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INTRODUCTION |
SREBPs1 control
the transcription of a number of genes encoding enzymes and proteins
involved in cholesterol and fatty acid metabolism (1). 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 (2-4). 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 (5). 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 (6). Once the nuclear form of SREBPs is
released into the cytoplasm, it is actively transported into the
nucleus in an importin
-dependent manner (7). In the
nucleus, the SREBPs are modified by polyubiquitin chains and rapidly
degraded by the 26 S proteasome (8). 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 18%
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 (9), 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 (10, 11).
Sumoylation requires a multiple-step reaction similar to that of
ubiquitin, but the specific enzymes are distinct from those involved in
ubiquitylation (12). 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 (13). 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 (14). Ubc9 is a SUMO-conjugating enzyme
(E2), receiving SUMO-1 from the E1 enzyme and transferring it to target
proteins (15). Most sumoylated proteins directly interact with Ubc9,
which catalyzes the sumoylation of such proteins (16). A recent report
showed that Ubc9 recognizes the consensus sequence that surrounds the acceptor lysine residue in sumoylation substrates (17), implying that
Ubc9 itself might play to a certain extent a ubiquitin E3-like role in
determining the substrate specificity (18). However, recent studies
have identified ubiquitin ligase (E3)-like ligases for sumoylation that
enhance SUMO-1 conjugation to target proteins in yeasts and mammals
(19).
In recent years, a growing number of SUMO-1 target proteins including
several transcription factors have been reported (20). 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 (21, 22), subcellular
localization (23), and antagonism to ubiquitylation. Thus, sumoylation
serves to enhance the stabilization of target proteins (24) and inhibit
DNA repair (25).
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 (26-28). In
contrast, sumoylation to p73
does not affect its transcriptional activity but rather its subcellular localization (29), whereas SUMO-1
modification attenuates the transcriptional activities of c-Myb,
c-Jun, and androgen receptor (30-32). 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.
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EXPERIMENTAL PROCEDURES |
Materials--
We obtained Redivue Pro-mix L-35S
in 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) and N-ethylmaleimide from Calbiochem; and iodoacetamide from
Fluka (Buchs, Switzerland).
Expression Plasmids--
The pSREBP-1a-(1-487) and the
pSREBP-2-(1-481) were described previously (33, 34). 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 (8). Expression plasmids pHisSUMO(GG) and pHisSUMO
GG
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 (8). 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).
Antibodies--
The anti-SREBP-1 polyclonal antibody RS005 and
the anti-SREBP-2 polyclonal antibody RS004 have been described
previously (8, 35). 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).
Cell Lines and Culture Conditions--
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 (8, 36).
Immunoprecipitation and Immunoblotting--
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 10% fetal bovine serum supplemented with 10% fetal
bovine serum. On day 1, the cells were transfected with 3 µg of
pHA-SUMO-1 using X-tremeGENE Q2 Transfection Reagent (Roche Applied
Science). After transfection, the cells were refed with medium A
containing 5% lipoprotein-deficient serum, 50 µM
pravastatin, and sodium mevalonate. After incubation for 48 h, the
cells were harvested with Buffer C* containing 20 mM
HEPES/KOH (pH 7.9), 20% glycerol, 1.5 mM
MgCl2, 300 mM NaCl, 0.5% Nonidet P-40, and 0.2 mM EDTA supplemented with a mixture of protease inhibitors,
10 µM MG-132, 20 mM
N-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 (8).
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 10% 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 5% lipoprotein-deficient serum
supplemented either with 1 µg/ml 25-hydroxycholesterol plus 10 µg/ml cholesterol (the sterol-loaded condition) or 50 µM pravastatin plus 50 µM 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, 1% Nonidet
P-40, 0.75% sodium deoxycholate, 1 mM dithiothreitol for
binding assays or Buffer C* for sumoylation assays supplemented with a
mixture of protease inhibitors, 10 µM MG-132, 20 mM N-ethylmaleimide, and 10 mM
iodoacetamide. After centrifugation at 13,000 × g for
10 min at 4 °C, the supernatant was incubated with 50 µl of a 50%
slurry of glutathione-Sepharose 4B or immunoprecipitated with the
indicated antibodies and 50 µl of a 50% slurry of Protein
A-Sepharose CL-4B. All resins were resolved on SDS-PAGE and
immunoblotted as described previously (8).
Northern Blotting--
HeLa cells (5 × 105
cells/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
(34, 37). Membranes transferring total RNA were hybridized with
radioactive cDNA probes, human hydroxymethylglutaryl (HMG)-CoA
synthase, LDL receptor, and glyceraldehyde-3-phosphate dehydrogenase.
Reporter Assays--
Reporter Assays were performed as described
previously (36). HEK293 cells were transfected with 0.2 µg of the
pLDLR (38) or the pG5Luc, 0.01 µg 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.
Pulse-Chase Experiments--
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 10% fetal bovine serum and then pulsed
with 200 µCi 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), 0.1% Triton X-100, 0.1% 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).
In Vitro Ubiquitylation Assay--
The ubiquitylation assay was
previously described (39). Substrate GST-SREBPs were prepared from
COS-1 cells transfected with pGSTSBPs. The cells were treated with 20 µM 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 µg of E2, and 8.0 µg of GST-Ub in
40 µl of the ubiquitylation buffer containing 50 mM
Tris/HCl (pH 7.5), 2 mM MgCl2, 1 mM
dithiothreitol, and 4 mM ATP for 3 h at 37 °C. The
reactions were terminated by the addition of the SDS sample buffer.
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RESULTS |
Posttranslational Modification of Endogenous SREBPs by Covalent
Attachment of SUMO-1--
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
(40), 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.

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Fig. 1.
Nuclear forms of SREBPs are covalently
modified by SUMO-1. A, potential SUMO-1 modification
sites in nuclear SREBPs. The amino acid sequences of the
sumoylation consensus motif and closest matches in human nuclear
SREBP-1a and SREBP-2 are listed. Numbering of the nuclear SREBPs amino
acid sequences is shown on the left. Potential sumoylation
sites are highlighted by boldface characters.
B, HeLa cells were transfected with an expression plasmid
encoding HA-SUMO-1. After 48 h of culture, the cell extracts were
subjected to immunoprecipitation (IP) with either preimmune
serum, anti-SREBP-1 (left), or anti-SREBP-2
(right) antibodies and im- munoblotting (IB)
with anti-HA (top) and SREBP-1/2 (bottom)
antibodies. The mobilities of the SUMO-1-modified and -unmodified
SREBPs are indicated on the right. The positions of
molecular mass standards are marked on the left.
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A Dominant Negative Form of SUMO-1 Induces the Expression of
SREBP-responsive Genes--
To determine whether the transcriptional
activities of endogenous SREBPs are regulated by sumoylation, either
SUMO-1(GG), a processed mature form of SUMO-1 (13), or SUMO-1
GG, a
dominant-negative form of SUMO-1 that is unable to attach target
proteins (42), 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-1
GG 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.

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Fig. 2.
A dominant-negative form of SUMO-1 induces
the expression of SREBP-responsive genes. HeLa cells in a 60-mm
dish were transfected with the indicated amounts of expression
plasmids, either His-SUMO-1(GG) (A) or His-SUMO-1 GG
(B). After transfection, the cells were cultured under
sterol-depleted conditions for 48 h. Total RNA (8 µg/lane) was
subjected to electrophoresis and blot hybridization with the indicated
32P-labeled probe as described under "Experimental
Procedures." The results were normalized to the signal generated from
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The same
results were obtained in three separate experiments. LDLR,
LDL receptor.
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Two Lys Residues (Lys123 and Lys418) in
SREBP-1a Are Sumoylated--
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 Lys418
completely 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, Lys123
and Lys418.

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Fig. 3.
Both Lys123 and
Lys123 in the nuclear form of SREBP-1a are modified by
SUMO-1. COS-1 cells were transfected with expression plasmids for
His-SUMO-1 and either wild-type or mutant versions of GST-SREBP-1a. GST
fusion proteins were purified with glutathione-Sepharose resins and
subjected to immunoblotting (IB) with anti-SUMO-1
(top) and SREBP-1a (bottom) antibodies.
A, the cells were transfected with expression plasmids for
mutant versions of GST-SREBP-1a lacking 3 or 4 lysine residues among
four potential sumoylation sites. B, mutant versions of
SREBP-1a containing one or both of two possible sumoylation sites,
Lys123 and Lys418, were expressed in the cells.
The mobilities of the SUMO-1-modified and -unmodified SREBP-1a are
indicated on the right. An asterisk marks the
nonspecific bands observed in all lanes.
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SREBP-2 Is Sumoylated at Lys464--
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 Lys464
serves as the sumoylation site in the nuclear form of SREBP-2.

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Fig. 4.
Lys464 is the sumoylation site in
the nuclear form of SREBP-2. COS-1 cells were cotransfected with
expression plasmids for His-SUMO-1 and either wild-type or mutant
versions (K420R and K464R) of SREBP-2. After 48 h of culture,
immunoprecipitates (IP) with anti-SREBP-2 antibodies were
subjected to immunoblotting (IB) with
anti-RGS(H)4 (top) and SREBP-2
(bottom) antibodies as described in the legend to Fig. 1.
The mobilities of the SUMO-1-modified and -unmodified SREBP-2 are
indicated on the right.
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Sumoylation Decreases the Transactivation Activities of
SREBPs--
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.

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Fig. 5.
Sumoylation of SREBPs reduces their
transactivation activities. HEK293 cells were cotransfected with
increasing amounts of expression plasmids encoding either wild-type or
mutant versions of SREBP-1a (A) or SREBP-2 (B) or
0.03 µg of expression plasmids encoding pHA-SUMO-1 and either
wild-type or mutant versions of pG4-FLSBP-1a (C) or
pG4-HisSBP-2 (D) along with 0.2 µg of the reporter
plasmids either pLDLR (A and B) or pG5Luc
(C and D) and 0.01 µg of pRL-CMV per 35-mm
dish. After 48 h of culture, luciferase assays were performed as
described under "Experimental Procedures." Promoter activities in
the absence of SREBP expression plasmids are set as 1. Data are
mean ± S.E. values of three independent experiments performed in
triplicate. Expression levels of sumoylated SREBPs were evaluated by
immunoprecipitation and immunoblotting with anti-SREBP-1/2 antibodies
in the cell extracts and shown inside the plot
area. An asterisk marks the nonspecific bands
observed in all lanes.
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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 (33, 41). 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 promoter that contains five consensus Gal4-binding sites, without
any co-regulatory transcription factors. Sumoylated SREBPs were shown
in the insets in Fig. 5, C and D. The
mutations at Lys123 and Lys418
resulted in a 1.9- and 2.1-fold increase in luciferase activities compared with Gal4-SREBP-1a, respectively (Fig. 5C).
Furthermore, Gal4-SREBP-1aK123R/K418R, which was no longer
sumoylated (Fig. 5C, inset), was more potent than
Gal4-SREBP-1aK123R and -K418R. Fig. 5D also shows that
Gal4-SREBP-2K464R significantly enhanced transcription of the reporter
gene to an extent elicited by double mutation in SREBP-1a. These
results indicate that SUMO-1 modification of SREBPs directly reduces
their own transcriptional activities. On the other hand, we cannot rule
out the possibility that sumoylation of SREBPs might influence their
interaction with co-regulatory factors or their DNA affinity.
Effect of Sumoylation of SREBPs on Their
Stability--
We previously reported that nuclear SREBPs are rapidly
degraded via the ubiquitin/26 S proteasome pathway (8). Since both sumoylation and ubiquitylation require a lysine residue of target proteins, we examined whether sumoylation competes with ubiquitylation, thereby affecting the ubiquitin-dependent degradation of
nuclear SREBPs. Pulse-chase experiments using COS-1 cells transfected with expression plasmids encoding either FLAG-tagged wild-type or K464R SREBP-2 were performed. Fig.
6A shows that the calculated half-life of wild-type and K464R SREBP-2 was ~2 h and that there were
no obvious differences in their stabilities. Similar results were
obtained when cells were transfected with expression plasmids encoding
either wild-type or mutant versions of FLAG-SREBP-1a (data not shown).
Taken together, these results indicate that sumoylation of SREBPs does
not influence their degradation through the ubiquitin/26 S proteasome
pathway.

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Fig. 6.
Effects of sumoylation of nuclear SREBPs on
their stability and ubiquitylation. A, COS-1 cells were
transfected with an expression plasmid encoding either wild-type or
K464R SREBP-2. After a 24-h incubation, the cells were trypsinized and
reseeded in 60-mm plates to normalize the transfection efficiency.
After further incubation for 24 h, pulse-chase experiments were
performed as described under "Experimental Procedures." At the
indicated time points, the cell lysates were subjected to
immunoprecipitation with anti-FLAG antibodies. All precipitates were
fractionated with SDS-PAGE and visualized by autoradiography. The same
results were obtained in three separate experiments. B,
COS-1 cells were transfected with an expression plasmid encoding either
wild-type or mutant versions of SREBPs. The purified GST-SREBPs from
the cells were subjected to an in vitro ubiquitylation
assay, as described under "Experimental Procedures."
Ubiquitin-modified SREBPs were detected by immunoblotting
(IB) with anti-SREBP-1/2 (top) and multiubiquitin
(bottom) antibodies. The mobilities of the ubiquitylated and
unmodified SREBPs are indicated on the right.
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To directly determine whether wild-type and mutant versions of SREBPs
are similarly modified by ubiquitin, we devised an in vitro
ubiquitylation assay for SREBPs. In this system, the GST-SREBPs expressed in COS-1 cells were conjugated with glutathione-Sepharose resins and then incubated with recombinant Ub-activating enzyme (E1)
and Ub-conjugating enzyme (E2) in the presence of GST-Ub and ATP. We
confirmed that Ubc4, but not UbcH7, collaborates as E2 for the in
vitro ubiquitylation assay of SREBPs (data not shown). As shown in
the top panels of Fig. 6B, in the
presence of Ubc4, the amounts of parental forms of SREBPs were
decreased (arrowheads), and high molecular mass smear bands
(closed bars) were observed by immunoblotting
with anti-SREBP antibodies. To confirm that these multiple smear bands
are indeed ubiquitin-modified forms of SREBPs, we performed
immunoblotting with anti-multiubiquitin antibodies. High
molecular mass smear bands were also detected with anti-multiubiquitin
antibodies (lower panels). Weak ubiquitylated smear bands were observed when UbcH7 was used as E2, which
probably reflected endogenously ubiquitylated SREBPs. These results
allow us to conclude that wild-type and mutant versions of SREBPs are similarly modified by ubiquitin and that sumoylation does not compete
with ubiquitylation in the SREBPs.
Sumoylation of SREBPs under Sterol-loaded or -depleted
Conditions--
Our next question was whether intracellular sterol
levels alter the rate of sumoylation of SREBPs. It is possible that
high sterol levels could accelerate sumoylation of nuclear SREBPs, thus
suppressing the transcription of SREBP-responsive genes. To investigate
this issue, we used M19 cells, which lacks site 2 protease (43),
resulting in the absence of endogenous nuclear SREBPs. In these cells,
one can determine sumoylation of exogenously expressed SREBPs under
either sterol-depleted or sterol-loaded conditions in the absence of
endogenous SREBPs. The cells were transfected with expression plasmids
encoding His-SUMO-1 and GST-SREBPs and cultured under either
sterol-depleted or sterol-loaded conditions. GST-SREBPs bound to
glutathione-Sepharose resins were subjected to immunoblotting analysis
with anti-SUMO-1 (Fig. 7, top)
and anti-SREBP-1/2 (bottom) antibodies. The amounts of
sumoylated and unmodified SREBPs under either sterol-loaded
(lanes 1 and 3) or sterol-depleted
(lanes 2 and 4) conditions were
similar. These results demonstrate that sterols do not alter the
sumoylation rate of SREBPs.

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Fig. 7.
Effects of sterols on sumoylation of
SREBPs. M19 cells were cotransfected with expression plasmids
encoding His-SUMO-1 and either GST-SREBP-1a (A) or -2 (B), and then cultured under either sterol-loaded
(lanes 1 and 3) or sterol-depleted
(lanes 2 and 4) conditions. GST-SREBPs
bound to glutathione-Sepharose resins were subjected to immunoblotting
(IB) with anti-SUMO-1 (top) and SREBP-1/2
(bottom) antibodies. The mobilities of the SUMO-1-modified
and -unmodified SREBPs are indicated on the right. The
positions of molecular mass standards are marked on the
left. The asterisks mark the nonspecific bands
observed in all lanes.
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The SUMO-conjugating Enzyme, Ubc9, Interacts with SREBPs--
To
investigate the direct interaction of SREBP with endogenous Ubc9, COS-1
cells were transfected with an expression plasmid encoding either FLAG
tag or FLAG-SREBP-1a/-2. The supernatants of cell lysates were
subjected to immunoprecipitation with anti-FLAG antibodies and
visualized by immunoblotting with anti-Ubc9 (Fig. 8A, middle) and
SREBP-1/2 (top) antibodies. Ubc9 was co-immunoprecipitated with FLAG-SREBPs (lanes 2 and 4),
indicating that Ubc9 is capable of interacting with the SREBPs. To
further investigate whether Ubc9 catalyzes sumoylation of SREBPs, COS-1
cells were transiently transfected with expression plasmids for
GST-SREBPs, His-SUMO-1, and either GFP or GFP-Ubc9(C93S), a dominant
negative form of Ubc9 (44). GST-SREBPs bound to glutathione-Sepharose
resins were subjected to immunoblotting with anti-SUMO-1
(top) and SREBP-1/2 (middle) antibodies.
Overexpression of GFP-Ubc9(C93S) inhibited sumoylation of SREBPs (Fig.
8B, lanes 6 (solid
bar) and 8 (solid arrowhead)). Thus, we conclude that the dominant negative
form of Ubc9 competes with the endogenous one, resulting in decreased modification of the nuclear SREBPs.

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Fig. 8.
Ubc9 interacts with SREBPs and catalyzes
attachment of SUMO-1 to SREBPs. A, COS-1 cells were
transfected with an expression plasmid encoding either FLAG tag
(lanes 1 and 3) or FLAG-SREBPs
(lanes 2 and 4). The cell lysates were
subjected to immunoprecipitation (IP) with anti-FLAG
antibodies and immunoblotting (IB) with anti-SREBP-1/2
(top) and Ubc9 (middle) antibodies. Expression of
endogenous Ubc9 was evaluated by immunoblotting in normalized whole
cell lysates (10 µg/lane) using anti-Ubc9 antibodies
(bottom). B, COS-1 cells were cotransfected with
expression plasmids for GST-SREBPs, His-SUMO-1, and either GFP
(lanes 5 and 7) or GFP-Ubc9 (C93S)
(lanes 6 and 8). GST-SREBPs bound to
glutathione-Sepharose resins were subjected to immunoblotting
(IB) with anti-SUMO-1 (top) and SREBP-1/2
(bottom). The asterisks mark the nonspecific
bands observed in all lanes. C, HeLa cells in a 60-mm dish
were transfected with the indicated amounts of expression plasmid for
His-Ubc9(C93S). After transfection, the cells were cultured under
sterol-depleted conditions for 48 h. Total RNA (8 µg/lane) was
subjected to Northern blot analysis as described in the legend to Fig.
2. The same results were obtained in three separate experiments.
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To examine whether the dominant negative form of Ubc9 is capable of
repressing sumoylation of endogenous SREBPs, thereby enhancing the
transcriptional activities, HeLa cells were transfected with an
expression plasmid for Ubc9(C93S). Northern blots of SREBP-responsive genes shown in Fig. 8C indicate that overexpression of
Ubc9(C93S) increased the amounts of HMG-CoA synthase and LDL receptor
mRNA in a dose-dependent manner, indicating that
blockade of sumoylation accelerated transcription of SREBP target
genes. These results suggest that Ubc9 interacts with SREBPs, thereby
enhancing the attachment of SUMO-1 to SREBPs and suppressing
their transcriptional activities.
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DISCUSSION |
The aim of this study was to identify posttranslational
modification of the SREBPs other than ubiquitylation and to elucidate the mechanisms that modulate SREBP activities through such
modifications. The major findings of the present study were 1) SUMO-1
modifies nuclear SREBPs in a Ubc9-dependent manner, 2)
residues Lys123 and Lys418 of SREBP-1a and
Lys464 of SREBP-2 can act as potential SUMO-1 acceptor
sites, and 3) SUMO-1 can negatively regulate the transactivation
function of SREBPs. These sumoylation motifs are all conserved in
vertebrate SREBPs whose sequences have been so far reported (U00968 for
human, U09103 for Chinese hamster, AF286470 for rat, and AY029224 for
chicken SREBP-1; U02031 for human, U12330 for Chinese hamster, and
AJ414379 for chicken SREBP-2).
Mutation analysis revealed that residues both Lys123 and
Lys418 are required for a full repression of the SREBP-1a
transcriptional activity, whereas Lys464 is sufficient for
a full repression of SREBP-2 to an extent comparable with that elicited
by double mutation in SREBP-1a (Fig. 5). It remains unknown why
SREBP-1a needs two sumoylation sites to control its transactivation
function. Interestingly, the amino acid sequence surrounding
Lys123 in SREBP-1a, PGIK123EESVP, perfectly
conforms to the consensus sequence, PX0-4(I/V)K(Q/T/S/L/E/P)EX0-3P, identified recently in the negative regulatory domain of several transcription factors and named the synergy control (SC) motif (45).
The putative SC/sumoylation sites in androgen receptor, glucocorticoid
receptor, and c-Myb were found to be functional (31, 32, 46). It is
hypothesized that an as yet unidentified SC factor might be
recruited to the SC motifs, thereby limiting the transcriptional
activities of certain transcription factors. The current finding that
covalent attachment of SUMO-1 at Lys123 in the SC motif
attenuates the transactivation function of SREBP-1a implies involvement
of such SC factor in a sumoylation-dependent manner.
However, whether the hypothetical SC factor recognizes unmodified SC
motifs or sumoylated motifs remains obscure, because sumoylation of the
SC motifs in SREBP-1a as well as androgen receptor and c-Myb attenuates
their transactivation capacity, whereas sumoylation of glucocorticoid
receptor elicits the opposite effect.
We found that the C-terminal domain containing ~90 amino acids of the
nuclear SREBPs exerts a transcriptional repression function when fused
to the Gal4 DNA binding domain plus the SREBP-1a activation domain
containing the N-terminal 50 amino acids (data not shown). In a
previous study, we indeed noticed that truncation of the domain in a
nuclear form of SREBP-1 somehow potentiated the transcriptional activities (2). The residues Lys418 of SREBP-1a and
Lys464 of SREBP-2 are located in this negative regulatory
domain and are likely to be involved in such repression. The amino acid
sequence surrounding Lys464 in SREBP-2,
VK464DEP, matches the consensus sumoylation site found in
the negative regulatory domain of four CCAAT/enhancer-binding protein
family members, which is evolutionally conserved in a variety of
vertebrate species (47). However, SUMO-1 attachment augmented the
inhibitory effect of the SREBP-2-negative regulatory domain but
decreased that of the CCAAT/enhancer-binding protein regulatory domain. In the case of SREBPs, covalent conjugation of SUMO-1 to
Lys418 or Lys464 could potentially influence
the flexibility of the C terminus as observed in sumoylated RanGAP1
(21). In support of this notion, the different migration patterns of
the sumoylated bands modified at Lys123 and
Lys418 in SREBP-1a (Fig. 3) suggest that sumoylation of
SREBPs might induce conformational changes in these proteins.
Sumoylation has been shown to be responsible for the translocation of
some proteins into discrete subnuclear matrix-associated structures,
called the promonocytic leukemia nuclear bodies (23, 48). Several
proteins, including Sp100, Daxx, CCAAT/enhancer-binding protein, and
ISG20, have been reported to be targeted to the nuclear bodies after
SUMO-1 modification (19); however, this does not necessarily mean that
all SUMO-1-conjugated proteins are destined to be localized in the
nuclear body (49). Interestingly, although the biological
function of the nuclear body is unclear, it has been
recently shown that sumoylation directly or indirectly promotes promonocytic leukemia degradation (50). Although we examined whether
mutations in the SUMO-1 sites affect the intracellular localization of
the GFP-tagged SREBPs, sumoylation did not induce significant changes
in the distribution of them in the nucleus (data not shown). Further
studies are required to investigate the role of SUMO-1 modification of
several target proteins including SREBPs on their subnuclear
localization in order to understand the biological functions of sumoylation.
Figs. 1, 3, and 4 show that the sumoylated SREBPs were barely detected
compared with unmodified SREBPs in these experiments. This does not
appear to be in line with the results of transactivation experiments
shown in Fig. 5, in which mutations in the SUMO-1 acceptor sites
resulted in marked increases of the transcriptional activities of
SREBPs, and the findings that expression of SREBP-responsive genes was
diminished by overexpression of SUMO-1 but increased by overexpression
of dominant negative SUMO-1 (Fig. 2). Sumoylated proteins are
considered as very transient structures resulting from a dynamic
equilibrium between SUMO-1-conjugated and -deconjugated forms (51).
Thus, sumoylation is likely to represent a mechanism for rapid and
reversible control, and target proteins are conjugated during a short
period of time, which is nevertheless sufficient to affect their
biological activities. So far, a number of mammalian desumoylating
enzymes, which remove SUMO-1 from its protein conjugates, have been
identified (19). The different patterns of subcellular or tissue
distribution of these enzymes may reflect the specific function of each
enzyme at its location and suggest that desumoylation, like
deubiquitylation, plays an important role in regulation of SUMO-1-mediated cellular processes.
The biological consequences of sumoylation and ubiquitylation are quite
different despite the facts that both SUMO-1 and ubiquitin are
conjugated to a lysine residue in target proteins and that their
modification machineries are mechanistically very similar. The addition
of a polyubiquitin chain to a lysine residue marks modified proteins
for a rapid degradation by the 26 S proteasome. In contrast,
sumoylation has been shown to increase the stability of I
B
by
antagonizing ubiquitylation (24). We previously reported that nuclear
SREBPs are degraded through the ubiquitin/26 S proteasome pathway (8).
In the present study, we provided experimental evidence that SUMO-1
modification of the SREBPs could not alter their stability (Fig.
6A) and that SUMO-1 conjugation did not compete with
ubiquitin modification (Fig. 6B). We devised a unique in vitro ubiquitylation assay, in which ubiquitin
conjugation occurred without the addition of a specific E3 ligase for
SREBPs in the presence of recombinant E1 and E2. It is likely that the GST-SREBPs prepared from cells treated with a proteasome inhibitor trapped an unidentified functional E3 ligase, which was
capable of attaching ubiquitin to the SREBPs. Based on these results, we conclude that lysine residues of sumoylation sites in the SREBPs are
at least not the major potential ubiquitylation sites. Taken together,
the independence of these two processes implies that inactivation of
SREBPs is doubly regulated by sumoylation and by their destruction
through the ubiquitin/26 S proteasome pathway. These results also
support the notion that the two independent processes might be required
for sufficient elimination of unexpected increases in the expression of
the SREBP-responsive genes to maintain homeostasis of lipid metabolism.
SREBPs are synthesized as membrane-bound precursors and
activated by a proteolytic process regulated by intracellular sterol levels. In addition to the proteolytic processing, a rise in
intracellular sterol levels, probably cholesterol levels in nuclear
membranes, might accelerate the rate of SUMO-1 conjugation to the
nuclear SREBPs, which in turn negatively regulates their
transactivation function to suppress the expression of genes encoding
enzymes for cholesterol biosynthesis. However, the results shown in
Fig. 7 clearly rule out this hypothesis. In a previous study, we also demonstrated that intracellular sterol levels do not alter the rapid
degradation of nuclear SREBPs through the ubiquitin/26 S proteasome
pathway (8). Taken together, these studies suggest that two independent
processes, ubiquitin-dependent degradation and
SUMO-1-mediated inactivation, are not regulated in response to changes
in lipid metabolism but act constitutively to control the
transcriptional activities of the nuclear SREBPs.
In conclusion, the importance of SUMO-1 modification for modulating the
transactivation function of nuclear SREBPs is now emerging. Our work
reports a new mechanism through which sumoylation can exert its
negative effect on SREBPs function and govern lipid metabolism.