From INSERM U135, Hormones, Gènes, and Reproduction, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre, France
Received for publication, July 17, 2002, and in revised form, December 18, 2002
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ABSTRACT |
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SUMO-1 (small ubiquitin-like modifier)
conjugation regulates the subcellular localization, stability, and
activity of a variety of proteins. We show here that SUMO-1
overexpression markedly enhances progesterone receptor (PR)-mediated
gene transcription. PR undergoes a sumoylation at lysine 388 located in
its N-terminal domain. However, sumoylation of the receptor is not
responsible for enhanced transcription because substitution of its
target lysine did not abolish the effect of SUMO-1 and even converted the receptor into a slightly more active transactivator. Furthermore estrogen receptor The progesterone receptor
(PR)1 is a transcription
factor belonging to the superfamily of nuclear receptors. It
contains two domains involved in transcription activation: the
constitutive activation function 1 (AF-1) localized in the N-terminal
region of the protein and the ligand-regulated activation function 2 (AF-2) corresponding to the ligand binding domain. X-ray diffraction analysis of receptor crystals have allowed a detailed study of the
molecular mechanisms involved in ligand-induced conformational changes
of the AF-2 region (1-9).
To activate transcription, steroid hormone receptors recruit
coactivators among which the p160 family seems to be of special importance. This family comprises SRC-1/NCoA-1 (steroid receptor coactivactor-1), SRC-2/TIF2/GRIP1 and SRC-3/TRAM-1/ACTR/AIB1/RAC3/pCIP (for reviews, see Refs. 10-11). CREB-binding protein (CBP) and p300
interact with both the nuclear receptors and the coactivators. Recruitment of histone acetylases allows local decondensation of
chromatin thus facilitating transcription (for reviews, see Refs.
12-14).
Aside from their regulation by specific ligands, nuclear receptors and
especially steroid hormone receptors are known to undergo covalent
modifications. Phosphorylation (for review, see Ref. 15) and
ubiquitinylation (15-20) have been shown to regulate their functions
and stability. Recently a new covalent modification of proteins and
especially of transcriptional regulators have been described: SUMO-1
has been found to be conjugated to a growing list of proteins: the Ran
GTPase-activating protein RanGAP1 (21-24), the NF SUMO-1 is a protein of 101 amino acids that has a low (18%) but
significant homology to ubiquitin. Modification by SUMO-1 (sumoylation)
has a marked similarity with ubiquitin conjugation reactions (for
reviews, see Refs. 36-37). It requires an E1-activating enzyme
(SAE1/SAE2 in mammals, Aos1/Uba2 in yeast) and the E2-conjugating enzyme Ubc9. E3 enzymes have been recently identified in yeast (38-39)
and in human cells (40-41) but do not appear to be required for SUMO
conjugation in vitro.
Whereas mono-ubiquitinylation has been described as specifically
involved in membrane receptor endocytosis (for review, see Refs. 42),
polyubiquitinylation addresses proteins to proteasomes where they are
degraded. In contrast, there is addition of a single SUMO-1 molecule to
target lysine(s). Whereas ubiquitinylation usually targets proteins to
degradation, sumoylation modifies their functional properties:
subcellular localization, stability, or ability to regulate gene transcription.
In this manuscript, we describe SUMO-1 conjugation to the progesterone
receptor and to its coactivator SRC-1. We also analyze the effect of
this modification on the subcellular localization and the
transcription-activating properties of these proteins.
Plasmids--
The plasmids encoding the full-length HA-tagged
SRC-1 and the deletion mutants pSG5-HA-SRC-1
All point mutation mutants were generated by site-directed mutagenesis
using PCR strategy. The generated fragments containing Lys/Arg
substitution were cloned into the pSG5-HA-SRC-1 vector by restriction
digestions. The multiple substituted mutants were obtained by
consecutive fragment exchanges using the restrictions sites
EcoRV, BamH1, BstEII, MscI,
and BglII. All the constructs were verified by DNA
sequencing on an automatic sequencer (ABI-Perkin 373A).
The expression vectors encoding the full-length progesterone receptor,
the human estrogen receptor Cell Culture, Transfections, and CAT Assays--
CV-1 and COS-7
cells were maintained in Dulbecco's modified Eagle's medium (DMEM)
(Invitrogen) supplemented with 10% fetal calf serum (Seromed). For CAT
assays and Western blotting analysis, transfections were performed by
the standard calcium phosphate precipitation method on CV-1 cells as
previously described in detail (43). Briefly, transfections were
performed on 6-well dishes with 330 µl of calcium phosphate
precipitate per well containing the reporter gene and expression
vectors as indicated for each experiment. Total DNA was normalized to
20 µg/ml of precipitate using salmon sperm DNA. The CAT activity was
measured with the CAT ELISA kit (Roche Molecular Biochemicals). Protein
concentrations were determined by using the BCA protein assay (Pierce),
and the CAT activity was corrected for protein content.
Preparation of Cell Extracts, Ni-NTA Precipitation, and Western
Blotting--
CV-1 cells seeded on 10-cm diameter Petri dishes were
transfected with the indicated expression vectors. For the preparation of whole cell extracts (WCE), 40 h after transfection, cells were washed with ice-cold phosphate-buffered saline and harvested in 1 ml of
lysis buffer supplemented with 50 mM sodium fluoride (34). Alternatively, for the nickel-affinity purification procedure (Ni-NTA),
the transfected cells were lysed in parallel in 1 ml of buffer A (6 M guanidinium-HCl, 100 mM
NaH2PO4, 10 mM imidazole, 10 mM Tris-HCl, pH 8). After sonication to reduce viscosity,
the clarified lysates were incubated for 2 h at room temperature
with 50 µl of Ni-NTA-coupled agarose beads (Qiagen) prewashed in
buffer A. The beads were washed twice with 1 ml of buffer B (8 M urea, pH 8, 100 mM
NaH2PO4, 20 mM imidazole, 10 mM Tris-HCl, pH 8) and once with 1 ml of buffer C (8 M urea pH 8, 100 mM
NaH2PO4, 20 mM imidazole, 10 mM Tris-HCl, pH 6.3). After a final wash with PBS, the
bound proteins were eluted by boiling in Laemmli loading buffer and
subjected to a 6.4% SDS-PAGE. The proteins were analyzed by Western
blotting using primary anti-PR monoclonal antibody Let126 (45) or
anti-HA 12CA5 monoclonal antibody (Roche Molecular Biochemicals) at a
concentration of 2 µg/ml. The ECL system (Amersham Biosciences) was
used for band detection.
Fluorescence Microscopy--
COS-7 cells were grown on 35-mm
dishes and were transfected with the expression vectors using
LipofectAMINE reagent (Invitrogen) according to the instructions of the
manufacturer. After different times, cells were fixed as previously
described (46). Incubations with the primary antibodies were performed
overnight at 4 °C in the presence of 1% goat serum. In single
labeling experiments, SRC-1 was detected with the rat monoclonal
anti-HA 3F10 (Roche Molecular Biochemicals) at 1 µg/ml. The secondary
antibody was the goat anti-rat Alexa 594 (Molecular Probes) (1/400).
For double labeling, SRC-1 was detected with the mouse monoclonal
anti-HA 12CA5 antibody (1 µg/ml) and endogenous SUMO-1 was detected
with the rabbit polyclonal anti-SUMO-1 FL-101 (Santa Cruz
Biotechnology) (2 µg/ml). The secondary antibodies sheep anti-mouse
CY3-conjugated antibody (Sigma) and Alexa 594 IgG (1/400) were
incubated with cells for 30 min at room temperature. Confocal images
were acquired using the LSM410 system on an Axiovert 135 M
Zeiss microscope (Carl Zeiss, Thornwood, NY) using excitation
wavelengths of 488 nm (for Alexa, green) and 543 nm (for CY3, red).
Sumoylation of the Progesterone Receptor--
To
examine if the progesterone receptor undergoes sumoylation, CV-1 cells
were transfected with a PR expression vector in the presence or absence
of a vector encoding His-tagged SUMO-1. Western blotting with an
anti-PR monoclonal antibody detected in cells expressing only PR and
not incubated with a ligand, a single protein band of ~115 kDa (Fig.
1A, WCE). Treatment
by the progesterone agonist R5020 provoked a decrease in receptor
migration previously shown to be due to its phosphorylation (47). In
contrast, in cells expressing both PR and SUMO-1, an additional band of ~155 kDa was detected in both hormone- or antiprogestin
(RU486)-treated and untreated cells.
To confirm that this band corresponded indeed to the sumoylated
receptor the His-tagged proteins were purified by chromatography on
nickel-charged agarose beads (Ni-NTA). The eluted proteins were
analyzed by Western blotting with an anti-PR antibody. The same band of
~155 kDa was observed in this experiment (Fig. 1A, Ni-NTA). Besides the sumoylated receptor minor bands
corresponding to the non-modified receptor species were also observed.
They were probably retained on the beads because of a low affinity of
receptor zinc fingers toward Ni-NTA.
SUMO-1 Enhances Progesterone Receptor-mediated Transcriptional
Activation--
To examine the effect of SUMO-1 on receptor-driven
transcription activation, CV-1 cells were cotransfected with an
expression vector encoding PR, the reporter gene
PRE2-TATA-CAT and increasing amounts of an expression
vector for SUMO-1.
As shown in Fig 1B, SUMO-1 expression markedly increased
hormone-induced transcription. There was no significant effect of SUMO-1 on transcription in the absence of hormone or in the presence of
the antagonists RU486 and ZK98299. The effect of SUMO-1 was thus
strictly hormone-dependent.
Receptor Sumoylation and SUMO-1 Enhancement of
Transcription--
We examined the possibility that receptor
sumoylation was responsible for the enhancement by SUMO-1 of
hormone-induced transactivation. Examination of the sequence of the
progesterone receptor showed a single sumoylation consensus at lysine
388 in the N-terminal domain of the protein. Indeed, compared with the
wild-type receptor, the mutant K388R was not sumoylated (Fig.
2A). (It should however be
noted that a very faint sumoylated band was still present in mutant
K388R. It may correspond to a second site of very weak affinity, and
which does not fit the target consensus). However the enhancement of
transcription of the reporter gene in the presence of hormone and of
SUMO-1 was also observed with the K388R mutant (Fig. 2B).
Furthermore, the non-sumoylated mutant was more active than the
wild-type receptor, in agreement with the recent data of Abdel-Hafiz
et al. (48). These observations suggested that sumoylation
of PR could not explain the overall effect of SUMO-1 on
receptor-mediated gene transcription. It should also be noted that
antiprogestin-complexed receptor, which does not provoke enhanced
transcription of target genes, is sumoylated in a manner similar to
that of hormone-complexed PR.
To further investigate the role of receptor sumoylation, we analyzed
SUMO-1 effect on ER Sumoylation of SRC-1 in Vivo--
The hormone-induced
transcriptional activation mediated by nuclear receptors involves a
multistep recruitment of several coactivators leading to the final
chromatin remodeling necessary for gene transcription. SRC-1 was the
first coactivator identified by its ligand-induced binding to the AF-2
region of the progesterone receptor (50). It has also been shown to be
a closely related partner of PR in vivo (51). We thus
examined the possibility that SRC-1 could be a target for SUMO-1 conjugation.
CV-1 cells were transfected with expression vectors
encoding the full-length HA-tagged SRC-1 in the presence or absence of an expression vector encoding His6-SUMO-1. Total cell
extracts were prepared and analyzed by Western blotting (Fig.
3A). In the absence of SUMO-1,
the anti-HA antibody detected a major band of ~175 kDa. In the cells
cotransfected with SUMO-1 expression vector three other bands were
observed (apparent molecular sizes of ~220, 245, and 260 kDa). These
bands probably corresponded to conjugation of SUMO-1 on one, two, or
three lysines of SRC-1, because SUMO-1 unlike ubiquitin is not able to
self-conjugate. The intensity of the bands decreased from the mono- to
the tri-conjugated forms. The latter was barely visible in several
experiments.
Mapping of SUMO-1 Modification Sites in SRC-1--
Examination of
the sequence of SRC-1 showed the presence of five consensus motifs
((I/L/V)KXE) for SUMO-1 conjugation (52-53). Interestingly,
all the potential target lysines were localized in protein-protein
interaction domains close to LXXLL motifs (Fig. 3B).
The lysines 732 and 774 were localized in the first nuclear receptor
interaction domain (NR1). The lysines 800 and 846 resided in the
CBP/p300 interaction domain. Lysine 1378 was located in the C-terminal
extremity of the protein corresponding to the second receptor-interacting domain (NR2). Interestingly only one of the consensus sequences (Lys-732) is conserved in all the three members of
the p160 family including SRC2/TIF2/GRIP1 and
SRC-3/TRAM1/ACTR/AIB1/RAC3. Two other motifs (Lys-800 and Lys-1378) are
conserved in two of these proteins; the remaining two motifs are
present only in SRC-1 (Fig. 3C).
Mutagenesis studies were undertaken to verify these predictions. CV-1
cells were cotransfected with expression vectors encoding various
deletion mutants of SRC-1 and His6-SUMO-1 (Fig.
4A). Total cell extracts and
Ni-NTA precipitates were analyzed by Western blotting using the anti-HA
antibody.
Removal of the nuclear interaction domain 1 (NR1) in mutant
We thus concluded that lysines 732 and 774 were the main sites of
SUMO-1 conjugation in SRC-1 whereas the other lysine(s) may serve as
weak affinity target sites.
SUMO-1 Modification of SRC-1 Enhances PR/SRC-1
Interaction in Vivo--
The two major sites of SUMO-1 conjugation
(Lys-732 and Lys-774) are located in the first nuclear receptor
interaction domain of SRC-1. This domain contains three
LXXLL motifs (named NR boxes), which are involved in
receptor-SRC-1 interaction (54-57). Residues flanking NR boxes on both
sides make significant contacts with the ligand binding domain of
receptors (58). These residues have also been shown to be important for
the preferential binding of various NR boxes to different nuclear
receptors and thus to determine a preference of the various receptors
for specific p160 coactivators (58). The sumoylation sites perfectly
flank NR box 3. This suggested that covalent binding of SUMO-1
molecule(s) could alter SRC-1-receptor interaction.
Few methods are available to study interactions between sumoylated
proteins since cell extracts must contain protein denaturing and
SH-blocking agents to prevent desumoylation (34). We thus used a
mammalian two-hybrid system to analyze the effect of sumoylation in vivo. CV-1 cells were cotransfected with a
Gal4 UAS-driven reporter gene, a vector encoding the
full-length PR fused to the Gal4 DBD
(DBDGal4-PR) and a vector encoding the SRC-1
sequence fused to the VP16 activation domain
(ADVP16-SRC-1). The transfected cells were incubated with
hormone (Fig. 5). The hormone-driven transcription observed in the presence of the DBDGal4-PR
construct was increased by SUMO-1 coexpression. However when
ADVP16-SRC-1 was added, SUMO-1 determined a markedly
stronger enhancement of transcription, showing that it increased
DBDGal4-PR interaction with ADVP16-SRC-1.
Mutation of lysines 732 and 734 (ADVP16-SRC-1 2R) hardly
modified the interaction between PR and SRC-1 but this interaction was
markedly affected in the non-sumoylated SRC-1 (ADVP16-SRC-1
5R), suggesting that the extent of sumoylation of SRC-1 plays a role in
receptor-coactivator interaction.
Effect of SUMO-1 on the Nucleocytoplasmic Trafficking of
SRC-1--
In previous studies, we have observed that the
intracellular localization of SRC-1 corresponds to a dynamic process,
the protein being initially addressed to the nucleus and thereafter
exported into the cytoplasm.2
In both nuclear and cytoplasmic compartments, SRC-1 is present in
dot-like structures. To analyze the effect of sumoylation, we
transfected cells with expression vectors encoding the wild-type SRC-1
or the mutant with five substituted arginines (5R-SRC-1), which cannot
be sumoylated. 24 h after transfection the wild-type SRC-1 was
predominantly present in the nucleus (Fig.
6A) where it colocalized with
endogenous SUMO-1. Forty-eight hours after transfection, SRC-1 was
mainly present in the cytoplasm, and only the few remaining nuclear
dots colocalized with endogenous SUMO-1 (Fig. 6B). In
contrast, the non-sumoylated 5R-SRC-1 mutant was mainly localized in
the cytoplasm already 24 h after transfection (only the few
remaining intranuclear SRC-1 stained dots colocalized with endogenous
SUMO-1) (Fig. 6A). The pattern observed with this mutant
24 h after transfection was very similar to that observed 48 h after transfection with wild-type receptor (Fig. 6B).
If sumoylation retards SRC-1 export from the nucleus, overexpression of
SUMO-1 should increase the nuclear residency time of SRC-1. This was
indeed observed in cells cotransfected with SUMO-1 where SRC-1 remained
in the nucleus even 48 h after transfection (Fig.
7A). No such nuclear retention
could be observed with the non-sumoylated 5R-SRC-1.
Finally, as previously observed by us in the case of
SRC-12 and others, proteasome inhibitors have been
reported to provoke the nuclear sequestration of various transcription
factors (59-62). Indeed in the presence of proteasome inhibitor
clasto-lactacystin, and 48 h after transfection, SRC-1 was still
present mainly in the nucleus presenting a hyperspeckled pattern. In
this case, there was a perfect colocalization between these speckles
and endogenous SUMO-1 (Fig. 7B).
Mutation of SUMO-1 Acceptor Sites in SRC-1 Does Not Alter Its
Effect on PR-mediated Transcription--
To obtain further insight
into the transcriptional role of SUMO-1 conjugation to SRC-1, we
studied the impact of mutations of SUMO-1 acceptor sites.
Expression vectors encoding PR and a reporter gene
(PRE2-TATA-CAT) were transfected into cells. Various
amounts of an expression vector encoding wild-type SRC-1 were
co-transfected and the cells treated by hormone. As previously
described (63-65), cotransfection of increasing amounts of SRC-1
enhanced the hormone-induced transcription.
If wild-type SRC-1 was replaced by non-sumoylated mutants (double
substituted K732R/K774R SRC-1 or 5×-substituted 5R-SRC-1), the effect
on the reporter gene transcription was very similar (Fig.
8A).
Since endogenous SUMO-1 protein may be limited in the cells, we
repeated the experiment in the presence of overexpressed SUMO-1 (Fig.
8B). Again, disruption of SUMO-1 target lysines in SRC-1 did
not modify its co-transcriptional activity.
SRC-1 Sumoylation, Ubiquitinylation, and
Stability--
SUMO-1 conjugation has been involved in the regulation
of the stability of several proteins (25, 66). Competition
between ubiquitinylation and sumoylation for the same lysines has been shown to prevent proteasome targeting and degradation. We have previously shown that SRC-1 is subjected to a proteasome-mediated proteolysis.3 We thus examined
the effect of sumoylation on ubiquitinylation. Ubiquitinylation
and sumoylated sites were clearly different since the non-sumoylated
5R-SRC-1 mutant underwent normal ubiquitinylation (Fig.
9A).
When both ubiquitin and SUMO-1 were co-expressed with SRC-1, the two
modified forms of SRC-1 were purified on nickel-charged agarose beads
(Fig. 9B) showing that SRC-1 can simultaneously be the
target of both modifications. Furthermore, SRC-1 sumoylation did not
constitute a mechanism for ubiquitin antagonism because even in the
presence of SUMO-1, the overall SRC-1 form was decreased in the
presence of ubiquitin.
Steroid hormone receptors undergo a variety of covalent
modifications which can modify their functional properties
(phosphorylation, ubiquitinylation, acetylation). In this manuscript,
we describe SUMO-1 conjugation to the progesterone receptor at lysine
388. The non-sumoylated PR mutant was still transcriptionally active and the gain of activity observed in this mutant suggested that the
sumoylation of the receptor may have a repression effect. Indeed, it
has recently been shown that the sumoylation of the PR is implicated in
the regulation of its autoinhibition and transrepression activities
(48). Sumoylation has different effects on different steroid receptors:
the estrogen receptor We have observed that overexpression of SUMO-1 very markedly enhanced
PR- and ER SUMO-1 modification of various proteins seems to play a major role in
their subcellular and especially intranuclear targeting. The
sumoylation of PML directs this protein into nuclear bodies (27-28,
69). Actually it has been shown that sumoylated PML is necessary for
the assembly and/or the stability of these intranuclear structures
(69). SUMO-1 conjugation to HIPK2 directs it into nuclear speckles
(30). The non-conjugated form of RanGAP1 is found in the cytoplasm.
Once modified by SUMO-1, it becomes localized in the nuclear pore
complex (22-23). Sumoylation of RanGAP1 is necessary for its
interaction with Ran GTP-binding protein RanBP2 (70). It must be
emphasized that the SAE (SUMO-1 activating) and Ubc9 (SUMO-1
conjugating) enzymes are mainly localized in the nucleus (52). We have
shown SRC-1 to localize initially in the nucleus and to be thereafter
exported into the cytoplasm.2 In both cellular
compartments, it is associated with dot-like structures. In the
non-sumoylated SRC-1 mutant, the time of residency in the nucleus is
markedly decreased. SUMO-1 conjugation thus seems to play a role in the
nuclear retention of SRC-1. These results suggest that sumoylation may
regulate the kinetics of SRC-1 subcellular localization and/or of the
assembly of transcriptional complexes. This may explain why we did not
observe any differences between the transcriptional activity of
wild-type and of non-sumoylated SRC-1. Indeed, at the time necessary
for the transcriptional assay, the different SRC-1 constructs have
recovered the same subcellular localization.
Several proteins have been shown to be stabilized by sumoylation. In
I A variety of transcriptional regulators undergo SUMO modification and
in several cases, their activity is modulated by this reaction. This is
the case of the tumor suppressor p53, which regulates the transcription
of a number of genes involved in cell cycle arrest and apoptosis
(33-35, 71-72). The transcriptional activator c-Jun (35), as well as
the transcriptional repressors or corepressors TEL (73),
HIPK2 (30), and Drosophila Tramtrack-69 (74)
also undergo sumoylation.
We have shown here that SUMO-1 overexpression very strongly enhances
PR-mediated gene transcription. However a similar effect was observed
when using non-sumoylated mutants of PR and SRC-1. This suggests that
besides its action on SRC-1 and PR, SUMO-1 exerts effects on other
proteins involved in gene transcription regulation by nuclear
receptors. Further studies will be necessary to define these unknown
and functionally important SUMO-1 targets.
(ER
)-driven transcription is also enhanced by
SUMO-1 overexpression contrasting with the absence of sumoylation of
this receptor. We thus analyzed SUMO-1 conjugation to the steroid receptor coactivator SRC-1. We showed that this protein contains two
major sites of conjugation at Lys-732 and Lys-774. Sumoylation was
shown to increase PR-SRC-1 interaction and to prolong SRC-1 retention
in the nucleus. It did not prevent SRC-1 ubiquitinylation and did not
exert a clear effect on the stability of the protein. Overexpression of
SUMO-1 enhanced PR-mediated gene transcription even in the presence of
non-sumoylated mutants of SRC-1. This observation suggests that
among the many protein partners involved in steroid hormone-mediated
gene regulation several are probably targets of SUMO-1 modification.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B inhibitor
I
B
(25), the promyelocytic leukemia protein PML (26-29), the
nuclear dot protein Sp100 (27), the homeodomain-interacting protein
kinase 2 HIPK2 (30), the topoisomerases I and II (31-32), the tumor
suppressor protein p53, and the protooncogene c-Jun (33-35).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-567, pSG5-HA-SRC-1
1-782, and pSG5-HA-SRC-1
1209-1440 have been previously
described (43). PCR was used to generate the expression vector for the
SRC-1 mutant pSG5-HA-SRC-1
785-1038, by inserting a
BamH1 site in position 2343 corresponding to the amino acid
782. The deletion was generated by replacing the wild-type
BamH1/Msc1 fragment with the PCR fragment digested by BamH1/MscI.
(pKSV-ER
), and their respective
reporter plasmids PRE2-TATA-CAT and
ERE2-TATA-CAT have been previously described (43). The
point mutation K388R was generated by PCR. The mutated fragment was
introduced into pSG5-hPR using MluI/MscI sites.
The construct was verified by sequencing. The expression vectors
encoding the ADVP16-SRC-1, PM-PR fusion proteins have been
described (43). For the ADVP16-SRC-1 2R and 5R mutants, the
SmaI/BglII fragments of pSG5-HA-SRC-1 2R and
pSG5-SRC-1 5R have been cloned, respectively, into the blunted
EcoRI/BamHI empty ADVP16 vector. The
His6-tagged SUMO-1 (pSG5-His6-SUMO-1) and the
His6-tagged ubiquitin (pSG5-His6-Ub) expression
vectors were kindly provided by Dr. S. Müller and Dr. D. Bohmann,
respectively (35, 44).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sumoylation of the progesterone
receptor; effect of SUMO-1 on PR-mediated gene transcription
activation. A, CV-1 cells were transfected with the
expression vector encoding the progesterone receptor in the presence or
absence the His6-tagged SUMO-1. The cells were grown for
24 h in the presence or absence of 10 nM R5020
(H) or 10 nM RU486 (RU) as indicated.
Total cell extracts (WCE) were analyzed by electrophoresis,
and PR was detected by Western blotting using the PR-specific let126
monoclonal antibody. The co-transfected CV-1 cells were lysed in buffer
containing guanidium-HCl (Ni-NTA), and the SUMO-1-modified proteins
were purified in parallel using Ni-NTA agarose beads as described under
"Experimental Procedures." Eluted proteins were separated by
electrophoresis and His6-SUMO-1 PR conjugates were detected
by Western blotting using the PR-specific Let126 monoclonal antibody.
WCE represent 15% of the input for Ni-NTA precipitation. Molecular
size markers are shown on the right, and the SUMO-1-modified
form of the PR is indicated by an asterisk. B,
CV-1 cells were transfected with the PRE2-TATA-CAT reporter
plasmid (5 µg/ml) along with pSG5-rPR (0.5 µg/ml) and increasing
amount of pSG5-His6-SUMO-1 expression vector. The cells
were treated 24 h before harvesting with 10 nM R5020
( ), 10 nM RU486 (
), 100 nM ZK98299 (
),
or left untreated (
). The results are means ± S.D. of three
independent determinations.
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Fig. 2.
Receptor sumoylation and SUMO-1 enhancement
of transcription. A, lysine 388 is the major
sumoylation site of PR. CV-1 cells were transfected with the expression
vector encoding the wild-type progesterone receptor (pSG5-hPR) or the
K388R mutant (pSG5-hPR K388R) in the presence or absence the
His6-tagged SUMO-1. The cells were grown in the presence of
10 nM R5020 for 24 h before harvesting. Total cell
extracts (WCE) were analyzed by electrophoresis and PR was
detected by Western blotting using the PR-specific let126 monoclonal
antibody. B, SUMO-1 enhances PR K388R-mediated gene
transcription activation. CV-1 cells were transfected with the
PRE2-TATA-CAT reporter plasmid (5 µg/ml) along with
pSG5-hPR (1 µg/ml) or pSG5-hPR K388R (1 µg/ml) as indicated and
increasing amount (1; 2.5, and 5 µg/ml) of
pSG5-His6-SUMO-1 expression vector. The cells were treated
with 10 nM R5020 (+) or untreated ( ) 24 h before
harvesting. The results are means ± S.D. of three independent
determinations. C, SUMO-1 enhances ER
-mediated gene
transcription activation. CV-1 cells were cotransfected with the
plasmid encoding the estrogen receptor ER
(0.5 µg/ml) with the
ERE2-TATA-CAT reporter plasmid (5 µg/ml) and increasing
amounts of pSG5-His6-SUMO-1 expression vector. The cells
were treated with 10 nM estradiol (
) or untreated (
)
24 h before harvesting. The results are means ± S.D. of
three independent determinations.
-stimulated gene transcription. Although ER
has previously been shown to be devoid of SUMO-1 conjugation consensus
site and to not undergo sumoylation in vitro (49), cotransfection with SUMO-1 did markedly enhance estradiol-driven reporter gene transcription (Fig. 2C). Enhancement of
transcription by SUMO-1 thus involves modification of proteins other
than the receptor.
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Fig. 3.
Covalent modification of SRC-1 by
SUMO-1. Putative sumoylation sites. A, CV-1
cells were transfected with the expression vector encoding the
HA-tagged SRC-1 in the presence or absence the His6-tagged
SUMO-1. Total cell extracts were separated by electrophoresis on a
6.4% SDS-PAGE and SRC-1 was immunoblotted with an anti-HA monoclonal
antibody. The SUMO-1-modified forms of SRC-1 are indicated by an
asterisk. B, a schematic representation shows the
sites corresponding to consensus sequences for SUMO-1 conjugation
(indicated by ). The functional domains identified in SRC-1 are also
represented: bHLH, helix-loop-helix motif; PAS,
Per Arnt-Sim motif; CBP/p300, CBP/p300
interaction domain; Q, glutamine rich domain; NR,
nuclear receptors interacting domains 1 and 2. The amino acids
corresponding to the putative target lysines are indicated
above. The localization of LXXLL motifs is
indicated with an asterisk. C, potential
sumoylation consensus motifs of the human SRC-1 based on the consensus
((I/L/V)KXE) (52-53) and conservation of this motif among
the members of the p160 coactivator family. SRC-2 designates the GRIP1
and TIF2 coactivators and SRC-3 designates AIB1/RAC3/ACTR/TRAM-1
proteins. The amino acids matching the consensus are indicated in
bold.
View larger version (38K):
[in a new window]
Fig. 4.
Identification of SUMO-1 conjugation sites in
SRC-1. A, schematic representation of the deletion
mutants of SRC-1 used for the determination of the SUMO-1-conjugated
sites. The residues conserved are indicated below the bold
lines. The putative (consensus-derived) SUMO-1 conjugated sites
are designated as in Fig. 3. B, CV-1 cells were transfected
with expression vector encoding the HA-tagged SRC-1 deletion mutants
respectively, in the presence or absence the His6-tagged
SUMO-1. Total cell extracts (WCE) were analyzed by 6.4%
SDS-PAGE and immunoblotted with anti-HA monoclonal antibody.
Alternatively, the same co-transfected CV-1 cells were lysed in buffer
containing guanidium-HCl (Ni-NTA), and the SUMO-1-modified proteins
were purified using Ni-NTA agarose beads as described under
"Experimental Procedures." Eluted proteins were separated by
electrophoresis and His6-SUMO-1 SRC-1 conjugates were
detected by Western blotting using the anti-HA monoclonal antibody. WCE
represent 15% of the input for Ni-NTA precipitation. C,
CV-1 cells were transfected with the expression vector encoding the
substitution Lys/Arg mutants of SRC-1, in the presence or absence the
His6-tagged SUMO-1. The total cell extracts (WCE) or
Ni-purified precipitates (Ni-NTA) were analyzed as in B. The
mutant name indicated the position of the substituted lysine with
arginine residue (5R corresponds to the substitution of the
five lysines).
1-781
completely abolished SUMO-1 conjugation (Fig. 4B). The mutants
785-1038 and
1209-1440, which retain this domain, were sumoylated. This result suggested that lysines 732 and 774 could be the
major sumoylation sites. Point mutations were used to verify this
hypothesis. When all five putative target lysines (Lys-732, Lys-774,
Lys-800, Lys-846, and Lys-1378) were substituted by arginines (5R
SRC-1), the sumoylation reaction was suppressed. Individual substitutions of Lys-800, Lys-846, and Lys-1378 did not affect SRC-1
overall modification by SUMO-1. In contrast, the double substitution on
Lys-732R and Lys-774R abolished sumoylation (Fig. 4C).
View larger version (19K):
[in a new window]
Fig. 5.
SUMO-1 expression enhances PR-SRC-1
interaction in vivo. The effect of SUMO-1 on the formation
of PR-SRC-1 complexes was analyzed by the two-hybrid system in CV-1
cells. The cells were co-transfected with the pG5-CAT reporter plasmid
(5 µg/ml) (Clontech), the vector encoding
ADVP16-SRC-1 ( ) or ADVP16-SRC-1 2R (
) or
ADVP16-SRC-1 5R (
) (2 µg/ml), the vector encoding the
full-length PR fused to the DBDGal4 (PM-PR) (0.2 µg/ml),
along with increasing amount of pSG5-His6-SUMO-1 expression
vector. The cells were treated with 10 nM R5020. In control
experiments, ADVP16-SRC-1 construct was replaced by
ADVP16 (
). The results are means ± S.D. of three
independent determinations.
View larger version (44K):
[in a new window]
Fig. 6.
Substitution of SUMO-1 target lysines
modifies the kinetics of the trafficking of SRC-1 between the nucleus
and the cytoplasm. Subcellular colocalization of wild-type
(WT) or mutated non-sumoylated (5R) SRC-1 with
SUMO-1. Wild-type or mutated HA-tagged SRC-1 expression vector was
transfected into COS-7 cells. The cells were fixed 24 h
(A) or 48 h after transfection (B), and
SRC-1 proteins were detected using the monoclonal anti-HA antibody
(red channel). The endogenous SUMO-1 was detected with the
anti-SUMO-1 FL-101 antibody (green channel). A merge of both
signals is shown in the right panels with overlapping
staining appearing in yellow.
View larger version (37K):
[in a new window]
Fig. 7.
Overexpression of SUMO-1 retains SRC-1 in the
nucleus. A, subcellular localization of p160 SRC-1 in the
presence of overexpressed SUMO-1. COS-7 cells were either transfected
with the HA-tagged SRC-1 expression vector alone (SRC-1 wt) or
co-transfected with a 4-fold excess of the His6-SUMO-1
expression vector (SRC-1 wt + SUMO-1). After 48 h, SRC-1 was
detected with the rat monoclonal anti-HA 3F10 antibody using confocal
fluorescence. The same experiment was repeated with non-sumoylated
5R-SRC-1 mutant (lower part of A). B,
subcellular colocalization of ectopically expressed HA-tagged SRC-1 wt
with endogenous SUMO-1 in the presence of the proteasome inhibitor
clasto-lactacystin. COS-7 cells were transfected with the wild-type
SRC-1 and treated with 50 µM clasto-lactacystin for the
last 24 h. Forty-eight hours after transfection, SRC-1 and SUMO-1
proteins were revealed as in Fig. 6. Bar, 5 µm. A merge of
both signals is shown in the right panels with overlapping
staining appearing in yellow.
View larger version (19K):
[in a new window]
Fig. 8.
Non-sumoylated mutants of SRC-1 enhance
PR-mediated transcription. CV-1 cells were transfected with the
PRE2-TATA-CAT reporter plasmid (2.5 µg/ml) along with
pSG5-rPR (0.2 µg/ml) and increasing amount of pSG5-HA vector encoding
respectively wild type ( ), K732R/K774R substitution mutant (
), or
5R mutant version (
) of SRC-1 (A) in the absence or in
the presence (B) of co-expressed
pSG5-His6-SUMO-1 vector (2.5 µg/ml). The cells were
treated with 10 nM R5020 24 h before harvesting. The
results are means ± S.D. of three independent
determinations.
View larger version (32K):
[in a new window]
Fig. 9.
SRC-1 can be both sumoylated and
ubiquitinated. A, CV-1 cells were transfected with the
expression vector encoding the HA-tagged wild-type SRC-1 or the
5R-SRC-1 mutant in the presence of the His6-tagged
ubiquitin expression vector (His6-Ub) (10 µg/ml). Total
cell extracts (WCE) were analyzed by electrophoresis on
6.4% SDS-PAGE and immunoblotted with anti-HA monoclonal antibody.
Alternatively, the same co-transfected CV-1 cells were lysed in buffer
containing guanidium-HCl (Ni-NTA). The ubiquitin-modified proteins were
purified using Ni-NTA agarose beads as described under "Experimental
Procedures." Eluted proteins were separated by electrophoresis, and
His6-SRC-1 conjugates were detected by Western blotting
using the anti-HA monoclonal antibody. WCE represent 15% of the input
for Ni-NTA precipitation. The ubiquitin conjugates of SRC-1 are
indicated with brackets. B, CV-1 cells were
transfected with the HA-tagged SRC-1, and the His6-tagged
SUMO-1 (10 µg/ml) expression vectors in the presence or absence of
the His6-tagged ubiquitin expression vector (5 µg/ml).
Molecular species of SRC-1 were detected in WCE or in Ni-NTA as
described in A. The SUMO-1-modified forms of SRC-1 are
indicated by an asterisk, the ubiquitin-conjugates are
indicated with brackets.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
is not sumoylated (49) whereas the androgen
receptor (AR) is sumoylated in its N-terminal domain in an
androgen-enhanced fashion (49). SUMO-1 conjugation of AR apparently
decreases its transcriptional activity as observed after substitution
of the sumoylated lysines. On the contrary this substitution has
no effect on AR-mediated transrepression (49). Ubc9, the conjugating
enzyme for SUMO-1, interacts with both AR and GR (49, 67-68). In
transiently transfected cells, coexpression of Ubc9 enhances
AR-dependent transcription. However, mutated Ubc9 having
lost its SUMO-1-ligating activity retains its effect on AR-mediated
transactivation (68). Thus, the role of Ubc9 in sumoylation reactions
does not explain its binding to AR and its effect on transactivation.
-mediated gene transcription. This effect was not related
to receptor sumoylation. We thus analyzed SUMO-1 modification of the
coactivator SRC-1. Two major sites of conjugation were localized. They
flanked at a distance of 20 amino acids a NR box situated in the
nuclear receptor-interacting region 1 (NR1). Since the function of the
nuclear receptor binding LXXLL motifs is known to be
modulated by adjacent amino acids (58), we examined the effect of
sumoylation on PR-SRC-1 interaction. Using a mammalian two-hybrid
system, we observed an increased interaction of PR with sumoylated
SRC-1. Substitution of the two lysines 732 and 734 showed little effect
on PR-SRC-1 interaction. In contrast, we observed a markedly decreased
interaction of PR with the non-sumoylated SRC-1 (5R mutant). The
significant difference between the two mutants underlines the
functional relevance of the NR box located at the C terminus of SRC-1,
close to the sumoylation target lysine 1378. These results suggest that
sumoylation may be implicated in the formation or stability of
receptor-coactivator complexes.
B
, SUMO-1 and ubiquitin compete for conjugation to the same
lysines. Thus I
B
sumoylation renders it resistant to signal-induced degradation. NF
B transcriptional activation is then
blocked (25). A similar mechanism has been observed for Mdm2, the E3
ubiquitin ligase for p53. Sumoylation stabilizes this protein (66). On
the contrary, sumoylation of SRC-1 does not compete with its
ubiquitinylation and has no clear effect on its stability.
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ACKNOWLEDGEMENTS |
---|
We thank Stephan Müller (Max Planck Institute, Martinsried, Germany) and Dirk Bohmann (European Molecular Biology Laboratory, Heidelberg, Germany) for the kind gift of the His6-tagged SUMO-1 and the His6-tagged ubiquitin expression vectors. We thank Chantal Carreaud-Aumas for excellent technical assistance.
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Note Added in Proof |
---|
While preparing this manuscript, SUMO-1 modification of the coactivator GRIP-1 has been described by Kotaja et al. (75). They have shown that the non-sumoylated mutant of the coactivator GRIP1 displayed half of the transcription enhancing activity of the wild-type protein. However, these authors have studied androgen receptor/GRIP1 interaction by using the isolated LBD domain of the receptor. As seen in the case of PR, N-terminal sumoylation also plays a role in these interactions.
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FOOTNOTES |
---|
* This work was supported by INSERM, the Association pour la Recherche sur le Cancer, the Ligue contre le Cancer, the Faculté de Médecine Paris-Sud, and the Fondation pour la Recherche Médicale.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: CNRS UPR9079, Oncogénèse,
différenciation et Transduction du signal, Institut André
Lwoff, 7 rue Guy Môquet, 94800 Villejuif, France.
§ Supported by the Association pour la Recherche sur le Cancer.
¶ To whom correspondence should be addressed: INSERM U 135 Hormones, Gènes et Reproduction, Hôpital de Bicêtre, 78 rue du Général Leclerc, 94275 Le Kremlin-Bicêtre cedex, France. Tel.: 33-1-45-21-33-29; Fax: 33-1-45-21-27-51; E-mail: u135@kb.inserm.fr.
Published, JBC Papers in Press, January 14, 2003, DOI 10.1074/jbc.M207148200
2 L. Amazit, Y. Alj, R. K. Tyagi, A. Chauchereau, H. Loosfelt, C. Pichon, J. Pantel, E. Foulon-Guinchard, P. Leclerc, E. Milgrom, and A. Guiochon-Mantel, manuscript in preparation.
3 L. Amazit, A. Chauchereau, Y. Alj, R. K. Tyagi, P. Leclerc, E. Milgrom, and A. Guiochon-Mantel, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PR, progesterone
receptor;
SRC-1, steroid receptor coactivator 1;
CREB, cAMP response
element-binding protein;
CBP, CREB-binding protein;
SUMO, small
ubiquitin-like modifier;
ER, estrogen receptor
;
HA, hemagglutinin epitope;
PRE, progesterone responsive element;
ERE, estrogen responsive element;
CAT, chloramphenicol acetyltransferase;
AD, activation domain;
DBD, DNA-binding domain;
Ub, ubiquitin;
WCE, whole cell extracts;
Ni-NTA, nickel nitrilotriacetic acid-charged
agarose beads;
R5020, 17,21-di-methyl-19-norpregna-4,9-dien-3,20-dione;
RU486, 17
-hydroxy-11
-(4-dimethylaminophenyl)-17
-(1-propynyl)-oestra-4,9-dien-3-one;
ZK98299, 11
-(4-dimethylaminophenyl)-17
-hydroxy-17
-(3-hydroxypropyl)-13
-methyl-4,9-gonadien-3-one;
DBD, DNA-binding domain;
LBD, ligand-binding domain.
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