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• W2045700225 abstract "Sterol regulatory element-binding proteins (SREBPs) are major transcription factors that activate the genes involved in cholesterol and fatty acid biosynthesis. We here report that the nuclear forms of SREBPs are modified by the small ubiquitin-related modifier (SUMO)-1. Mutational analyses identified two major sumoylation sites (Lys123 and Lys418) in SREBP-1a and a single site (Lys464) in SREBP-2. Mutant SREBPs lacking one or two sumoylation sites exhibited increased transactivation capacity on an SREBP-responsive promoter. Overexpression of SUMO-1 reduced whereas its dominant negative form increased mRNA levels of SREBP-responsive genes. Nuclear SREBPs interacted with the SUMO-1-conjugating enzyme Ubc9, and overexpression of a dominant negative form of Ubc9 increased the mRNA levels of SREBP-responsive genes. Pulse-chase experiments revealed that sumoylation did not affect the degradation of SREBPs through the ubiquitin-proteasome pathway. In vitroubiquitylation assay showed no competition between ubiquitin and SUMO-1 for the same lysine. Considered together, our results indicate that SUMO-1 modification suppresses the transactivation capacity of nuclear SREBPs in a manner different from the negative regulatory mechanism mediated by proteolysis. Sterol regulatory element-binding proteins (SREBPs) are major transcription factors that activate the genes involved in cholesterol and fatty acid biosynthesis. We here report that the nuclear forms of SREBPs are modified by the small ubiquitin-related modifier (SUMO)-1. Mutational analyses identified two major sumoylation sites (Lys123 and Lys418) in SREBP-1a and a single site (Lys464) in SREBP-2. Mutant SREBPs lacking one or two sumoylation sites exhibited increased transactivation capacity on an SREBP-responsive promoter. Overexpression of SUMO-1 reduced whereas its dominant negative form increased mRNA levels of SREBP-responsive genes. Nuclear SREBPs interacted with the SUMO-1-conjugating enzyme Ubc9, and overexpression of a dominant negative form of Ubc9 increased the mRNA levels of SREBP-responsive genes. Pulse-chase experiments revealed that sumoylation did not affect the degradation of SREBPs through the ubiquitin-proteasome pathway. In vitroubiquitylation assay showed no competition between ubiquitin and SUMO-1 for the same lysine. Considered together, our results indicate that SUMO-1 modification suppresses the transactivation capacity of nuclear SREBPs in a manner different from the negative regulatory mechanism mediated by proteolysis. sterol regulatory element-binding protein basic helix-loop-helix-leucine zipper endoplasmic reticulum SREBP cleavage-activating protein ubiquitin-activating enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase hydroxymethylglutaryl low density lipoprotein ubiquitin hemagglutinin synergy control glutathioneS-transferase SREBPs1 control the transcription of a number of genes encoding enzymes and proteins involved in cholesterol and fatty acid metabolism (1Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1110) Google Scholar). These transcription factors belong to a large class of transcription factors containing a basic helix-loop-helix leucine zipper (bHLH-Zip) motif. The SREBP family comprises three subtypes: SREBP-1a and SREBP-1c, which are generated by alternative splicing, mainly regulating lipogenic gene expression, and SREBP-2 governing cholesterol metabolism. Unlike other members of the bHLH-Zip transcription factors, the SREBPs are synthesized as membrane-bound precursors on the endoplasmic reticulum (ER) and activated by a two-step proteolytic process (2Sato R. Yang J. Wang X. Evans M.J. Ho Y.K. Goldstein J.L. Brown M.S. J. Biol. Chem. 1994; 269: 17267-17273Abstract Full Text PDF PubMed Google Scholar, 3Wang X. Sato R. Brown M.S. Hua X. Goldstein J.L. Cell. 1994; 77: 53-62Abstract Full Text PDF PubMed Scopus (859) Google Scholar, 4Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar). The precursor proteins contain an N-terminal transcriptional activation domain with a bHLH-Zip motif and a C-terminal regulatory domain separated by two transmembrane regions. The C-terminal regulatory domain associates with SREBP cleavage-activating protein (SCAP), an ER membrane protein with eight membrane-spanning segments, which contains a sterol-sensing domain (5Hua X. Nohturfft A. Goldstein J.L. Brown M.S. Cell. 1996; 87: 415-426Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar). An SREBP·SCAP complex remains on the ER membrane as long as intracellular cholesterol levels are high, whereas in cells depleted of cholesterol ER-derived membrane vesicles containing this complex moves to the Golgi, where a sequential cleavage of the SREBPs by site 1 and site 2 protease occurs, releasing the active nuclear forms (6Espenshade P.J. Cheng D. Goldstein J.L. Brown M.S. J. Biol. Chem. 1999; 274: 22795-22804Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Once the nuclear form of SREBPs is released into the cytoplasm, it is actively transported into the nucleus in an importin औ-dependent manner (7Nagoshi E. Imamoto N. Sato R. Yoneda Y. Mol. Biol. Cell. 1999; 10: 2221-2233Crossref PubMed Scopus (103) Google Scholar). In the nucleus, the SREBPs are modified by polyubiquitin chains and rapidly degraded by the 26 S proteasome (8Hirano Y. Yoshida M. Shimizu M. Sato R. J. Biol. Chem. 2001; 276: 36431-36437Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). In the presence of proteasome inhibitors, ALLN and lactacystin, the stabilized nuclear SREBPs are capable of enhancing their responsive gene expression. Thus, ubiquitylation of the nuclear SREBPs and the subsequent turnover play important roles in regulation of lipid metabolism. Posttranslational modification of a variety of cellular proteins has been variably linked to protein phosphorylation and acetylation other than ubiquitylation. SUMO-1, a 101-amino acid protein bearing 187 identity with ubiquitin but with a remarkably similar secondary structure, has been recently identified. SUMO-1 differs from ubiquitin in its surface-charge distribution, eliciting its specificity (9Jin C. Shiyanova T. Shen Z. Liao X. Int. J. Biol. Macromol. 2001; 28: 227-234Crossref PubMed Scopus (27) Google Scholar), and does not have a consensus sumoylation motif, (I/V/L)KX(E/D), in its molecule, explaining why SUMO-1 does not make multichain forms (10Yeh E.T. Gong L. Kamitani T. Gene (Amst.). 2000; 248: 1-14Crossref PubMed Scopus (420) Google Scholar, 11Hochstrasser M. Cell. 2001; 107: 5-8Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). Sumoylation requires a multiple-step reaction similar to that of ubiquitin, but the specific enzymes are distinct from those involved in ubiquitylation (12Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (516) Google Scholar). SUMO-1 is synthesized as a precursor with the C-terminal extension of several amino acids, which needs to be processed to expose the C-terminal Gly97 residue that is essential for conjugation to target proteins (13Saitoh H. Pu R.T. Dasso M. Trends. Biochem. Sci. 1997; 22: 374-376Abstract Full Text PDF PubMed Scopus (126) Google Scholar). Then the processed SUMO-1 is recognized as a substrate by SUMO-activating enzyme (E1), which is a heterodimer consisting of SAE1 (also called Uba2) and SAE2 (also called Aos1) subunits (14Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (445) Google Scholar). Ubc9 is a SUMO-conjugating enzyme (E2), receiving SUMO-1 from the E1 enzyme and transferring it to target proteins (15Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Most sumoylated proteins directly interact with Ubc9, which catalyzes the sumoylation of such proteins (16Desterro J.M. Thomson J. Hay R.T. FEBS Lett. 1997; 417: 297-300Crossref PubMed Scopus (304) Google Scholar). A recent report showed that Ubc9 recognizes the consensus sequence that surrounds the acceptor lysine residue in sumoylation substrates (17Sampson D.A. Wang M. Matunis M.J. J. Biol. Chem. 2001; 276: 21664-21669Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar), implying that Ubc9 itself might play to a certain extent a ubiquitin E3-like role in determining the substrate specificity (18Bernier-Villamor V. Sampson D.A. Matunis M.J. Lima C.D. Cell. 2002; 108: 345-356Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). However, recent studies have identified ubiquitin ligase (E3)-like ligases for sumoylation that enhance SUMO-1 conjugation to target proteins in yeasts and mammals (19Kim K.I. Baek S.H. Chung C.H. J. Cell. Physiol. 2002; 191: 257-268Crossref PubMed Scopus (134) Google Scholar). In recent years, a growing number of SUMO-1 target proteins including several transcription factors have been reported (20Muller S. Hoege C. Pyrowolakis G. Jentsch S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 202-210Crossref PubMed Scopus (652) Google Scholar). In contrast to ubiquitylation, which usually marks proteins for rapid degradation, sumoylation is involved in the regulation of protein functions through changes in protein-protein interactions (21Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1010) Google Scholar, 22Seeler J.S. Marchio A. Losson R. Desterro J.M. Hay R.T. Chambon P. Dejean A. Mol. Cell. Biol. 2001; 21: 3314-3324Crossref PubMed Scopus (107) Google Scholar), subcellular localization (23Zhong S. Muller S. Ronchetti S. Freemont P.S. Dejean A. Pandolfi P.P. Blood. 2000; 95: 2748-2752Crossref PubMed Google Scholar), and antagonism to ubiquitylation. Thus, sumoylation serves to enhance the stabilization of target proteins (24Desterro J.M. Rodgriguez M.S. Hay R.T. Mol. Cell. 1998; 2: 233-239Abstract Full Text Full Text PDF PubMed Scopus (919) Google Scholar) and inhibit DNA repair (25Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Crossref PubMed Scopus (1751) Google Scholar). The effects of SUMO-1 modification of transcription factors are very diverse, depending on the nature of transcription factors. SUMO-1 modification induces nuclear relocalization of p53 and enhances the DNA-binding ability of heat shock transcription factor 1 and 2, resulting in increased transcriptional activities (26Fogal V. Gostissa M. Sandy P. Zacchi P. Sternsdorf T. Jensen K. Pandolfi P.P. Will H. Schneider C. Del Sal G. EMBO J. 2000; 19: 6185-6195Crossref PubMed Scopus (321) Google Scholar, 27Goodson M.L. Hong Y. Rogers R. Matunis M.J. Park-Sarge O.K. Sarge K.D. J. Biol. Chem. 2001; 276: 18513-18518Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 28Hong Y. Rogers R. Matunis M.J. Mayhew C.N. Goodson M.L. Park-Sarge O.K. Sarge K.D. J. Biol. Chem. 2001; 276: 40263-40267Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). In contrast, sumoylation to p73α does not affect its transcriptional activity but rather its subcellular localization (29Minty A. Dumont X. Kaghad M. Caput D. J. Biol. Chem. 2000; 275: 36316-36323Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar), whereas SUMO-1 modification attenuates the transcriptional activities of c-Myb, c-Jun, and androgen receptor (30Muller S. Berger M. Lehembre F. Seeler J.S. Haupt Y. Dejean A. J. Biol. Chem. 2000; 275: 13321-13329Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar, 31Poukka H. Karvonen U. Janne O.A. Palvimo J.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14145-14150Crossref PubMed Scopus (371) Google Scholar, 32Bies J. Markus J. Wolff L. J. Biol. Chem. 2002; 277: 8999-9009Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Thus, SUMO-1 modification might be a common mechanism that regulates specific activities of transcriptional factors. Here we report a novel posttranslational modification of SREBP-1 and SREBP-2 by the covalent attachment of two molecules and a single molecule of the SUMO-1 protein, respectively. We show that sumoylation does not affect ubiquitylation and the stability but does modulate transcriptional activities of the SREBPs. Our results point to another mechanism through which the SREBPs activities are attenuated in conjunction with degradation by the ubiquitin/26 S proteasome pathway. We obtained Redivue Pro-mix L-35Sin vitro cell labeling mix, glutathione-Sepharose 4B, and Protein A-Sepharose CL-4B from Amersham Biosciences; protease inhibitor mixture and lipoprotein-deficient serum from Sigma; MG-132 (benzyloxycarbonyl-Leu-Leu-Leu-CHO) andN-ethylmaleimide from Calbiochem; and iodoacetamide from Fluka (Buchs, Switzerland). The pSREBP-1a-(1–487) and the pSREBP-2-(1–481) were described previously (33Inoue J. Sato R. Maeda M. J. Biochem. (Tokyo). 1998; 123: 1191-1198Crossref PubMed Scopus (58) Google Scholar, 34Sato R. Okamoto A. Inoue J. Miyamoto W. Sakai Y. Emoto N. Shimano H. Maeda M. J. Biol. Chem. 2000; 275: 12497-12502Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Expression plasmids pFLSBP-1a-(2–487) and pFLSBP-2-(2–481) were constructed by inserting fragments coding amino acids 2–487 of human SREBP-1a and 2–481 of SREBP-2 into the pCMV-3Flag (Sigma), respectively. To generate expression plasmid pGSTSBP-1a-(2–487) and pGSTSBP-2-(2–481), the fragments were transferred to the pME-GST(6P-3), which was kindly provided by Dr. Tezuka (Institute of Medical Science, University of Tokyo). To generate the expression plasmid pG4-FLSBP-1a-(2–487), the fragment coding amino acids 2–487 of SREBP-1a was ligated into the expression plasmid pM-Flag, which was constructed by inserting a GAL4 DNA-binding domain expression vector, pM (Clontech). The expression plasmid pG4-HisSBP-2-(2–481) was constructed by inserting the fragment for amino acids 2–481 of SREBP-2 into the expression plasmid pM-His, which was engineered to contain the in-frame N-terminal His epitope tag MRGS(H)6. All expression plasmids encoding SREBP mutants were synthesized by a PCR-assisted method using the site-directed mutagenesis kit following the instructions provided by the supplier (Stratagene, La Jolla, CA). The pEGFP-SUMO-1 was a kind gift from Dr. Minoru Yoshida (RIKEN). The pHA-SUMO-1 was kindly provided by Dr. Chiba (Tokyo Metropolitan Institute of Medical Science). The expression plasmid pHisSUMO was constructed by inserting a fragment encoding human SUMO-1 cloned into the pME-His (8Hirano Y. Yoshida M. Shimizu M. Sato R. J. Biol. Chem. 2001; 276: 36431-36437Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Expression plasmids pHisSUMO(GG) and pHisSUMOΔGG were generated by ligating PCR-generated fragments. To construct expression plasmids pHisUbc9 and pGFPUbc9, a fragment of human Ubc9 was amplified by reverse transcriptase-PCR and inserted into the pME-His and the pME-GFP (8Hirano Y. Yoshida M. Shimizu M. Sato R. J. Biol. Chem. 2001; 276: 36431-36437Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). The expression plasmid pHisUbc9(C93S) was generated using the site-directed mutagenesis kit. To generate the pG5Luc reporter plasmid, five copies of Gal4 binding sites in the pG5CAT (Clontech) were transferred to the pGL3-Basic (Promega, Madison, WI). The anti-SREBP-1 polyclonal antibody RS005 and the anti-SREBP-2 polyclonal antibody RS004 have been described previously (8Hirano Y. Yoshida M. Shimizu M. Sato R. J. Biol. Chem. 2001; 276: 36431-36437Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 35Sato R. Miyamoto W. Inoue J. Terada T. Imanaka T. Maeda M. J. Biol. Chem. 1999; 274: 24714-24720Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). The anti-RGS(H)4 monoclonal antibody was purchased from Qiagen (Hilden, Germany); the anti-FLAG monoclonal antibody M2 and anti-GST polyclonal antibody were obtained from Sigma; anti-Ubc9 polyclonal antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); anti-multiubiquitin monoclonal antibody was from Medical & Biological Laboratories Co. (Nagoya, Japan); anti-HA monoclonal antibody was from BabCO (Richmond, CA); and anti-GMP-1 (anti-SUMO-1) monoclonal antibody was from Zymed Laboratories (San Francisco, CA). HeLa, COS-1, HEK293 and M19 cells and site 2 protease null mutant Chinese hamster ovary cells, which were kindly provided by Dr. Chang (Dartmouth College, Hanover, NH), were cultured as described previously (8Hirano Y. Yoshida M. Shimizu M. Sato R. J. Biol. Chem. 2001; 276: 36431-36437Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 36Nakahara M. Fujii H. Maloney P.R. Shimizu M. Sato R. J. Biol. Chem. 2002; 277: 37229-37234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). For detection of sumoylated endogenous SREBPs, HeLa cells (20 100-mm dishes) were set up on day 0 in medium A (Dulbecco's modified Eagle's medium; Sigma) supplemented with 107 fetal bovine serum supplemented with 107 fetal bovine serum. On day 1, the cells were transfected with 3 ॖg of pHA-SUMO-1 using X-tremeGENE Q2 Transfection Reagent (Roche Applied Science). After transfection, the cells were refed with medium A containing 57 lipoprotein-deficient serum, 50 ॖmpravastatin, and sodium mevalonate. After incubation for 48 h, the cells were harvested with Buffer C* containing 20 mmHEPES/KOH (pH 7.9), 207 glycerol, 1.5 mmMgCl2, 300 mm NaCl, 0.57 Nonidet P-40, and 0.2 mm EDTA supplemented with a mixture of protease inhibitors, 10 ॖm MG-132, 20 mmN-ethylmaleimide, and 10 mm iodoacetamide. After centrifugation at 13,000 × g for 10 min at 4 °C, the supernatant was immunoprecipitated by the Seize Classis X Protein A immunoprecipitation kit (Pierce) following the instructions provided by the supplier and resolved on SDS-PAGE and immunoblotted as described previously (8Hirano Y. Yoshida M. Shimizu M. Sato R. J. Biol. Chem. 2001; 276: 36431-36437Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). COS-1 and M19 cells were set up on day 0. On day 1, the COS-1 cells were transfected using the DEAE-dextran methods and then refed with medium A supplemented with 107 fetal bovine serum. M19 cells were transfected using LipofectAMINE (Invitrogen) and then refed with medium B (a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium) containing 57 lipoprotein-deficient serum supplemented either with 1 ॖg/ml 25-hydroxycholesterol plus 10 ॖg/ml cholesterol (the sterol-loaded condition) or 50 ॖm pravastatin plus 50 ॖm sodium mevalonate (the sterol-depleted condition). After incubation for 48 h, the cells were harvested with a radioimmune precipitation buffer containing 50 mm Tris/HCl (pH 7.8), 150 mm NaCl, 5 mm EDTA, 15 mm MgCl2, 17 Nonidet P-40, 0.757 sodium deoxycholate, 1 mm dithiothreitol for binding assays or Buffer C* for sumoylation assays supplemented with a mixture of protease inhibitors, 10 ॖm MG-132, 20 mmN-ethylmaleimide, and 10 mmiodoacetamide. After centrifugation at 13,000 × g for 10 min at 4 °C, the supernatant was incubated with 50 ॖl of a 507 slurry of glutathione-Sepharose 4B or immunoprecipitated with the indicated antibodies and 50 ॖl of a 507 slurry of Protein A-Sepharose CL-4B. All resins were resolved on SDS-PAGE and immunoblotted as described previously (8Hirano Y. Yoshida M. Shimizu M. Sato R. J. Biol. Chem. 2001; 276: 36431-36437Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). HeLa cells (5 × 105cells/60-mm dish) transfected with various expression plasmids were cultured under the sterol-depleted condition for 48 h and then harvested. Northern blot analysis was performed as described previously (34Sato R. Okamoto A. Inoue J. Miyamoto W. Sakai Y. Emoto N. Shimano H. Maeda M. J. Biol. Chem. 2000; 275: 12497-12502Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 37Inoue J. Kumagai H. Terada T. Maeda M. Shimizu M. Sato R. Biochem. Biophys. Res. Commun. 2001; 283: 1157-1161Crossref PubMed Scopus (29) Google Scholar). Membranes transferring total RNA were hybridized with radioactive cDNA probes, human hydroxymethylglutaryl (HMG)-CoA synthase, LDL receptor, and glyceraldehyde-3-phosphate dehydrogenase. Reporter Assays were performed as described previously (36Nakahara M. Fujii H. Maloney P.R. Shimizu M. Sato R. J. Biol. Chem. 2002; 277: 37229-37234Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). HEK293 cells were transfected with 0.2 ॖg of the pLDLR (38Sato R. Inoue J. Kawabe Y. Kodama T. Takano T. Maeda M. J. Biol. Chem. 1996; 271: 26461-26464Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar) or the pG5Luc, 0.01 ॖg of the pRL-CMV, an expression plasmid encoding Renilla luciferase (Promega), and the indicated amounts of various expression plasmids. After incubation for 48 h, the Dual-LuciferaseTM Reporter System (Promega) was used to determine luciferase activities. On day 0, monolayers of COS-1 cells were set up and transfected on day 1 with wild-type or mutant SREBPs. One day after transfection, the cells were trypsinized and reseeded to normalize the transfection efficiency. On day 3, the cells were preincubated for 1 h with methionine/cysteine-free medium A (Invitrogen) supplemented with 107 fetal bovine serum and then pulsed with 200 ॖCi of L-35S cell labeling mix for 1 h. After the pulse period, the cells were incubated for 1 h and then washed with phosphate-buffered saline and refed with a prewarmed complete medium. After each chase period, the cells were harvested and lysed with a cold lysis buffer containing 10 mm Tris/HCl (pH 7.4), 0.17 Triton X-100, 0.17 SDS, and 2 mm EDTA. The immunoprecipitates with anti-FLAG antibodies were visualized by autoradiography. Exposed filters were quantitatively analyzed on FluorImage Analyzer with Image Gauge (Fuji Film). The ubiquitylation assay was previously described (39Murata S. Minami Y. Minami M. Chiba T. Tanaka K. EMBO Rep. 2001; 21: 1133-1138Crossref Scopus (469) Google Scholar). Substrate GST-SREBPs were prepared from COS-1 cells transfected with pGSTSBPs. The cells were treated with 20 ॖm MG-132 for the last 12 h of the culture and then lysed. The GST-SREBPs immobilized on the glutathione-Sepharose 4B were incubated with 40 ng of E1, 0.4 ॖg of E2, and 8.0 ॖg of GST-Ub in 40 ॖl of the ubiquitylation buffer containing 50 mmTris/HCl (pH 7.5), 2 mm MgCl2, 1 mmdithiothreitol, and 4 mm ATP for 3 h at 37 °C. The reactions were terminated by the addition of the SDS sample buffer. Analysis of the amino acid sequences of the nuclear forms of human SREBPs revealed that SREBP-1a contains four matches to the consensus sumoylation sequence centered around Lys123, Lys381, Lys418, and Lys470, and that SREBP-2 contains two matches, centered around Lys420 and Lys464 (Fig.1A). To examine whether the endogenous SREBPs are modified by SUMO-1, HeLa cells were transiently transfected with an expression plasmid for HA-SUMO-1 and cultured under sterol-depleted conditions for 48 h to increase in the amount of the nuclear SREBPs. The nuclear extracts treated with isopeptidase inhibitors, N-ethylmaleimide and iodoacetamide, were subjected to immunoprecipitation and immunoblot analysis. In the case of SREBP-1a, anti-HA antibodies recognized several slower migrating bands in the immunoprecipitates with anti-SREBP-1 antibodies (Fig.1B, left top). Anti-SREBP-1 antibodies also detected weaker bands for SUMO-1-modified forms above the parental form of SREBP-1a (left bottom). In the case of SREBP-2, anti-HA antibodies recognized a single band in the immunoprecipitates with anti-SREBP-2 antibodies (right top), and anti-SREBP-2 antibodies detected a faint SUMO-1-modified form above the parental form of SREBP-2 (right bottom). These results demonstrated that endogenous SREBPs are modified by SUMO-1. Based on the fact that SUMO-1 can be covalently attached to a lysine residue only in a monomeric form (40Tatham M.H. Jaffray E. Vaughan O.A. Desterro J.M. Botting C.H. Naismith J.H. Hay R.T. J. Biol. Chem. 2001; 276: 35368-35374Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar), our findings suggest that more than two residues of lysine among four potential sites in SREBP-1a and a single residue of lysine between two potential sites in SREBP-2 are modified. To determine whether the transcriptional activities of endogenous SREBPs are regulated by sumoylation, either SUMO-1(GG), a processed mature form of SUMO-1 (13Saitoh H. Pu R.T. Dasso M. Trends. Biochem. Sci. 1997; 22: 374-376Abstract Full Text PDF PubMed Scopus (126) Google Scholar), or SUMO-1ΔGG, a dominant-negative form of SUMO-1 that is unable to attach target proteins (42Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), was overexpressed in HeLa cells. Northern blot analyses for SREBP-responsive genes shown in Fig.2 indicate that overexpression of SUMO-1(GG) decreased the amounts of HMG-CoA synthase and LDL receptor mRNA in a dose-dependent manner, suggesting that sumoylated endogenous SREBPs (Fig. 1B) down-regulate transcription of their target genes (Fig. 2A). In contrast, overexpression of SUMO-1ΔGG increased the amounts of HMG-CoA synthase and LDL receptor mRNA in a dose-dependent manner, indicating that block of sumoylation accelerated transcription of SREBP target genes (Fig. 2B). These results indicate that sumoylation of endogenous SREBPs can affect regulation of the SREBP-responsive gene expression. To determine which Lys residues in SREBP-1a are sumoylated, we cotransfected COS-1 cells with expression plasmids for His-SUMO-1 and either wild-type or mutant versions of GST-SREBP-1a. GST-SREBP-1a bound to glutathione-Sepharose resins were subjected to immunoblotting with anti-SUMO-1 antibodies (Fig.3, A and B,top panels). Three sumoylated bands were detected in cells transfected with an expression plasmid for wild-type SREBP-1a, whereas a single sumoylated band was detected in cells expressing either SREBP-1a K381R/K418R/K470R or K123R/K381R/K470R (Fig.3A, top, lanes 1,2, and 4). No bands were observed after the removal of four lysine residues in potential sumoylation sites (lane 6). Another immunoblotting analysis with anti-SREBP-1a antibodies confirmed that all sumoylated bands observed in the top panel were derived from sumoylated SREBP-1a (bottom). It is likely that the most slowly migrating sumoylated band in GST-SREBP-1a (top,lane 1) represents doubly sumoylated proteins at Lys123 and Lys418. Fig. 3B also shows that mutation at both Lys123 and Lys418completely abolished sumoylation of SREBP-1a (lane 10). Mutation of one of two possible sumoylation sites, Lys123 and Lys418, resulted in a single sumoylated band (lanes 8 and 9), confirming that these residues are major sumoylation sites. Taken together, these results clearly demonstrate that the molecule of SREBP-1a contains two possible sumoylation sites, Lys123and Lys418. To determine which Lys residues of two putative sumoylation sites in SREBP-2 are modified, COS-1 cells were transiently transfected with expression plasmids for His-SUMO-1 and either wild-type or mutant versions of SREBP-2. Fig. 4 shows that a slowly migrating SUMO-1-conjugated band (∼83 kDa) was detected when either wild-type or K420R SREBP-2 was expressed together with His-SUMO-1. These results clearly indicate that Lys464serves as the sumoylation site in the nuclear form of SREBP-2. To assess the potential consequences of sumoylation of SREBPs, we examined whether sumoylation influences the transcriptional activities of SREBPs. HEK293 cells were cotransfected with a reporter plasmid, pLDLR, containing the promoter region of human low density lipoprotein receptor gene, and expression plasmids encoding either wild-type or mutant versions of SREBPs. The cells were cultured under the sterol-loaded condition to suppress the processing of endogenous SREBPs, and luciferase assays were carried out. As shown in Fig.5A, both SREBP-1aK123R and -K418R significantly increased luciferase activities compared with wild-type SREBP-1a. Double mutation markedly activated the transcription of the reporter gene. Fig. 5B also shows that SREBP-2K464R markedly activated transcription of the reporter gene compared with wild-type SREBP-2. Similar results were obtained using a reporter plasmid containing the promoter region of the human HMG-CoA synthase gene (data not shown). These results indicate that sumoylation of both SREBP-1a and SREBP-2 negatively regulates their transcriptional activities and that sumoylation at both Lys123 and Lys418 of SREBP-1a coordinately attenuates the transcription. SREBPs require co-regulatory transcription factors, such as Sp1 and CBF/NF-Y, that bind DNA sequences adjacent to the SREBP binding sites and enhance the transcriptional activities of SREBPs by forming complexes with SREBPs (33Inoue J. Sato R. Maeda M. J. Biochem. (Tokyo). 1998; 123: 1191-1198Crossref PubMed Scopus (58) Google Scholar, 41Osborne T.F. J. Biol. Chem. 2000; 275: 32379-32382Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Based on these early findings, we speculated that sumoylation might influence the interaction between SREBPs and these co-regulatory factors. Alternatively, it is possible that modification by SUMO-1 may weaken the affinity between SREBPs and their responsive DNA sequences. Instead of focusing on these possibilities, we evaluated whether sumoylation of SREBPs directly resulted in inactivation of these proteins. Accordingly, we used the heterologous Gal4 system with a reporter plasmid, pG5Luc. In this assay the luciferase gene transcription is driven by a" @default.
• W2045700225 created "2016-06-24" @default.
• W2045700225 creator A5006022288 @default.
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