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INTRODUCTION |
Glucocorticoids are steroid hormones essential for life that are
produced by the adrenal glands and affect all tissues of the body.
Synthetic derivatives of glucocorticoids are widely used in the
treatment of a number of inflammatory diseases. These hormones are
necessary for normal growth and development, liver and immune
functions, and in mediating stress responses (1-3). Glucocorticoids
mediate their actions by binding to the glucocorticoid receptor
(GR),1 a member of the
superfamily of steroid/thyroid/retinoic acid proteins that function as
ligand-dependent transcription factors. Like other members
of this superfamily, GR contains a number of structure motifs including
a DNA binding domain, ligand binding domain, and two transactivation
motifs AF1 and AF2 (4). Upon ligand binding, the cytoplasmic GR becomes
activated and translocates to the nucleus where it can bind to specific
DNA elements termed glucocorticoid response elements (GREs), causing
stimulation of transcription. Alternatively, activated GR can interact
with other transcription factors such as NF-
B or AP-1 to repress
gene expression activated by these pro-inflammatory transcription
factors (5-8).
The sensitivity of cells to glucocorticoids has been shown to be
dependent on the number of GRs found in the cell (9-11). Upon ligand
binding the GR undergoes a process of homologous down-regulation that
has been extensively studied (9-15). GR levels are regulated at
multiple levels including mRNA and protein (16, 17). At the RNA
level, several studies have shown that glucocorticoid treatment
decreases GR mRNA levels by 50-80% in many different tissues
(18-20). This occurs via transcriptional mechanisms requiring an
intragenic element within the GR gene itself (20). Additionally, early
studies showed that hormone treatment leads to a subsequent decrease in
the ability of the receptor to bind hormone, but the mechanism for this
phenomena was poorly understood. Dense amino acid labeling of
3H-labeled steroid-bound receptor suggested that hormone
treatment influenced receptor protein turnover (12). Generation of
GR-specific antibodies permitted analysis of GR protein and revealed
that ligand binding of GR led to a reduction in receptor protein levels (10, 19). Studies examining the half-life of the GR protein, separate
from any ligand effects on RNA, have shown that ligand occupation of
the receptor significantly decreases GR protein half-life from 18 to
9 h. Recently, the phosphorylation state of the receptor has been
shown to be an important determinant of receptor stability, and a
phospho-deficient mutant of mouse GR does not undergo
ligand-dependent down-regulation (21).
Because phosphorylation has been shown to act as a signal for protein
recognition by the ubiquitin and proteasome pathways for protein
degradation, we considered the possibility that the proteasome mediates
GR down-regulation (22-25). Proteins such as IB
, Fos, and the
tumor suppresser p53 have been shown to be regulated by this pathway
with phosphorylation playing a critical role (26-28). Phosphorylation
is thought to be the signal to allow substrate recognition by enzymes
in the ubiquitination pathway. The ubiquitin pathway consists of
ubiquitin-activating enzymes (UBAs), E2 ubiquitin-conjugating enzymes
(UBCs), and E3 ubiquitin-ligase enzymes. Phosphorylation allows
recognition by E2 and/or E3 molecules that covalently add the 76-amino
acid protein ubiquitin to lysine residue(s) of the targeted protein
(26, 29, 30). Poly-ubiquitination of a protein allows it to be
recognized by the multisubunit protein complex known as the proteasome,
which degrades the protein into small peptides and amino acids. Recent
development of specific inhibitors of the proteasome has permitted
identification of its central role in the turnover of both short lived
and long lived proteins (31-34).
The glucocorticoid receptor has been shown recently to interact with an
E2-conjugating protein and two E3-ligase proteins found within the
ubiquitin enzymatic pathway, but the functional consequences of GR
interaction with these proteins remain unclear. We report here that
inhibition of the proteasome by MG-132 abolishes dexamethasone-induced
mouse glucocorticoid receptor protein down-regulation. By using the
proteasome inhibitors MG-132 and 
-lactone, we demonstrate enhanced
hormone responsiveness by use of a glucocorticoid response element-containing reporter construct. By immunoprecipitation of GR
protein, we have visualized higher molecular weight forms of the
glucocorticoid receptor that we have identified as ubiquitinated forms
of GR. Mutagenesis of lysine 426, found in a candidate PEST degradation
motif within the glucocorticoid receptor, abrogated ligand-dependent down-regulation, enhanced hormone
responsiveness, and was found to be unresponsive to proteasome
inhibition. Together these findings suggest a mechanism for termination
of glucocorticoid-dependent glucocorticoid receptor
signaling via protein degradation through the proteasome pathway.
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EXPERIMENTAL PROCEDURES |
Materials--
Dexamethasone
(1,4-pregnadien-9-fluoro-16-methyl-11,17,21-triol-3,20-dione)
(Dex) was purchased from Steraloids (Wilton, NH). MG-132
(benzyloxycarbonyl-Leu-Leu-Leu-aldehyde) and clasto-latacystin -lactone were purchased from Calbiochem. N-Ethylmaleimide
was purchased from Pierce. [14C]Chloramphenicol (40-60
mCi/mmol) was obtained from PerkinElmer Life Sciences. Acetyl coenzyme
A, Mini Complete tablets, Pefabloc, and aprotinin protease inhibitors
were purchased from Roche Molecular Biochemicals. Peroxidase-labeled
anti-rabbit and anti-mouse antibodies and the ECL chemiluminescent
detection reagents were purchased from Amersham Pharmacia Biotech. All
other reagents were obtained from Sigma.
Recombinant Plasmids--
Wild type and mutant mouse GR were
expressed in the pSV2Rec vector under the control of the SV40 promoter
(35). pCMV5-mGR was also used in some experiments, and detailed
descriptions of this expression vector are available (36). Mutation of
mouse glucocorticoid receptor cDNA from Lys-426 to K426A was done
by polymerase chain reaction site-directed mutagenesis using the QuickChange Site-directed mutagenesis kit from Stratagene (Cedar Creek,
TX). Primers used were 5'-ACG GGA CCA CCT CCC GCA CTC TGC CTG GTG
TGC-3' and 5'-GCA CAC CAG GCA GAG TGC GGG AGG TGG TCC CGT 
3'.
GRE2-TATA-CAT and GRE2-TATA-Luc reporter
plasmids have been previously described (37, 38). The c-Myc-tagged
ubiquitin expression vector (wt-H6M-Ub) was a kind gift of Ron Kopito
(39).
Cell Culture and Transfection--
COS-1 (African Green monkey
kidney) cells were grown in Dulbecco's modified Eagle's medium with
high glucose medium supplemented with 2 mM glutamine, 10%
(v/v) of a 1:1 mixture of heat-inactivated fetal calf/calf serum
(Summit Biotechnology, Ft. Collins, CO) and 100 IU/ml penicillin and
100 µg/ml streptomycin. HeLa cells were grown in monolayer cultures
in Joklik's minimum essential medium and harvested with Versene as
described previously (40). All cultures were maintained in 5%
CO2 humidified atmosphere at 37 °C and passaged every
3-4 days. For transfection of cells, cells were plated 24 h prior
to transfection and allowed to grow to 50-70% confluency. Cells were
washed with Opti-MEM (Life Technologies, Inc.) and transfected using
DMRIEC (Life Technologies, Inc.) or TransIT transfection reagent from
Panvera Corp. (Madison, WI) according to the manufacturers'
recommendations. The precipitates were incubated with cells for 5 h. The Opti-MEM media was removed, and media containing
dextran-charcoal stripped serum were added. Plasmid DNA was prepared
using Qiagen Maxiprep kits (Valencia, CA).
Luciferase Assays--
Cells were transfected as described
above. Following transfections, media were changed to media containing
dextran/charcoal-stripped serum, and cells were allowed to recover for
12-16 h and then treated with vehicle or dexamethasone for the times
indicated. Cells were harvested and lysates prepared, and luciferase
assay was performed using an Enhanced Luciferase Assay Kit from BD
PharMingen (Franklin Lakes, NJ). Luciferase activity was measured
using a Microtiter Plate Luminometer and Revelation 4.06 Software
(Dynex Technologies). Luciferase activity was calculated per µg of
protein for each sample.
Chloramphenicol Acetyltransferase (CAT) Assays--
Cells were
transfected as described above. HeLa cells were washed with ice-cold
1× PBS and incubated with Versene at 37 °C for 15 min. Cells were
spun down for 5 min and taken up in 0.25 M EDTA (pH 8.0).
Lysates were prepared by tip sonication, and endogenous CAT activity
was inactivated by heating to 65 °C for 15 min. Protein
concentration was determined by the method of Bradford, using a reagent
kit from Bio-Rad. Equal amounts of protein were incubated in reactions
with acetyl coenzyme A and 0.1 µCi of
[14C]chloramphenicol. Reactions were terminated by ethyl
acetate extraction, and CAT activities were determined by thin layer
chromatography (TLC) separation of acetylated products from substrate
chloramphenicol. Plates were visualized by PhosphorImager (Molecular
Dynamics, Inc. Sunnyvale, CA) and analyzed using ImageQuant software.
Activity was expressed as percent chloramphenicol acetylation. In some experiments CAT activity was determined by terminating reactions by the
mixed xylene method as described by Promega (Madison, WI) and utilized
a standard curve of CAT activity from supplied protein.
Western Blot Analysis--
In protein half-life studies using
the mouse GR vector pSV2wRec and mutant GR GRK426A cellular extracts
were prepared from transiently transfected COS-1 cells pretreated with
1 µM cycloheximide for 1 h and then treated with
vehicle or 100 nM dexamethasone. Cells were washed
with cold 1× PBS and harvested by scraping with a rubber spatula into
PBS, and cells were centrifuged. The cell pellet was taken up in RIPA
buffer (20 mM Tris (pH 7.5), 2 mM EDTA, 150 mM NaCl, 0.05% Triton X-100 (pH 7.5)) containing protease inhibitors 1 µg/ml aprotinin, Pefabloc, and Mini Complete protease inhibitor tablet from Roche Molecular Biochemicals. Protein levels were
quantitated using the Lowry method (Bio-Rad). Approximately 100 µg of
protein per sample was resolved by 8% SDS-polyacrylamide gel,
transferred to nitrocellulose membrane, and stained with Ponceau S
(0.5% in 1% acetic acid) to evaluate loading equivalency and transfer
efficiency. BenchMark Prestained Protein ladders from Life
Technologies, Inc., were used to determine molecular size of visualized
bands. Membranes were placed in a blocking solution of 10%
nonfat milk (10 mM Tris-HCl (pH 7.4), 150 mM
NaCl, 0.05% Tween 20) for 1 h at room temperature or overnight in
a cold room. Membranes were then washed two times for 15 min with 1%
nonfat milk buffer (10 mM Tris-HCl, 150 mM
NaCl, 0.05% Tween 20) and then incubated for 1 h with anti-GR
epitope-purified polyclonal antibody 1857 at a dilution of 1:2000.
After two 15-min washes, the membrane was then incubated with 1% milk
buffer with peroxidase-labeled goat anti-rabbit antibody at a dilution
of 1:15,000 (ECL, Amersham Pharmacia Biotech). The membrane was washed
as described and reacted for 1 min with chemiluminescent reagents
commercially supplied (ECL). Blots were processed for autoradiography,
and signals were quantitated by using NIH Image densitometry software.
Immunoprecipitations--
Cellular lysates from COS-1 cells were
prepared in immunoprecipitation buffer (10 mM Tris (pH
7.4), 150 mM NaCl, 2 mM EDTA, 0.5% sodium
deoxycholate, and 0.5% Nonidet P-40) containing protease inhibitors 1 µg/ml aprotinin and Pefabloc, Mini Complete protease inhibitor tablet
from Roche Molecular Biochemicals, and 5 mM
N-ethylmaleimide (26). Addition of
N-ethylmaleimide to isolated cellular extracts inhibits
isopeptidase activities that may otherwise affect the detection of
ubiquitinated proteins (26). To immunoprecipitate GR, a 1:1 mixture of
antibodies 57 and 59, epitope-specific rabbit polyclonal antibodies,
were added to 200 µg of cellular extract, and samples were rotated at
4 °C overnight (41, 42). Protein A-Sepharose was added and samples
were rotated for 2 h. Sepharose was pelleted, and samples were
washed 5× in a large volume of IP buffer. Samples were taken up in
Laemmli sample buffer and boiled for 5 min to dissociate immune
complexes from Sepharose. Equal volumes of samples were run on SDS-PAGE
and BenchMark Prestained Protein ladders from Life Technologies, Inc.,
were used to determine molecular size of visualized bands.
Immunoprecipitated proteins were visualized using GR-specific peptide
antibody 1857, as described above. Blots were also probed with
anti-c-Myc antibody from Babco (Richmond, CA) to detect Myc-tagged
ubiquitin GR conjugates. Immunoprecipitated GR was run on SDS-PAGE
gels, and blots were probed with an anti-ubiquitin antibody that was
the generous gift of Dr. Arthur Haas (43).
Statistical Analyses--
Statistical significance was
determined using a t test with a confidence level of 0.05. Western blot data was determined for significance by using pixel data
from NIH Image Software, and CAT assay data were visualized using
PhosphorImager technology and ImageQuant software from Molecular
Dynamics, Inc. (Sunnyvale, CA).
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RESULTS |
Dexamethasone-induced Mouse GR Protein Down-regulation Is Blocked
by Proteasome Inhibition in Transiently Transfected COS-1
Cells--
Glucocorticoid receptor ligand-dependent
down-regulation is thought to be a complex process involving regulation
of both GR receptor gene expression and receptor protein. Regulation at
the RNA and the protein levels occurs simultaneously and has been demonstrated in numerous studies using transcriptional or translational inhibitors. With the development of GR-specific antibodies, it has been
shown in a number of tissues and cell types that in the presence of
ligand GR protein becomes destabilized and receptor levels decrease
(10, 13, 14, 19). Interestingly, disruption of GR interaction with heat
shock protein 90 (Hsp90) also leads to GR protein down-regulation, and
inhibition of the proteasome function has recently been shown to block
this down-regulation (44).
Thus we wished to determine the role of the proteasome in
ligand-dependent down-regulation of the glucocorticoid
receptor. To address this issue the COS-1 cell line (which is devoid of endogenous GR) was transfected with pSV2Rec, a mouse GR expression vector. Following transfection cells were pretreated with the proteasomal inhibitor MG-132
(benzyloxycarbonyl-Leu-Leu-Leu-aldehyde) or vehicle for 1 h
and then were treated with vehicle or dexamethasone. Cellular extracts
were isolated, and Western blot analysis of GR was performed with
peptide-specific antibodies. Glucocorticoid treatment caused a profound
decrease in the levels of GR protein (Fig.
1, 1st versus
2nd lanes) that was inhibited by the inclusion of MG-132
(Fig. 1, 2nd versus 4th lanes).
Densitometric analysis of three separate experiments revealed that
MG-132 abrogated the repressive effect of dexamethasone on GR protein
levels. Dexamethasone treatment for 12 h caused GR protein levels
to fall on average to 42% of control, whereas cells pretreated with
MG-132 maintained normal receptor levels. The ability of the proteasome
inhibitors to block ligand-dependent glucocorticoid
receptor protein down-regulation suggests that the GR is degraded by
the ubiquitin-proteasome pathway.
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Fig. 1.
Glucocorticoid receptor-dependent
down-regulation is blocked by the proteasome inhibitor MG-132 in COS-1
cells. COS-1 cells were transiently transfected with 4 µg of the
mouse glucocorticoid (GR) expression vector pSV2Rec. Cells
were treated with vehicle or 1 µM MG-132 for 1 h and
then treated with dexamethasone (Dex) for 12 h. Whole
cell lysates were prepared, and 100 µg was separated by SDS-PAGE
electrophoresis. Blots were analyzed by Western blot using antipeptide
hGR antibody 57. Western blot shown is representative of typical
results. Densitometry was determined using NIH IMAGE 1.6 software, and
the results of three independent experiments are shown (*,
p < 0.05 versus basal).
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Proteasome Inhibition Enhances GR Transcriptional Activity in
Transiently Transfected COS-1 Cells--
To ascertain if the observed
receptors preserved by proteasome inhibition were functional, assays
were performed using glucocorticoid-responsive reporter genes. COS-1
cells were transiently transfected with pSV2Rec GR expression vector
and hormonally responsive GRE2-TATA-CAT. Transfected cells
were pretreated with MG-132 and then treated with various doses of
dexamethasone. Dexamethasone treatment induced reporter activity,
whereas MG-132 alone had little effect on activity (Fig.
2A). In contrast cells treated
with MG-132 and dexamethasone showed significantly increased reporter
activity compared with cells treated with Dex alone. Percent CAT
conversion levels increased 3-4-fold in cells treated with MG-132 and
Dex. Similar observations were made when cells were treated with
clasto-lactacystin -lactone (-lactone), a second more specific
inhibitor of the proteasome (Fig. 2B). These data suggest
the receptors maintained by proteasome inhibition during glucocorticoid
treatment are functional and further suggest that hormone
responsiveness may be limited by glucocorticoid receptor protein
down-regulation via the proteasome pathway.
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Fig. 2.
Glucocorticoid induction of reporter activity
is enhanced by pretreatment with proteasome inhibitors in transfected
COS-1. A, COS-1 cells were transiently transfected with
0.5 µg of pSV2Rec mouse glucocorticoid receptor expression vector and
0.5 µg of glucocorticoid-responsive reporter GRE2-CAT.
Cells were treated with vehicle or 1 µM MG-132 for 1 h and then treated with 1 µM, 100 nM, and 10 nM Dex for 16 h. Lysates were prepared for CAT. Equal
µg of extract were used in each reaction. B, COS-1 cells
were transfected with 100 ng of pSV2Rec GR expression vector and 1 µg
of GRE2-CAT and pretreated as above with 1 µM
-lactone and then Dex at 1 µM. Data are presented as
percent CAT conversion and represent the mean of three independent
experiments. *, p < 0.05 versus fold
induction. Con, control.
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Proteasome Inhibition Enhances Endogenous hGR Transactivation
in HeLa Cells--
Because studies in transfected cells overexpressing
glucocorticoid receptor suggested that proteasome inhibition blocked
glucocorticoid-induced down-regulation and maintained functional
responsiveness, we wished to determine whether the proteasome plays a
similar role in cells that contain endogenous human GR. The HeLa cell
line was utilized to address this question because these cells express
hGR endogenously, and the down-regulation of glucocorticoid receptor
protein by treatment with dexamethasone has been well documented in
this cell line (9, 14). Down-regulation of GR protein levels has been
proposed to be the limiting factor in HeLa cells responsiveness to
glucocorticoids (11, 15). To examine if inhibiting the proteasome
protein degradation pathway affected GR responsiveness in HeLa cells,
we again used the proteasome inhibitor MG-132 and analyzed the effects
of proteasome inhibition on hormone responsiveness in HeLa cells
transfected with a hormone-responsive dimer GRE driving the CAT
reporter gene. Following transfection, cells were pretreated with
MG-132 and then were treated with dexamethasone. Cells treated with
MG-132 demonstrated only a slightly higher percent CAT conversion than
control, whereas dexamethasone markedly induced activity over the
control (Fig. 3). In contrast, HeLa cells
treated with a combination of MG-132 and dexamethasone showed significantly induced reporter activity as compared with control. Thus,
inhibition of the proteasome was also effective in enhancing glucocorticoid responsiveness in HeLa cells harboring endogenous glucocorticoid receptor. These findings suggest that glucocorticoid responsiveness in HeLa cells is limited by the process of GR
degradation involving the proteasome pathway.
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Fig. 3.
Endogenous human glucocorticoid receptor
transcriptional activity is enhanced by pretreatment with the
proteasome inhibitor MG-132. HeLa cells were transiently
transfected with 1 µg of GRE2-CAT reporter construct.
Following recovery from transfection cells were pretreated with vehicle
Me2SO or 1 µM MG-132 for 1 h and then
treated with vehicle or 100 nM Dex for 16 h. Cellular
lysates were prepared, and CAT activity was assayed. Data shown are of
percent CAT conversion, and the data shown represent three independent
transfections. Data shown are representative of three independent
experiments. *, p < 0.05 versus fold
induction.
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Immunoprecipitation of Mouse Glucocorticoid Receptor Identified
Higher Molecular Weight Ubiquitinated Species of GR--
The process
of protein degradation by the proteasome involves enzymes of the
ubiquitination pathway, which add ubiquitin molecule(s) to a protein
and which effectively target that protein for degradation by the
proteasome. Because our studies indicated that proteasome inhibition
blunted dexamethasone-induced GR protein down-regulation and enhanced
transcriptional response, we wished to determine whether GR is subject
to ubiquitination. To determine whether GR is ubiquitinated, COS-1
cells were co-transfected with pCMV-GR and the Myc-tagged wild type
ubiquitin expression vector. Transfected cells were treated with
vehicle or dexamethasone in the absence and presence of MG-132;
cellular extracts were prepared, and samples were immunoprecipitated
using a mixture of epitope-specific GR antibodies. Immunoprecipitated
samples were analyzed by Western blot using antibodies to GR and to the
c-Myc-tagged ubiquitin. By using GR-specific antibody 57, the ~97-kDa
band of GR was seen. It should be noted that under these experimental
conditions dexamethasone not only alters GR turnover but also decreases
transcription of the GR gene and promotes GR mRNA degradation (13,
20). Additionally, longer film exposures revealed higher molecular
mass forms of GR, at ~180 kDa and greater, that were
consistently observed in a striated pattern suggesting the addition of
multiple ubiquitin molecules (Fig. 4).
The higher molecular weight proteins were more prominent in extracts
from MG-132-treated cells. When the same blots were stripped and then
probed with anti-c-Myc antibody to detect the tagged c-Myc-ubiquitin,
corresponding proteins were seen at 180 kDa in a striated pattern that
was particularly apparent in cells treated with MG-132. Because
immunoprecipitations with antibodies specific to GR were used as the
initial selection, these higher molecular weight forms of GR probably
represent forms of GR that have a number of lysines modified with
mono-ubiquitin or specific lysines that are poly-ubiquitinated as seen
for other proteins (39, 45, 46). These findings suggest that GR is targeted for degradation by ubiquitination and then are recognized and
degraded by the proteasome complex. We conclude that the GR is
ubiquitinated. To determine how GR is targeted for degradation, we
undertook analysis of GR for any degradation motifs.
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Fig. 4.
Glucocorticoid receptor
immunoprecipitation. COS-1 cells were transiently transfected with
4 µg of pCMV-GR and 4 µg of wt-H6M-Ub ubiquitin (Ub)
expression vectors. Following transfection cells were allowed to
recover and were treated with vehicle or 1 µM MG-132 for
1 h followed by treatment with 100 nM Dex for 16 h. Whole cell lysate was prepared in the presence of protease
inhibitors and 5 mM N-ethylmaleimide.
Approximately 200 µg of cellular lysate was used for
immunoprecipitation with the antisera to peptide 57 and 59 which
recognize epitopes to hGR. Immunoprecipitated samples were separated by
SDS-PAGE electrophoresis, and blots were probed with antibodies to GR
and anti-Myc ubiquitin.
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Analysis of GR for Potential Degradation Motifs Identified a PEST
Element--
Knowledge of the mechanism involved in protein
recognition by the enzymes of ubiquitin pathway remains unclear, but a
number of motifs have been shown to be important in substrate
recognition by E2/E3 proteins (25). Furthermore, current research
suggests that phosphorylation is a critical signal for the rapid
degradation of a number of proteins. A number of rapidly degraded
proteins contain PEST regions that consist of the amino acids Pro
(P), Glu (E), Ser (S), and Thr
(T). PEST regions contain phosphorylation sites, are
regions of hydrophilic amino acids, and are flanked by Lys, Arg, and
His (47, 48). Analysis of the mouse glucocorticoid receptor using a
PEST-FIND program allowed identification of a PEST motif from amino
acids 407-426 (Fig. 5). This region has a PEST-FIND score of +18.3, and on a scale from 50 to +50 a value above +5 is thought to be indicative of possible functional PEST motif
(48). For example two proteins, IB and Fos, are known to be
degraded by the proteasome have PEST scores of 5.9 and 10.1, respectively (27, 49, 50). The potential PEST motif in GR could help
explain the rapid destabilization of GR after ligand binding.
Interestingly, both the rat and human glucocorticoid receptor also
contain this destabilizing motif (Fig. 5). PEST-FIND analysis
calculated a score of +18.3 for the rat GR and +16.1 for the human GR.
To address directly the relevance of this analysis, we targeted this
region in the mouse GR for site-directed mutagenesis at the candidate
site for ubiquitination.
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Fig. 5.
PEST-FIND analysis identified a degradation
motif within the GR. To identify possible degradation motifs
within the GR, the PEST-FIND program was run (47, 48). The
structure of the GR and the identified PEST motif are shown. Note the
identified lysine shown in bold was the site targeted for
site-directed mutagenesis. Similar PEST sequences were found in the rat
and human glucocorticoid receptors. TA, transactivation;
DBD, DNA binding domain; LBD, ligand binding
domain.
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Mutagenesis of Lysine Residue Abrogates GR Protein
Ligand-dependent Down-regulation--
The identified PEST
region of GR at amino acids 407-426 was next targeted for
site-directed mutagenesis. The candidate site for ubiquitination at
Lys-426 was mutated to alanine and the mutation confirmed by DNA
sequencing. Previous half-life studies have shown that GR protein
levels fall to 50% in the presence of 100 nM dexamethasone in about 9 h, whereas in the absence of ligand levels fall to 50%
in 18 h (21). To determine the effect of this lysine mutation on
GR ligand-dependent down-regulation similar half-life
studies were performed. COS-1 cells were transiently transfected with wild type GR or mutant K426A GR expression vector and treated with
cycloheximide prior to hormone administration. This generates an
intracellular pool of GR protein and allows us to examine changes in GR
protein levels without analyzing the effects of glucocorticoid on the
repression of GR gene transcription. Cells were then treated with
dexamethasone, and cellular extracts were prepared for Western blot
analysis (Fig. 6A). Wild type
GR levels were lower due to ligand-dependent
down-regulation. In a separate transfection mutant K426A GR did not
undergo ligand-dependent down-regulation, and receptor
levels remained relatively unchanged by treatment with dexamethasone.
Densitometry of three independent experiments determined that wild type
GR levels fell to an average of ~50% of control levels (Fig.
6B), whereas the K426A GR levels remained constant despite
the presence of hormone. The failure of K426A GR to undergo ligand-dependent down-regulation suggests that the lysine
within the identified PEST region serves as an acceptor ubiquitin site that plays a prominent role in GR turnover. Mutagenesis of one or more
lysine residues has also been shown to alter the protein turnover rates
of IB and yeast a-factor receptor (29, 51). We next
considered if mutagenesis of Lys-426 had any effects on GR
function.
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Fig. 6.
K426A mutant does not undergo
ligand-dependent down-regulation. A, COS-1
cells were transfected with wild type (Wt), or in a separate
experiment K426A, and 16-24 h later were treated with 1 µM cycloheximide (Cyclo) for 1 h and then
with 100 nM Dex or left untreated for 12 h.
Whole cell lysates were prepared, and proteins were separated on 8%
acrylamide gel and transferred to nitrocellulose membrane. GR was
visualized using anti-GR 1857 and using an ECL kit (Amersham Pharmacia
Biotech). A typical Western blot analysis of wild type GR and mutant
Lys-426 GR is shown. B, densitometry of three independent
experiments as determined by NIH IMAGE software. *, p < 0.05 versus fold induction.
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Transcriptional Activation by Lysine 426 Mutant Is
Enhanced--
The maintenance of normal levels of K426A GR protein
observed in the half-life studies prompted us to examine whether this mutant receptor incapable of down-regulation at the protein level had
altered transcriptional activation in response to hormone. In this
study a glucocorticoid-responsive reporter was transiently transfected
with either wild type or mutant K426A GR and treated with dexamethasone
for 12, 24, 48, and 72 h. Cells expressing K426A displayed
increased reporter activity over wild type mouse GR at all times tested
(Fig. 7A). Levels of
luciferase activity for mutant K426A were consistently 2-3.6-fold
higher than that of wild type, with fold activity increasing as the
duration of the experiment increased from 12 to 48 h. Western blot
analysis indicated that after 12 or 24 h of dexamethasone
treatment, K426A mutant GR protein levels were similar to those of the
untreated controls (Fig. 7B), probably reflecting the fact
that K426A mutant is not undergoing down-regulation at these early time
points. In contrast, wild type GR protein levels treated with
dexamethasone were undergoing ligand-dependent
down-regulation after 12 and 24 h when compared with untreated
controls. With extended hormone treatment we do see a reduction in both
wild type and mutant GR reflecting the transcriptional component of
ligand down-regulation described previously (13-16). Also as expected
in these transient transfection experiments, we observed in the
controls a time-dependent decrease in protein expression
from transfected plasmids. Thus, mutagenesis of Lys-426 enhanced both
glucocorticoid receptor levels, and hormone responsiveness suggests
lysine 426 plays a role in limiting GR hormone responsiveness.
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Fig. 7.
K426A GR mutant displays enhanced
transcriptional activation over that of wild type GR.
A, COS-1 cells were transiently transfected with wild type
mouse GR or K426A mutant GR and GRE2Luc. Cells were then
treated with 100 nM Dex or vehicle (C) for 12, 24, 48, and 72 h. Cell extracts were prepared for luciferase
activity determination. Luciferase assay was done using an Enhanced
Luciferase Assay Kit from BD PharMingen (Franklin Lakes, NJ) using a
Microtiter Plate Luminometer and Revelation 4.06 Software (Dynex
Technologies). Luciferase activity was calculated per µg of protein
for each sample, and results are presented as relative light
units (RLU)/µg protein. B, Western blot
analysis was done concurrently with luciferase assays. Cell lysates
were prepared, and 20 µg of protein was separated by SDS-PAGE. Blots
were analyzed by Western blot using antipeptide hGR antibody 57. The
Western blot shown is representative of typical results seen in three
separate experiments.
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Proteasome Inhibition Failed to Enhance the Transcriptional
Activity of the K426A Mutant GR--
To ascertain if proteasome
inhibition enhanced K426A mutant GR transcriptional activity as seen in
previous experiments with wild type GR, CAT assays were performed using
a glucocorticoid-responsive reporter. COS-1 cells were transiently
transfected with wild type GR or K426A mutant GR expression vectors and
hormone-responsive GRE2-TATA-CAT. Transfected cells were
pretreated with MG-132 and then treated with dexamethasone.
Dexamethasone treatment induced reporter activity of both wild type and
mutant K426A GR, but the K426A mutant displayed enhanced activity over
wild type GR (Fig. 8). When cells were
pretreated with MG-132 and then treated with dexamethasone, wild type
GR displayed enhanced activity as seen previously. In contrast, cells
expressing K426A GR and treated with MG-132 and dexamethasone failed to
significantly enhance transcription, although some small increases were
seen in each of the experiments performed. These data demonstrate that
Lys-426 is a critical amino acid involved in maintaining GR hormone
responsiveness. Mutagenesis of this site leads to enhanced hormone
responsiveness and also blocks the additional induction of
transcriptional activity seen in the presence of the proteasomal
inhibitor MG-132.
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Fig. 8.
Proteasome inhibition did not enhance mutant
GR K426A dexamethasone induction of reporter activity in transfected
COS-1. COS-1 cells were transiently transfected with 0.25 µg of
pSV2Rec mouse glucocorticoid receptor expression vector and 0.25 µg
of glucocorticoid-responsive reporter GRE2-CAT. Cells were
treated with vehicle or 1 µM MG-132 for 1 h and then
treated with 10 nM Dex for 12 h. Lysates were prepared
for CAT. Equal µg of extract were used in each reaction. Data are
presented as percent CAT conversion and represent the mean of three
independent experiments. *, p < 0.05.
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DISCUSSION |
Upon ligand binding the glucocorticoid receptor undergoes a rapid
decline of receptor levels that has been well demonstrated in a number
of glucocorticoid receptor target tissues (9, 12, 19, 52). The rapid
decrease in receptor levels is due to a dramatic decrease in GR protein
half-life in the presence of ligand that is thought to diminish
cellular responsiveness to ligand (10-15). Glucocorticoid receptor
protein levels have also been shown to decline when cells are treated
by other agents that disrupt GR interactions with heat shock proteins
by either heat shock or geldanamycin (44, 53). In the case of heat
shock-mediated degradation of the glucocorticoid receptor, it was shown
to be an ATP-dependent process. Unlike other proteases, the
need for ATP is a hallmark of proteasome activity suggesting that the
ubiquitin-dependent pathway of proteolysis may be involved
(53). Whitesell and Cook (44) were able to block geldanamycin-induced
degradation by use of the proteasome inhibitor lactacystin. These
researchers suggested that GR is targeted for destruction by the
ubiquitin pathway. In this study we demonstrate that inhibition of the
proteasome by the peptide aldehyde MG-132 blocked GR
ligand-dependent down-regulation in COS-1 cells transfected
with GR cDNA. Similar results have been demonstrated in experiments
with transfected estrogen receptor (ER), where proteasome inhibition by
MG-132 was observed to block estrogen-induced degradation of ER
although ER does not have an obvious PEST element.
Ligand-dependent down-regulation was also shown to be
blocked by MG-132 in studies of the progesterone receptor and the aryl
hydrocarbon receptor (AhR) (54, 55).
Our study of COS-1 cells transiently transfected with GR and HeLa
cells, containing endogenous hGR, demonstrated that proteasome inhibition by MG-132 or -lactone leads to enhanced GR
transactivation in reporter assays. These experiments reveal that the
elevated levels of GR protein seen by Western blot, in the presence of MG-132 and dexamethasone, induced greater reporter activity than cells
treated with either agent alone. The elevation of hormone-induced reporter expression suggests that hormone responsiveness can be significantly affected by inhibition of GR degradation. Proteasome inhibition elevates reporter activity of other transcription factors such as the AhR, Sp1, and p53 (56-58). Like GR, the AhR is a
cytoplasmic protein that upon ligand binding enters the nucleus. The
enhanced transcriptional activity of AhR in the presence of MG-132 was found to be consistent with the elevated levels of AhR in the nucleus
(58). Similar to AhR, the enhancement of GR transcriptional activity
observed in the presence of dexamethasone and MG-132 may be due to a
greater fraction of total cellular GR being in the nucleus, as well as
the decrease in GR degradation. In contrast, in transcriptional
activity assays of the estrogen and thyroid receptors, it was
found that proteasomal inhibition decreased ligand-induced
transcriptional activity (56, 59). The reasons for this discrepancy are
not clear, but it has been suggested that the ER co-activator
complex formation may be disrupted by proteasome inhibition (56). In
the case of the thyroid receptor, it was suggested that proteasomal
activity may be necessary to modify thyroid receptor by proteolysis to
produce a transcriptionally active form of the receptor (59). Whereas
AhR, Sp1, and p53 do contain PEST elements with scores greater than
10.0, the significance of the lower scoring PEST element of the thyroid
receptor and the lack of an element in the estrogen is unclear.
This suggests that additional mechanisms perhaps involving the
degradation of receptor-associated co-activators or co-repressors
may prevail.
A number of steroid receptors have now been identified as being
ubiquitinated and targeted for destruction by the proteasome in a
process involving ligand binding by the receptor (56, 60, 61). We
present evidence that the GR is ubiquitinated, although previously
published attempts (44) to detect ubiquitinated forms of GR had been
unsuccessful. When COS-1 cells were transfected with GR cDNA and
treated with MG-132, higher molecular weight forms of
immunoprecipitated GR protein were seen by Western blot analysis. These
data suggest a mechanism of ligand-dependent
down-regulation for GR involving targeting for degradation by the
proteasome. To identify the possible phosphorylation and ubiquitination
sites involved in this mechanism, we analyzed the GR protein for
candidate degradation motifs. By using the PEST-FIND computer program,
we identified a conserved PEST element found in the mouse, rat, and human GR. PEST elements have been shown to be important in the turnover
of a number of proteins including IB, -cyclins, and c-Fos (27,
62-64). These elements contain phosphorylation sites and are usually
flanked by a lysine residue, a candidate site for ubiquitination. It
remains unclear whether these regions allow recognition by kinases or
members of the ubiquitination pathway (48). The identified region in
the mouse glucocorticoid receptor contains the identified
phosphorylation site Ser-412 or peptide 23 (65-67) and is flanked by a
lysine residue at position 426, a possible site of ubiquitination. To
determine the significance of this region, Lys-426 was mutated to
alanine by site-directed mutagenesis. In half-life studies using the
protein synthesis inhibitor cycloheximide, we observed by Western blot
that wild type GR underwent dexamethasone-induced down-regulation, but
the mutant K426A GR did not. These data suggest that residue Lys-426 is
critical in the ligand-dependent regulation of GR. In the
case of IB, mutagenesis of two critical lysine residues caused
the protein to be resistant to signal-regulated degradation (29). The
K426A glucocorticoid receptor mutant also displayed enhanced ability to
transactivate the hormone-responsive GRE2-luciferase reporter when compared with wild type glucocorticoid receptor. Western
blot revealed that dexamethasone treatment of the mutant GR
K426A-expressing cells did not lead to protein down-regulation when
compared with untreated controls at 12 and 24 h. In contrast, wild
type GR-expressing cells treated with dexamethasone were observed
undergoing ligand-dependent down-regulation at 12 and 24 h when compared with vehicle-treated controls. With extended hormone treatment we did observe down-regulation of wild type and
mutant GR probably reflecting the transcriptional component of ligand
down-regulation previously studied (13-16). As expected in these
transient transfection experiments, we also observed a
time-dependent decrease in protein expression from both
transfected plasmids and the growth of non-transfected cells during the
extended experimental protocol.
The transcriptional activity of the K426A mutant GR also displayed
enhanced responsiveness compared with wild type GR in studies using the
hormone-responsive GRE2-CAT reporter. In contrast to wild
type GR, the K426A mutant did not display enhanced transcriptional activity in the presence of the proteasomal inhibitor MG-132. Preliminary studies of the K426A mutant GR have surprisingly indicated that this mutant is still ubiquitinated, but quantitation of the relative amounts occurring in cells has been difficult. These findings
suggest that alternative ubiquitination sites besides K426A may be
present on GR or that mutagenesis of Lys-426 could affect overall GR
ubiquitination levels. Unidentified alternative ubiquitination sites
also may have a role in ligand-independent turnover of GR.
Nevertheless, our data clearly show that Lys-426 is an important amino
acid involved in GR hormone responsiveness, and its mutagenesis leads
to a more potent GR. Lys-426 is also a critical amino acid for the
proteasomal inhibitor enhancement of GR transcriptional activation, and
mutagenesis of this site successfully blocked the transcriptional
enhancement of GR by proteasomal inhibition.
The identified PEST region of the glucocorticoid receptor also contains
possible phosphorylation sites including the identified site Ser-412,
first identified as peptide 23. Interestingly, Ser-412 was identified
as a site of ligand-dependent phosphorylation (67). Phosphorylation status has also been shown to affect the half-life of
GR in studies using a phospho-deficient GR, and a mutant containing all
eight phosphorylation sites mutated to alanine did not undergo ligand-dependent down-regulation (21). Other members of the steroid receptor superfamily have been shown to be phosphoproteins (68). Recently, phosphorylation of a specific serine residue has been
implicated in the degradation of the progesterone receptor, and
proteasome inhibition blocked degradation by
ligand-dependent and -independent mechanisms (60). The
kinases involved in phosphorylation of ligand-bound GR, and the E2 and
E3 ligases that recognize and ubiquitinate GR, are unknown. The
proteins UBC9, E6AP, and RSP5 have been shown to interact with GR and
have been shown to act as co-activators of GR, but their ability to
ubiquitinate GR has not been demonstrated (65). The role of the Ser-412
phosphorylation site in ligand-dependent down-regulation,
however, requires further investigation.
The clinical significance of glucocorticoid receptor protein
down-regulation is evident when considering that without
down-regulation there exists the potential for overstimulation when
circulating levels of hormone are high. Conversely, the effects of long
term glucocorticoid treatment could lead to GR down-regulation leaving cells unresponsive to hormone treatment (11). We provide evidence for
the first time that the proteasome is involved in
ligand-dependent GR down-regulation and that proteasomal
inhibition enhances GR transcriptional activity. In the presence of
proteasomal inhibitors, we visualized higher molecular weight forms of
GR representing ubiquitinated GR. We identified a PEST degradation
element (residues 407-426) in GR, and mutagenesis of lysine 426 effectively blocked ligand-dependent down-regulation. This
mutant also demonstrated elevated transcriptional activity. Together
these results suggest a mechanism for termination of
steroid-dependent GR signaling by protein degradation
through the proteasome pathway.
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed. Tel.: 919-541-1564;
Fax: 919-541-1367; E-mail: cidlowski@niehs.nih.gov.
