The Cystic Fibrosis Transmembrane Conductance Regulator Is Regulated by a Direct Interaction with the Protein Phosphatase 2A
2005; Elsevier BV; Volume: 280; Issue: 50 Linguagem: Inglês
10.1074/jbc.m507308200
ISSN1083-351X
AutoresWilliam R. Thelin, Mehmet Kesımer, Robert Tarran, Silvia M. Kreda, Barbara R. Grubb, John K. Sheehan, M. Jackson Stutts, Sharon L. Milgram,
Tópico(s)Tracheal and airway disorders
ResumoThe cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed at the apical surface of epithelia. Although the regulation of CFTR by protein kinases is well documented, channel deactivation by phosphatases is not well understood. We find that the serine/threonine phosphatase PP2A can physically associate with the CFTR COOH terminus. PP2A is a heterotrimeric phosphatase composed of a catalytic subunit and two divergent regulatory subunits (A and B). The cellular localization and substrate specificity of PP2A is determined by the unique combination of A and B regulatory subunits, which can give rise to at least 75 different enzymes. By mass spectrometry, we identified the exact PP2A regulatory subunits associated with CFTR as Aα and B′ϵ and find that the B′ϵ subunit binds CFTR directly. PP2A subunits localize to the apical surface of airway epithelia and PP2A phosphatase activity co-purifies with CFTR in Calu-3 cells. In functional assays, inhibitors of PP2A block rundown of basal CFTR currents and increase channel activity in excised patches of airway epithelia and in intact mouse jejunum. Moreover, PP2A inhibition in well differentiated human bronchial epithelial cells results in a CFTR-dependent increase in the airway surface liquid. Our data demonstrate that PP2A is a relevant CFTR phosphatase in epithelial tissues. Our results may help reconcile differences in phosphatase-mediated channel regulation observed for different tissues and cells. Furthermore, PP2A may be a clinically relevant drug target for CF, which should be considered in future studies. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed at the apical surface of epithelia. Although the regulation of CFTR by protein kinases is well documented, channel deactivation by phosphatases is not well understood. We find that the serine/threonine phosphatase PP2A can physically associate with the CFTR COOH terminus. PP2A is a heterotrimeric phosphatase composed of a catalytic subunit and two divergent regulatory subunits (A and B). The cellular localization and substrate specificity of PP2A is determined by the unique combination of A and B regulatory subunits, which can give rise to at least 75 different enzymes. By mass spectrometry, we identified the exact PP2A regulatory subunits associated with CFTR as Aα and B′ϵ and find that the B′ϵ subunit binds CFTR directly. PP2A subunits localize to the apical surface of airway epithelia and PP2A phosphatase activity co-purifies with CFTR in Calu-3 cells. In functional assays, inhibitors of PP2A block rundown of basal CFTR currents and increase channel activity in excised patches of airway epithelia and in intact mouse jejunum. Moreover, PP2A inhibition in well differentiated human bronchial epithelial cells results in a CFTR-dependent increase in the airway surface liquid. Our data demonstrate that PP2A is a relevant CFTR phosphatase in epithelial tissues. Our results may help reconcile differences in phosphatase-mediated channel regulation observed for different tissues and cells. Furthermore, PP2A may be a clinically relevant drug target for CF, which should be considered in future studies. Cystic fibrosis (CF) 2The abbreviations used are: CFcystic fibrosisCFTRcystic fibrosis transmembrane conductance regulatorPKAcAMP-dependent protein kinaseAKAPA kinase-anchoring proteinHBEhuman bronchial epithelialMSmass spectrometryTES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidPCLpericiliary liquid. is an autosomal lethal disease characterized by abnormal ion transport in epithelial tissues. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an apical membrane chloride channel. The regulation of CFTR by the cAMP-dependent protein kinase (PKA) and other protein kinases has been extensively documented. PKA can phosphorylate the CFTR regulatory domain (R domain) on at least 11 serine residues (1Cheng S.H. Rich D.P. Marshall J. Gregory R.J. Welsh M.J. Smith A.E. Cell. 1991; 66: 1027-1036Abstract Full Text PDF PubMed Scopus (532) Google Scholar, 2Seibert F.S. Tabcharani J.A. Chang X.B. Dulhanty A.M. Mathews C. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1995; 270: 2158-2162Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 3Chang X.B. Tabcharani J.A. Hou Y.X. Jensen T.J. Kartner N. Alon N. Hanrahan J.W. Riordan J.R. J. Biol. Chem. 1993; 268: 11304-11311Abstract Full Text PDF PubMed Google Scholar). In vivo, PKA phosphorylation increases CFTR open probability and the number of channels in the plasma membrane (4Gadsby D.C. Nairn A.C. Physiol. Rev. 1999; 79 (Suppl. 1): 77-107Crossref PubMed Scopus (369) Google Scholar, 5Ameen N.A. Marino C. Salas P.J. Am. J. Physiol. 2003; 284 (-C438): C429Crossref PubMed Scopus (35) Google Scholar). Work from our laboratory and others has demonstrated that PKA and other regulatory proteins are compartmentalized in close proximity to CFTR. The cellular machinery capable of generating cAMP, including the adenosine receptor and membrane-bound adenylate cyclase are present with CFTR in apical membrane patches (6Huang P. Lazarowski E.R. Tarran R. Milgram S.L. Boucher R.C. Stutts M.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14120-14125Crossref PubMed Scopus (176) Google Scholar). A kinase-anchoring proteins (AKAPs) target PKA to protein complexes with CFTR (7Sun F. Hug M.J. Lewarchik C.M. Yun C.H. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 8Sun F. Hug M.J. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 14360-14366Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 9Huang P. Trotter K. Boucher R.C. Milgram S.L. Stutts M.J. Am. J. Physiol. 2000; 278 (-C422): C417Crossref PubMed Google Scholar), and the disruption of the PKA/AKAP interaction abolishes the ability of PKA to activate CFTR in response to physiologic stimuli (9Huang P. Trotter K. Boucher R.C. Milgram S.L. Stutts M.J. Am. J. Physiol. 2000; 278 (-C422): C417Crossref PubMed Google Scholar). In addition, the phosphodiesterase PDE4D is also present with CFTR in patch preparations and forms a cAMP diffusion barrier at the apical plasma membrane (10Barnes A.P. Livera G. Huang P. Sun C. O'Neal W.K. Conti M. Stutts M.J. Milgram S.L. J. Biol. Chem. 2005; 280: 7997-8003Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Other CFTR regulatory molecules including protein kinase C and the AMP-activated protein kinase are found in protein complexes associated directly with CFTR (11Liedtke C.M. Yun C.H. Kyle N. Wang D. J. Biol. Chem. 2002; 277: 22925-22933Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 12Hallows K.R. Raghuram V. Kemp B.E. Witters L.A. Foskett J.K. J. Clin. Invest. 2000; 105: 1711-1721Crossref PubMed Scopus (184) Google Scholar). cystic fibrosis cystic fibrosis transmembrane conductance regulator cAMP-dependent protein kinase A kinase-anchoring protein human bronchial epithelial mass spectrometry 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid periciliary liquid. Less is known about the ability of serine/threonine phosphatases to regulate CFTR activity or how they are compartmentalized with CFTR. Work from many laboratories suggests that multiple phosphatases, including PP2A, PP2B, PP2C, and alkaline phosphatase, are involved in the deactivation of CFTR (13Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274 (-C1410): C1397Crossref PubMed Google Scholar, 14Hwang T.C. Horie M. Gadsby D.C. J. Gen. Physiol. 1993; 101: 629-650Crossref PubMed Scopus (93) Google Scholar, 15Travis S.M. Berger H.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11055-11060Crossref PubMed Scopus (59) Google Scholar, 16Reddy M.M. Quinton P.M. Am. J. Physiol. 1996; 270 (-C480): C474Crossref PubMed Google Scholar, 17Fischer H. Illek B. Machen T.E. Pflugers Arch. 1998; 436: 175-181Crossref PubMed Scopus (39) Google Scholar). In vitro, PP2A and PP2C are most effective in dephosphorylating purified CFTR and recombinant R domain. Furthermore, exogenous PP2A and PP2C inactivate CFTR in excised membrane patches (13Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274 (-C1410): C1397Crossref PubMed Google Scholar, 18Berger H.A. Travis S.M. Welsh M.J. J. Biol. Chem. 1993; 268: 2037-2047Abstract Full Text PDF PubMed Google Scholar). In human sweat ducts, cardiac myocytes, 3T3 fibroblasts, and Hi-5 insect cells, inhibitors of PP2A increase CFTR channel activity (14Hwang T.C. Horie M. Gadsby D.C. J. Gen. Physiol. 1993; 101: 629-650Crossref PubMed Scopus (93) Google Scholar, 16Reddy M.M. Quinton P.M. Am. J. Physiol. 1996; 270 (-C480): C474Crossref PubMed Google Scholar, 18Berger H.A. Travis S.M. Welsh M.J. J. Biol. Chem. 1993; 268: 2037-2047Abstract Full Text PDF PubMed Google Scholar, 19Yang I.C. Cheng T.H. Wang F. Price E.M. Hwang T.C. Am. J. Physiol. 1997; 272 (-C155): C142Crossref PubMed Google Scholar). Likewise, PP2B inhibitors stimulate CFTR in NIH 3T3 fibroblasts (17Fischer H. Illek B. Machen T.E. Pflugers Arch. 1998; 436: 175-181Crossref PubMed Scopus (39) Google Scholar). However, the contribution of PP2A and PP2B to CFTR deactivation may vary in different cell types (15Travis S.M. Berger H.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11055-11060Crossref PubMed Scopus (59) Google Scholar, 20Zhu T. Dahan D. Evagelidis A. Zheng S. Luo J. Hanrahan J.W. J. Biol. Chem. 1999; 274: 29102-29107Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In baby hamster kidney cells, PP2C co-immunoprecipitates with exogenously expressed CFTR (20Zhu T. Dahan D. Evagelidis A. Zheng S. Luo J. Hanrahan J.W. J. Biol. Chem. 1999; 274: 29102-29107Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Additionally, PP2C overexpression in Fischer rat thyroid cells decreases CFTR chloride conductance (15Travis S.M. Berger H.A. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11055-11060Crossref PubMed Scopus (59) Google Scholar). However, the ability of endogenous PP2C to regulate CFTR in native epithelial tissues is unclear, since no PP2C inhibitors have been identified. To date, no single phosphatase has been demonstrated to be both necessary and sufficient to completely down-regulate CFTR channel activity, suggesting that CFTR is dephosphorylated by multiple phosphatases. Here, we present evidence for a direct interaction between CFTR and the serine/threonine phosphatase PP2A. This interaction involves the COOH terminus of CFTR and the PP2A B′ϵ regulatory subunit. PP2A localizes to the apical cell surface, where it negatively regulates CFTR channel activity in Calu-3 cells and intact mouse jejunum. Furthermore, inhibition of PP2A increases the airway surface liquid in primary human bronchial epithelial (HBE) cells by a mechanism requiring CFTR channel activity. We conclude that PP2A is indeed a relevant CFTR phosphatase in epithelial tissues. Cell Culture and Immunofluorescence—Calu-3 cells were maintained as described previously (21Mohler P.J. Kreda S.M. Boucher R.C. Sudol M. Stutts M.J. Milgram S.L. J. Cell Biol. 1999; 147: 879-890Crossref PubMed Scopus (166) Google Scholar). Human airway epithelial cells were obtained from freshly excised bronchial specimens from normal subjects by protease digestion, seeded directly as primary cultures on 12-mm Transwell Col membranes (T-Col; Costar) in modified BEGM media under ALI conditions, and studied when fully differentiated (3-5 weeks). Cultures with transepithelial resistances (Rt) > 300 ohms cm2 were studied. Immunofluorescence and confocal microscopy were performed as described previously using anti-PP2A subunit antibodies (22Kreda S.M. Mall M. Mengos A. Rochelle L. Yankaskas J. Riordan J.R. Boucher R.C. Mol. Biol. Cell. 2005; 16: 2154-2167Crossref PubMed Scopus (222) Google Scholar). Monoclonal anti-catalytic subunit, monoclonal anti-A regulatory subunit, and polyclonal anti-B′ subunit were acquired from Upstate Biotechnology, Inc. (Lake Placid, NY). Affinity Purification of CFTR-interacting Proteins—Peptides corresponding to residues 1375-1401, 1411-1441, 1451-1476, and 1471-1480 of human CFTR synthesized with an N-terminal biotin tag (Genemed Synthesis) were resuspended in 50 mm Tris-Cl (pH 7.4). 20 nmol of the peptides were immobilized on 100 μl of streptavidin-agarose (Sigma) and incubated with Calu-3 cell lysates. Lysates were prepared by incubating twenty 100-mm dishes of Calu-3 cells in binding buffer (50 mm Tris-Cl, 150 mm NaCl, 0.2% CHAPS, and protease (Roche Applied Science) and phosphatase (Sigma) inhibitor mixtures) at 4 °C for 1 h. Following ultracentrifugation, the soluble fraction was precleared over empty streptavidin beads and incubated with CFTR peptides conjugated to streptavidin beads for 1 h. The bound fractions were washed extensively in binding buffer, eluted with 5% formic acid, and lyophilized. Prior to MS analysis, the protein samples were reduced, alkylated, and digested with proteomics grade trypsin (Sigma). The peptides were analyzed by liquid chromatography-MS/MS on a Q-Tof micro (Waters, Manchester, UK). All data were acquired using Masslynx 4.0 software and than processed using Proteinlynx module. The processed data were searched against updated NCBInr and Sprot data bases using the Mascot search engine. Mascot probability-based Mowse individual ion scores of >46 were accepted as indicating identity or extensive homology (p < 0.05). The MS/MS spectrum scores between 20 and 45 were examined individually, with the acceptance criteria being that the parent and fragment ion masses were within the calibrated tolerance limits and that the spectrum contained an extended series of consecutive y- or b- ions. Small scale experiments analyzed by Western blot were performed using lysates from two 100-mm dishes of Calu-3 cells and 2 nmol of CFTR peptide. In Vitro Binding Assays—Binding assays were performed as described previously, with several exceptions (21Mohler P.J. Kreda S.M. Boucher R.C. Sudol M. Stutts M.J. Milgram S.L. J. Cell Biol. 1999; 147: 879-890Crossref PubMed Scopus (166) Google Scholar, 23Scott R.O. Thelin W.R. Milgram S.L. J. Biol. Chem. 2002; 277: 22934-22941Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Briefly, the cDNA sequence of the human PP2A B′ϵ subunit was amplified by PCR and cloned into the BamHI and XhoI sites of pET-28C (Novagen, Madison, WI). Recombinant B′ϵ subunit was produced in either rabbit reticulocyte lysates (Promega, Madison, WI) or BL21 Escherichia coli (Stratagene, La Jolla, CA). For reticulocyte lysates, the PP2A B′ϵ subunit was purified using Ni2+-nitrilotriacetic acid beads (Qiagen) in binding buffer (50 mm Tris-Cl (pH 7.4), 150 mm NaCl, 0.1% Triton X-100). To remove any reticulocyte proteins that co-purify with the B′ϵ subunit, the beads were washed three times in binding buffer plus 2 m urea followed by three washes in binding buffer. The B′ϵ subunit was eluted with binding buffer plus 10 mm EDTA. Bacterially, expression of B′ϵ subunit was performed as previously described (24Smith F.D. Oxford G.S. Milgram S.L. J. Biol. Chem. 1999; 274: 19894-19900Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Purified PP2A core dimer consisting of the catalytic and A regulatory subunit was obtained from Promega (Madison, WI). Purified proteins were then incubated with indicated CFTR peptides immobilized to streptavidin beads in binding buffer plus 0.2% bovine serum albumin for 1 h at 4°C. Following extensive washing, the bound proteins were eluted, resolved by SDS-PAGE, and analyzed by Western blot or phosphorimaging analysis. Co-immunoprecipitation and Phosphatase Assays—Calu-3 cells were scraped into a hypotonic lysis buffer (150 mm Tris-Cl, 10 mm NaCl, and protease inhibitors) and physically disrupted by Dounce homogenization on ice. The lysates were centrifuged at 500 × g for 10 min to remove nuclei and unbroken cells. The supernatant was subsequently centrifuged at 100,000 × g for 1 h to pellet cell membranes. Membranes were resuspended in binding buffer for 1 h on ice. CFTR was immunoprecipitated using CFTR 596 antibodies (gift of Dr. J. Riordan, University of North Carolina, Chapel Hill, NC) or isotype-matched control antibodies covalently conjugated to Protein G dynabeads. Bound proteins were washed extensively and analyzed by Western blot using specific antibodies for PP2A subunits or for phosphatase activity using a PP2A immunoprecipitation phosphatase assay kit (Upstate Biotechnology). CFTR Currents in Outside-out Membrane Patches of Calu-3 Cells—The procedures were essentially as described previously (6Huang P. Lazarowski E.R. Tarran R. Milgram S.L. Boucher R.C. Stutts M.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14120-14125Crossref PubMed Scopus (176) Google Scholar). Briefly, CFTR Cl- channel activity was recorded at a membrane potential of 30 mV with a 6-8-megaohm resistance for an open pipette. Both the pipette and the bath solutions were the same as described previously (6Huang P. Lazarowski E.R. Tarran R. Milgram S.L. Boucher R.C. Stutts M.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14120-14125Crossref PubMed Scopus (176) Google Scholar). CFTR channel activity was recorded digitally (PClamp software) for 300 s following patch excision. PP2A inhibitors or protein kinase A inhibitor was included in the pipette solution as indicated. CFTR Currents in Mouse Jejunum—Details of this approach have been described previously (25Grubb B.R. Am. J. Physiol. 1995; 268 (-G513): G505PubMed Google Scholar, 26Grubb B.R. Methods Mol. Med. 2002; 70: 525-535PubMed Google Scholar). Briefly, sections of the midportion of mouse jejunum were studied under short circuit current (Isc) conditions during a 90-min recording. A constant voltage pulse (1-5 mV, 1-s duration) was applied to the tissue every minute (Physiologic Instruments, San Diego, CA). Potential difference and resistance were calculated by Ohm's law from the changes in Isc in response to the voltage pulse. Tissues were treated with 100 μm endothall or vehicle. Confocal Microscopy Measurement of Periciliary Liquid (PCL)—The technique has been described in detail (27Tarran R. Grubb B.R. Parsons D. Picher M. Hirsh A.J. Davis C.W. Boucher R.C. Mol. Cell. 2001; 8: 149-158Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). Briefly, phosphate-buffered saline (20 μl) containing 2 mg/ml Texas Red-dextran (10 kDa; Molecular Probes, Inc., Eugene, OR) and benzamil (10-4 m) was added to cultured bronchial epithelium, and excess was aspirated to bring PCL height to ∼7 μm. The CFTR-specific inhibitor CFTRinh172 was also included where noted at a concentration of 10-5 m. To measure the average height of the PCL, five predetermined points (one central and four 2 mm from the edge of the culture) were XZ-scanned as previously described (27Tarran R. Grubb B.R. Parsons D. Picher M. Hirsh A.J. Davis C.W. Boucher R.C. Mol. Cell. 2001; 8: 149-158Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). For all studies, perfluorocarbon was added mucosally to prevent evaporation of the PCL, and the culture was placed on the stage of the confocal microscope over a serosal reservoir (80 μl of TES-buffered Ringer). Okadaic acid was added to the apical surface as a dry powder in perfluorocarbon (5 μg of okadaic acid/25 ml of perfluorocarbon/cm2 of culture). Perfluorocarbon has no effect on PCL height or ion transport, as previously described (27Tarran R. Grubb B.R. Parsons D. Picher M. Hirsh A.J. Davis C.W. Boucher R.C. Mol. Cell. 2001; 8: 149-158Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). PP2A Physically Associates with the COOH Terminus of CFTR—The COOH terminus of CFTR mediates protein-protein interactions with PDZ proteins (7Sun F. Hug M.J. Lewarchik C.M. Yun C.H. Bradbury N.A. Frizzell R.A. J. Biol. Chem. 2000; 275: 29539-29546Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar, 28Cheng J. Moyer B.D. Milewski M. Loffing J. Ikeda M. Mickle J.E. Cutting G.R. Li M. Stanton B.A. Guggino W.B. J. Biol. Chem. 2002; 277: 3520-3529Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 29Short 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 (401) Google Scholar, 30Wang S. Yue H. Derin R.B. Guggino W.B. Li M. Cell. 2000; 103: 169-179Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), the adaptor protein AP-2 (31Weixel K.M. Bradbury N.A. J. Biol. Chem. 2000; 275: 3655-3660Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), and AMP kinase (12Hallows K.R. Raghuram V. Kemp B.E. Witters L.A. Foskett J.K. J. Clin. Invest. 2000; 105: 1711-1721Crossref PubMed Scopus (184) Google Scholar) to regulate cell surface stability, membrane trafficking, and channel activity. In the present study, we asked whether the highly conserved CFTR COOH terminus can interact with additional proteins that regulate channel function. We chose to focus on the last 25 amino acids of CFTR (encompassing residues 1451-1476), which precede, but do not include, the PDZ binding motif. Previous studies suggest that this region is important for CFTR trafficking and channel activity (32Ostedgaard L.S. Randak C. Rokhlina T. Karp P. Vermeer D. Ashbourne Excoffon K.J. Welsh M.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1937-1942Crossref PubMed Scopus (54) Google Scholar); however, no protein interactions have been reported. We immobilized biotinylated CFTR-(1451-1476) peptides on streptavidin-agarose beads, incubated the peptides in cell lysates prepared from Calu-3 cells, and eluted the bound proteins with formic acid. Using nanoliquid chromatography MS/MS, we identified the serine/threonine protein phosphatase PP2A associated with CFTR-(1451-1476). PP2A dephosphorylates CFTR in vitro and decreases channel activity in multiple cell systems (14Hwang T.C. Horie M. Gadsby D.C. J. Gen. Physiol. 1993; 101: 629-650Crossref PubMed Scopus (93) Google Scholar, 16Reddy M.M. Quinton P.M. Am. J. Physiol. 1996; 270 (-C480): C474Crossref PubMed Google Scholar, 18Berger H.A. Travis S.M. Welsh M.J. J. Biol. Chem. 1993; 268: 2037-2047Abstract Full Text PDF PubMed Google Scholar, 19Yang I.C. Cheng T.H. Wang F. Price E.M. Hwang T.C. Am. J. Physiol. 1997; 272 (-C155): C142Crossref PubMed Google Scholar). Recently, Vastiau et al. (33Vastiau A. Cao L. Jaspers M. Owsianik G. Janssens V. Cuppens H. Goris J. Nilius B. Cassiman J.J. FEBS Lett. 2005; 579: 3392-3396Crossref PubMed Scopus (17) Google Scholar) also identified an interaction between the regulatory domain of CFTR and PP2A, suggesting that PP2A anchoring to CFTR may involve multiple contacts. PP2A is a major cellular phosphatase that regulates many protein targets. PP2A functions as a heterotrimeric complex composed of a catalytic subunit and two regulatory subunits, A and B (34Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1541) Google Scholar). The specificity of PP2A is determined by the unique combination of regulatory subunits associated with the catalytic subunit. The A regulatory subunit is encoded by one of two genes α and β, which are 86% identical. The A regulatory subunit is tightly associated with the catalytic subunit and functions as a scaffold to recruit the B regulatory subunit. The B regulatory subunit is highly divergent in comparison with the other PP2A subunits. The B regulatory subunit is divided into the B, B′, B″, and B‴ families, which are encoded by at least 14 different genes, some of which produce as many as five splice variants. The diversity of regulatory subunits gives rise to over 75 distinct PP2A enzymes. By mass spectrometry, we observe peptides from all three PP2A subunits (Fig. 1A). Importantly, the MS/MS spectra provide amino acid sequence information that allowed us to precisely identify the PP2A regulatory subunits associated with CFTR-(1451-1476) as Aα and B′ϵ (Fig. 1B). By Western blot, we confirmed that the PP2A catalytic, A regulatory, and B′ regulatory subunits associate with CFTR-(1451-1476) but not other CFTR C-terminal peptides (Fig. 2A). Furthermore, these PP2A subunits also co-precipitate with endogenous CFTR from Calu-3 cell membranes (Fig. 2B), consistent with an interaction in vivo. The PP2A Bα subunit, which is structurally unrelated to the B′ family, does not co-purify with CFTR-(1451-1476) or co-immunoprecipitate with endogenous CFTR. In addition, we asked whether PP2A phosphatase activity purified with full-length CFTR. CFTR immunoprecipitates were assayed for PP2A using a colorimetric phosphatase assay. We find that a PP2A-like activity specifically co-precipitates with CFTR, but not an IgG control (Fig. 2C). Characteristic of PP2A, the phosphatase activity was inhibited by 1 μm okadaic acid or 1 μm endothall, potent inhibitors of PP2A (IC50 = 0.1 and 90 nm, respectively). Although okadaic acid and endothall can also inhibit PP1 (IC50 = 10 nm and 5 μm, respectively), previous studies have found no evidence to support the ability of PP1 to physically associate with CFTR, to dephosphorylate CFTR in vitro, or regulate channel activity (13Luo J. Pato M.D. Riordan J.R. Hanrahan J.W. Am. J. Physiol. 1998; 274 (-C1410): C1397Crossref PubMed Google Scholar, 18Berger H.A. Travis S.M. Welsh M.J. J. Biol. Chem. 1993; 268: 2037-2047Abstract Full Text PDF PubMed Google Scholar, 20Zhu T. Dahan D. Evagelidis A. Zheng S. Luo J. Hanrahan J.W. J. Biol. Chem. 1999; 274: 29102-29107Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Although PP2C has been shown to be associated with CFTR in baby hamster kidney cells, the assay buffer does not contain Mg2+, which is required for PP2C activity. Taken together, these data suggest that the phosphatase activity associated with CFTR is PP2A. The PP2A B′ϵ Subunit Binds Directly to the COOH Terminus of CFTR—It is clear that multiple phosphatases can regulate CFTR channel activity. However, the degree to which PP2A contributes to this regulation varies in different functional assays and cell systems. Given the diversity of PP2A enzymes, we were intrigued by the possibility that the B regulatory subunit may, in part, account for these differences. Therefore, we asked whether the PP2A B′ϵ subunit could interact directly with CFTR. We find that PP2A B′ϵ subunit expressed and purified from reticulocyte lysates binds to CFTR-(1451-1476) but not other CFTR C-terminal peptides (Fig. 3A). NHERF-1, previously shown to interact with the extreme COOH terminus of CFTR, is enriched by the CFTR-(1471-1480) peptide, demonstrating the specificity of this assay (Fig. 3A). Although we do not find the catalytic or A subunit associated with the purified B′ϵ subunit from reticulocyte lysates (data not shown), we cannot rule out the possibility that these subunits are present at low levels and may be influencing binding to CFTR-(1451-1476). Using bacterially expressed, purified B′ϵ subunit, we observe direct and dose-dependent binding to CFTR-(1451-1476) (Fig. 3B). Furthermore, we also find that the core PP2A dimer, composed of the catalytic and A regulatory subunit, do not bind to CFTR-(1451-1476) unless the B′ϵ subunit is present (Fig. 3C). Whereas it is not clear whether the B′ϵ subunit alone is necessary and sufficient for targeting PP2A to CFTR, these data suggest that the B′ϵ subunit may play a critical role in the binding specificity. PP2A Localizes to the Apical Cell Surface of Airway Epithelia—If PP2A is a physiologically relevant CFTR phosphatase, we reasoned that its subcellular localization should partially overlap with CFTR. Given the large number of PP2A substrates, we expect the ubiquitously expressed catalytic and tightly coupled A regulatory subunits to have a broad cellular distribution. However, specific B regulatory subunits, which target the PP2A enzyme to distinct subcellular compartments, should have a more restricted localization. Using a pan-B′ antibody, which recognizes all five gene products (α, β, γ, δ, and ϵ), we examined the localization of PP2A in the human airway. The B′ subunits exhibit a broad subcellular distribution in the human airway (Fig. 4). We find that B′ staining strongly localizes to the apical membrane of ciliated cells in superficial epithelia and gland ducts, consistent with the localization of CFTR in these tissues. Although we cannot unambiguously attribute the apical localization to the B′ϵ subunit, it is likely that this staining reflects the distribution of one or a combination of the cytosolic B′ subunits, B′α, B′β, B′δ, and B′ϵ (35McCright B. Rivers A.M. Audlin S. Virshup D.M. J. Biol. Chem. 1996; 271: 22081-22089Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). In addition, we observe staining of perinuclear membranes and nuclear speckles, which is consistent with the observation that the B′γ and B′δ localize to the nucleus (35McCright B. Rivers A.M. Audlin S. Virshup D.M. J. Biol. Chem. 1996; 271: 22081-22089Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 36Tarran R. Button B. Picher M. Paradiso A.M. Ribeiro C.M. Lazarowski E.R. Zhang L. Collins P.L. Pickles R.J. Fredburg J.J. Boucher R.C. J. Biol. Chem. 2005; 280: 35751-35759Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). As we expected, the PP2A catalytic and A regulatory subunits are more broadly localized in airway epithelia (data not shown) but clearly localized to the apical cell surface of ciliated cells. The antibodies directed against the catalytic and A regulatory subunits detect both gene products for each subunit. Thus, these antibodies label every PP2A molecule that we expect to give
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