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• W2019180408 abstract "We report here that ubiquitin ligase Nedd4-2 regulates amino acid transporter ATA2 activity on the cell surface. We first found that a proteasome inhibitor MG132 increased the uptake of α-(methylamino)isobutyric acid, a model substrate for amino acid transport system A, in 3T3-L1 adipocytes as well as the preadipocytes. Transient expression of Nedd4-2 in Xenopus oocytes and Chinese hamster ovary cells down-regulated the ATA2 transport activity induced by injected cRNA and transfected cDNA, respectively. Neither the Nedd4-2 mutant with defective catalytic domain nor c-Cbl affected the ATA2 activity significantly. RNA-mediated interference of Nedd4-2 increased the ATA2 activity in the cells, and this was associated with decreased polyubiquitination of ATA2 on the cell surface membrane. Immunofluorescent analysis of Nedd4-2 in the adipocytes stably transfected with the enhanced green fluorescent protein (EGFP)-tagged ATA2 showed the co-localization of Nedd4-2 and EGFP-ATA2 in the plasma membrane but not in the perinuclear ATA2 storage site, supporting the idea that the primary site for the ubiquitination of ATA2 is the plasma membrane. These data suggest that ATA2 on the plasma membrane is subject to polyubiquitination by Nedd4-2 with consequent endocytotic sequestration and proteasomal degradation and that this process is an important determinant of the density of ATA2 functioning on the cell surface. We report here that ubiquitin ligase Nedd4-2 regulates amino acid transporter ATA2 activity on the cell surface. We first found that a proteasome inhibitor MG132 increased the uptake of α-(methylamino)isobutyric acid, a model substrate for amino acid transport system A, in 3T3-L1 adipocytes as well as the preadipocytes. Transient expression of Nedd4-2 in Xenopus oocytes and Chinese hamster ovary cells down-regulated the ATA2 transport activity induced by injected cRNA and transfected cDNA, respectively. Neither the Nedd4-2 mutant with defective catalytic domain nor c-Cbl affected the ATA2 activity significantly. RNA-mediated interference of Nedd4-2 increased the ATA2 activity in the cells, and this was associated with decreased polyubiquitination of ATA2 on the cell surface membrane. Immunofluorescent analysis of Nedd4-2 in the adipocytes stably transfected with the enhanced green fluorescent protein (EGFP)-tagged ATA2 showed the co-localization of Nedd4-2 and EGFP-ATA2 in the plasma membrane but not in the perinuclear ATA2 storage site, supporting the idea that the primary site for the ubiquitination of ATA2 is the plasma membrane. These data suggest that ATA2 on the plasma membrane is subject to polyubiquitination by Nedd4-2 with consequent endocytotic sequestration and proteasomal degradation and that this process is an important determinant of the density of ATA2 functioning on the cell surface. Amino acid transport system A is a Na+-dependent active transport system for neutral amino acids expressed in most tissues (1Christensen H.N. Methods Enzymol. 1989; 173: 576-616Crossref PubMed Scopus (102) Google Scholar). A unique characteristic of this system is its ability to recognize N-alkylated amino acids as substrates (2Christensen H.N. Oxender D.L. Liang M. Vatz K.A. J. Biol. Chem. 1965; 240: 3609-3616Abstract Full Text PDF PubMed Google Scholar). α-(Methylamino)isobutyric acid (MeAIB) 2The abbreviations used are: MeAIB, α-(methylamino)isobutyric acid; ATA, amino acid transporter A; 2DG, 2-deoxy-d-glucose; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; RNAi, RNA-mediated interference; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; CHO, Chinese hamster ovary; siRNA, small interfering RNA; GLUT, glucose transporter. 2The abbreviations used are: MeAIB, α-(methylamino)isobutyric acid; ATA, amino acid transporter A; 2DG, 2-deoxy-d-glucose; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; RNAi, RNA-mediated interference; E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin ligase; CHO, Chinese hamster ovary; siRNA, small interfering RNA; GLUT, glucose transporter. is commonly used as a model substrate for system A. Among the various amino acid transport systems known to function in mammalian cells, system A is best known for its regulation (3Haussinger D. Lang F. Kilberg M.S. Mammalian Amino Acid Transport: Mechanisms and Control. Plenum Press, New York1992: 113-130Crossref Google Scholar, 4Kilberg M.S. Bracy D.S. Handlogten M.E. Fed. Proc. 1986; 45: 2438-2441PubMed Google Scholar, 5Shotwell M.A. Kilberg M.S. Oxender D.L. Biochim. Biophys. Acta. 1983; 737: 267-284Crossref PubMed Scopus (355) Google Scholar). Recently, several groups including us have established the molecular identity of the amino acid transport system A (6Hatanaka T. Huang W. Ling R. Prasad P.D. Sugawara M. Leibach F.H. Ganapathy V. Biochim. Biophys. Acta. 2001; 1510: 10-17Crossref PubMed Scopus (96) Google Scholar, 7Hatanaka T. Huang W. Wang H. Sugawara M. Prasad P.D. Leibach F.H. Ganapathy V. Biochim. Biophys. Acta. 2000; 1467: 1-6Crossref PubMed Scopus (130) Google Scholar, 8Reimer R.J. Chaudhry F.A. Gray A.T. Edwards R.H. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7715-7720Crossref PubMed Scopus (163) Google Scholar, 9Sugawara M. Nakanishi T. Fei Y.J. Huang W. Ganapathy M.E. Leibach F.H. Ganapathy V. J. Biol. Chem. 2000; 275: 16473-16477Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 10Sugawara M. Nakanishi T. Fei Y.J. Martindale R.G. Ganapathy M.E. Leibach F.H. Ganapathy V. Biochim. Biophys. Acta. 2000; 1509: 7-13Crossref PubMed Scopus (124) Google Scholar, 11Varoqui H. Zhu H. Yao D. Ming H. Erickson J.D. J. Biol. Chem. 2000; 275: 4049-4054Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 12Wang H. Huang W. Sugawara M. Devoe L.D. Leibach F.H. Prasad P.D. Ganapathy V. Biochem. Biophys. Res. Commun. 2000; 273: 1175-1179Crossref PubMed Scopus (97) Google Scholar, 13Yao D. Mackenzie B. Ming H. Varoqui H. Zhu H. Hediger M.A. Erickson J.D. J. Biol. Chem. 2000; 275: 22790-22797Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). These studies have identified three distinct transporter proteins that are responsible for system A transport activity in mammalian cells, and all three transporters are capable of mediating the Na+-coupled uptake of the system A model substrate MeAIB. The three transporters are known as amino acid transporter A (ATA)1 (also known as SNAT1), ATA2 (SNAT2), and ATA3 (SNAT4). These transporters belong to the solute-linked carrier family SLC38 (14Mackenzie B. Erickson J.D. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 784-795Crossref PubMed Scopus (407) Google Scholar). ATA1 and ATA2 possess similar functional characteristics but show distinct tissue expression pattern. ATA1 is expressed primarily in the placenta and brain, whereas ATA2 is expressed ubiquitously in mammalian tissues. ATA3 is functionally distinguishable from ATA1 and ATA2, and its expression is restricted to the liver. It is generally believed that ATA2 represents system A, which is known for its regulatory features. There is also evidence to indicate that ATA2 corresponds to system A activity in adipocytes (15Hyde R. Christie G.R. Litherland G.J. Hajduch E. Taylor P.M. Hundal H.S. Biochem. J. 2001; 355: 563-568Crossref PubMed Scopus (72) Google Scholar, 16Ritchie J.W. Baird F.E. Christie G.R. Stewart A. Low S.Y. Hundal H.S. Taylor P.M. Cell. Physiol. Biochem. 2001; 11: 259-270Crossref PubMed Scopus (35) Google Scholar). Recently, we reported the regulation of ATA2 in adipocytes by insulin and in diabetes. 3Hatanaka, T., Hatanaka, Y., Tsuchida, J.-i., Ganapathy, V., and Setou, M. (October 18, 2006) J. Biol. Chem., 10.1074/jbc.M604534200. 3Hatanaka, T., Hatanaka, Y., Tsuchida, J.-i., Ganapathy, V., and Setou, M. (October 18, 2006) J. Biol. Chem., 10.1074/jbc.M604534200. In detail, we showed that insulin accelerated the translocation of ATA2 from the trans-Golgi network storage site to the plasma membrane and that the steady-state levels of ATA2 mRNA decreased in diabetes. The insulin-modulated translocation does not occur via a common endosomal pathway that is available for other plasma membrane proteins but via a pathway that is specific for ATA2. It is the balance between insertion into and sequestration from the plasma membrane that determines the density of the transporter on the cell surface that is responsible for measurable transport function. Very little is known at this time on the molecular events involved in the degradation of ATA2 subsequent to sequestration from the plasma membrane. Membrane proteins are frequently degraded in lysosomes, but there are some examples of transporter proteins being degraded by the ubiquitin-proteasome system (18Malik B. Schlanger L. Al-Khalili O. Bao H.F. Yue G. Price S.R. Mitch W.E. Eaton D.C. J. Biol. Chem. 2001; 276: 12903-12910Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 19Xia X. Roundtree M. Merikhi A. Lu X. Shentu S. Lesage G. J. Biol. Chem. 2004; 279: 44931-44937Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 20Malik B. Yue Q. Yue G. Chen X.J. Price S.R. Mitch W.E. Eaton. D.C. Am. J. Physiol. 2005; 289: F107-F116Crossref PubMed Scopus (54) Google Scholar). The ubiquitin-proteasome system is responsible for the disposal of many short-lived proteins in eukaryotic cells, and the process is initiated by covalent tagging of the target protein with a polyubiquitin chain on the lysine residue (21Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6879) Google Scholar). Here, we investigated the internalization and degradation pathway of ATA2 in 3T3-L1 adipocytes as well as preadipocytes. These studies show that an E3 ubiquitin ligase Nedd4-2 ligates ATA2 with the ubiquitin chain as the sorting signal for endocytosis, and then the ubiquitin-conjugated ATA2 is degraded by proteasomes. This is rather a specific internalization and degradation pathway for this membrane transporter than a general bulk pathway such as the lysosomal degradation. Materials—Mouse ATA2 (mATA2) cDNA was cloned from mouse kidney cDNA library as described previously.3 3T3-L1 murine fibroblasts were from Health Science Research Resources Bank (Osaka, Japan). CHO-K1 cells were provided from Cell Resource Center for Biomedical Research, Tohoku University (Sendai, Japan). Mouse Nedd4-2 cDNA, cell culture media, Lipofectamine 2000, and Alexa568-conjugated secondary antibodies were purchased from Invitrogen. [14C]MeAIB was from American Radiolabeled Chemicals (St. Louis, MO). 2-Deoxy-d-[14C]glucose (14C-labeled 2DG) was purchased from Moravek Biochemicals (Brea, CA). MG132 was purchased from the Peptide Institute Inc. (Osaka, Japan). The anti-GFP monoclonal antibody (JL-8) and the polyclonal antibodies against Nedd4 and c-Cbl were obtained from BD Biosciences (San Jose, CA). The anti-ubiquitin monoclonal antibody (1B3) was purchased from Medical Biological Laboratories (Nagoya, Japan). The horseradish peroxidase-conjugated secondary goat anti-rabbit IgG antibody was from Jackson ImmunoResearch (West Grove, PA). Mouse Nedd4 cDNA was purchased from DNAform (Ibaragi, Japan). Cell Culture—3T3-L1 fibroblasts (preadipocytes) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum, and differentiation was induced according to established protocols as described previously (22Bose A. Cherniack A.D. Langille S.E. Nicoloro S.M. Buxton J.M. Park J.G. Chawla A. Czech M.P. Mol. Cell. Biol. 2001; 21: 5262-5275Crossref PubMed Scopus (57) Google Scholar, 23Harrison S.A. Buxton J.M. Clancy B.M. Czech M.P. J. Biol. Chem. 1990; 265: 20106-20116Abstract Full Text PDF PubMed Google Scholar).3 Briefly, cells were allowed to reach confluence at least 2 days before the induction of differentiation. Differentiation was induced (on day 0) with high glucose DMEM, 10% fetal bovine serum containing 0.25 μm dexamethasone, 5 μg/ml insulin, and 500 μm methylisobutylxanthine. After 48 h (day 2), the cells were fed with high glucose DMEM, 10% fetal bovine serum containing 5 μg/ml insulin. After an additional 48 h (day 4), the cells were re-fed every 2 days with high glucose DMEM, 10% fetal bovine serum. All media were supplemented with 2 mm glutamine, 50 units of penicillin/ml, and 50 μg of streptomycin/ml. Differentiation was monitored by noting the accumulation of lipid droplets, which typically began by day 4 of differentiation. Cells were considered fully differentiated between days 8 and 12. CHO-K1 cells were cultured according to the protocol of the provider. Uptake Experiments in 3T3-L1 Cells—Before the uptake experiments, 3T3-L1 cells were fed with serum-free DMEM with or without MG132 (10 μm) for 4 h and then incubated with or without insulin (1 μm) in uptake buffer, pH 7.4, for 30 min. Uptake experiments were carried out following the previously described protocol.3 The uptake buffer was 25 mm Tris/HEPES, pH 8.0, for MeAIB (7Hatanaka T. Huang W. Wang H. Sugawara M. Prasad P.D. Leibach F.H. Ganapathy V. Biochim. Biophys. Acta. 2000; 1467: 1-6Crossref PubMed Scopus (130) Google Scholar, 9Sugawara M. Nakanishi T. Fei Y.J. Huang W. Ganapathy M.E. Leibach F.H. Ganapathy V. J. Biol. Chem. 2000; 275: 16473-16477Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar) or HEPES/Tris, pH 7.4, for 2DG, and the buffer contained 140 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, and 0.8 mm MgSO4. The uptake experiment was performed in 3T3-L1 cells at 37 °C for 30 min with [14C]MeAIB or 14C-labeled 2DG as the substrate. The concentration of the radiolabeled substrate was 3.6 μm for MeAIB and 10 μm for 2DG. The culture medium was removed by aspiration, and the cells were washed once with the uptake buffer. 0.25 ml of uptake buffer containing radiolabeled substrate (MeAIB or 2DG) was added to the wells and incubated for 30 min at 37 °C. Uptake was terminated by aspirating the buffer and subsequently washing the cells twice with ice-cold fresh uptake buffer. The cells were then lysed with 0.25 ml of 1% SDS in 0.2 N NaOH, and the lysate was transferred to scintillation vials for quantification of radioactivity. Carrier-mediated uptake of the substrate was calculated by subtracting the uptake measured in the presence of an excess amount of unlabeled substrate (10 mm) from the uptake measured in the absence of unlabeled substrate. Enhanced Green Fluorescent Protein (EGFP)-tagged ATA2 Expression in Mammalian Cells—CHO-K1 cells that transiently express ATA2 tagged with EGFP were established using the BD Living Colors pEGFP vector system from BD Biosciences Clontech (Palo Alto, CA), as described previously.3 Using the same cDNA construct (EGFP-ATA2), 3T3-L1 cells that stably express EGFP-tagged ATA2 were also established, as described previously.3 We have named this cell line EGFPATA2 3T3-L1 cells. Co-expression of ATA2 and E3 Ubiquitin Ligase in Xenopus laevis Oocytes and Uptake Measurements—We followed the protocol for the preparation of cRNAs and the oocytes as described previously (24Hatanaka T. Nakanishi T. Huang W. Leibach F.H. Prasad P.D. Ganapathy V. Ganapathy M.E. J. Clin. Investig. 2001; 107: 1035-1043Crossref PubMed Scopus (62) Google Scholar, 25Nakanishi T. Hatanaka T. Huang W. Prasad P.D. Leibach F.H. Ganapathy M.E. Ganapathy V. J. Physiol. (Lond.). 2001; 532: 297-304Crossref Scopus (166) Google Scholar, 26Hatanaka T. Huang W. Nakanishi T. Bridges C.C. Smith S.B. Prasad P.D. Ganapathy M.E. Ganapathy V. Biochem. Biophys. Res. Commun. 2002; 291: 291-295Crossref PubMed Scopus (82) Google Scholar, 27Hatanaka T. Haramura M. Fei Y.J. Miyauchi S. Bridges C.C. Ganapathy P.S. Smith S.B. Ganapathy V. Ganapathy M.E. J. Pharmacol. Exp. Ther. 2004; 308: 1138-1147Crossref PubMed Scopus (119) Google Scholar). Briefly, capped cRNAs from rat ATA2 (9Sugawara M. Nakanishi T. Fei Y.J. Huang W. Ganapathy M.E. Leibach F.H. Ganapathy V. J. Biol. Chem. 2000; 275: 16473-16477Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), mouse Nedd4-2, and human c-Cbl cDNAs were synthesized using the mMESSAGE mMACHINE kit (Ambion, Austin, TX) as described previously. Mature oocytes (stage IV or V) from X. laevis were isolated by treatment with collagenase A (1.6 mg/ml), manually defolliculated, and maintained at 18 °C in modified Barth's medium supplemented with 10 mg/ml gentamycin. On the following day oocytes were injected with 25 ng of ATA2 cRNA and 25 ng of Nedd4-2 cRNA or c-Cbl cRNA. Water-injected oocytes served as controls. The oocytes were used for MeAIB uptake experiments 4 days after cRNA injection. Uptake of [14C]MeAIB into oocytes was measured in a 24-well microtiter plate as described previously (28Fei Y.J. Prasad P.D. Leibach F.H. Ganapathy V. Biochemistry. 1995; 34: 8744-8751Crossref PubMed Scopus (72) Google Scholar). Briefly, 10 oocytes were incubated with the labeled substrate at room temperature for 60 min in the desired uptake buffer (100 mm NaC1, 2 mm KCl, 1 mm MgC12, and 1 mm CaC12 buffered with 10 mm Hepes/Tris, pH 8.0). Concentration of labeled amino acids was 7.2 μm. Uptake was terminated by washing the oocytes with ice-cold uptake medium four times. Each oocyte was then dissolved in 0.2 ml of 10% SDS, and radioactivity associated with the oocyte was determined by liquid scintillation spectrometry. Each experiment was repeated three times, and similar results were obtained each time. Transient Co-expression of ATA2 and E3 Ubiquitin Ligase in CHO-K1 Cells—Plasmids encoding Nedd4 and Nedd4-2 in which conserved cysteine in the catalytic active site HECT domain was mutated to serine (CS mutant) (29Magnifico A. Ettenberg S. Yang C. Mariano J. Tiwari S. Fang S. Lipkowitz S. Weissman A.M. J. Biol. Chem. 2003; 278: 43169-43177Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) were generated by a site-directed mutagenesis kit (QuikChange XL, Stratagene, La Jolla, CA). We mutated the cysteine 854 or 822 to serine in the HECT domain of Nedd4 or Nedd4-2 and obtained the corresponding CS mutants, Nedd4 C854S or Nedd4-2 C822S. The cDNAs of EGFP-ATA2 and E3 ubiquitin ligase were co-transfected in CHO cells with Lipofectamine 2000 as described previously.3 Uptake experiments were performed by following the protocol in 3T3-L1 cells as described above using the 100 μm MeAIB and 20 min of incubation. Cell Lysis and Immunoprecipitation—Cells were lysed with Triton X-100 lysis buffer containing 50 mm Tris-Cl, pH 7.4, 150 mm NaCl, 1% Triton X-100, 2 mm EDTA, protease inhibitor mixture (Complete, Roche Applied Science). An agarose-conjugated anti-GFP rat monoclonal antibody (Medical Biological Laboratories) was added to lysates and incubated at 4 °C for 1.5 h. The agarose-conjugated antibody was washed thoroughly (four times) with Triton X-100 lysis buffer and subjected to the Western blot analysis using the anti-ubiquitin or anti-GFP antibody. Biotinylation of cell surface proteins was performed by the method described by Rotmann et al. (30Rotmann A. Strand D. Martine U. Closs E.I. J. Biol. Chem. 2004; 279: 54185-54192Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) for the study of the internalization of the cationic amino acid transporter with slight modifications.3 Briefly, the preadipocytes stably expressing EGFPATA2 protein were grown to confluence and differentiated into adipocytes in 10-cm dishes. After the experimental treatment, the cells were rinsed with ice-cold phosphate-buffered saline (PBS) containing 0.1 mm CaCl2 and 1 mm MgCl2 (PBS+) and incubated in the same solution supplemented with 0.5 mg/ml sulfosuccinimidobiotin (EZ-Link sulfo-NHS-SS-Biotin: Pierce) for 30 min at 4 °C. The cells were then rinsed with the quenching solution in the cell surface protein biotinylation and purification kit (Pierce) once and Tris-buffered saline twice to quench any unbound biotin. The cells were then lysed by the addition of 1 ml of radioimmunoprecipitation assay buffer (100 mm Tris/HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing protease inhibitors (Complete EDTA-free; Roche Diagnostics) for 30 min at 4 °C. After removal of the cellular debris, protein concentrations of the lysates were determined using the Bradford method. 1 mg of the lysate proteins were batch-extracted overnight at 4 °C using avidin-coated agarose beads (immobilized NeutrAvidin, Pierce) and then released from the beads by incubation in the SDS-PAGE sample buffer (50 mm Tris/HCl, pH 6.8, 2% SDS, 100 mm dithiothreitol, 10% glycerol, 0.001% bromphenol blue, 5 min at 95 °C). Western Blot Analysis—Cell lysates, immunoprecipitated proteins, or cell surface proteins were separated in 6-10% SDS-PAGE and then blotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were probed with the appropriate primary antibodies. The bound primary antibodies were detected with the corresponding horseradish peroxidase-conjugated secondary antibodies. Signal was visualized using ECL kit (Amersham Biosciences). Stealth siRNA Treatment—We obtained two Stealth siRNA for mouse Nedd4-2 from Invitrogen. Each siRNA is a 25-bp duplex oligoribonucleotide with a sense strand corresponding to nucleotides 1755-1779 or 2336-2360 of the reported mouse Nedd4-2 coding sequence (GenBank™ accession number BC039746). The sense sequences of Nedd4-2 siRNA 1755 and 2336 are 5′-AAACUCUCUGGAGUACGGAACAGCC-3′ and 5′-UUCAGAUCCACUUGGUAUGUCUGCC-3′, respectively. Nedd4-2-scrambled control-siRNA 1755 and 2336 with sense strands 5′-AAACAUCCUCGGUUGAGCAAAGGCC-3′ and 5′-UUCCUGACUACACGUUAUGUGUGCC-3′ are the control stealth siRNAs for Nedd4-2 siRNA 1755 and 2336, respectively. 3T3-L1 preadipocytes were transfected by 20 nm of the Nedd4-2 siRNA or the control siRNA in the antibiotic-free growth medium using Lipofectamine 2000 per the instructions of the manufacturer. After siRNA treatment (24 h), the medium containing the Nedd4-2 siRNA or the control siRNA was changed to fresh medium. 24 h after the medium change (i.e. 48 h after the initiation of RNAi treatment), the cells were used for MeAIB uptake experiments or immunoprecipitation as described above and in the figure legends. Immunofluorescent Analysis and Confocal Laser Scanning Microscopy—3T3-L1 adipocytes were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline for 15 min. Coverslips were incubated with diluted Nedd4-2 antibody for 1 h and with diluted Alexa568-conjugated secondary antibodies in 2% skim milk, 0.1% Triton X-100, 0.02% SDS, phosphate-buffered saline for 30 min. Images were taken with upright confocal laser scanning microscope Carl Zeiss LSM5 PASCAL (Carl Zeiss, Oberkochen, Germany). Proteasome Inhibition Increased ATA2 Transport Activity by Increasing the Amount of ATA2 Protein in 3T3-L1 Adipocytes and Preadipocytes—To determine the effect of the proteasome inhibition on ATA2 transport activity, we treated 3T3-L1 adipocytes or preadipocytes with a proteasome inhibitor MG132 and then measured the uptake of MeAIB, a specific substrate for ATA2 into the cells (Fig. 1A). In adipocytes as well as preadipocytes, the treatment with MG132 for 4 h accelerated MeAIB uptake significantly. The effects of insulin and MG132 were additive. The uptake of 2DG, a specific substrate for the members of the facilitated glucose transporter family (GLUTs) did not show any change after MG132 treatment either with or without insulin (data not shown). We also used the 3T3-L1 cell line stably transfected with the EGFP-tagged ATA2 (EGFPATA2 3T3-L1 cells) to observe the changes of intracellular localization of ATA2. The establishment of this cell line has been described previously.3 In adipocytes as well as preadipocytes, the fluorescent signals associated with EGFP-ATA2 were markedly increased after treatment with MG132 (Fig. 1B). We compared the fluorescence signals under identical conditions in cells treated with or without MG132. We first optimized the experimental conditions in MG132-treated cells to avoid the saturation of signals and then applied identical conditions to nontreated cells. Strong signals were detected at the perinuclear site in MG132-treated cells. Under these conditions, the signals from nontreated cells were barely detectable. We also analyzed the steady-state levels of the ATA2 fusion protein by Western blot using anti-GFP antibody with whole cell lysates from EGFP-ATA2 3T3-L1 adipocytes and preadipocytes treated with or without MG132 (Fig. 1C). The band corresponding to 74 kDa (indicated with the arrow in Fig. 1C) was the intact EGFP-ATA2 protein as described previously,3 and the signal intensity of this band was increased by MG132 treatment in adipocytes as well as in preadipocytes. In addition to the band corresponding to the size of the intact, unmodified EGFPATA2, there were other protein bands with a range of higher molecular sizes whose intensities were also increased markedly in MG132-treated cells. ATA2 Protein as a Target for the Ubiquitin-Proteasome Pathway in 3T3-L1 Cells—To determine whether EGFP-ATA2 fusion protein is subject to ubiquitination, we treated the 3T3-L1 adipocytes with or without MG132 and then immunoprecipitated the proteins in the cell lysate with anti-GFP antibody. The proteins in the immunoprecipitate were then separated by SDS-PAGE and then immunoblotted with either anti-ubiquitin antibody or anti-GFP antibody. The presence of the ladder positive to the anti-ubiquitin antibody was detected by Western blot with the immunoprecipitate from MG132-treated cells but not from control cells (Fig. 2). The bands detected with the anti-ubiquitin antibody were of the size ranging from 90 to 250 kDa, suggesting varying degrees of ubiquitination of the fusion protein. In contrast, the proteins detected obviously by the anti-GFP antibody were smaller than 100 kDa including the intact EGFP-ATA2. After the long exposure time for ECL detection, the signals of the proteins with larger molecular sizes were faintly detectable in the blot of the immunoprecipitate from the MG132-treated cells (data not shown). This difference of detectability with the anti-ubiquitin or anti-GFP antibody was supposed to be due to the stoichiometry for polyubiquitination of a target protein EGFP-ATA2 and ubiquitins. Influence of Ubiquitin Ligase on ATA2 Transport Function upon Co-expression in Xenopus Oocytes—To determine the effect of ubiquitination on ATA2, we co-expressed ATA2 with either Nedd4-2 (a HECT domain E3) or c-Cbl (a RING domain E3) in Xenopus oocytes and then monitored the transport function of ATA2 by measuring the Na+-dependent uptake of MeAIB (Fig. 3). This co-expression system has been used by several investigators to determine the molecular identity of the ubiquitin ligase that interacts with any given target protein (31Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Pratt J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (579) Google Scholar, 32Konstas A.A. Shearwin-Whyatt L.M. Fotia A.B. Degger B. Riccardi D. Cook D.I. Korbmacher C. Kumar S. J. Biol. Chem. 2002; 277: 29406-29416Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 33Boehmer C. Henke G. Schniepp R. Palmada M. Rothstein J.D. Broer S. Lang F. J. Neurochem. 2003; 86: 1181-1188Crossref PubMed Scopus (99) Google Scholar). Co-expression of Nedd4-2 with ATA2 down-regulated ATA2-mediated MeAIB uptake, whereas c-Cbl did not affect MeAIB uptake significantly. Influence of Ubiquitin Ligase on ATA2 Transport Function upon Co-expression in CHO Cells—To investigate the interaction between ATA2 and an E3 ubiquitin ligase in further detail, we co-transfected the cDNAs of ATA2 and Nedd4, Nedd4-2, or c-Cbl in CHO cells. ATA2-specific transport activity was measured by MeAIB uptake (Fig. 4). Ligase-defective cysteine-toserine mutants in the HECT domain (CS mutants) of Nedd4 and Nedd4-2 were used to determine the involvement of the catalytic activity of ligase in observed changes in ATA2 transport function. MeAIB uptake induced by ATA2 cDNA was found to be decreased markedly by co-transfection with Nedd4-2 in CHO cells, an effect not seen with Nedd4-2 CS mutant. The effect of Nedd4 on ATA2 transport function was minimal compared with that of Nedd4-2. We confirmed by Western blot using appropriate antibodies that Nedd, Nedd4-2, their corresponding CS mutants, and c-Cbl were expressed at comparable levels in these cells (data not shown), showing that the observed differences in the transport function of ATA2 were not due to differences in the expression levels of these ligases. Effect of Knockdown of Nedd4-2 on the Activity and Ubiquitination of ATA2 in 3T3-L1 Cells—To evaluate the physiological relevance of endogenous Nedd4-2 in ubiquitination/internalization/degradation of ATA2, we knocked down the endogenous Nedd4-2 in 3T3-L1 preadipocytes by RNAi and then monitored the transport function of ATA2 by uptake measurements and the extent of ubiquitination of the EGFP-ATA2 fusion protein by Western blot (Figs. 5 and 6). We first confirmed the RNAi-induced down-regulation of Nedd4-2 protein by Western blot using cell lysates prepared from cells treated with two independent Nedd4-2-specific siRNAs or with the corresponding nonspecific scrambled siRNAs (Fig. 5A). Knockdown effect for Nedd4-2 siRNA 1755 or 2336 normalized with α-tubulin was 86 and 88%. The down-regulation of Nedd4-2 by two independent siRNAs resulted in the significant increase in the transport function of ATA2 as evident from the increase in MeAIB uptake (Fig. 5B). Insulin treatment enhanced the extent of increase of MeAIB uptake induced by both Nedd4-2 siRNAs. With the proteins immunoprecipitated by the anti-GFP antibody from" @default.
• W2019180408 created "2016-06-24" @default.
• W2019180408 creator A5007914982 @default.
• W2019180408 creator A5014819067 @default.
• W2019180408 creator A5033241262 @default.
• W2019180408 date "2006-11-01" @default.
• W2019180408 modified "2023-10-15" @default.
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