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• W2020217051 abstract "To attenuate injury during cholestasis, adaptive changes in bile acid transporter expression in the liver provide alternative bile acid excretory pathways. Apical sodium-dependent bile acid transporter (ASBT) (SLC10A2), only expressed in the liver on the cholangiocyte apical membrane, is rapidly regulated in response to inflammation and bile acids. Here, we studied the mechanisms controlling ASBT protein levels in cholangiocytes to determine whether ASBT expression is regulated by ubiquitination and disposal through the proteasome. Protein turnover assays demonstrated that ASBT is an unstable and short-lived protein. Treatment with MG-132, a proteasome inhibitor, causes time-dependent increased ASBT levels and increased intracellular accumulation of ASBT. In cells cotransfected with green fluorescent protein-tagged ASBT and hemagglutinin-tagged ubiquitin, we demonstrated coimmunoprecipitation and colocalization of ASBT and ubiquitin. Interleukin-1β (IL-1β) induced down-regulation of ASBT is abrogated by a JNK inhibitor and is accompanied by an increase in ASBT polyubiquitin conjugates and a reduced ASBT half-life. In phosphorylation-deficient S335A and T339A mutants, the ASBT half-life is markedly prolonged, IL-1β-induced ASBT ubiquitination is significantly reduced, and IL-1β fails to increase ASBT turnover. These results indicate that ASBT undergoes ubiquitin-proteasome degradation under basal conditions and that ASBT proteasome disposal is increased by IL-1β due to JNK-regulated serine/threonine phosphorylation of ASBT protein at both Ser-335 and Thr-339. These studies are the first report of regulation of a bile acid transporter expression by the ubiquitin-proteasome pathway. To attenuate injury during cholestasis, adaptive changes in bile acid transporter expression in the liver provide alternative bile acid excretory pathways. Apical sodium-dependent bile acid transporter (ASBT) (SLC10A2), only expressed in the liver on the cholangiocyte apical membrane, is rapidly regulated in response to inflammation and bile acids. Here, we studied the mechanisms controlling ASBT protein levels in cholangiocytes to determine whether ASBT expression is regulated by ubiquitination and disposal through the proteasome. Protein turnover assays demonstrated that ASBT is an unstable and short-lived protein. Treatment with MG-132, a proteasome inhibitor, causes time-dependent increased ASBT levels and increased intracellular accumulation of ASBT. In cells cotransfected with green fluorescent protein-tagged ASBT and hemagglutinin-tagged ubiquitin, we demonstrated coimmunoprecipitation and colocalization of ASBT and ubiquitin. Interleukin-1β (IL-1β) induced down-regulation of ASBT is abrogated by a JNK inhibitor and is accompanied by an increase in ASBT polyubiquitin conjugates and a reduced ASBT half-life. In phosphorylation-deficient S335A and T339A mutants, the ASBT half-life is markedly prolonged, IL-1β-induced ASBT ubiquitination is significantly reduced, and IL-1β fails to increase ASBT turnover. These results indicate that ASBT undergoes ubiquitin-proteasome degradation under basal conditions and that ASBT proteasome disposal is increased by IL-1β due to JNK-regulated serine/threonine phosphorylation of ASBT protein at both Ser-335 and Thr-339. These studies are the first report of regulation of a bile acid transporter expression by the ubiquitin-proteasome pathway. ASBT 1The abbreviations used are: ASBT, apical sodium-dependent bile acid transporter; NRC, normal rat cholangiocytes; ALLN, N-acetyl-leuleu-norleucinal; GFP, green fluorescent protein; HA, hemagglutinin; CHX, cycloheximide; MAPK, mitogen-activated protein kinases; JNK, c-Jun N-terminal kinase; MEK, MAPK kinase; PBS, phosphate-buffered saline; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone.1The abbreviations used are: ASBT, apical sodium-dependent bile acid transporter; NRC, normal rat cholangiocytes; ALLN, N-acetyl-leuleu-norleucinal; GFP, green fluorescent protein; HA, hemagglutinin; CHX, cycloheximide; MAPK, mitogen-activated protein kinases; JNK, c-Jun N-terminal kinase; MEK, MAPK kinase; PBS, phosphate-buffered saline; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; TPCK,l-1-tosylamido-2-phenylethyl chloromethyl ketone. mediates bile acid absorption from the lumen of the terminal ileum, renal tubules, and bile ducts (1Alpini G. Glaser S.S. Rodgers R. Phinizy J.L. Robertson W.E. Lasater J. Caligiuri A. Tretjak Z. LeSage G.D. Gastroenterology. 1997; 113: 1734-1740Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 2Shneider B.L. J. Pediatr. Gastroenterol. Nutr. 2001; 32: 407-417Crossref PubMed Scopus (124) Google Scholar, 3Lazaridis K.N. Pham L. Tietz P. Marinelli R.A. deGroen P.C. Levine S. Dawson P.A. LaRusso N.F. J. Clin. Investig. 1997; 100: 2714-2721Crossref PubMed Scopus (225) Google Scholar). Bile acids are the major products of cholesterol catabolism and function in the processes of bile secretion, intestinal absorption of lipids and lipid-soluble nutrients, and cholesterol elimination from the body (4Meier P.J. Stieger B. Annu. Rev. Physiol. 2002; 64: 635-661Crossref PubMed Scopus (465) Google Scholar). ASBT plays a key role in the recovery of bile acids from the intestinal lumen or renal tubules to prevent loss of bile acids via stool and urine, respectively (2Shneider B.L. J. Pediatr. Gastroenterol. Nutr. 2001; 32: 407-417Crossref PubMed Scopus (124) Google Scholar, 5Shneider B.L. Fox V.L. Schwarz K.B. Watson C.L. Ananthanarayanan M. Thevananther S. Christie D.M. Hardikar W. Setchell K.D. Mieli-Vergani G. Suchy F.J. Mowat A.P. Hepatology. 1997; 25: 1176-1183Crossref PubMed Scopus (95) Google Scholar). The role of ASBT in bile ducts is less well known. ASBT may function primarily following liver injury since the recently described ASBT knockout mouse (6Dawson P.A. Haywood J. Craddock A.L. Wilson M. Tietjen M. Kluckman K. Maeda N. Parks J.S. J. Biol. Chem. 2003; 278: 33920-33927Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) is grossly indistinguishable from wild-type mice, yet ASBT is up-regulated in liver injury due to bile duct ligation (7Lee J. Azzaroli F. Wang L. Soroka C.J. Gigliozzi A. Setchell K.D. Kramer W. Boyer J.L. Gastroenterology. 2001; 121: 1473-1484Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). ASBT, expressed on the apical membrane of cholangiocytes, is posed to absorb bile acids from ductal bile. Absorbed bile acid molecules are returned via the peribiliary plexus to the hepatic sinusoids, again removed by hepatocytes to be secreted into bile (8Hofmann A.F. Dig. Dis. Sci. 1989; 34: 16S-20SCrossref PubMed Scopus (37) Google Scholar). We have proposed that the exchange of bile acids between cholangiocytes and hepatocytes (termed cholehepatic shunting) potentially prevents bile acid-induced liver injury due to extrahepatic biliary obstruction by maintaining bile acid flux and preventing intracellular bile acid accumulation in the liver (9Alpini G. Glaser S. Alvaro D. Ueno Y. Marzioni M. Francis H. Baiocchi L. Stati T. Barbaro B. Phinizy J.L. Mauldin J. Lesage G. Gastroenterology. 2002; 123: 1226-1237Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Recently, the inflammatory cytokine IL-1β has been shown to down-regulate ASBT in the terminal ileum (10Chen F. Ma L. Sartor R.B. Li F. Xiong H. Sun A.Q. Shneider B. Gastroenterology. 2002; 123: 2005-2016Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Dysregulation of the ASBT adaptation to cholestasis (due to increased expression of IL-1β) could blunt the compensatory up-regulation of ASBT in response to cholestasis and promote bile acid-induced liver damage (9Alpini G. Glaser S. Alvaro D. Ueno Y. Marzioni M. Francis H. Baiocchi L. Stati T. Barbaro B. Phinizy J.L. Mauldin J. Lesage G. Gastroenterology. 2002; 123: 1226-1237Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar).Recent data demonstrate that the ubiquitin-proteasome degradation system affects the activity of some membrane transporters (11Hicke L. Trends Cell Biol. 1999; 9: 107-112Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 12Keitel V. Nies A.T. Brom M. Hummel-Eisenbeiss J. Spring H. Keppler D. Am. J. Physiol. 2003; 284: G165-G174Crossref PubMed Scopus (112) Google Scholar, 13Hirano K. Zuber C. Roth J. Ziak M. Am. J. Pathol. 2003; 163: 111-120Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 14Wang L. Soroka C.J. Boyer J.L. J. Clin. Investig. 2002; 110: 965-972Crossref PubMed Scopus (143) Google Scholar, 15Bebok Z. Varga K. Hicks J.K. Venglarik C.J. Kovacs T. Chen L. Hardiman K.M. Collawn J.F. Sorscher E.J. Matalon S. J. Biol. Chem. 2002; 277: 43041-43049Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 16Skach W.R. Kidney Int. 2000; 57: 825-831Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The system is responsible for the disposal of many of the short-lived proteins in eukaryotic cells (17Imai J. Yashiroda H. Maruya M. Yahara I. Tanaka K. Cell Cycle. 2003; 2: 585-590Crossref PubMed Scopus (62) Google Scholar, 18Hatakeyama S. Nakayama K.I. J. Biochem. (Tokyo). 2003; 134: 1-8Crossref PubMed Scopus (47) Google Scholar, 19Horak J. Biochim. Biophys. Acta. 2003; 1614: 139-155Crossref PubMed Scopus (62) Google Scholar). The ubiquitin-proteasome pathway targets proteins for degradation via covalent tagging of the substrate protein with a polyubiquitin chain (20Schwartz D.C. Hochstrasser M. Trends Biochem. Sci. 2003; 28: 321-328Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). This degradation pathway is implicated in the regulation of many short-lived proteins involved in essential cellular functions, including cell cycle control, transcription regulation, signal transduction, and protein translocation (17Imai J. Yashiroda H. Maruya M. Yahara I. Tanaka K. Cell Cycle. 2003; 2: 585-590Crossref PubMed Scopus (62) Google Scholar, 21Aguilar R.C. Wendland B. Curr. Opin. Cell Biol. 2003; 15: 184-190Crossref PubMed Scopus (145) Google Scholar, 22Voorhees P.M. Dees E.C. O'Neil B. Orlowski R.Z. Clin. Cancer Res. 2003; 9: 6316-6325PubMed Google Scholar, 23Strous G.J. van Kerkhof P. Mol. Cell. Endocrinol. 2002; 197: 143-151Crossref PubMed Scopus (46) Google Scholar, 24Kostova Z. Wolf D.H. EMBO J. 2003; 22: 2309-2317Crossref PubMed Scopus (361) Google Scholar). The proteins degraded by this pathway are covalently modified on lysine residues by fixation of a 8-kDa polypeptide, called ubiquitin, in a three-step process (18Hatakeyama S. Nakayama K.I. J. Biochem. (Tokyo). 2003; 134: 1-8Crossref PubMed Scopus (47) Google Scholar). In the first step, ubiquitin is activated by an ubiquitin-activating enzyme. The activated ubiquitin is subsequently transferred to an ubiquitin carrier protein. Finally, ubiquitin-protein ligase catalyzes the covalent binding of ubiquitin to the target protein. Following this process, multiubiquitinated proteins are rapidly degraded by the 26 S proteasome.We speculated that the initial ASBT down-regulation due to ileal inflammation or due to IL-1β in vitro (10Chen F. Ma L. Sartor R.B. Li F. Xiong H. Sun A.Q. Shneider B. Gastroenterology. 2002; 123: 2005-2016Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) is caused by enhanced ASBT disposal by the ubiquitin-proteasome pathway. Here we present results demonstrating that ASBT is an unstable protein that is rapidly degraded. Moreover, we show that the rapid IL-1β-dependent reduction of ASBT in cholangiocytes is due to increased ASBT disposal through the ubiquitin-proteasome pathway. IL-1β-mediated down-regulation of ASBT expression requires phosphorylation of ASBT since mutation of two ASBT phosphorylation sites motifs reduces the rate of ASBT disposal under basal conditions and markedly reduces IL-1β-dependent ubiquitination and disposal of ASBT. These results indicate that the proteasome plays an important role in the regulation of ASBT protein level.EXPERIMENTAL PROCEDURESMaterials and Plasmids—Dulbecco's modified Eagle's medium was purchased from Invitrogen. Fetal bovine serum was obtained from Sigma. IL-1β was purchased from R&D Systems Inc. (Minneapolis, MN), and ubiquitin antibody, MG-132, phenylmethylsulfonyl fluoride, N-acetyl-leu-leu-norleucinal (ALLN), lactacystin, TPCK, TLCK, aprotinin, SP600125, SB203580, and U0126 were purchased from Calbiochem. β-actin monoclonal antibodies, cycloheximide (CHX), and routine research reagents were purchased from Sigma. The green fluorescent protein (GFP) and hemagglutinin (HA) mouse monoclonal antibodies were from Santa Cruz Biotechnology Inc. ASBT antiserum was a gift from Dr. Paul Dawson. Wild-type rat ASBT cDNA was subcloned into a GFP vector, pEGFPN2 (gift from Dr. An-Qiang Sun, Mount Sinai School of Medicine, New York, NY). The pME18S-FLAG-tagged ASBT was constructed as follows. pME18S-FLAG-ASBT was generated by subcloning the ASBT cDNA into the EcoRI/XhoI sites of pME18s-FLAG plasmid. ASBT construct was assembled by PCR using the following synthetic oligonucleotides as primers: 5′-ATCTGTGAATTCGATAACTCCTCCGTCTGTTCCCCAAATGC-3′ and 5′-CAGACTCTCGAGCTATTTCTCATCTGGTTGAAATCCCTTGTTTG-3′forpME18s-FLAG-ASBT. The PCR products were inserted into the EcoRI/XhoI sites of pME18S-FLAG.Site-directed Mutagenesis—ASBT-S335A, ASBT-T339A, and double mutant (ST/AA) were made using rat ASBT-GFP as template. The QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to convert codons for potential sorting determinant residues to alanine residues according to the manufacturer's directions with minor modifications as described previously (25Sun A.Q. Salkar R. Sachchidanand Xu S. Zeng L. Zhou M.M. Suchy F.J. J. Biol. Chem. 2003; 278: 4000-4009Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The positive clones were verified by DNA sequencing.Cell Culture and Transfection—Mz-ChA-1 cholangiocarcinoma cell line (human gall bladder in origin) was a gift from Dr. Fitz (University of Texas Southwestern, Dallas, TX). Cells were maintained in complete Dulbecco's modified Eagle's medium (containing 10% (v/v) fetal bovine serum, 50 units/ml penicillin, 50 μg/ml streptomycin, and 2 mm l-glutamine). Transient DNA transfection was carried out by LipofectAMINE 2000-mediated transfection (Invitrogen) according to the manufacturer's directions. Briefly, 1 × 105 cells were plated on a 6-well plate. The next day, cells were ∼70% confluent and were transfected with 2 μg of DNA. On the following day, transfected Mz-ChA-1 cells were split at 1:3 ratios in complete medium, and the transfected cell lines were selected by growth in the antibiotic G418 (1 mg/ml) (Invitrogen). 10–15 days after transfection, stable cell lines were obtained by subcloning individual colonies. Positive clones were selected by immunofluorescence microscopy, and expression was subsequently tested by Western blotting. All of the cells were maintained in a humidified incubator at 37 °C under 5% CO2 atmosphere.The HEK 293 cells, a human embryonic kidney cell line, were maintained in Dulbecco's modified Eagle's medium/nutrient mixture F12 (supplemented with 10% fetal calf serum), 1% penicillin/streptomycin, and 1% glutamate and incubated at 37 °C under 5% CO2. The HEK 293 cells were plated in 6-well culture dishes for Western blotting. 24 h after plating, HEK 293 cells were cotransfected with expression plasmids by the calcium phosphate coprecipitation method. Briefly, 200 μl of 300 mm CaCl2, 200 μl of 2× Hepes-buffered saline and a total 4 μg of plasmid were mixed gently and then added to the culture medium in 6-well dishes. Transfection efficiency into HEK 293 cells with calcium phosphate coprecipitation was very high, and up to 50–80% of cells were successfully transfected. Normal rat cholangiocytes (NRC) were isolated and cultured as we described previously (26Alpini G. Phinizy J.L. Glaser S. Francis H. Benedetti A. Marucci L. LeSage G. Am. J. Physiol. 2003; 284: G1066-G1073Crossref PubMed Scopus (27) Google Scholar).Western Blot Analysis—Cells were rinsed with ice-cold PBS and resuspended into the lysis buffer (1% Triton X-100, 50 mm Tris-HCl, pH 7.4, 150 mm NaCl, and protease inhibitor mixture (Roche Applied Science). Following a brief sonication, cell extracts were centrifuged to remove cell debris. Protein concentration was measured by the method of Bradford with bovine serum albumin as a standard. Cell lysates (50 μg) were then separated through 10% SDS-PAGE and blotted onto nitrocellulose membrane. The membrane was incubated for 1 h at room temperature in Tris-buffered saline/Tween 20 (20 mm Tris-HCl, pH 7.4,150 mm NaCl, 0.05% Tween 20) with 5% nonfat milk. The membrane was then incubated with anti-GFP antibody (1:2000) for 1 h in Tris-buffered saline/Tween 20. The detection system was horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences). Specific binding of the antibody was visualized by the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences) according to the manufacturer's instructions. The intensity of the bands was determined by scanning video densitometry using the ChemiImager™ 5500 low light imaging system (Alpha Innotech Corp., San Leandro, CA).Immunoprecipitation—Cells were lysed at intervals using Nonidet P-40 lysis buffer containing 50 mm Tris-Cl, pH 7.8, 150 mm NaCl, 1% Nonidet P-40, 10 mm EDTA, protease inhibitor mixture, 1 mm sodium orthovanadate, and 5 mm sodium fluoride. HA and GFP monoclonal antibodies were added to 500 μg of lysates and incubated at 4 °C for 3 h. Protein G plus agarose beads (Santa Cruz Biotechnology) were added to the antigen-antibody mixture and further incubated overnight at 4 °C. Beads were washed thoroughly (four times) with phosphate-buffered saline containing 0.1% Nonidet P-40 and 0.1% SDS and subjected to Western blot analysis using anti-HA or anti-GFP monoclonal antibody.ASBT Turnover Assays—For half-life analysis, Mz-ChA-1 cells stably expressing ASBT-GFP were treated with CHX. Mz-ChA-1 cells expressing ASBT-GFP were plated in 10-cm dishes. Overnight cultures at near confluency (90–100%) were treated with 100 μg/ml CHX to block protein synthesis. Cells were harvested in lysis buffer at the indicated time points. Protein levels were then examined by Western blotting for GFP as described above.Immunofluorescence—ASBT-GFP and HA-ubiquitin coexpression was assessed via indirect immunofluorescence. Mz-ChA-1 cells were seeded in 5% FBS and Dulbecco's modified Eagle's medium:Ham's F-12 on Lab-Tek Chamber slides (Nalge Nunc International). Slides were then washed with PBS, fixed in–20 °C methanol for 10 min, air-dried, and washed in 0.3% Tween/PBS for 5 min. Slides were blocked with 1% normal sheep serum in 1× antibody dilution buffer (1% bovine serum albumin in 0.3% Tween/PBS) for 1 h and incubated with 1:200 HA antibody overnight at 4 °C in a humid chamber to prevent evaporation of the antibody solution. After washing in 0.3% Tween/PBS for 10 min (3×), slides were reprobed with goat anti-mouse Alexa 594 (1:500; Molecular Probes, Eugene, OR) for 2 h at room temperature in a dark humid chamber and then washed in 0.3% Tween in PBS for subsequent washing steps. Slides were stained with 4′,6-diamidino-2-phenylindole, mounted with Gel/Mount (Biomeda, Foster City, CA) and visualized. Samples without primary antibody were used as control. Images were acquired with a Nikon TE2000-U microscope outfitted with epifluorescence and a Cascade digital camera (Roper Scientific, Inc., Tucson, AZ), and images were acquired and processed using Metamorph software version 6.0 (Universal Imaging, Downingtown, PA).Statistical Analysis—Data are presented as means ± S.D. Statistical significance was determined by Student's t test. We assigned significance at p < 0.05.RESULTSASBT Is a Short-lived Protein—To study the stability of ASBT protein, we determined the ASBT protein half-life in Mz-ChA-1 cells stably expressing ASBT-GFP. Whole cell extracts were isolated from cells at various times following the addition of 100 μg/ml CHX. As shown in Fig. 1A, the inhibition of protein synthesis resulted in the loss of ASBT by 12 h with over half of the protein decayed by 6 h. The mean half-life for ASBT-GFP was found to be 5.7 ± 1.2 h in three experiments. ASBT-GFP is labile as compared with the expressed GFP protein in Mz-ChA-1 cells (Fig. 1B). Furthermore, Mz-ChA-1 cells expressing ASBT-FLAG (Fig. 1C) had a similar half-life as compared with ASBT-GFP (6.3 ± 1.2 versus 5.7 ± 1.2 h, respectively). Since expressed GPF protein is stable and FLAG-tagged and GFP-tagged ASBT are similarly unstable, it is unlikely that the GFP tag alters ASBT stabilityProteasome Inhibitors Cause a Time- and Dose-dependent Accumulation of ASBT—To demonstrate the role of the proteasome in the degradation of ASBT, the effect of protease inhibitors on the steady-state levels of ASBT was assessed by immunoblot analysis of Mz-ChA-1 cells stably expressing wild-type ASBT-GFP. Although the protease inhibitors aprotinin, TLCK, TPCK, and phenylmethylsulfonyl fluoride did not significantly affect ASBT expression, the selective proteasome inhibitors, lactacystin, ALLN, and MG-132, led to a marked increase in steady-state levels of ASBT (Fig. 2A). Treatment with MG-132 induced a time-dependent increase in ASBT (Fig. 2B). Increased ASBT was evident as early as 4 h following treatment and progressively accumulated during the 24 h following the application of MG-132. In the presence of this inhibitor for 8 h, an upsmearing of ASBT-GFP to higher molecular weight forms was observed, consistent with accumulation of ubiquitinated ASBT (27Longva K.E. Blystad F.D. Stang E. Larsen A.M. Johannessen L.E. Madshus I.H. J. Cell Biol. 2002; 156: 843-854Crossref PubMed Scopus (318) Google Scholar). Concentrations of MG-132 as low as 5 μm induced significant increases in ASBT within 6 h of application (Fig. 2C), with higher concentrations of MG-132 resulting in higher levels of ASBT immunoreactivity. Also, fluorescent microscopy showed that exposure to MG-132 increased the total cellular GFP fluorescence signal (Fig. 2D). A similar increase in cellular GFP fluorescence was obtained using the highly specific proteasome inhibitors lactacystin and ALLN (data not shown). From these results, it is concluded that accumulation of post-translational modified ASBT is induced only by proteasome-specific protease inhibitors.Fig. 2ASBT is targeted to the proteasome. A, cells expressing wild-type ASBT-GFP were incubated with Me2SO (DMSO), 1 mm phenylmethylsulfonyl fluoride (PMSF), 10 μm lactacystin, 100 μm ALLN, 10 μm MG-132, 200 μm aprotinin, 100 μm TLCK, or 100 μm TPCK. Steady-state levels of ASBT-GFP were measured after 6 h. 50 μg of extract was separated by SDS-PAGE, transferred to nitrocellulose, and subjected to Western blot analysis using anti-GFP monoclonal antibody. B, proteasome inhibition causes a time-dependent increase in ASBT immunore-activity. Cells expressing wild-type ASBT-GFP were treated with 10 μm proteasome inhibitor MG-132 and analyzed by Western blot analysis for ASBT-GFP immunoreactivity 2, 4, 6, of 8 h following treatment. Upsmearing of ASBT-GFP to higher molecular weight forms is noted with 8 h of MG-132 treatment (arrow). C, proteasome inhibition causes a dose-dependent increase in ASBT immunoreactivity. Cells expressing wild-type ASBT-GFP were treated with 0–50 μm proteasome inhibitor MG-132 and analyzed by Western blot analysis for ASBT-GFP immunoreactivity 6 h following treatment. D, proteasome inhibition causes an increase in ASBT-GFP fluorescence signal in Mz-ChA-1 cells. Cells expressing Wild-type ASBT-GFP were treated with 10 μm proteasome inhibitor MG-132 and analyzed by fluorescence microscopy for ASBT-GFP fluorescence signal before (upper panel) and after 6 h (lower panel) following treatment (imaged using ×40 objective). E, proteasome inhibition causes an increase in endogenous ASBT protein. NRC cells were treated with MG-132 for 24 h and were analyzed by Western blot using a specific ASBT antiserum.View Large Image Figure ViewerDownload (PPT)Since NRC cells express significant amounts of endogenous ASBT protein, the influence of MG-132 on endogenous ASBT level was analyzed. Western blot analysis using a specific ASBT antiserum demonstrated that treatment of NRC cells with MG-132 (Fig. 2E) resulted in a 2-fold increase of endogenous ASBT protein levels. These results indicate that both endogenously and exogenously expressed ASBT proteins are degraded by the ubiquitin-proteasome pathway.ASBT Protein Is Ubiquitinated—To determine the intracellular distribution of ASBT-GFP, fluorescence microscopy was used to monitor the expression of GFP-tagged ASBT after exposure to a proteasome inhibitor (Fig. 3A). Control experiments indicate that the presence of an N-terminal GFP tag does not alter the kinetics of ASBT degradation (data not shown). In untreated cells, ASBT-GFP fluorescence was detected in the plasma membrane and intracellularly in a reticular distribution. Exposure to the proteasome inhibitor, MG-132, increased total cellular ASBT-GFP fluorescence and increased the fluorescence signal at a distinct site adjacent to the nucleus. After 16 h of exposure to the inhibitor, nearly all of the ASBT-GFP fluorescence was present in a single large, juxtanuclear structure that appears to impinge upon and to distort the contour of the nuclear envelope. These findings are consistent with previous studies that show accumulation of ubiquitinated conjugates in a vesicle as a consequence of over-whelming the proteasome (28Tsirigotis M. Zhang M. Chiu R.K. Wouters B.G. Gray D.A. J. Biol. Chem. 2001; 276: 46073-46078Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). In both the presence and the absence of MG-132, the green fluorescent signal was observed throughout the cytoplasm when only GFP was transfected into Mz-ChA-1 cells (data not shown).Fig. 3Ubiquitinated ASBT molecules accumulate. A, the time course of ASBT accumulation. Mz-ChA-1 cells stably expressing low levels of ASBT-GFP were incubated in the presence of 10 μm MG-132 for the times indicated. Note the redistribution of ASBT from the plasma membrane to intracellular structures after 4 h of MG-132 treatment (×60 objective). B, colocalization of ASBT-GFP with HA-tagged ubiquitin (HA-Ub). Mz-ChA-1 cells were transiently cotransfected with ASBT-GFP and HA-tagged-ubiquitin cDNA and incubated in the presence of 10 μm MG-132 for 12 h. Cells were imaged by fluorescence microscopy for GFP and immunofluorescence for HA. ASBT-GFP and HA-ubiquitin fluorescence are primarily intracellular, and the merged image of ASBT-GFP and HA-ubiquitin fluorescence signals reveals strong colocalization (×40 objective). C, ASBT-GFP and HA-tagged ubiquitin coimmunoprecipitate. GFP-tagged ASBT or HA-tagged ubiquitin was transfected, or both were cotransfected in HEK 293 cells. After 24 h, cells were treated with 10 μm MG-132 for 6 h. Immunoprecipitates were collected with antibodies against HA (left panel) or GFP (right panel) and blotted with antibodies against the HA (lower panels) or GFP (upper panels). Initial immunoblots were performed in GFP immunoprecipitates blotted for HA and HA immunoprecipitates blotted for GFP. Membranes were then stripped and then reblotted for the other antibody.View Large Image Figure ViewerDownload (PPT)We investigated whether or not ubiquitin was present in the GFP-tagged ASBT-containing structure that is formed in response to proteasome inhibition. Mz-ChA-1 cells were transiently transfected with GFP-tagged ASBT together with excess plasmid encoding HA-tagged ubiquitin and analyzed by immunofluorescence microscopy using antibody to HA. The majority of HA-tagged ubiquitin protein colocalized with GFP-ASBT (Fig. 3B). We next determined whether GFP-tagged ASBT and HA-tagged ubiquitin coimmunoprecipitate. GFP-tagged ASBT and HA-tagged ubiquitin were transiently expressed in HEK293 cells, and immunoprecipitates were then collected with anti-GFP or anti-HA antibodies and subjected to immunoblot analysis with antibodies directed against the HA epitope or GFP (Fig. 3C). In cells cotransfected with GFP-tagged ASBT and HA-tagged ubiquitin, GFP-ASBT and larger molecular weight forms of GFP-ASBT (consistent with ubiquitin-conjugated ASBT and labeled ubiquitin-ASBT) were detected in HA and GFP immunoprecipitates (Fig. 3C, upper panels). In cells transfected with only GFP-ASBT, GFP-ASBT and ubiquitin-ASBT were detected in GFP but not HA immunoprecipitates (Fig. 3C, upper panels). Ubiquitin-HA was detected in HA immunoprecipitates only in cells expressing HA-tagged ubiquitin (Fig. 3C, lower left panel). Ubiquitin-HA was detected in GFP immunoprecipitates only in cells cotransfected with GFP-tagged ASBT and HA-tagged ubiquitin but not in single transfections (Fig. 3C, lower right panel). The data show that GFP-tagged ASBT and HA-tagged ubiquitin coimmunoprecipitate. Similar GFP immunoblots of GFP immunoprecipitates from cells cotransfected with GFP-tagged ASBT and HA-tagged ubiquitin as compared with cells transfected with GFP-ASBT alone (Fig. 3C, upper right panel) show that endogenous and expressed ubiquitin modifies ASBT in a similar way.IL-1β Induces JNK-dependent Ubiquitination and Disposal of ASBT—We first sought to determine whether the IL-1β down-regulation of ASBT is due to increased proteasome-mediated degradation. Treatment with 1 ng/ml IL-1β for 4 h reduced ASBT protein levels by 70% as compared with saline control (Fig. 4A, first and second lane). IL-1β regulates the activity of a variety of target genes and transcription" @default.
• W2020217051 created "2016-06-24" @default.
• W2020217051 creator A5028466877 @default.
• W2020217051 creator A5036944657 @default.
• W2020217051 creator A5046091589 @default.
• W2020217051 creator A5050463501 @default.
• W2020217051 creator A5081735024 @default.
• W2020217051 creator A5083371790 @default.
• W2020217051 date "2004-10-01" @default.
• W2020217051 modified "2023-10-09" @default.
• W2020217051 title "Degradation of the Apical Sodium-dependent Bile Acid Transporter by the Ubiquitin-Proteasome Pathway in Cholangiocytes" @default.
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