Title: The Deubiquitinating Enzyme USP10 Regulates the Post-endocytic Sorting of Cystic Fibrosis Transmembrane Conductance Regulator in Airway Epithelial Cells
Abstract: The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is a cyclic AMP-regulated chloride channel and a regulator of other ion channels and transporters. In epithelial cells CFTR is rapidly endocytosed from the apical plasma membrane and efficiently recycles back to the plasma membrane. Because ubiquitination targets endocytosed CFTR for degradation in the lysosome, deubiquitinating enzymes (DUBs) are likely to facilitate CFTR recycling. Accordingly, the aim of this study was to identify DUBs that regulate the post-endocytic sorting of CFTR. Using an activity-based chemical screen to identify active DUBs in human airway epithelial cells, we demonstrated that Ubiquitin Specific Protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and its trafficking in the post-endocytic compartment. small interference RNA-mediated knockdown of USP10 increased the amount of ubiquitinated CFTR and its degradation in lysosomes, and reduced both apical membrane CFTR and CFTR-mediated chloride secretion. Moreover, a dominant negative USP10 (USP10-C424A) increased the amount of ubiquitinated CFTR and its degradation, whereas overexpression of wt-USP10 decreased the amount of ubiquitinated CFTR and increased the abundance of CFTR. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR. The cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC transporter superfamily, is a cyclic AMP-regulated chloride channel and a regulator of other ion channels and transporters. In epithelial cells CFTR is rapidly endocytosed from the apical plasma membrane and efficiently recycles back to the plasma membrane. Because ubiquitination targets endocytosed CFTR for degradation in the lysosome, deubiquitinating enzymes (DUBs) are likely to facilitate CFTR recycling. Accordingly, the aim of this study was to identify DUBs that regulate the post-endocytic sorting of CFTR. Using an activity-based chemical screen to identify active DUBs in human airway epithelial cells, we demonstrated that Ubiquitin Specific Protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and its trafficking in the post-endocytic compartment. small interference RNA-mediated knockdown of USP10 increased the amount of ubiquitinated CFTR and its degradation in lysosomes, and reduced both apical membrane CFTR and CFTR-mediated chloride secretion. Moreover, a dominant negative USP10 (USP10-C424A) increased the amount of ubiquitinated CFTR and its degradation, whereas overexpression of wt-USP10 decreased the amount of ubiquitinated CFTR and increased the abundance of CFTR. These studies demonstrate a novel function for USP10 in facilitating the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR. The endocytosis, endocytic recycling, and endosomal sorting of numerous transport proteins and receptors are regulated by ubiquitination (1.Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (965) Google Scholar, 2.Clague M.J. Urbé S. Trends Cell Biol. 2006; 16: 551-559Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 3.Mukhopadhyay D. Riezman H. Science. 2007; 315: 201-205Crossref PubMed Scopus (966) Google Scholar, 4.Love K.R. Catic A. Schlieker C. Ploegh H.L. Nat. Chem. Biol. 2007; 3: 697-705Crossref PubMed Scopus (183) Google Scholar, 5.Williams R.L. Urbé S. Nat. Rev. 2007; 8: 355-368Crossref Scopus (569) Google Scholar, 6.Millard S.M. Wood S.A. J. Cell Biol. 2006; 173: 463-468Crossref PubMed Scopus (53) Google Scholar). Ubiquitin, an 8-kDa protein, is conjugated to target proteins via a series of steps that includes ubiquitin-activating enzymes (E1), 2The abbreviations used are: E1ubiquitin-activating enzymeE2ubiquitin-conjugating enzymeE3ubiquitin ligaseCFTRcystic fibrosis transmembrane conductance regulatorUSP10ubiquitin-specific protease-10DUBdeubiquitinating enzymeENaCepithelial sodium channelABCATP-binding cassetteHAhemagglutininRTreverse transcriptionsiRNAsmall interference RNAUbVMEubiquitin-vinyl methyl esterLAMP-1lysosome-associated protein-1. ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) (1.Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (965) Google Scholar). Proteins that are ubiquitinated in the plasma membrane are internalized and are either deubiquitinated and recycle back to the plasma membrane or, via interactions with the endosomal sorting complexes required for transport machinery, are delivered to the lysosome for degradation (1.Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (965) Google Scholar, 2.Clague M.J. Urbé S. Trends Cell Biol. 2006; 16: 551-559Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 3.Mukhopadhyay D. Riezman H. Science. 2007; 315: 201-205Crossref PubMed Scopus (966) Google Scholar, 4.Love K.R. Catic A. Schlieker C. Ploegh H.L. Nat. Chem. Biol. 2007; 3: 697-705Crossref PubMed Scopus (183) Google Scholar, 5.Williams R.L. Urbé S. Nat. Rev. 2007; 8: 355-368Crossref Scopus (569) Google Scholar, 6.Millard S.M. Wood S.A. J. Cell Biol. 2006; 173: 463-468Crossref PubMed Scopus (53) Google Scholar, 7.Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1145) Google Scholar). Sorting of ubiquitinated plasma membrane proteins for either the lysosomal pathway or for the recycling pathway is regulated, in part, by the removal of ubiquitin by deubiquitinating enzymes (DUBs) (1.Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (965) Google Scholar, 2.Clague M.J. Urbé S. Trends Cell Biol. 2006; 16: 551-559Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 3.Mukhopadhyay D. Riezman H. Science. 2007; 315: 201-205Crossref PubMed Scopus (966) Google Scholar, 4.Love K.R. Catic A. Schlieker C. Ploegh H.L. Nat. Chem. Biol. 2007; 3: 697-705Crossref PubMed Scopus (183) Google Scholar, 5.Williams R.L. Urbé S. Nat. Rev. 2007; 8: 355-368Crossref Scopus (569) Google Scholar, 6.Millard S.M. Wood S.A. J. Cell Biol. 2006; 173: 463-468Crossref PubMed Scopus (53) Google Scholar). Thus, the balance between ubiquitination and deubiquitination regulates the plasma membrane abundance of several membrane proteins, including the epithelial sodium channel (ENaC), the epidermal growth factor receptor, the transforming growth factor-β receptor, and the cytokine receptor γ-c (8.Boulkroun S. Ruffieux-Daidie D. Vitagliano J.J. Poirot O. Charles R.P. Lagnaz D. Firsov D. Kellenberger S. Staub O. Am. J. Physiol. Renal Physiol. 2008; 295: F889-F900Crossref PubMed Scopus (60) Google Scholar, 9.Butterworth M.B. Edinger R.S. Ovaa H. Burg D. Johnson J.P. Frizzell R.A. J. Biol. Chem. 2007; 282: 37885-37893Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 10.Fakitsas P. Adam G. Daidié D. van Bemmelen M.X. Fouladkou F. Patrignani A. Wagner U. Warth R. Camargo S.M. Staub O. Verrey F. J. Am. Soc. Nephrol. 2007; 18: 1084-1092Crossref PubMed Scopus (117) Google Scholar, 11.Gesbert F. Malardé V. Dautry-Varsat A. Biochem. Biophys. Res. Commun. 2005; 334: 474-480Crossref PubMed Scopus (23) Google Scholar, 12.Urbé S. McCullough J. Row P. Prior I.A. Welchman R. Clague M.J. Biochem. Soc. Trans. 2006; 34: 754-756Crossref PubMed Scopus (21) Google Scholar, 13.Wicks S.J. Grocott T. Haros K. Maillard M. ten Dijke P. Chantry A. Biochem. Soc. Trans. 2006; 34: 761-763Crossref PubMed Scopus (55) Google Scholar, 14.Wicks S.J. Haros K. Maillard M. Song L. Cohen R.E. Dijke P.T. Chantry A. Oncogene. 2005; 24: 8080-8084Crossref PubMed Scopus (149) Google Scholar). ubiquitin-activating enzyme ubiquitin-conjugating enzyme ubiquitin ligase cystic fibrosis transmembrane conductance regulator ubiquitin-specific protease-10 deubiquitinating enzyme epithelial sodium channel ATP-binding cassette hemagglutinin reverse transcription small interference RNA ubiquitin-vinyl methyl ester lysosome-associated protein-1. CFTR is rapidly endocytosed from the plasma membrane and undergoes rapid and efficient recycling back to the plasma membrane in human airway epithelial cells, with >75% of endocytosed wild-type CFTR recycling back to the plasma membrane (15.Ameen N. Silvis M. Bradbury N.A. J. Cyst. Fibros. 2007; 6: 1-14Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 16.Gentzsch M. Chang X.B. Cui L. Wu Y. Ozols V.V. Choudhury A. Pagano R.E. Riordan J.R. Mol. Biol. Cell. 2004; 15: 2684-2696Crossref PubMed Scopus (181) Google Scholar, 17.Swiatecka-Urban A. Brown A. Moreau-Marquis S. Renuka J. Coutermarsh B. Barnaby R. Karlson K.H. Flotte T.R. Fukuda M. Langford G.M. Stanton B.A. J. Biol. Chem. 2005; 280: 36762-36772Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 18.Swiatecka-Urban A. Talebian L. Kanno E. Moreau-Marquis S. Coutermarsh B. Hansen K. Karlson K.H. Barnaby R. Cheney R.E. Langford G.M. Fukuda M. Stanton B.A. J. Biol. Chem. 2007; 282: 23725-23736Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). A study published several years ago demonstrated that, although ubiquitination did not regulate CFTR endocytosis, ubiquitination reduced the plasma membrane abundance of CFTR in BHK cells by redirecting CFTR from recycling endosomes to lysosomes for degradation (19.Sharma M. Pampinella F. Nemes C. Benharouga M. So J. Du K. Bache K.G. Papsin B. Zerangue N. Stenmark H. Lukacs G.L. J. Cell Biol. 2004; 164: 923-933Crossref PubMed Scopus (280) Google Scholar). However, neither the E3 ubiquitin ligase(s) responsible for the ubiquitination of CFTR nor the DUB(s) responsible for the deubiquitination of CFTR in the endocytic pathway have been identified in any cell type. Moreover, the effect of the ubiquitin status of CFTR on its endocytic sorting in human airway epithelial cells has not been reported. Thus, the goals of this study were to determine if the ubiquitin status regulates the post-endocytic sorting of CFTR in polarized airway epithelial cells, and to identify the DUBs that deubiquitinate CFTR. Approximately 100 DUBs have been identified in the human genome and are classified into five families based on sequence similarity and mechanism of action (1.Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (965) Google Scholar, 2.Clague M.J. Urbé S. Trends Cell Biol. 2006; 16: 551-559Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 3.Mukhopadhyay D. Riezman H. Science. 2007; 315: 201-205Crossref PubMed Scopus (966) Google Scholar, 4.Love K.R. Catic A. Schlieker C. Ploegh H.L. Nat. Chem. Biol. 2007; 3: 697-705Crossref PubMed Scopus (183) Google Scholar, 5.Williams R.L. Urbé S. Nat. Rev. 2007; 8: 355-368Crossref Scopus (569) Google Scholar, 6.Millard S.M. Wood S.A. J. Cell Biol. 2006; 173: 463-468Crossref PubMed Scopus (53) Google Scholar, 20.Lorenzo M.E. Jung J.U. Ploegh H.L. J. Virol. 2002; 76: 5522-5531Crossref PubMed Scopus (80) Google Scholar, 21.Schlieker C. Korbel G.A. Kattenhorn L.M. Ploegh H.L. J. Virol. 2005; 79: 15582-15585Crossref PubMed Scopus (130) Google Scholar). To identify DUBs that regulate the deubiquitination of CFTR from this large class of enzymes, we chose an activity-based, chemical probe screening approach developed by Dr. Hidde Ploegh (4.Love K.R. Catic A. Schlieker C. Ploegh H.L. Nat. Chem. Biol. 2007; 3: 697-705Crossref PubMed Scopus (183) Google Scholar, 21.Schlieker C. Korbel G.A. Kattenhorn L.M. Ploegh H.L. J. Virol. 2005; 79: 15582-15585Crossref PubMed Scopus (130) Google Scholar, 22.Borodovsky A. Ovaa H. Kolli N. Gan-Erdene T. Wilkinson K.D. Ploegh H.L. Kessler B.M. Chem. Biol. 2002; 9: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). This approach utilizes a hemagglutinin (HA)-tagged ubiquitin probe engineered with a C-terminal modification incorporating a thiol-reactive group that forms an irreversible, covalent bond with active DUBs. Using this approach we demonstrated in polarized human airway epithelial cells that ubiquitin-specific protease-10 (USP10) is located in early endosomes and regulates the deubiquitination of CFTR and thus its trafficking in the post-endocytic compartment. These studies demonstrate a novel function for USP10 in promoting the deubiquitination of CFTR in early endosomes and thereby enhancing the endocytic recycling of CFTR. The role of DUBs in the intracellular trafficking of CFTR was studied in human airway epithelial cells (CFBE41o− cells, homozygous for the ΔF508 mutation) stably expressing wt-CFTR. Details on the stable transfection and characterization of CFBE41o− cells expressing wt-CFTR (hereafter called CFBE cells) have been described in detail by several laboratories (17.Swiatecka-Urban A. Brown A. Moreau-Marquis S. Renuka J. Coutermarsh B. Barnaby R. Karlson K.H. Flotte T.R. Fukuda M. Langford G.M. Stanton B.A. J. Biol. Chem. 2005; 280: 36762-36772Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 18.Swiatecka-Urban A. Talebian L. Kanno E. Moreau-Marquis S. Coutermarsh B. Hansen K. Karlson K.H. Barnaby R. Cheney R.E. Langford G.M. Fukuda M. Stanton B.A. J. Biol. Chem. 2007; 282: 23725-23736Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 23.Bebok Z. Collawn J.F. Wakefield J. Parker W. Li Y. Varga K. Sorscher E.J. Clancy J.P. J. Physiol. 2005; 569: 601-615Crossref PubMed Scopus (154) Google Scholar). CFBE cells between passages 18 and 27 were maintained in minimal essential medium supplemented with 50 μg/ml penicillin, 50 μg/ml streptomycin, 2 mm l-glutamine, 10% fetal bovine serum, 2 μg/ml puromycin, and 5 μg/ml plasmocin in a 5% CO2-95% air incubator at 37 °C. To establish confluent, polarized monolayers, 1 × 106 cells were seeded onto 24-mm Transwell permeable supports (0.4-μm pore size, Corning, Corning, NY) coated with Vitrogen plating medium containing human fibronectin (10 μg/ml, Collaborative Biomedical Products, Bedford, MA), Vitrogen 100 (1%, Collagen, Palo Alto, CA), and bovine serum albumin (10 μg/ml, Sigma-Aldrich) and grown in an air-liquid interface culture at 37 °C for 6–9 days, as described (17.Swiatecka-Urban A. Brown A. Moreau-Marquis S. Renuka J. Coutermarsh B. Barnaby R. Karlson K.H. Flotte T.R. Fukuda M. Langford G.M. Stanton B.A. J. Biol. Chem. 2005; 280: 36762-36772Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). To identify active DUBs in CFBE cells we used a chemical probe screening approach described in detail by Dr. Hidde Ploegh (4.Love K.R. Catic A. Schlieker C. Ploegh H.L. Nat. Chem. Biol. 2007; 3: 697-705Crossref PubMed Scopus (183) Google Scholar, 21.Schlieker C. Korbel G.A. Kattenhorn L.M. Ploegh H.L. J. Virol. 2005; 79: 15582-15585Crossref PubMed Scopus (130) Google Scholar, 22.Borodovsky A. Ovaa H. Kolli N. Gan-Erdene T. Wilkinson K.D. Ploegh H.L. Kessler B.M. Chem. Biol. 2002; 9: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). The chemical structure of the probe utilized in this study is presented in Fig. 1 by Borodovsky et al. (22.Borodovsky A. Ovaa H. Kolli N. Gan-Erdene T. Wilkinson K.D. Ploegh H.L. Kessler B.M. Chem. Biol. 2002; 9: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). Briefly, cells were lysed in radioimmunoprecipitation assay buffer (25 mm Tris-HCl, pH 7.6, 10 mm NaCl, 1% Nonidet P-40 (IGEPAL), 1% sodium deoxycholate, 0.1% SDS), and 0.1 μg of the HA-UbVME probe was added to 20 μg of the post-nuclear supernatant obtained by low speed (10,000 × g) centrifugation of cell lysates or to early endosomal fractions (isolated as described below) isolated from CFBE cells. The HA-UbVME probe forms an irreversible, covalent bond with active DUBs. Identification of DUBs covalently linked to the HA-UbVME probe was achieved by immunoprecipitation of the HA-UbVME·DUB complex(s) using an anti-HA monoclonal antibody (Santa Cruz Biotechnology) followed by SDS-PAGE and Western blot analysis using specific anti-DUB antibodies (see "Antibodies and Reagents"). The specificity of the HA-UbVME probe for active DUBs was confirmed with the addition of N-ethylmaleimide (10 μm), which inhibits cysteine protease DUBs, during the labeling reaction (4.Love K.R. Catic A. Schlieker C. Ploegh H.L. Nat. Chem. Biol. 2007; 3: 697-705Crossref PubMed Scopus (183) Google Scholar, 21.Schlieker C. Korbel G.A. Kattenhorn L.M. Ploegh H.L. J. Virol. 2005; 79: 15582-15585Crossref PubMed Scopus (130) Google Scholar, 22.Borodovsky A. Ovaa H. Kolli N. Gan-Erdene T. Wilkinson K.D. Ploegh H.L. Kessler B.M. Chem. Biol. 2002; 9: 1149-1159Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). To determine if USP10 is expressed in early endosomes, differential centrifugation and fractionation techniques were used to isolate early endosomes from CFBE cells using a protocol adapted from Butterworth et al. (9.Butterworth M.B. Edinger R.S. Ovaa H. Burg D. Johnson J.P. Frizzell R.A. J. Biol. Chem. 2007; 282: 37885-37893Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Briefly, polarized CFBE cells, grown on 24-mm permeable membrane supports, were scraped into phosphate-buffered saline, pelleted, and resuspended in 600 μl of HEPES buffer (250 mm sucrose, 10 mm HEPES, 0.5 mm EDTA at pH 7.4 containing protease inhibitors (Roche Applied Science)). The cells were homogenized with a Dounce homogenizer and passed through a 22-gauge needle 20 times. Following a low speed spin (3000 × g), the post-nuclear supernatant was diluted 1:1 with 62% sucrose in HEPES buffer and placed at the bottom of a 4.4-ml ultracentrifuge tube (Sorvall, Ashville, NC). 1.5 ml of 35% sucrose in HEPES buffer was layered on top followed by 1.5 ml of 25% sucrose in HEPES buffer and 0.5 ml of HEPES buffer. The gradients were centrifuged in a TH-660 rotor at 167,000 × g for 75 min at 4 °C, and the interfaces were collected to isolate the early endosomal fractions. Western blot analysis for various Rab GTPases was used to confirm purity of the early endosomal fraction. Rab5a and early endosome antigen-1 served as markers for the early endosomal fraction, whereas LAMP-1 and actin served as negative controls. To assess the amount of ubiquitinated CFTR in CFBE cells, a protocol was adapted from Urbe et al. (24.Urbé S. Sachse M. Row P.E. Preisinger C. Barr F.A. Strous G. Klumperman J. Clague M.J. J. Cell Sci. 2003; 116: 4169-4179Crossref PubMed Scopus (153) Google Scholar). Briefly, polarized CFBE cells were lysed in boiling lysis buffer (2% SDS, 1 mm EDTA, 50 mm sodium fluoride, and Complete Protease Inhibitor Mixture (Roche Applied Science)) preheated to 100 °C. The lysates were transferred to screw-cap tubes, incubated for 10 min at 100 °C, and cooled to room temperature, and the lysates were diluted by the addition of four volumes of the dilution buffer (2.5% Triton X-100, 12.5 mm Tris, pH 7.5, 187.5 mm NaCl, and Complete Protease Inhibitor Mixture (Roche Applied Science)). After pelleting cell debris by low speed centrifugation (3000 × g), the lysates were immunoprecipitated overnight at 4 °C with 5 μg of anti-CFTR antibody (clone M3A7, Upstate Biotechnology) complexed with Protein G-agarose. Immunoprecipitated complexes were washed three times with dilution buffer (2% Triton X-100, 0.4% SDS, 10 mm Tris, pH 7.5, 150 mm NaCl), once with a high salt wash buffer (200 mm NaCl, 400 mm NaOAc), and once more with the dilution buffer before preparation for SDS-PAGE and Western blot analysis using a ubiquitin antibody that recognizes mono-, multi-, and polyubiquitin additions (FK2 ubiquitin clone, Biomol) or a ubiquitin antibody that recognizes only polyubiquitin additions (FK1 ubiquitin clone, Biomol). The quantitation of ubiquitinated CFTR was calculated as the signal obtained with the ubiquitin antibody normalized for immunoprecipitated CFTR detected with the CFTR antibody (clone 24-1, R&D Systems). To determine if CFTR interacts with USP10 in the early endosomal fractions, USP10 was immunoprecipitated from early endosomal fractions isolated from the CFBE cell lysate by methods described previously in detail (18.Swiatecka-Urban A. Talebian L. Kanno E. Moreau-Marquis S. Coutermarsh B. Hansen K. Karlson K.H. Barnaby R. Cheney R.E. Langford G.M. Fukuda M. Stanton B.A. J. Biol. Chem. 2007; 282: 23725-23736Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Briefly, CFBE cells were lysed in immunoprecipitation buffer containing 150 mm NaCl, 50 mm Tris (pH 7.2), 0.1% IGEPAL (Sigma), 5 mm MgCl2, 5 mm EDTA, 1 mm EGTA, 30 mm NaF, 1 mm Na3VO4, and Complete Protease Inhibitor Mixture (Roche Applied Science), and USP10 was immunoprecipitated by incubation with 5 μg of a polyclonal USP10 antibody (Bethyl Laboratories) and protein A-agarose complex. Immunoprecipitated proteins were eluted from the protein A-agarose complexes by incubation at 100 °C for 3 min in Laemmli sample buffer (Bio-Rad) containing 80 mm dithiothreitol. Immunoprecipitated proteins were separated by SDS-PAGE using 15% gels (Bio-Rad) and analyzed by Western blot analysis. The biochemical determination of apical membrane CFTR was performed by domain selective cell surface biotinylation using EZ-LinkTM Sulfo-NHS-LC-Biotin (Pierce), as described previously in detail (25.Moyer B.D. Loffing J. Schwiebert E.M. Loffing-Cueni D. Halpin P.A. Karlson K.H. Ismailov II Guggino W.B. Langford G.M. Stanton B.A. J. Biol. Chem. 1998; 273: 21759-21768Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 26.Swiatecka-Urban A. Duhaime M. Coutermarsh B. Karlson K.H. Collawn J. Milewski M. Cutting G.R. Guggino W.B. Langford G. Stanton B.A. J. Biol. Chem. 2002; 277: 40099-40105Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Reverse transcription (RT)-PCR studies were conducted to examine the endogenous expression of USP10 in CFBE cells, as previously described in detail (18.Swiatecka-Urban A. Talebian L. Kanno E. Moreau-Marquis S. Coutermarsh B. Hansen K. Karlson K.H. Barnaby R. Cheney R.E. Langford G.M. Fukuda M. Stanton B.A. J. Biol. Chem. 2007; 282: 23725-23736Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 27.Swiatecka-Urban A. Boyd C. Coutermarsh B. Karlson K.H. Barnaby R. Aschenbrenner L. Langford G.M. Hasson T. Stanton B.A. J. Biol. Chem. 2004; 279: 38025-38031Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). For USP10, triplicate reactions of each cDNA sample were incubated at 95° C for 10 min, followed by 35 cycles of 15 s at 95° C and 1 min at 60° C using TaqMan Gene Expression Assay primers (Applied Biosystems) for human USP10. RT-PCR products were run on an low melting point agarose gel to confirm product size, subcloned into pCR4-TOPO (Invitrogen), and submitted for sequence analysis to confirm the identity of the products. Q-RT-PCR studies were conducted to examine the effect of siUSP10 on USP10 mRNA expression using a protocol published previously in detail (18.Swiatecka-Urban A. Talebian L. Kanno E. Moreau-Marquis S. Coutermarsh B. Hansen K. Karlson K.H. Barnaby R. Cheney R.E. Langford G.M. Fukuda M. Stanton B.A. J. Biol. Chem. 2007; 282: 23725-23736Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Predesigned TaqMan Gene Expression Assay Q-RT-PCR primers (Applied Biosystems) for human USP10 were employed. The cDNA generated during RT was quantified (NanoDrop, NanoDrop Technologies), and data were expressed as percent change in USP10 mRNA expression. Plasmids containing GFP-wt-USP10 and GFP-USP10 (C424A) were a generous gift from Dr. Susanna Chiocca (European Institute of Oncology, IFOM-IEO campus (28.Soncini C. Berdo I. Draetta G. Oncogene. 2001; 20: 3869-3879Crossref PubMed Scopus (141) Google Scholar)). All constructs were sequence verified upon receipt by ABI PRISM dye terminator cycle sequencing (Applied Biosystems, Foster City, CA). Transient transfections of CFBE cells with GFP-wt-USP10 and USP10-C424A were conducted using Effectene (Qiagen, Valencia, CA) according to the manufacturer's instructions. USP10 expression was selectively reduced using siRNA for USP10 purchased from Qiagen, by methods described previously (18.Swiatecka-Urban A. Talebian L. Kanno E. Moreau-Marquis S. Coutermarsh B. Hansen K. Karlson K.H. Barnaby R. Cheney R.E. Langford G.M. Fukuda M. Stanton B.A. J. Biol. Chem. 2007; 282: 23725-23736Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 29.Wolde M. Fellows A. Cheng J. Kivenson A. Coutermarsh B. Talebian L. Karlson K. Piserchio A. Mierke D.F. Stanton B.A. Guggino W.B. Madden D.R. J. Biol. Chem. 2007; 282: 8099-8109Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). In brief, CFBE cells were seeded at 0.1 × 106 on 24-mm Transwell permeable membrane supports and cultured for 3 days. On day 4, post-seeding, cells were transfected with either 5, 15, or 50 nm siRNA for USP10 or a scrambled, control siRNA with HiPerfect transfection reagent according to the manufacturer's protocol (Qiagen). Sequences for siRNAs are: siUSP10 sense (5′-CACAGCUUCUGUUGACUCUTT-3′) and antisense (5′-AGAGUCAACAGAAGCUGUGTT-3′), and siNegative scrambled sense (5′-UUCUCCGAACGUGUCACGU-3′) and antisense (5′-ACGUGACACGUUCGGAGAA-3′). Cells were studied on day 8 post-seeding (i.e. 4 days after transfection with siRNA). Co-localization studies were conducted to confirm Western blot studies demonstrating that endogenous USP10 is expressed in early endosomes. Briefly, CFBE cells seeded at 0.1 × 106 on collagen-coated, glass-bottom Mat-Tek dishes, were infected 24 h after seeding with a baculovirus expressing a eGFP-Rab5a-eGFP plasmid (Organelle LightsTM Endosomes-GFP, Molecular Probes, Invitrogen), according to the manufacturer's instructions, and fixed for immunolabeling 96 h post-infection, as described previously (30.Bomberger J.M. Parameswaran N. Hall C.S. Aiyar N. Spielman W.S. J. Biol. Chem. 2005; 280: 9297-9307Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). USP10 was visualized by indirect immunofluorescence using a polyclonal antibody for USP10 (Bethyl Laboratories, Montgomery, TX) followed by an Alexa 586-labeled secondary antibody. Z-stack images (0.4-μm sections) of labeled cells were acquired with a Nikon Sweptfield confocal microscope (Apo TIRF 100× oil immersion 1.49 numerical aperture objective) fitted with a QuantEM:512sc camera (Photometrics, Tucson, AZ) and Elements 2.2 software (Nikon, Inc.) to reconstruct and render three-dimensional images. Experiments were repeated three times, with five fields imaged for each experiment. Ussing chamber measurements of CFTR-mediated chloride secretion were performed as described previously (31.Swiatecka-Urban A. Moreau-Marquis S. Maceachran D.P. Connolly J.P. Stanton C.R. Su J.R. Barnaby R. O'Toole G.A. Stanton B.A. Am. J. Physiol. Cell Physiol. 2006; 290: C862-C872Crossref PubMed Scopus (60) Google Scholar). Endocytic and recycling assays were performed in CFBE cells as described previously (17.Swiatecka-Urban A. Brown A. Moreau-Marquis S. Renuka J. Coutermarsh B. Barnaby R. Karlson K.H. Flotte T.R. Fukuda M. Langford G.M. Stanton B.A. J. Biol. Chem. 2005; 280: 36762-36772Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 18.Swiatecka-Urban A. Talebian L. Kanno E. Moreau-Marquis S. Coutermarsh B. Hansen K. Karlson K.H. Barnaby R. Cheney R.E. Langford G.M. Fukuda M. Stanton B.A. J. Biol. Chem. 2007; 282: 23725-23736Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 31.Swiatecka-Urban A. Moreau-Marquis S. Maceachran D.P. Connolly J.P. Stanton C.R. Su J.R. Barnaby R. O'Toole G.A. Stanton B.A. Am. J. Physiol. Cell Physiol. 2006; 290: C862-C872Crossref PubMed Scopus (60) Google Scholar). For both assays, the plasma membrane proteins were first biotinylated at 4 °C using EZ-LinkTM Sulfo-NHS-SS-Biotin (Pierce). For the endocytic assay, cells were warmed to 37 °C for 5 min after biotinylation, and GSH was applied at 4 °C to reduce the disulfide bonds between proteins labeled with Sulfo-NHS-SS-Biotin in the plasma membrane. Biotinylated proteins that were endocytosed during the 5-min period at 37 °C are not reduced by GSH, which is impermeant to the plasma membrane, and thus reside in the endosomal compartment. Cells were lysed, and biotinylated proteins were isolated using streptavidin-agarose beads, eluted into SDS sample buffer, and separated by 7.5% SDS-PAGE. For recycling assays, cells were warmed to 37 °C for 5 min after biotinylation to load endocytic vesicles with biotinylated proteins. Cells were then cooled immediately to 4 °C, and the disulfide bonds on Sulfo-NHS-SS-Biotin-labeled proteins remaining in the plasma membrane were reduced by GSH at 4 °C. Subsequently, cells were either lysed or warmed again to 37 °C for 5 min (to allow endocytosed and biotinylated CFTR to recycle to the plasma membrane). Cells were then cooled again to 4 °C, and the disulfide bonds on the proteins biotinylated with Sulfo-NHS-SS-Biotin remaining in the plasma membrane were reduced with GSH. The recycling of endocytosed CFTR was calculated as the difference between the amount of biotinylated CFTR after the first and second GSH treatments. The antibodies used were: mouse anti-human CFTR C terminus antibody (clone 24-1, R&D systems, Minneapolis, MN); mouse anti-CFTR antibody (clone M3A7, Upstate Biotechnology, Lake Placid, NY); mouse anti-EEA1 antibody, mouse anti-ezrin antibody, mouse anti-Rab5 antibody, mouse anti-LAMP-1 antibody, mouse anti-actin antibody, mouse anti-GFP antibody (BD Biosciences, San Jose, CA); mouse anti-HA antibody (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-ubiquitin (clones FK2 and FK1) antibodies (Biomol, Plymouth Meeting, PA); rabbit anti-USP10 antibody, rabbit a