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• W2023681929 abstract "The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl− channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl− current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR. The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl− channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl− current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR. cystic fibrosis transmembrane conductance regulator CFTR regulatory domain protein kinase A protein kinase A anchoring protein PSD-95/Disc-large/ZO-1 protein kinase inhibitor ERM-binding phosphoprotein 50 Na+/H+ exchange regulatory factor NHE3 kinase A regulatory protein polyacrylamide gel electrophoresis glutathione S-transferase phosphate-buffered saline polyvinylidene difluoride The cystic fibrosis transmembrane conductance regulator (CFTR)1 is the basis of the cAMP-activated anion conductance pathway at the apical membranes of epithelial cells (1.Quinton P.M Physiol. Rev. 1999; 79 Suppl. 1: 3-22Crossref Scopus (307) Google Scholar). In secretory epithelia, CFTR is often the rate-determining step in salt and water transport, accounting for impairments in fluid secretion observed in patients having CFTR mutations. To date, more than 800 mutations in CFTR have been observed in patients with cystic fibrosis, but relatively few of these (∼5%) are found in the regulatory domain of CFTR (CFTR data base, available on the World Wide Web), perhaps because of its functional importance. The key regulatory pathway determining CFTR activity involves elevation of cAMP and activation of protein kinase A (PKA) (2.Gadsby D.C. Nairn A.C Physiol. Rev. 1999; 79 Suppl. 1: 77-107Crossref Scopus (369) Google Scholar). Nine consensus sites for PKA phosphorylation lie in the central R domain region of CFTR. An important issue in the regulation of epithelial cell secretion has been the specificity of this process. The receptors for cAMP-mediated secretory agonists as well as the associated adenylate cyclase are localized to the basolateral membrane, yet the principal target of activated PKA, the CFTR, is apically localized. In recent years, it has become apparent that the selective actions of signaling mediators that do not inherently possess substrate specificity (e.g. PKA) are conferred by the formation of regulatory complexes that provide privileged access of regulators to their substrates (3.Pawson T. Scott J.D Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1891) Google Scholar). Indeed, it is now appreciated that different PKA isoforms are compartmentalized in the soluble and particulate cell fractions and that the association of PKA with cytoskeletal or membrane structures is mediated primarily by a physical association between the type II regulatory subunits (RII subunits) of PKA and protein kinase A anchoring proteins (AKAPs). AKAPs are thought to sequester the regulatory and catalytic components of PKA in proximity to their substrates and thereby confer the needed specificity. Indeed, a model Cl− secretory epithelium, T84, was found to express both RI and RII isoforms of PKA; and in these cells, about 23 of total PKA activity was due to RII that was localized on cellular structures (4.Singh A.K. Tasken K. Walker W. Frizzell R.A. Watkins S.C. Bridges R.J. Bradbury N.A. Am. J. Physiol. 1998; 275: C562-C570Crossref PubMed Google Scholar). These findings raise the possibility that PKA may regulate CFTR via compartmental restrictions that are based on protein interactions and that this arrangement may lead to phosphorylation of CFTR at specific sites within the protein. PDZ domain proteins are emerging as important organizing centers for regulatory complexes, and these scaffold-based regulatory proteins are often polarized to specific sites in polarized epithelial cells (5.Fanning A.S. Anderson J.M Curr. Opin. Cell Biol. 1999; 11: 432-439Crossref PubMed Scopus (273) Google Scholar). For example, the Na+/H+ exchanger regulatory factor (NHERF, also termed ezrin-binding phosphoprotein 50 or EBP50) was identified initially from its ability to confer PKA-mediated inhibition of the apical Na+/H+ exchanger in rabbit renal brush border membranes (6.Weinman E.J. Steplock D. Wang Y. Shenolikar S J. Clin. Invest. 1995; 95: 2143-2149Crossref PubMed Scopus (311) Google Scholar). The human homologue of NHERF, EBP50, binds to members of the ERM (ezrin-radixin-moesin) family of proteins (7.Reczek D. Berryman M. Bretscher A. J. Cell Biol. 1997; 139: 169-179Crossref PubMed Scopus (517) Google Scholar). The C terminus of CFTR corresponds to a PDZ interaction motif (TRL), and it binds to the first PDZ domain of EBP50 with high affinity (8.Short 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 (399) Google Scholar, 9.Hall R.A. Ostedgaard L.S. Premont R.T. Blitzer J.T. Rahman N. Welsh M.J. Lefkowitz R.J Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8496-8501Crossref PubMed Scopus (375) Google Scholar, 10.Wang S. Raab R.W. Schatz P.J. Guggino W.B. Li M. FEBS Lett. 1998; 427: 103-108Crossref PubMed Scopus (249) Google Scholar). It has been proposed that the EBP50-CFTR association, together with other proteins that are sequestered in a regulatory complex through these physical interactions, may provide the basis for functional interactions observed between CFTR and other ion channels (11.Schwiebert E.M. Benos D.J. Egan M.E. Stutts M.J. Guggino W.B Physiol. Rev. 1999; 79 Suppl. 1: 145-166Crossref Scopus (379) Google Scholar). Recently, Mohler et al. (12.Mohler 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). have provided evidence that a Yes-kinase-associated protein interacts with the second PDZ domain of EBP50. Accordingly, CFTR may bring other regulators, such as this Src family kinase, into its vicinity though these interactions. The functional impact of CFTR-EBP50 interactions is not fully understood, but the interaction of this PDZ domain protein with ezrin is of interest concerning its possible role in the regulation of CFTR by PKA. The PKA-dependent regulation of NHE3 is mediated by EBP50/NHERF (13.Weinman E.J. Steplock D. Tate K. Hall R.A. Spurney R.F. Shenolikar S J. Clin. Invest. 1998; 101: 2199-2206Crossref PubMed Scopus (89) Google Scholar), which is a known ezrin-binding protein. In addition, protein overlay methods have implicated an interaction between ezrin and the regulatory subunit of PKA (14.Dransfield D.T. Bradford A.J. Smith J. Martin M. Roy C. Mangeat P.H. Goldenring J.R EMBO J. 1997; 16: 35-43Crossref PubMed Scopus (267) Google Scholar). Accordingly, ezrin's interaction with EBP50 may localize PKA in close proximity to CFTR. The purpose of this study was to examine this hypothesis using both protein interaction assays and functional measurements of CFTR activity. Our findings show that CFTR is part of a regulatory complex in human airway cells and that this complex contains ezrin and both the catalytic and regulatory subunits of PKA. Disruption of these interactions blocks CFTR- and PKA-specific substrate phosphorylation reactions. Ezrin was found at the apical membrane domain of both airway and intestinal secretory epithelia, and it bound RII in protein overlay and co-immunoprecipitation experiments. Finally, in patch clamp experiments, the functional activation of CFTR by PKA could be disrupted by conditions that interfere with ezrin binding of RII. These findings provide physical and functional evidence that PKA regulation of CFTR is AKAP-mediated, and they suggest that ezrin is a CFTR-associated AKAP in secretory epithelial cells. Ht31 and Ht31P peptides (15.Carr D.W. Stofko-Hahn R.E. Fraser I.D. Bishop S.M. Acott T.S. Brennan R.G. Scott J.D J. Biol. Chem. 1991; 266: 14188-14192Abstract Full Text PDF PubMed Google Scholar, 16.Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar) were obtained from Genemed Synthesis (San Francisco, CA). Protein A/G-agarose beads and molecular weight markers were obtained from Life Technologies, Inc. PKA catalytic subunit, cAMP, and cAMP-agarose were purchased from Sigma. Renaissance Chemiluminescence Reagent Plus and [γ-32P]ATP (3000 Ci/mmol) were obtained from NEN Life Science Products. PKA inhibitor peptide (residues 5–24) (PKI) and the SignaTECTTM cAMP-dependent protein kinase assay system were purchased from Promega (Madison, WI). Protease inhibitor tablets and restriction endonucleases were from Roche Molecular Biochemicals. Phosphatase inhibitors were from Alomone (Jerusalem, Israel). Other reagent grade chemicals were obtained from Sigma. Monoclonal anti-PKA catalytic subunit antibody was obtained from Transduction Laboratories (Lexington, KY). Polyclonal antibody against PKA type IIα regulatory subunit (RIIα) was purchased form Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Monoclonal anti-CFTR antibodies were from Genzyme (Framingham, MA). Monoclonal anti-GST and anti-ezrin antibodies were obtained from Sigma. Calu-3 cells were grown in Dulbecco's modified Eagle's medium/Ham's F-12 medium containing 15% fetal bovine serum. Cells were incubated in a humidified atmosphere containing 5% CO2 at 37 °C. T84 cells were grown under similar conditions, except that the medium contained 10% fetal bovine serum. For confocal microscopy, Calu-3 or T84 cells were seeded onto Costar Transwell cell culture inserts, and the culture media were changed every 2 days. The apical medium bathing Calu-3 cells was removed after several days in culture, and the cells were maintained at an air interface until use. Confocal microscopy (see below) was performed after 14–21 days in culture. Confluent cell monolayers were scraped into buffer (10 mm Tris·HCl (pH 7.4), 50 mmNaCl, 1 mm EDTA, and protease inhibitors) and homogenized using a Dounce type homogenizer. Postnuclear supernatants were obtained by centrifugation (14,000 × g for 1 min), and from this supernatant, cytosolic and membrane fractions were obtained by centrifugation at 100,000 × g for 60 min. Cytosolic proteins were concentrated by precipitation using 10% trichloroacetic acid and then resuspended in lysis buffer (50 mm HEPES (pH 7.4), 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 10% glycerol). Membrane fractions were resuspended in lysis buffer directly. Full-length ezrin cDNA (a gift from Dr. T. Hunter, Salk Institute) was amplified by polymerase chain reaction using the following primers: 5′-GAATTCCCGAAACCAATCAATGTC and 5′-GATATCTTACAGGGGCCTCGAACTCGTC, which resulted in the generation of an EcoRI site at the N terminus and an EcoRV site at the C terminus of ezrin (sites indicated by underlining). The polymerase chain reaction product was cloned into pCR2.1 (Invitrogen). Fidelity of the polymerase chain reaction product was confirmed by DNA sequencing. Ezrin cDNA was released by digestion of pCREzrin with EcoRI and EcoRV and then inserted into pGEX-4T-1 (Amersham Pharmacia Biotech) atEcoRI and SmaI sites. Expression and purification of GST-ezrin fusion protein or GST protein in bacteria followed the manufacturer's instructions (Amersham Pharmacia Biotech). In attempts to detect endogenous protein kinase activity associated with CFTR, precleared Calu-3 cell lysates (∼3 mg of protein) were mixed with 1 μg of anti-CFTR R domain antibody or control antibody (anti-GST) for 1.5 h at 4 °C in lysis buffer. Twenty μl of washed protein G-Sepharose beads were added to each immunoprecipitation and incubated for 1 h at 4 °C with gentle rotation. Immunocomplexes with protein G-Sepharose beads were precipitated by centrifugation at 12,000 ×g for 10 s and washed three times with 1 ml of lysis buffer and once with phosphorylation buffer (50 mmTris·HCl (pH 7.5), 10 mm MgCl2, 0.1 mg/ml bovine serum albumin). Where indicated, 4 μm Ht31 was added to CFTR immunoprecipitates to disrupt AKAP-RII subunit interactions (3.Pawson T. Scott J.D Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1891) Google Scholar). The control peptide, Ht31P, inserts a proline to disrupt the helical structure of Ht31; it was used at the same concentration. Samples were immediately subjected to phosphorylation reactions. For other co-precipitation experiments, Calu-3 cell lysates or subfractions (∼500 μg) were mixed with appropriate experimental or control antibodies at 4 °C for 1.5 h in lysis buffer with gentle rotation. Twenty μl of washed protein A- or protein G-Sepharose beads were then added to each immunoprecipitate and incubated for 1 h at 4 °C. Immunocomplexes were pelleted by centrifugation at 12,000 × g for 10 s and washed four times with 1 ml of lysis buffer. Pellets were resuspended in Laemmli sample buffer and resolved by SDS-PAGE. Phosphorylation of immunoprecipitated CFTR was performed as described previously (17.Bradbury N.A. Clark J.A. Watkins S.C. Widnell C.C. Smith H.S.T. Bridges R.J Am. J. Physiol. 1999; 276: L659-L668Crossref PubMed Google Scholar). Briefly, immunoprecipitated CFTR was phosphorylated by endogenous kinase or by adding 5 units of PKA catalytic subunit in the presence of 0.15 μm[γ-32P]ATP (3000 Ci/mmol). Phosphorylation reactions were performed in the presence or absence of 10 μm cAMP and incubated at 37 °C in phosphorylation buffer for 10 min followed by two washes with lysis buffer. Phosphorylation reaction products were resolved by 7.5% SDS-PAGE and processed for autoradiography or phosphor imaging (Bio-Rad). Phosphorylation of biotinylated kemptide (amino acid sequence LRRASLG) was performed according to the manufacturer's instructions (Promega). 32P incorporation was quantified by liquid scintillation counting. Expression, purification, and radiolabeling of RIIα were performed as described previously (18.Hausken Z.E. Coghlan V.M. Hastings C.A. Reimann E.M. Scott J.D J. Biol. Chem. 1994; 269: 24245-24251Abstract Full Text PDF PubMed Google Scholar). Briefly, a plasmid containing the mouse RIIα cDNA (a gift of Dr. J. D. Scott, Vollum Institute) was transformed into Escherichia coli BL21 (DE3) competent cells (Novagen). Expression of RIIα was achieved by adding a final concentration of 1 mmisopropyl-β-d-thiogalactopyranoside to the bacterial culture, with incubation for 4 h at 37 °C. The bacteria were harvested by centrifugation and resuspended in PBS with protease inhibitors. Resuspended bacteria were lysed by nitrogen cavitation and then centrifuged at 10,000 × g for 15 min. The supernatant was mixed with 10 g of ammonium sulfate at 4 °C for 15 min. Precipitated proteins were separated from soluble material by centrifugation at 10,000 × g for 15 min and then resuspended in PBS containing protease inhibitors. The resuspended material was mixed with 5 ml of cAMP-agarose for 16 h at 4 °C. Nonspecific binding was removed by washing with high salt buffer. Bound RIIα was eluted with a solution containing 25 mm cAMP. Purified RIIα was radiolabeled by incubation with [γ-32P]ATP and the catalytic subunit of PKA in phosphorylation buffer for 1 h at 30 °C. Separation of labeled RIIα from free ATP was achieved using desalting columns (Pierce). 10 μg of GST-ezrin fusion protein and GST protein were resolved by 7.5% SDS-PAGE and transferred to PVDF membrane. The membrane was blocked in blocking buffer (5% nonfat milk, 10 mm Tris (pH 8.0), 150 mm NaCl) containing 1% bovine serum albumin for 3 h at room temperature and then incubated for 4 h with 100,000 cpm/ml of 32P-labeled RIIα in blocking buffer containing 0.1% bovine serum albumin. For competition assays, the membrane was incubated with labeled RIIα and 4 μm of Ht31 or Ht31P peptide. Membrane was washed three times in TBST (10 mm Tris (pH 8.0), 150 mmNaCl, 0.05% Tween 20), and the label was visualized by autoradiography. Samples were resolved by SDS-PAGE and transferred to PVDF membranes. Unbound sites were blocked for 1 h at room temperature with 5% (w/v) skim milk powder in TBS (TBST lacking Tween 20). Membranes were incubated 1 h at room temperature with the appropriate primary antibodies. The membranes were then washed four times for 5 min each with TBST and incubated for 1 h with 2 μg/ml horseradish peroxidase-conjugated secondary antibodies (Sigma) in TBST with 10% fetal bovine serum. The blots were washed five times for 5 min each with TBST, and reactive bands were visualized by Renaissance Chemiluminescence (NEN Life Science Products). Samples were exposed to x-ray film (Eastman Kodak Co.). Calu-3 cells were seeded onto glass coverslips and used 1–3 days after seeding. Coverslips were placed in a chamber that was perfused at a rate of 7–15 ml/min with a solution of the following composition 120 mm NaCl, 25 mm NaHCO3, 0.4 mmKH2PO4, 1.6 mmK2HPO4, 1 mm MgCl2, 1.5 mm CaCl2, and 5 mm glucose. This solution was gassed with 5% CO2, 95% O2 and heated to 37 °C. The perfusion chamber was mounted on the stage of an inverted microscope (Nikon Diaphot). Patch pipettes were pulled from borosilicate glass tubing (Warner Instrument Corp., Hartford, CT) and filled with a solution composed of 95 mm potassium gluconate, 30 mm KCl, 1.2 mmNaH2PO4, 4.8 mmNa2HPO4, 1 mm MgCl2, 5 mm glucose, 0.5 mm EGTA, 1 mm ATP, and 0.1 mm GTP. Average pipette resistance was 2–4 megaohms. Pipettes were mounted to the headstage of an EPC7 patch clamp amplifier (List Medical Instruments, Darmstadt, Germany) and advanced by a mechanical micromanipulator (Narishige, Japan). After establishing the whole-cell configuration, membrane voltage was measured in the current clamp mode of the amplifier. Then the cells were voltage-clamped to −40 mV, and the whole-cell current was recorded. To precisely measure membrane conductance, G m, impedance analysis was performed. For this purpose, four sine waves (203.45, 406.9, 813.8, and 1627.6 Hz) were superimposed onto the clamp voltage. The resulting currents were fed into a four channel lock-in amplifier (Quad synchron detector; Physiologisches Institut, Freiburg, Germany), and the respective real (R i) and imaginary (I i) parts of the currents were sampled onto a computer's hard disc. On-line analysis was performed using the program Biocap (Physiologisches Institut, Freiburg). The use of four frequencies allowed us to closely monitor changes in four parameters: pipette capacitance, access conductance, membrane conductance, and membrane capacitance. Here, we report on changes in G m. Filter-grown Calu-3 or T84 cells were fixed in 2% paraformaldehyde in PBS for 10 min followed by a permeabilization with a mixture of 2% paraformaldehyde and 0.1% Triton X-100 in PBS for 10 min. The cells were then washed three times in PBS containing 0.5% bovine serum albumin and 0.15% glycine at pH 7.4 (buffer A). This was followed by a 30-min incubation with purified goat serum at 25 °C and three additional washes with buffer A. Cells were incubated for 1 h with a primary antibody (monoclonal IgG1 against ezrin) followed by three washes in buffer A and an incubation with fluorescein isothiocyanate-labeled secondary antibody (Alexa 488; Molecular Probes, Inc.). The cells were then washed six times in buffer A and mounted on a glass coverslip using a synthetic resin (Gelvatol, Mosanto). Coverslips were placed on a horizontal stage and imaged using a Leica TCS-NT confocal microscope. Images were collected using a 100× plan-apochromatic oil immersion objective; pixel size in the x, y, andz axes were calibrated to satisfy Nyquist sampling (0.1-μmx/y, 0.2-μm z). Serial scans were collected using a 488-nm laser line to optimally excite the Alexa 488 fluorochrome used for labeling. Image stacks were exported to ImageSpace (Molecular Dynamics, Inc.) for subsequent reconstruction and processing. Final presentation of images uses a pseudocolor representation encompassing the black and white intensity range 0–255 as illustrated with the final images. CFTR was immunoprecipitated from Calu-3 cell lysates with an antibody against the CFTR regulatory domain (CFTR-RD). The immunoprecipitate was incubated under phosphorylation conditions with [γ-32P]ATP, which included the addition of phosphatase inhibitors (see “Experimental Procedures”), separated on SDS-PAGE, and visualized by autoradiography. In the presence of cAMP, a major diffuse band (characteristic of CFTR) with a molecular mass of ∼180 kDa was phosphorylated by a protein kinase that was co-immunoprecipitated with CFTR (Fig. 1,lane 3). We examined the effect of cAMP on the endogenous kinase activity by incubating the CFTR immunoprecipitate with [γ-32P]ATP and 10 μm cAMP. The addition of cAMP promoted the phosphorylation of CFTR relative to control experiments performed in its absence (Fig. 1, comparelanes 3 and 4). The increase in γ-32P incorporation into CFTR with the addition of cAMP was ∼50% as determined by densitometry. As is routine procedure for CFTR identification (19.Cheng 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 (1416) Google Scholar), this band was strongly phosphorylated by the addition of the catalytic subunit of PKA to the immunoprecipitate (Fig.1, lane 1). In contrast, a Calu-3 cell immunoprecipitate generated using an irrelevant antibody (anti-GST monoclonal) did not reproduce this phosphoprotein signal (Fig. 1,lane 2). To confirm that the band phosphorylated by the endogenous kinase was CFTR, we also probed the immunoprecipitate with an anti-CFTR C terminus antibody; this immunoblot identified a similar 180-kDa diffuse band (data not shown). Next, we used a PKA-specific substrate peptide and PKI to determine whether the endogenous kinase associated with CFTR is PKA. Two experiments were performed. First, we tested whether the endogenous kinase associated with CFTR is able to phosphorylate the PKA-specific substrate peptide, kemptide. As shown in Fig.2 A, the endogenous kinase precipitated by anti-CFTR-RD phosphorylated kemptide. This phosphorylation was also stimulated by cAMP (Fig. 2 B). The PKA activity associated with the CFTR immunoprecipitate was about 4-fold higher than that of the immunoprecipitate obtained with a control antibody (Fig. 2 A). Second, we tested the effect of PKI on the ability of the endogenous kinase to phosphorylate kemptide. As shown in Fig. 2 A, the phosphorylation of kemptide by the endogenous kinase associated with CFTR was blocked by PKI. PKI-insensitive phosphorylation (∼10% of total phosphorylation) may reflect background activity of contaminating kinases. Taken together, these results strongly suggest that the kinase activity associated with CFTR is PKA. To determine whether PKA is physically associated with CFTR, we performed co-immunoprecipitations in which a CFTR-RD antibody was mixed with Calu-3 cell lysates and then precipitated with protein G-agarose beads. After SDS-PAGE and transfer to PVDF membrane, this immunoprecipitate was probed with anti-PKA antibodies. Fig.3 A shows that RIIα of PKA was present in the CFTR immunoprecipitate. In contrast, Calu-3 cell lysates immunoprecipitated with an irrelevant antibody did not yield a positive signal on RII immunoblots (Fig. 3 A). A similar experiment was done to probe for the catalytic subunit of PKA (PKAc) in CFTR immunoprecipitates. As shown in Fig. 3 B, the PKAc subunit was also identified in the CFTR immunoprecipitate. Since both immunoprecipitation and immunoblotting were performed using antibodies raised in mice, the heavy and light chains of the precipitating antibody were also detected. These results demonstrate that protein kinase A type II is physically associated with CFTR. Cell fractionation was employed to investigate the distribution of CFTR, RIIα, and PKAc expression in Calu-3 and T84 epithelia. In these experiments, cells were homogenized and subjected to centrifugation at 14,000 × g for 1 min. The resultant postnuclear supernatant was further separated at 100,000 × g for 60 min. A membrane fraction was prepared by suspending the pellet in lysis buffer, while a cytosolic fraction was made by concentration of the supernatant (See “Experimental Procedures”). As expected, CFTR was identified predominantly in the membrane fraction of Calu-3 and T84 cells (Fig. 4). The CFTR expression level is much higher in Calu-3 cells than that in T84 cells (Fig. 4, compareupper and lower panels), consistent with previous biochemical observations (17.Bradbury N.A. Clark J.A. Watkins S.C. Widnell C.C. Smith H.S.T. Bridges R.J Am. J. Physiol. 1999; 276: L659-L668Crossref PubMed Google Scholar). RIIα and PKAc were mainly present in the membrane fraction of Calu-3 and T84 cells, although these proteins could be also detected in the cytosolic fraction. These results provide evidence that PKAII co-immunoprecipitates with CFTR and that CFTR is a substrate for this kinase. Since the association of PKAII with subcellular structures is generally mediated by AKAP(s) (20.Rubin C.S. Biochim. Biophys. Acta. 1994; 1224: 467-479PubMed Google Scholar), we reasoned that an AKAP might link PKAII to CFTR. To test this hypothesis, we used Ht31, an amphipathic peptide that corresponds to the RII binding motif of a human thyroid AKAP (15.Carr D.W. Stofko-Hahn R.E. Fraser I.D. Bishop S.M. Acott T.S. Brennan R.G. Scott J.D J. Biol. Chem. 1991; 266: 14188-14192Abstract Full Text PDF PubMed Google Scholar), to determine whether a similar RII binding motif is responsible for linking PKAII to CFTR. CFTR immunoprecipitation from Calu-3 cell lysates was performed in the presence or absence of Ht31. Fig.5 shows that preincubation of the immunoprecipitate with Ht31 decreased the phosphorylation level of CFTR that was due to endogenous PKA activity. The control peptide, Ht31P, did not alter CFTR phosphorylation. These data suggest that one or more AKAPs mediate the observed association of protein kinase activity with CFTR. Previous studies (14.Dransfield D.T. Bradford A.J. Smith J. Martin M. Roy C. Mangeat P.H. Goldenring J.R EMBO J. 1997; 16: 35-43Crossref PubMed Scopus (267) Google Scholar,21.Lamprecht G. Weinman E.J. Yun C.-H.C. J. Biol. Chem. 1998; 273: 29972-29978Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) have suggested that ezrin, a cytoskeleton-associated protein with a molecular mass of about 80 kDa, is an RII-binding protein. To determine whether ezrin can function as an AKAP in Calu-3 cells, we performed RIIα overlay assays in which 10 μg of GST-ezrin fusion protein and GST protein alone were resolved by SDS-PAGE, transferred to PVDF membrane, and probed with 32P-labeled RIIα. Fig.6 A shows that RIIα binds GST-ezrin but not GST. This binding could be blocked by the addition of 4 μm Ht31 in the overlay (Fig. 6 A,right panel). To function as an AKAP, ezrin should also interact with RIIα in vivo. To assess this, we performed co-immunoprecipitation experiments using ezrin and RIIα antibodies. In Calu-3 cell lysates, however, we were unable to detect the regulatory subunit of PKA in an ezrin immunoprecipitate (data not shown). This lack of consistency between the RIIα overlay of ezrin and the RIIα co-precipitation with ezrin prompted us to examine other cell fractions. Using the cytosolic fraction, we could detect RIIα in the ezrin immunoprecipit" @default.
• W2023681929 created "2016-06-24" @default.
• W2023681929 creator A5012552638 @default.
• W2023681929 creator A5026190252 @default.
• W2023681929 creator A5049859124 @default.
• W2023681929 creator A5090316903 @default.
• W2023681929 date "2000-05-01" @default.
• W2023681929 modified "2023-10-16" @default.
• W2023681929 title "Protein Kinase A Associates with Cystic Fibrosis Transmembrane Conductance Regulator via an Interaction with Ezrin" @default.
• W2023681929 cites W1497015848 @default.
• W2023681929 cites W1530102523 @default.
• W2023681929 cites W1560760512 @default.
• W2023681929 cites W1581529107 @default.
• W2023681929 cites W1821092064 @default.
• W2023681929 cites W1954895018 @default.
• W2023681929 cites W1965144258 @default.
• W2023681929 cites W1966232883 @default.
• W2023681929 cites W1968578988 @default.
• W2023681929 cites W1969039665 @default.
• W2023681929 cites W1971571944 @default.
• W2023681929 cites W1971584001 @default.
• W2023681929 cites W1972172786 @default.
• W2023681929 cites W1975971473 @default.
• W2023681929 cites W1986116780 @default.
• W2023681929 cites W1990628555 @default.
• W2023681929 cites W1991550774 @default.
• W2023681929 cites W1999318646 @default.
• W2023681929 cites W2000990440 @default.
• W2023681929 cites W2001449914 @default.
• W2023681929 cites W2008207415 @default.
• W2023681929 cites W2010691166 @default.
• W2023681929 cites W2024028178 @default.
• W2023681929 cites W2037348714 @default.
• W2023681929 cites W2047233384 @default.
• W2023681929 cites W2049583947 @default.
• W2023681929 cites W2051446765 @default.
• W2023681929 cites W2057032201 @default.
• W2023681929 cites W2061780012 @default.
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