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• W2034143917 abstract "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. 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. Cystic fibrosis (CF) 4The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; NBD1, nucleotide-binding domain; TBB, 4,5,6,7-tetrabromobenzotriazole; BHK, baby hamster kidney; NMDG, N-methyl-d-glucamine; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; PBS, phosphate-buffered saline; DPC, diphenylamine 2-carboxylate; AMPK, AMP-activated kinase; NDPK, nucleoside diphosphate kinase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; HBE, human bronchial epithelial.4The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; NBD1, nucleotide-binding domain; TBB, 4,5,6,7-tetrabromobenzotriazole; BHK, baby hamster kidney; NMDG, N-methyl-d-glucamine; PKC, protein kinase C; PKA, cAMP-dependent protein kinase; PBS, phosphate-buffered saline; DPC, diphenylamine 2-carboxylate; AMPK, AMP-activated kinase; NDPK, nucleoside diphosphate kinase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid; MES, 4-morpholineethanesulfonic acid; HBE, human bronchial epithelial. is a common autosomal recessive multisystem disease resulting from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) (1Kerem B. Rommens J.M. Buchanan J.A. Markiewicz D. Cox T.K. Chakravarti A. Buchwald M. Tsui L.C. Science. 1989; 245: 1073-1080Crossref PubMed Scopus (3140) Google Scholar). The molecular mechanisms that control CFTR function are complex and incompletely understood (2Riordan J.R. Annu. Rev. Physiol. 2005; 67: 701-718Crossref PubMed Scopus (190) Google Scholar). CFTR belongs to the ATP-binding cassette family of transmembrane pumps involved in ATP-driven substrate transport. Uniquely among ATP-binding cassette proteins, CFTR is an ion channel, but from the transport characteristics of other chloride channels it remains unclear why CFTR requires two ATP-binding domains if passive chloride transport is its sole function. Deletion of F508 (ΔF508) from the first nucleotide-binding domain (NBD1) of CFTR is by far the commonest pathogenic mutation (80-90% of alleles) and induces a multisystem disease (3Welsh M.J. Ramsey B.W. Accurso F. Cutting G.R. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill Inc., New York2001: 5121-5188Google Scholar). The resultant clinical features are difficult to reconcile with defects in ion transport alone (4Tabary O. Escotte S. Couetil J.P. Hubert D. Dusser D. Puchelle E. Jacquot J. J. Immunol. 2000; 164: 3377-3384Crossref PubMed Scopus (86) Google Scholar, 5Tirouvanziam R. de Bentzmann S. Hubeau C. Hinnrasky J. Jacquot J. Peault B. Puchelle E. Am. J. Respir. Cell Mol. Biol. 2000; 23: 121-127Crossref PubMed Scopus (215) Google Scholar, 6Tirouvanziam R. Khazaal I. Peault B. Am. J. Physiol. 2002; 283: L445-L451Crossref PubMed Scopus (72) Google Scholar, 7Mehta A. Pediatr. Pulmonol. 2005; 39: 292-298Crossref PubMed Scopus (72) Google Scholar) because ΔF508-CFTR perturbs inflammation, cell metabolism and multiple ion-channel physiology. ΔF508 attenuates CFTR biosynthesis (8Cheng S.H. Gregory R.J. Marshall J. Paul S. Souza D.W. White G.A. O'Riordan C.R. Smith A.E. Cell. 1990; 63: 827-834Abstract Full Text PDF PubMed Scopus (1398) Google Scholar), cell surface expression (9Lukacs G.L. Chang X.B. Bear C. Kartner N. Mohamed A. Riordan J.R. Grinstein S. J. Biol. Chem. 1993; 268: 21592-21598Abstract Full Text PDF PubMed Google Scholar), and channel gating (10Dalemans W. Barbry P. Champigny G. Jallat S. Dott K. Dreyer D. Crystal R.G. Pavirani A. Lecocq J.P. Lazdunski M. Nature. 1991; 354: 526-528Crossref PubMed Scopus (553) Google Scholar), but it remains unknown how loss of F508 leads to defective gating because the F508 residue lies remote from both the channel pore and the site of ATP binding on NBD1 (11Sheppard D.N. Welsh M.J. Physiol. Rev. 1999; 79: S23-S45Crossref PubMed Scopus (782) Google Scholar, 12Lewis H.A. Zhao X. Wang C. Sauder J.M. Rooney I. Noland B.W. Lorimer D. Kearins M.C. Conners K. Condon B. Maloney P.C. Guggino W.B. Hunt J.F. Emtage S. J. Biol. Chem. 2005; 280: 1346-1353Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 13Lewis H.A. Buchanan S.G. Burley S.K. Conners K. Dickey M. Dorwart M. Fowler R. Gao X. Guggino W.B. Hendrickson W.A. Hunt J.F. Kearins M.C. Lorimer D. Maloney P.C. Post K.W. Rajashankar K.R. Rutter M.E. Sauder J.M. Shriver S. Thibodeau P.H. Thomas P.J. Zhang M. Zhao X. Emtage S. EMBO J. 2004; 23: 282-293Crossref PubMed Scopus (329) Google Scholar). Neither is it established how ΔF508 CFTR alters the function of unrelated proteins, including other epithelial ion channels (14Stutts M.J. Canessa C.M. Olsen J.C. Hamrick M. Cohn J.A. Rossier B.C. Boucher R.C. Science. 1995; 269: 847-850Crossref PubMed Scopus (948) Google Scholar). CFTR is part of a macromolecular complex in the apical membrane of epithelia (2Riordan J.R. Annu. Rev. Physiol. 2005; 67: 701-718Crossref PubMed Scopus (190) Google Scholar) comprising (among others) a number of protein kinases (see below), syntaxins, ezrinbinding phosphoprotein 50 (15Short D.B. Trotter K.W. Reczek D. Kreda S.M. Bretscher A. Boucher R.C. Stutts M.J. Milgram S.L. J. Biol. Chem. 1998; 273: 19797-19801Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar), and CAP70 (16Wang S. Raab R.W. Schatz P.J. Guggino W.B. Li M. FEBS Lett. 1998; 427: 103-108Crossref PubMed Scopus (247) Google Scholar). Provided ATP is available for NBD binding, CFTR channel function is activated by protein kinases such as PKA and PKC (17Zhu T. Hinkson D.A. Dahan D. Evagelidis A. Hanrahan J.W. Methods Mol. Med. 2002; 70: 99-109PubMed Google Scholar) but is inhibited by AMP-activated kinase (AMPK) (18Hallows K.R. McCane J.E. Kemp B.E. Witters L.A. Foskett J.K. J. Biol. Chem. 2003; 278: 998-1004Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 19Hallows K.R. Raghuram V. Kemp B.E. Witters L.A. Foskett J.K. J. Clin. Investig. 2000; 105: 1711-1721Crossref PubMed Scopus (178) Google Scholar). CFTR also binds nucleoside diphosphate kinase (NDPK), which regulates AMPK without the need for AMP (20Crawford R.M. Treharne K.J. Arnaud-Dabernat S. Daniel J.Y. Foretz M. Voillet B. Mehta A. FASEB J. 2007; 21: 88-98Crossref PubMed Google Scholar, 21Crawford III, R.M. Treharne K.J. Arnaud-Dabernat S. Daniel J.Y. Foretz M. Voillet B. Mehta A. Mol. Cell. Biol. 2006; 26: 5921-5931Crossref PubMed Scopus (13) Google Scholar, 22Crawford R.M. Treharne K.J. Best O.G. Muimo R. Riemen C.E. Mehta A. Biochem. J. 2005; 392: 201-209Crossref PubMed Scopus (18) Google Scholar). We observed that the crystal structure for the NBD1 domain shows that F508 is located on the surface of NBD1 where it is accessible for protein-protein interactions (12Lewis H.A. Zhao X. Wang C. Sauder J.M. Rooney I. Noland B.W. Lorimer D. Kearins M.C. Conners K. Condon B. Maloney P.C. Guggino W.B. Hunt J.F. Emtage S. J. Biol. Chem. 2005; 280: 1346-1353Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 13Lewis H.A. Buchanan S.G. Burley S.K. Conners K. Dickey M. Dorwart M. Fowler R. Gao X. Guggino W.B. Hendrickson W.A. Hunt J.F. Kearins M.C. Lorimer D. Maloney P.C. Post K.W. Rajashankar K.R. Rutter M.E. Sauder J.M. Shriver S. Thibodeau P.H. Thomas P.J. Zhang M. Zhao X. Emtage S. EMBO J. 2004; 23: 282-293Crossref PubMed Scopus (329) Google Scholar, 23Thibodeau P.H. Brautigam C.A. Machius M. Thomas P.J. Nat. Struct. Mol. Biol. 2005; 12: 10-16Crossref PubMed Scopus (146) Google Scholar). However, no such molecular interactions have yet been assigned to the F508 residue or its adjacent region in wild-type CFTR, and this report investigates whether regulatory proteins interact with this region in a F508-dependent manner. We hypothesized that an explanation for the multisystem nature of CF may reside in differences between proteins bound to wild-type and ΔF508 CFTR. The amino acid sequence of CFTR adjacent to F508 (bold-face) contains a consensus sequence (KENIIFGVSYDEYR) for phosphorylation by protein kinase CK2 (formerly known as casein kinase 2) (24Meggio F. Marin O. Pinna L.A. Cell. Mol. Biol. Res. 1994; 40: 401-409PubMed Google Scholar), with a potential target serine located at serine 511 (italic underline). This protein kinase has many unusual features and a diverse range of targets making it an attractive candidate for study in CF cells (25Wang H. Davis A. Yu S. Ahmed K. Mol. Cell. Biochem. 2001; 227: 167-174Crossref PubMed Scopus (84) Google Scholar, 26Litchfield D.W. Biochem. J. 2003; 369: 1-15Crossref PubMed Scopus (1004) Google Scholar, 27Meggio F. Pinna L.A. FASEB J. 2003; 17: 349-368Crossref PubMed Scopus (1090) Google Scholar). For example, CK2 inhibits NDPK, and NDPK not only binds CFTR but also controls AMPK (20Crawford R.M. Treharne K.J. Arnaud-Dabernat S. Daniel J.Y. Foretz M. Voillet B. Mehta A. FASEB J. 2007; 21: 88-98Crossref PubMed Google Scholar, 21Crawford III, R.M. Treharne K.J. Arnaud-Dabernat S. Daniel J.Y. Foretz M. Voillet B. Mehta A. Mol. Cell. Biol. 2006; 26: 5921-5931Crossref PubMed Scopus (13) Google Scholar, 22Crawford R.M. Treharne K.J. Best O.G. Muimo R. Riemen C.E. Mehta A. Biochem. J. 2005; 392: 201-209Crossref PubMed Scopus (18) Google Scholar). CK2 is a heterotetramer containing two 47-kDa α subunits (catalytic) and two 26-kDa β subunits (regulatory) that targets over 300 proteins linking its promiscuous activity to essential cellular functions (27Meggio F. Pinna L.A. FASEB J. 2003; 17: 349-368Crossref PubMed Scopus (1090) Google Scholar). CK2 can also use either ATP or GTP as the phosphate donor for kinase activity toward multiple substrates thus adding to the complexity (27Meggio F. Pinna L.A. FASEB J. 2003; 17: 349-368Crossref PubMed Scopus (1090) Google Scholar). Unlike most protein kinases, CK2 is often described as constitutively active, but CK2 function may nevertheless be regulated, first by restricting its subcellular localization and second by modification of the regulatory interactions between the α and β subunits. Specific inhibitors are correspondingly either directed toward the nucleotide-binding α subunit (exploiting its unique structure) or to the site of polyamine binding on the acidic groove in the regulatory β subunit. We investigated whether CK2 interacts functionally with the F508 region of CFTR and whether deletion of F508 disrupts binding of this signaling molecule using immunofluorescence, inhibitor studies, electrophysiology, and site-directed mutagenesis of Ser-511 in CFTR. Cell Culture—16HBE14o- and CFBE41o-cells were cultured to confluence in medium M199 containing 10% fetal calf serum, 2 mml-glutamine, 5% antibiotics, and 5% Fungizone. Calu-3, Hep-G2, T84, and BHK cells were cultured in Dulbec-co's modified Eagle's medium with the same supplements. Ussing Chambers—After mounting Calu-3 epithelia (Rt > 200 Ω; where Rt is transepithelial resistance) in Ussing chambers, basolateral membranes were permeabilized with amphotericin B (10 μm) and an outwardly directed Cl- gradient imposed as detailed previously (28Collett A. Ramminger S.J. Olver R.E. Wilson S.M. Am. J. Physiol. 2002; 282: L621-L630Google Scholar). Cl- current was stimulated with 10 μm forskolin, and 4,5,6,7-tetrabromo-benzotriazole (TBB) or diphenylamine 2-carboxylate (DPC) was added as indicated. Single Channel Patch Clamp Studies—CFTR Cl- channels were recorded in either cell-attached or excised inside-out membrane patches. For cell-attached recordings, the pipette solution contained the following: 140 mm N-methyl-d-glucamine (NMDG), 3 mm MgCl2, 10 mm TES, and 1 mm cesium EGTA, pH 7.3, with HCl ([Cl-], 147 mm). The bath solution contained the following: 137 mm NaCl, 4 mm KCl, 3 mm MgCl2, and 10 mm TES, pH 7.3, with NaOH ([Cl-], 147 mm) and was maintained at 37 °C; pipette potential was +50 mV. For excised recordings, the pipette (extracellular) solution contained the following: 140 mm NMDG, 140 mm aspartic acid, 5 mm CaCl2, 2 mm MgSO4, and 10 mm TES, pH 7.3, with Tris ([Cl-], 10 mm). The bath (intracellular) solution contained the following: 140 mm NMDG, 3 mm MgCl2, 1 mm cesium EGTA, and 10 mm TES, pH 7, with HCl ([Cl-], 147 mm; [Ca2+]free, <10-8m), and was maintained at 37 °C; voltage was -50 mV. In cell-attached recordings, CFTR Cl- channels were activated with forskolin (20 μm) and in excised inside-out membrane patches by PKA (75 nm) and ATP (1 mm). CFTR Cl- channels were filtered at 500 Hz, digitized at 5 kHz, and digitally filtered at 100 Hz. To measure i, Gaussian curves were fit to current amplitude histograms. To measure Po, we created lists of open and closed times and calculated Po as described (29Scott-Ward T.S. Li H. Schmidt A. Cai Z. Sheppard D.N. Mol. Membr. Biol. 2004; 21: 27-38Crossref PubMed Scopus (40) Google Scholar). The number of channels in a membrane patch was determined from the maximum number of simultaneous channel openings observed during an experiment (29Scott-Ward T.S. Li H. Schmidt A. Cai Z. Sheppard D.N. Mol. Membr. Biol. 2004; 21: 27-38Crossref PubMed Scopus (40) Google Scholar). Xenopus Oocyte Studies—Oocytes were isolated and microinjected as described (30Mall M. Hipper A. Greger R. Kunzelmann K. FEBS Lett. 1996; 381: 47-52Crossref PubMed Scopus (132) Google Scholar). In brief, cRNAs for CFTR, cDNAs encoding human wt-CFTR or ΔF508-CFTR were linearized in pBluescript or pTLN with NotI and in vitro transcribed using T7, T3, or SP6 promotor and polymerase (Promega). After isolation from adult Xenopus laevis frogs (Horst Kähler, Hamburg, Germany), oocytes were dispersed and defolliculated with collagenase (45 min, type A; Roche Applied Science). Subsequently, oocytes were rinsed and stored at 18 °C in ND96 buffer as follows: 96 mm NaCl, 2 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 5mm HEPES, sodium pyruvate 2.5, pH 7.55, supplemented with theophylline (0.5 mm) and gentamicin (5 mg/liter). Two-electrode Voltage-clamp—Oocytes were injected with cRNA (1-10 ng) in 47 nl of double-distilled water (Nanoliter Injector WPI, Germany). Water-injected oocytes served as controls. 2-4 days after injection, oocytes were impaled with two electrodes (Harvard Apparatus), which had resistances of <1 megohms when filled with 2.7 mol/liter KCl. Using two bath electrodes and a virtual ground head stage, the voltage drop across Rserial was effectively zero. Membrane currents were measured by voltage-clamping the oocytes (Warner oocyte clamp amplifier OC725C) in intervals from -90 to +30 mV, in steps of 10 mV, each at 1 s. Conductances were calculated using Ohm's law. Oocytes were perfused continuously with physiological solutions at a rate of 5-10 ml/min. All experiments were conducted at room temperature (22 °C). For the poly(E:Y) peptide, 43 nl of poly(E:Y) peptide (Sigma) was injected into CFTR-expressing oocytes reaching a final concentration of 10-20 μm. Currents were assessed 3-24 h after injection. For the KENIIF/KENII peptide, peptides were injected into CFTR-expressing oocytes to a final concentration of 100 nm. Currents were assessed 20 h after injection. Immunofluorescence—Nasal ciliated epithelial cells harvested from the inferior turbinate of patients undergoing unrelated surgery (approved by local ethical committee) were maintained in cell culture medium M199 prior to fixation in 4% paraformaldehyde. Cells were permeabilized using 1% Triton X-100, washed three times in PBS, and then blocked in 1 mm glycine for 15 min, followed by 5% donkey serum for 15 min. Pelleted cells were resuspended in PBS containing primary antibodies (goat anti-CK2α (Santa Cruz Biotechnology) and mouse anti-CFTR NBD1 (Neomarkers) at a 1:100 dilution) and incubated at room temperature, with shaking, overnight. After three washes in PBS, pelleted cells were resuspended in PBS containing fluorescein isothiocyanate-labeled anti-goat and rhodamine-labeled anti-mouse IgG secondary antibodies (1:100; Jackson ImmunoResearch). After a 2-h incubation, with shaking, the cells were washed five times in PBS and resuspended in 15 μl of anti-fade mountant (6% n-propyl gallate in 70% glycerol, 100 mm Tris/HCl, pH 7.4) for mounting on glass slides. Coverslips were sealed with nail varnish for image capture using a Zeiss 510 laser scanning confocal microscope. Immunoblotting—Immunoblotting was carried out as described previously (21Crawford III, R.M. Treharne K.J. Arnaud-Dabernat S. Daniel J.Y. Foretz M. Voillet B. Mehta A. Mol. Cell. Biol. 2006; 26: 5921-5931Crossref PubMed Scopus (13) Google Scholar). Briefly, blotted membranes were blocked for 30 min in TBS/Tween with 5% milk powder; and anti-NBD1 antibody (Neomarkers) was applied for 1.5 h followed by four 15-min washes, and then horseradish peroxidase-labeled anti-mouse secondary antibody was applied at a 1:5000 dilution for 45 min followed by four 15-min washes. Bound horseradish peroxidase was visualized using a chemilu-minescence system and exposure to x-ray film. Kinase Assays—5 μg of protein (or 50 μgin10× samples) samples were assayed for CK2 activity (27Meggio F. Pinna L.A. FASEB J. 2003; 17: 349-368Crossref PubMed Scopus (1090) Google Scholar). Briefly, [γ-32P]ATP or [γ-32P]GTP was used as phosphate donor to detect endogenous CK2 activity directed toward the specific target peptide RRRADDSDDDDD (gift of F. Meggio, S. Sarno, and L. Pinna). 10 μm TBB specified the activity. Cloning of NBD1 from CFTR—To clone an NBD1 construct (amino acids 351-665 of human CFTR), we used full-length cftr cDNA (gift; C. Boyd, Edinburgh, Scotland) as template and designed the following primers (forward, 5′-ctcgagatgtggtatgactctcttgga-3′; reverse, 5′-tcagttagccatcagtttacacgccggcg-3′) incorporating XhoI and NotI restriction sites 5′ and 3′, respectively. PCR was performed with these primers and template (92 °C for 1 min, 38 °C for 2.5 min, and 46 °C for 3.5 min, for 35 cycles); the product (∼950-base fragment) was gel-purified and placed into a standard ligation reaction with XhoI/Not-digested pcDNA3.1-His+ (His+-tagging plasmid vector (Invitrogen)). A small amount of this ligation reaction was transformed into JM109 competent Escherichia coli bacteria. The transformation mixture was shake-incubated for 4 h, then 20 μl plated onto 100 μg/ml ampicillin-LB plates, and incubated overnight. Ampicillin-resistant bacterial colonies were used to inoculate 50 ml of ampicillin-LB broth and shake-incubated overnight. pcDNA3.1-His+ plasmid cDNAs encoding NBD1 of CFTR were isolated from the bacterial culture using alkaline lysis (Qiagen) and verified by DNA sequencing using the ABI Prism DNA sequencer. Purification of NBD1 from Bacteria—One liter of LB broth, inoculated with E. coli containing pcDNA3.1-His+ plasmid cDNA encoding NBD1, was grown for 10-12 h in the presence of 100 μg/ml ampicillin. The bacteria were pelleted for 10 min at 4000 rpm. The pellet was resuspended in 10 ml of lysis buffer (50 mm NaPO4, 0.3 m NaCl, 8 m urea, 10 mm imidazole, 2% Tween 20, pH 8 with protease inhibitors), and sonicated three times with 15-s bursts. DNA and insoluble proteins were pelleted by centrifugation at 13,000 rpm for 5 min. The supernatant was added to 5 ml of start buffer (0.2 m NaPO4, 0.5 m NaCl and 10 mm imidazole, pH 7.4) and applied to a Ni2+-Sepharose affinity column (Bio-Rad), which was pre-equilibrated in start buffer. Sample was run through the column three times, and the column was washed with a further 10 column volumes of start buffer; His-tagged NBD1 was eluted with 7 ml of elution buffer (0.2 m NaPO4, 0.5 m NaCl, 500 mm imidazole, pH 7.4), and 0.5-ml fractions were collected by gravity flow. Column fractions were screened for NBD1 protein by Brad-ford assay and Western blotting using anti-NBD1 antibody. Purity of NBD1 was verified by Coomassie Blue and Silver stain (supplemental Fig. S4). Generation of the S511A Mutant in the NBD1 Region of CFTR—The cftr gene-specific primers, forward, 5′-tcatctttggtgttgcctatgatgaatat-3′, and reverse, 5′-atattcatcataggcaacaccaaagatga-3′, were used to introduce a Ser to Ala point mutation at the 511 site in the CFTR NBD1 region using PfuTurbo DNA polymerase in a PCR-based method (Quickchange SDM kit, Invitrogen). Once the mutation had been generated and verified by DNA sequencing, the mutant was digested and subsequently ligated into our expression vector. The mutant was then transformed into a competent JM109 E. coli cell line for expression. Amplification and protein purification as described for the NBD1 wild-type fragment. In Vivo Phosphorylation of CFTR—Hep-G2 cells were transfected with vectors encoding either NBD1 alone or full-length CFTR. 48 h after transfection, some plates were treated with TBB (10 μm) to specify CK2 phosphorylation, and all cultures were maintained for 12 h in the presence of cell-permeable inhibitors of PKA (100 μm), PKC (10 μm), and calmodulin-dependent protein kinase II (1 μm; all from Calbiochem) to reduce background phosphorylation by other kinases. Cells were lysed into RIPA buffer containing phosphatase inhibitors, and CFTR was purified and enriched by immunoprecipitation. Transfected Hep-G2 cells showed consistent levels of CFTR expression with all mutants and transcripts (Fig. 4C, lower blots). Co-immunoprecipitation of CK2 with CFTR Mutants—BHK cells were transfected with vectors encoding wild-type, ΔF508, S511A, or S511D CFTR. 48 h after transfection, cells were harvested, and membranes were isolated as described previously (52Crawford R.M. Treharne K.J. Best O.G. Riemen C.E. Muimo R. Gruenert D.C. Arnaud-Dabernat S. Daniel J.Y. Mehta A. Cell. Signal. 2006; 18: 1595-1603Crossref PubMed Scopus (15) Google Scholar). Membranes were solubilized in RIPA buffer containing protease and phosphatase inhibitors, and CFTR was immunoprecipitated using antibody irreversibly linked to protein G-Sepharose beads. A minimal quantity of beads was used to normalize the amount of CFTR precipitated from each transfection. Immunoprecipitations were stringently washed three times in RIPA buffer and once in TBST with 0.5 m NaCl. Samples were separated by SDS-PAGE using the Novex (Invitrogen) system with 4-12% BisTris gels and MES buffer, blotted onto nitrocellulose, and probed for CFTR and CK2 as described above. CK2 and CFTR in a Differentiated Human Epithelium—Using immunofluorescence and confocal microscopy, we tested whether the subcellular localization of CK2 coincides with the known apical distribution of CFTR in biopsies of normal and homozygous ΔF508 CF ciliated human nasal epithelial cells. Fig. 1 demonstrates that CK2α is not only enriched at the apical membrane but also colocalizes with CFTR in wild-type but not ΔF508 CF epithelial cells (CFTR antibody specificity is shown in supplemental Fig. S1). Levels of CFTR protein are reported to be reduced when F508 is deleted (31Penque D. Mendes F. Beck S. Farinha C. Pacheco P. Nogueira P. Lavinha J. Malho R. Amaral M.D. Lab. Investig. 2000; 80: 857-868Crossref PubMed Scopus (92) Google Scholar); thus, the absence of CFTR staining at the apical membrane of the CF cell is not surprising. However, the enrichment of CK2 at the apical membrane in wild-type cells and the lack of CK2 where CFTR levels are depleted are novel results and suggest that CK2 localization is somehow dependent on CFTR. cAMP-dependent Chloride Transport Is Dependent on CK2—CFTR regulates cAMP-dependent fluid and electrolyte transport across epithelia (3Welsh M.J. Ramsey B.W. Accurso F. Cutting G.R. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill Inc., New York2001: 5121-5188Google Scholar). To investigate CK2-dependent regulation of a classical CFTR function, we tested the effects of pharmacological CK2 inhibition on forskolin-stimulated, apical membrane Cl- currents (ICl(apical)) with the Ussing chamber technique (28Collett A. Ramminger S.J. Olver R.E. Wilson S.M. Am. J. Physiol. 2002; 282: L621-L630Google Scholar). Fig. 2A demonstrates that preincubation of Calu-3 epithelia (a robust model for CFTR-mediated anion transport reviewed in 32) with the CK2-selective inhibitor TBB (33Sarno S. Reddy H. Meggio F. Ruzzene M. Davies S.P. Donella-Deana A. Shugar D. Pinna L.A. FEBS Lett. 2001; 496: 44-48Crossref PubMed Scopus (306) Google Scholar) decreased ICl(apical) with half-maximal inhibition at ∼15 μm that causes potent inhibition of CK2 activity in mammalian cells (33Sarno S. Reddy H. Meggio F. Ruzzene M. Davies S.P. Donella-Deana A. Shugar D. Pinna L.A. FEBS Lett. 2001; 496: 44-48Crossref PubMed Scopus (306) Google Scholar). ICl(apical) was similarly abrogated in 16HBE14o-bronchial epithelia (32Gruenert D.C. Willems M. Cassiman J.J. Frizzell R.A. J. Cyst. Fibros. 2004; 3: 191-196Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) (HBE; supplemental Fig. S2A). TBB targets the ATP-binding site on CK2α and is without effect on 30 other protein kinases, including PKA, PKC, and AMPK (33Sarno S. Reddy H. Meggio F. Ruzzene M. Davies S.P. Donella-Deana A. Shugar D. Pinna L.A. FEBS Lett. 2001; 496: 44-48Crossref PubMed Scopus (306) Google Scholar). Nevertheless, we determined whether this agent might interact with NBD1 and found that TBB (50 nm to 1 mm) did not compete with ATP for NBD1 binding (supplemental Fig. S2B). To understand better how TBB inhibits CFTR-mediated Cl- transport, we studied the effects of TBB on the single-channel activity of CFTR using the patch clamp technique. Fig. 2B, panel a, demonstrates that addition of TBB (10 μm) to the solution bathing an intact cell caused a prompt inhibition of channel activity in a cell-attached membrane patch (compare expanded trace 1 with expanded traces 2 and 3 in Fig. 2B, panels a and d). TBB mediated its effects in two ways. First, the drug caused a dramatic time-dependent prolongation of the closed time interval between bursts of channel openings and, hence, decreased open probability (Po) to zero (Fig. 2B, panel e, histogram, left). Second, the drug caused a small decrease in current flow through individual channels (Fig. 2B, panel f, histogram, right). Interestingly, Fig. 2B (panels b-f, middle traces and histograms) shows that when TBB (10 μm) is directly added to the intracellular solution using an excised inside-out membrane patch, the drug has little effect on channel gating and hence Po, but it still reduced current flow through individual channels. Two conclusions can be drawn from these data. First, the small effect of TBB on CFTR-mediated current flow most likely represents the direct interaction of TBB with CFTR. Second, the effect of TBB on CFTR channel gating that is observed in intact cells, but lost on patch excision (summary histogram for Po), suggests that TBB exerts its effect via a CFTR-interacting protein. Given that TBB is a selective inhibitor of CK2, the simplest interpretation of the data is that CK2 regulates CFTR channel gating in intact cells. Wild-type (but Not ΔF508) CFTR Expression Generates a CK2-sensitive Chloride Current—Next, we confirmed that a structurally unrelated peptide inhibitor of CK2 (poly(E:Y) peptide 4:1) that targets the CK2 β subunit also inhibited forskolin-stimulated CFTR current. First, we used the Xenopus oocyte expres" @default.
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• W2034143917 title "Protein Kinase CK2, Cystic Fibrosis Transmembrane Conductance Regulator, and the ΔF508 Mutation" @default.
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