified by mass and by the metastable loss of HPO3 and H3PO4 from the parent ions. Peptide sequence and phosphorylation at CFTR residues 660Ser, 737Ser, and 795Ser were confirmed using MALDI/PSD analysis. Peptide sequences and phosphorylation at CFTR residues 700Ser, 712Ser, 768Ser, and 813Ser were deduced from peptide mass, metastable fragment ion formation, and PKA consensus sequences. Peptide sequence and phosphorylation at residue 753Ser was confirmed using MALDI/CID analysis. This is the first report of phosphorylation of 753Ser in full-length CFTR.     

Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia

The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl(-) current. Mutation of four in vivo phosphorylation sites, Ser(660), Ser(737), Ser(795), and Ser(813) (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser(660) or Ser(813) alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser(660) nor Ser(813) alone increased current to wild-type levels; both residues were required. Changing Ser(737) to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser(737) may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.     

Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel.

CFTR, the protein associated with cystic fibrosis, is phosphorylated on serine residues in response to cAMP agonists. Serines 660, 737, 795, and 813 were identified as in vivo targets for phosphorylation by protein kinase A. The SPQ fluorescence assay revealed that mutagenesis of any one of these sites did not affect Cl- channel activity. Indeed, concomitant mutagenesis of three of the four sites still resulted in cAMP-responsive Cl- channel activity. However, mutagenesis of all four sites abolished the response. One interpretation of these results is that the CFTR Cl- channel is blocked by the R domain and that phosphorylation on serines by protein kinase A electrostatically repels the domain, allowing passage of Cl-. The four phosphorylation events appear to be degenerate: no one site is essential for channel activity, and, at least in the case of serine 660, phosphorylation at one site alone is sufficient for regulation of Cl- channel activity.     

Computational studies reveal phosphorylation-dependent changes in the unstructured R domain of CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel that is mutated in cystic fibrosis, an inherited disease of high morbidity and mortality. The phosphorylation of its ∼ 200 amino acid R domain by protein kinase A is obligatory for channel gating under normal conditions. The R domain contains more than ten PKA phosphorylation sites. No individual site is essential but phosphorylation of increasing numbers of sites enables progressively greater channel activity. In spite of numerous studies of the role of the R domain in CFTR regulation, its mechanism of action remains largely unknown. This is because neither its structure nor its interactions with other parts of CFTR have been completely elucidated. Studies have shown that the R domain lacks well-defined secondary structural elements and is an intrinsically disordered region of the channel protein. Here, we have analyzed the disorder pattern and employed computational methods to explore low-energy conformations of the R domain. The specific disorder and secondary structure patterns detected suggest the presence of molecular recognition elements (MoREs) that may mediate phosphorylation-regulated intra- and inter-domain interactions. Simulations were performed to generate an ensemble of accessible R domain conformations. Although the calculated structures may represent more compact conformers than occur in vivo, their secondary structure propensities are consistent with predictions and published experimental data. Equilibrium simulations of a mimic of a phosphorylated R domain showed that it exhibited an increased radius of gyration. In one possible interpretation of these findings, by changing its size, the globally unstructured R domain may act as an entropic spring to perturb the packing of membrane-spanning sequences that constitute the ion permeability pathway and thereby activate channel gating.     

Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C

We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.     

PKC-mediated stimulation of amphibian CFTR depends on a single phosphorylation consensus site. insertion of this site confers PKC sensitivity to human CFTR

Mutations of the CFTR, a phosphorylation-regulated Cl(-) channel, cause cystic fibrosis. Activation of CFTR by PKA stimulation appears to be mediated by a complex interaction between several consensus phosphorylation sites in the regulatory domain (R domain). None of these sites has a critical role in this process. Here, we show that although endogenous phosphorylation by PKC is required for the effect of PKA on CFTR, stimulation of PKC by itself has only a minor effect on human CFTR. In contrast, CFTR from the amphibians Necturus maculosus and Xenopus laevis (XCFTR) can be activated to similar degrees by stimulation of either PKA or PKC. Furthermore, the activation of XCFTR by PKC is independent of the net charge of the R domain, and mutagenesis experiments indicate that a single site (Thr665) is required for the activation of XCFTR. Human CFTR lacks the PKC phosphorylation consensus site that includes Thr665, but insertion of an equivalent site results in a large activation upon PKC stimulation. These observations establish the presence of a novel mechanism of activation of CFTR by phosphorylation of the R domain, i.e., activation by PKC requires a single consensus phosphorylation site and is unrelated to the net charge of the R domain.     

Role of Tyrosine Phosphorylation in the Muscarinic Activation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl⁻) channel, which plays an important role in physiological anion and fluid secretion, and is defective in several diseases. Although its activation by PKA and PKC has been studied extensively, its regulation by receptors is less well understood. To study signaling involved in CFTR activation, we measured whole-cell Cl⁻ currents in BHK cells cotransfected with GPCRs and CFTR. In cells expressing the M3 muscarinic acetylcholine receptor, the agonist carbachol (Cch) caused strong activation of CFTR through two pathways; the canonical PKA-dependent mechanism and a second mechanism that involves tyrosine phosphorylation. The role of PKA was suggested by partial inhibition of cholinergic stimulation by the specific PKA inhibitor Rp-cAMPS. The role of tyrosine kinases was suggested by Cch stimulation of 15SA-CFTR and 9CA-CFTR, mutants that lack 15 PKA or 9 PKC consensus sequences and are unresponsive to PKA or PKC stimulation, respectively. Moreover the residual Cch response was sensitive to inhibitors of the Pyk2 and Src tyrosine kinase family. Our results suggest that tyrosine phosphorylation acts on CFTR directly and through inhibition of the phosphatase PP2A. Results suggest that PKA and tyrosine kinases contribute to CFTR regulation by GPCRs that are expressed at the apical membrane of intestinal and airway epithelia.     

A short segment of the R domain of cystic fibrosis transmembrane conductance regulator contains channel stimulatory and inhibitory activities that are separable by sequence modification

The regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) contains consensus phosphorylation sites for cAMP-dependent protein kinase (PKA) that are the basis for physiological regulation of the CFTR chloride channel. A short peptide segment in the R domain with a net negative charge of B9 (amino acids 817-838, NEG2) and predicted helical tendency is shown to play a critical role in CFTR chloride channel function. Deletion of NEG2 from CFTR completely eliminates the PKA dependence of channel activity. Exogenous NEG2 peptide interacts with CFTR to exert both stimulatory and inhibitory effects on the channel function. The NEG2 peptide with sequence scrambled to remove helical tendencies also inhibits channel function, but does not stimulate. Similar results are found for a NEG2 peptide whose helical structure is disrupted by a proline residue. When six of the negatively charged carboxylic acid residues are replaced by their cognate amides, reducing net negative charge to B3, but increasing helical propensity as assessed by circular dichroism, the peptide stimulates CFTR channel function, but does not inhibit. We speculate that the NEG2 region interacts with other cytosolic domains of CFTR to control opening and closing transitions of the chloride channel.     

Mechanism of activation of Xenopus CFTR by stimulation of PKC

PKA-mediated phosphorylation of the regulatory (R) domain plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA, and dependent on the cell type. In the present study, we expressed Xenopus CFTR (XCFTR) and hCFTR in Xenopus oocytes and examined their responses (i.e., macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately sixfold that of PKA stimulation. In contrast, with hCFTR, the response to PKC was 90% of the response to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted cysteine accessibility method, we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately fivefold increase in single-channel open probability, with a minor (30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in P_o. In both instances, there were no changes in the number of channels in the membrane. We speculate that in animals other than humans, PKC stimulation may be the dominant mechanism for activation of CFTR. chloride channel; channel regulation; cystic fibrosis transmembrane conductance regulator gating; cystic fibrosis; phosphorylation; protein kinase A     

Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3

NEG2, a short C-terminal segment (817–838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, Lys-946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of Asp-835, Asp-836, or Glu-838 of NEG2 to prevent the channel activation by PKA. Arg-764 or Arg-766 of the Ser-768 phosphorylation site of the R domain is proposed to promote the channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A, and E838A but also K946A reduced the PKA-dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. However, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity whereas D835R/D836R/E838R/K946D/H950D was fractionally misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing, and Ser-768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2, and the Ser-768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.     

CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices

The regulatory (R) region of the cystic fibrosis transmembrane conductance regulator (CFTR) is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the nucleotide-binding domains (NBDs) of CFTR are required for channel gating. We report NMR studies in the absence and presence of NBD1 that provide structural details for the isolated R region and its interaction with NBD1 at residue-level resolution. Several sites in the R region with measured fractional helical propensity mediate interactions with NBD1. Phosphorylation reduces the helicity of many R-region sites and reduces their NBD1 interactions. This evidence for a dynamic complex with NBD1 that transiently engages different sites of the R region suggests a structural explanation for the dependence of CFTR activity on multiple PKA phosphorylation sites.     

Functional roles of nonconserved structural segments in CFTR's NH2-terminal nucleotide binding domain

The cystic fibrosis transmembrane conductance regulator (CFTR), encoded by the gene mutated in cystic fibrosis patients, belongs to the family of ATP-binding cassette (ABC) proteins, but, unlike other members, functions as a chloride channel. CFTR is activated by protein kinase A (PKA)-mediated phosphorylation of multiple sites in its regulatory domain, and gated by binding and hydrolysis of ATP at its two nucleotide binding domains (NBD1, NBD2). The recent crystal structure of NBD1 from mouse CFTR (Lewis, H.A., S.G. Buchanan, S.K. Burley, K. Conners, M. Dickey, M. Dorwart, R. Fowler, X. Gao, W.B. Guggino, W.A. Hendrickson, et al. 2004. EMBO J. 23:282-293) identified two regions absent from structures of all other NBDs determined so far, a "regulatory insertion" (residues 404-435) and a "regulatory extension" (residues 639-670), both positioned to impede formation of the putative NBD1-NBD2 dimer anticipated to occur during channel gating; as both segments appeared highly mobile and both contained consensus PKA sites (serine 422, and serines 660 and 670, respectively), it was suggested that their phosphorylation-linked conformational changes might underlie CFTR channel regulation. To test that suggestion, we coexpressed in Xenopus oocytes CFTR residues 1-414 with residues 433-1480, or residues 1-633 with 668-1480, to yield split CFTR channels (called 414+433 and 633+668) that lack most of the insertion, or extension, respectively. In excised patches, regulation of the resulting CFTR channels by PKA and by ATP was largely normal. Both 414+433 channels and 633+668 channels, as well as 633(S422A)+668 channels (lacking both the extension and the sole PKA consensus site in the insertion), were all shut during exposure to MgATP before addition of PKA, but activated like wild type (WT) upon phosphorylation; this indicates that inhibitory regulation of nonphosphorylated WT channels depends upon neither segment. Detailed kinetic analysis of 414+433 channels revealed intact ATP dependence of single-channel gating kinetics, but slightly shortened open bursts and faster closing from the locked-open state (elicited by ATP plus pyrophosphate or ATP plus AMPPNP). In contrast, 633+668 channel function was indistinguishable from WT at both macroscopic and microscopic levels. We conclude that neither nonconserved segment is an essential element of PKA- or nucleotide-dependent regulation.     

Conformation, independent of charge, in the R domain affects cystic fibrosis transmembrane conductance regulator channel openings

The R domain of cystic fibrosis transmembrane conductance regulator (CFTR), when phosphorylated, undergoes conformational change, and the chloride channel opens. We investigated the contribution of R domain conformation, apart from the changes induced by phosphorylation, to channel opening, by testing the effect of the peptidyl-prolyl isomerase, cyclophilin A, on the CFTR channel. When it was applied after the channel had been opened by PKA phosphorylation, cyclophilin A increased the open probability of wild-type CFTR (from P(o) = 0.197 +/- 0.010 to P(o) = 0.436 +/- 0. 029) by increasing the number of channel openings, not open time. Three highly conserved proline residues in the R domain, at positions 740, 750, and 759, were considered as candidate targets for cyclophilin A. Mutations of these prolines to alanines (P3A mutant) resulted in a channel unresponsive to cyclophilin A but with pore properties similar to the wild type, under strict control of PKA and ATP, but with significantly increased open probability (P(o) = 0.577 +/- 0.090) compared to wild-type CFTR, again due to an increase in the number of channel openings and not open time. Mutation of each of the proline residues separately and in pairs demonstrated that all three proline mutations are required for maximal P(o). When P3A was expressed in 293 HEK cells and tested by SPQ assay, chloride efflux was significantly increased compared to cells transfected with wild-type CFTR. Thus, treatments favoring the trans-peptidyl conformation about conserved proline residues in the R domain of CFTR affect openings of CFTR, above and beyond the effect of PKA phosphorylation.     

Defective formation of PKA/CnA-dependent annexin 2-S100A10/CFTR complex in DeltaF508 cystic fibrosis cells

Cystic fibrosis (CF) is characterised by impaired epithelial ion transport and is caused by mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/PKA and ATP-regulated chloride channel. We recently demonstrated a cAMP/PKA/calcineurin (CnA)-driven association between annexin 2 (anx 2), its cognate partner –S100A10 and cell surface CFTR. The complex is required for CFTR and outwardly rectifying chloride channel function in epithelia. Since the cAMP/PKA-induced Cl⁻ current is absent in CF epithelia, we hypothesized that the anx 2–S100A10/CFTR complex may be defective in CFBE41o cells expressing the commonest F508del-CFTR (ΔF-CFTR) mutation. Here, we demonstrate that, despite the presence of cell surface ΔF-CFTR, cAMP/PKA fails to induce anx 2–S100A10/CFTR complex formation in CFBE41o− cells homozygous for F508del-CFTR. Mechanistically, PKA-dependent serine phosphorylation of CnA, CnA–anx 2 complex formation and CnA-dependent dephosphorylation of anx 2 are all defective in CFBE41o− cells. Immunohistochemical analysis confirms an abnormal cellular distribution of anx 2 in human and CF mouse epithelia.

Thus, we demonstrate that cAMP/PKA/CnA signaling pathway is defective in CF cells and suggest that loss of anx 2–S100A10/CFTR complex formation may contribute to defective cAMP/PKA-dependent CFTR channel function.     

Interaction of the protein phosphatase 2A with the regulatory domain of the cystic fibrosis transmembrane conductance regulator channel

A direct interaction of the regulatory domain (R domain) of the cystic fibrosis transmembrane conductance regulator protein (CFTR) with PR65, a regulatory subunit of the protein phosphatase 2A (PP2A), was shown in yeast two hybrid, pull-down and co-immunoprecipitation experiments. The R domain could be dephosphorylated by PP2A in vitro. Overexpression of the interacting domain of PR65 in Caco-2 cells, as well as treatment with okadaic acid, showed a prolonged deactivation of the chloride channel. Taken together our results show a direct and functional interaction between CFTR and PP2A.     

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.The cystic fibrosis transmembrane regulator (CFTR)² gene is mutated in patients with cystic fibrosis. CFTR has an adapted ABC transporter structural motif thereby creating an anion channel at the apical surface of secretory epithelia (1). The consequent CFTR-mediated ion transport is tightly controlled by ATP binding and phosphorylation by protein kinase A (PKA). However, a number of other protein kinases including PKC, Ca²⁺/calmodulin-dependent kinase, and cGMP-dependent kinase also control the activity of CFTR (2–4). These kinases converge on the regulatory domain of CFTR that is unique not only within the large ABC transporter family but among all known sequences, and may be considered as a “phosphorylation control module” (3). Regulation of CFTR by an inhibitory kinase, the adenosine monophosphate-dependent kinase (AMPK), has been described recently but the regulatory sites within CFTR, the mechanism of regulation, and the physiological relevance have all remained obscure (5–8). Additionally, CFTR mutation is linked to inflammation and a lack of functional CFTR expression has itself been suggested to up-regulate AMPK activity in epithelial cells carrying the cystic fibrosis (CF) defect. Pharmacologic AMPK activation was shown to inhibit secretion of inflammatory mediators (9). Thus AMPK may play multiple roles in CF pathophysiology making the mechanism of interaction an important problem in biology.AMPK is a ubiquitous serine/threonine kinase that exists as a heterotrimer with a catalytic α subunit and regulatory β and γ subunits, each with multiple isoforms. In response to metabolic depletion and a consequent increase in the cellular AMP to ATP ratio, AMPK phosphorylates numerous proteins and activates catabolic pathways that generate ATP, whereas inhibiting cell growth, protein biosynthesis, and a number of other ATP-consuming processes, thereby operating as a cellular “low-fuel” sensor (10, 11). AMPK also controls signaling pathways involved in apoptosis, cell cycle, and tissue inflammation (12). Because AMPK is a cellular metabolic sensor that inhibits CFTR and limits cAMP activated Cl^– secretion, a coupling of membrane transport by CFTR to the cellular metabolism has been proposed (13). However, AMPK activity can also increase without detectable changes in the cytosolic AMP to ATP ratio, suggesting a contribution of additional AMP-independent signals for regulation of CFTR by AMPK (14). Drugs used to combat type 2 diabetes, such as phenformin and metformin, act in this manner to activate AMPK, AMP-independently. It is also likely that cytosolic AMP is compartmentalized depending on the distribution of AMP generating enzymes such as phosphodiesterases that convert cAMP to AMP. The concept of spatiotemporal control of cAMP signaling by anchored protein complexes is well established (15). CFTR is known to form such macromolecular complexes with a number of interacting partners (16–18). For example, competitive interaction of EBP50-PKA and Shank2-PDE4D with CFTR has been demonstrated recently (19). In addition, Barnes and co-workers (20) demonstrated that phosphodiesterase 4D generates a cAMP diffusion barrier local to the apical membrane of the airway epithelium. It is therefore likely that activator pathways through cAMP and inhibitory AMP/AMPK signaling occur in a local CFTR-organized compartment. Here we explore the functional links between CFTR, inhibition of phosphodiesterases, and AMPK focusing on the effects of mutating putative AMPK targets within the R domain on CFTR function.