Sterol Regulatory Element-binding Proteins Are Negatively Regulated through SUMO-1 Modification Independent of the Ubiquitin/26 S Proteasome Pathway

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. 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Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). and by amino of SREBP-1a and of SREBP-2 into the and the to the which by of of the the amino of SREBP-1a into the which by a domain The by the for amino of SREBP-2 into the which to contain the N-terminal encoding SREBP synthesized by a the the by the The a from Yoshida The by of The by a encoding SUMO-1 into the (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). and generated by and a of Ubc9 by and into the and the (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 generated the the of sites in the to the The and the have been (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, R. J. T. T. M. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). The from the and from from from from and from and cells and site 2 protease which by as (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, M. H. Shimizu M. Sato R. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of sumoylated cells on in A modified with with the cells with of the cells with A containing and for the cells with containing and with a of protease inhibitors, and for the by the A the by the and on and as (8Hirano Y. Yoshida M. Shimizu M. Sato R. J. Biol. 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Chem. 1996; Full Text Full Text PDF PubMed Scopus Google or the of the an encoding and the of for the to of cells and on 1 with or the cells and to the the cells for 1 with A with and with of for 1 the the cells for 1 and with and with a the cells and with a containing and 2 The with by on with The ubiquitylation assay S. Y. M. T. EMBO 2001; 21: Scopus Google Scholar). from cells with The cells with for the of the and The on the with of of and of in of the ubiquitylation containing 2 1 and for The by the of the of the amino acid of the nuclear forms of SREBPs revealed that SREBP-1a contains to the consensus sumoylation sequence and and that SREBP-2 contains two and the SREBPs are modified by SUMO-1, cells with an for and for to in the of the nuclear The nuclear with inhibitors, and to and In the of recognized several in the with for forms the form of SREBP-1a In the of recognized a single in the with and a form the form of SREBP-2 These results that SREBPs are modified by on the that SUMO-1 be to a lysine residue in a form E. Desterro J.M. C.H. Hay R.T. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), our that than two of lysine sites in SREBP-1a and a single residue of lysine between two sites in SREBP-2 are the transcriptional of SREBPs are by a processed 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 a form of SUMO-1 that is to target proteins T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), in analyses for SREBP-responsive genes in indicate that overexpression of the of and mRNA in a that sumoylated SREBPs transcription of their target genes In overexpression of increased the of and mRNA in a that of sumoylation transcription of SREBP target genes These results indicate that sumoylation of SREBPs affect regulation of the SREBP-responsive gene expression. which in SREBP-1a are cells with for and or of to to with A and sumoylated in cells with an for whereas a single sumoylated in cells SREBP-1a or and the of lysine in sumoylation sites with that sumoylated in the from sumoylated SREBP-1a is that the sumoylated in sumoylated proteins and that and sumoylation of SREBP-1a of one of two sumoylation and in a single sumoylated and that are major sumoylation together, results that the of SREBP-1a contains two sumoylation which of two sumoylation sites in SREBP-2 are cells with for and or of SREBP-2. that a or SREBP-2 with These results indicate that as the sumoylation site in the nuclear form of SREBP-2. the of sumoylation of sumoylation the transcriptional of cells with a containing the of low density lipoprotein and encoding or of The cells the to the of and in and increased with activated the transcription of the that activated transcription of the gene with SREBP-2. results a containing the of the gene not These results indicate that sumoylation of SREBP-1a and SREBP-2 their transcriptional and that sumoylation and of SREBP-1a the SREBPs transcription factors, such as and that to the SREBP sites and enhance the transcriptional of SREBPs by with SREBPs J. Sato R. M. J. Biochem. PubMed Scopus Google Scholar, J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). on that sumoylation might the between SREBPs and it is that modification by SUMO-1 the between SREBPs and their responsive of on sumoylation of SREBPs directly in of the with a In this assay the gene transcription is by a that contains consensus transcription SREBPs in the in and D. The and in a and in with which no sumoylated than and that transcription of the gene to an extent by in These results indicate that SUMO-1 modification of SREBPs directly their transcriptional the other the that sumoylation of SREBPs might their with factors or their We reported that nuclear SREBPs are rapidly degraded the 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). sumoylation and ubiquitylation a lysine residue of target sumoylation with ubiquitylation, the degradation of nuclear Pulse-chase experiments cells with encoding or SREBP-2 that the of and SREBP-2 and that no in their results cells with encoding or of not together, results indicate that sumoylation of SREBPs does not their degradation through the S proteasome pathway. directly and of SREBPs are modified by ubiquitin, an in vitroubiquitylation assay for In this the in cells with and with enzyme and enzyme in the presence of and We that but not as for the in ubiquitylation assay of SREBPs not in the of in the presence of the of forms of SREBPs and by with that are forms of with with as which These results to that and of SREBPs are modified by ubiquitin and that sumoylation does not with ubiquitylation in the intracellular sterol levels the of sumoylation of is that sterol levels sumoylation of nuclear the transcription of SREBP-responsive genes. this which site 2 protease R.B. D. J. J. Brown M.S. Goldstein J.L. Mol. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), in the of nuclear In one sumoylation of SREBPs or in the of The cells with encoding and and or to to with and The of sumoylated and SREBPs 1 and or 2 and These results that not the sumoylation of the of SREBP with Ubc9, cells with an encoding or The of to with and by with and Ubc9 with 2 and that Ubc9 is capable of with the Ubc9 catalyzes sumoylation of cells with for and or a dominant negative form of Ubc9 L. Kamitani T. E.T. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). to to with and Overexpression of sumoylation of SREBPs and Thus, that the dominant negative form of Ubc9 with the in modification of the nuclear the dominant negative form of Ubc9 is capable of sumoylation of enhancing the transcriptional cells with an for of SREBP-responsive genes in indicate that overexpression of increased the of and mRNA in a that of sumoylation transcription of SREBP target genes. These results that Ubc9 with enhancing the of SUMO-1 to SREBPs and their transcriptional The of this to modification of the SREBPs other than ubiquitylation and to the that SREBP through such The major of the SUMO-1 nuclear SREBPs in a and of SREBP-1a and of SREBP-2 as SUMO-1 acceptor and SUMO-1 the transactivation of These sumoylation are in SREBPs have been reported for for for and for for for for revealed that and are for a of the SREBP-1a transcriptional whereas is for a of SREBP-2 to an extent with that by in SREBP-1a remains why SREBP-1a needs two sumoylation sites to control its transactivation the amino acid sequence in to the consensus identified recently in the negative regulatory domain of several transcription factors and the synergy control motif D. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). The sites in and to be H. U. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, J. J. L. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, N. D. 2002; PubMed Scopus Google Scholar). is that an as might be to the the transcriptional of certain transcription The that of SUMO-1 in the motif the transactivation of SREBP-1a of such in a However, the recognizes or sumoylated remains sumoylation of the in SREBP-1a as as and their transactivation whereas sumoylation of the We that the C-terminal domain containing amino of the nuclear SREBPs a transcriptional to the domain the SREBP-1a activation domain containing the N-terminal amino not In a that of the domain in a nuclear form of the transcriptional (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). The of SREBP-1a and of SREBP-2 are in this negative regulatory domain and are to be involved in such The amino acid sequence in the consensus sumoylation site in the negative regulatory domain of protein family which is in a variety of J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). However, SUMO-1 the of the regulatory domain but that of the protein regulatory In the of conjugation of SUMO-1 to or the of the as in sumoylated (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). In of this the different of the sumoylated modified and in SREBP-1a that sumoylation of SREBPs might changes in Sumoylation has been to be for the of proteins into called the nuclear S. S. S. Dejean A. 2000; PubMed Google Scholar, T. H. J. Biol. 1997; PubMed Scopus Google Scholar). including and have been reported to be to the nuclear SUMO-1 modification (19Kim K.I. Baek S.H. Chung C.H. J. Cell. Physiol. 2002; 191: 257-268Crossref PubMed Scopus (134) Google this does not that proteins are to be in the nuclear J.S. A. A. S. E. M. A. T. Melchior F. Dejean A. EMBO J. 2002; 21: PubMed Scopus Google Scholar). the of the nuclear is it has been recently that sumoylation directly or degradation C. G. L. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). in the SUMO-1 sites affect the intracellular of the sumoylation did not changes in the of in the nucleus not studies are to the role of SUMO-1 modification of several target proteins including SREBPs on their in to the functions of and that the sumoylated SREBPs with SREBPs in does not to be in with the results of transactivation experiments in in which in the SUMO-1 acceptor sites in of the transcriptional of and the that of SREBP-responsive genes by overexpression of SUMO-1 but increased by overexpression of dominant negative SUMO-1 proteins are as from a between and forms I. E.S. Dohmen R.J. Mol. 2000; PubMed Scopus (107) Google Scholar). Thus, sumoylation is to a mechanism for rapid and and target proteins are a of which is to affect their a number of which SUMO-1 from its protein have been identified (19Kim K.I. Baek S.H. Chung C.H. J. Cell. Physiol. 2002; 191: 257-268Crossref PubMed Scopus (134) Google Scholar). The different of subcellular or of enzymes the specific of enzyme its and that an important role in regulation of cellular The of sumoylation and ubiquitylation are different the that SUMO-1 and ubiquitin are to a lysine residue in target proteins and that their modification are The of a polyubiquitin to a lysine residue marks modified proteins for a rapid degradation by the 26 S In sumoylation has been to the of by ubiquitylation J.M. M.S. Hay R.T. Mol. Cell. 2: Full Text Full Text PDF PubMed Scopus Google Scholar). We reported that nuclear SREBPs are degraded through the 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 that SUMO-1 modification of the SREBPs not their and that SUMO-1 conjugation did not with ubiquitin modification We a ubiquitylation in which ubiquitin conjugation the of a specific ligase for SREBPs in the presence of E1 and is that the from cells with a proteasome an which capable of ubiquitin to the on that lysine of sumoylation sites in the SREBPs are not the major ubiquitylation together, the of two that of SREBPs is by sumoylation and by their through the S proteasome pathway. These results the that the two might be for of in the of the SREBP-responsive genes to of lipid metabolism. SREBPs are synthesized as membrane-bound precursors and activated by a proteolytic process by intracellular sterol In to the proteolytic a in intracellular sterol cholesterol levels in nuclear might the of SUMO-1 conjugation to the nuclear which in their transactivation to the of genes encoding enzymes for cholesterol biosynthesis. However, the results in this In a that intracellular sterol levels not the rapid degradation of nuclear SREBPs through the 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). together, studies that two degradation and are not in to changes in lipid metabolism but to control the transcriptional of the nuclear In the of SUMO-1 modification for the transactivation of nuclear SREBPs is a mechanism through which sumoylation its negative on SREBPs and lipid metabolism. We of and Yoshida for the and We are to for the