From the
Pediatric and Reproductive Endocrinology
Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, the
¶Human Retrovirus Section, Center for Cancer
Research, NCI-Frederick, National Institutes of Health, Frederick, Maryland
21702, and the ||Department of Pathology and
Program in Molecular Biology, University of Colorado Health Sciences Center,
Denver, Colorado 80262
Received for publication, March 19, 2003 , and in revised form, April 25, 2003.
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ABSTRACT |
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INTRODUCTION |
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After entering the nucleus, GR binds as a homodimer to specific DNA
enhancer elements, the glucocorticoid response element (GREs) in the promoter
regions of glucocorticoid target genes
(2). Promoter-bound GR
,
via its two transactivational domains, attracts histone acetyltranferase
co-activators and chromatin remodeling complexes, which help transmit signals
from the activated ligand-bound GR
to the transcription initiation
complex (7). After dissociating
from DNA, GR
is exported into the cytoplasm, becoming again fully
competent for ligand binding and signal transmission
(8).
Several mechanisms regulate the nuclear export of GR. The
CRM1/exportin and the classic nuclear export signal (NES)mediated nuclear
export machineries were postulated to be involved in GR
nuclear export,
based on evidence that leptomycin B, an inhibitor of these systems, abrogated
the rapid nuclear to cytoplasmic translocation and cytoplasmic retention of
GR
; however, no classic NES(s) are evident in the GR
molecule
(6,
9). The
calcium-calreticulin-mediated, classic NES-independent nuclear export system,
on the other hand, was also reported to be involved in the nuclear export and
cytoplasmic retention of GR
(10,
11). Reassembly of the
GR
in the heterocomplex with hsps may not be sufficient to relocate
GR
in the cytoplasm, since such complexes are also observed in the
nucleus both before and after withdrawal of the ligand
(8). All three main domains of
GR
, i.e. the amino-terminal, DNA-binding (DBD), and
ligand-binding (LBD) domains, seem to be involved in nuclear export of this
molecule. A serine residue at position 226 of GR
located in the
amino-terminal domain is necessary for phosphorylation by the c-Jun
NH2-terminal kinase to facilitate the nuclear export of the
GR
, while a 67-amino acid region in the DBD is sufficient to support
calreticulin-mediated nuclear export
(9,
12). In addition, we
previously reported that removal of the LBD from GR
resulted in
constitutive localization of this peptide in the nucleus, indicating that the
LBD also contributes to nuclear to cytoplasmic translocation of GR
(13).
14-3-3 family proteins constitute a highly conserved family present in high abundance in all eukaryotic cells. They consist of nine isotypes from at least 7 distinct genes in vertebrates and regulate important biologic activities by directly binding to and altering the subcellular localization and/or stability of key molecules in several signaling cascades (1416). For example, 14-3-3 proteins regulate the apoptosis pathway by binding BAD and affect the intracellular signaling of several growth factors, including insulin, by interacting with important molecules of their cascades, such as Raf-1, insulin receptor substrate 1 (IRS1), and the forkhead transcription factors (1721). 14-3-3 proteins also influence other signaling events through physical interaction with members of the protein kinase C family proteins Cbl and polyoma middle-T antigen (15). In addition, 14-3-3 proteins play a critical role in the progression/arrest of the cell cycle by binding to Cdc25C, Wee1, Cyclin B1, and possibly Chk1 (15, 16). Binding of 14-3-3 to Cdc25C segregates the latter into the cytoplasm and eliminates its phosphatase activity from the nucleus, thus inhibiting cells from progressing through the G2/M check-point (2224).
14-3-3 proteins bind the phosphorylated serine or threonine residues of
their partner proteins located within a specific amino acid sequence,
RSXpSXP, identified as a "high affinity 14-3-3-binding
motif" (25). They
contain nine -helical structures and form a homo- or heterodimer
through their NH2-terminal portions
(2527).
Their central third to fifth
-helices create a binding pocket for a
phosphorylated serine/threonine residue, and the C-terminal seventh to ninth
helices determine the specificity to target peptide motifs
(25,
26). 14-3-3 proteins contain
one classic NES in their ninth helix, which helps localize 14-3-3/partner
protein complexes in the cytoplasm
(26,
28).
Recent research indicated that GR formed complexes with 14-3-3
proteins and Raf-1 (29).
Although an early study reported that GR
LBD interacted with
14-3-3
in a ligand-dependent fashion in a yeast two-hybrid assay, a
subsequent report indicated that GR
was associated with 14-3-3
proteins, both in the ligand-free and -bound conditions
(29,
30). To further investigate
the functional contribution of 14-3-3 to the biologic activity of GR
,
we examined the subcellular localization and transactivation properties of
GR
in a cell line used in its wild type 14-3-3
replete and in
its mutant type 14-3-3
-deficient forms
(31). We found that endogenous
14-3-3
helps localize ligand-free GR
in the cytoplasm and
contributes to nuclear export of GR
after withdrawal of ligand. In
addition, endogenous 14-3-3
suppresses ligand-activated
GR
-induced transactivation of a glucocorticoid-responsive promoter.
These results indicate that 14-3-3
functions as a negative regulator of
the glucocorticoid signaling pathway by shifting the subcellular circulation
of this receptor toward the cytoplasm.
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MATERIALS AND METHODS |
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pLexA-GRLBD and -GR
LBD, which express the LexA DBD fusions of
the human GR
LBD or GR
LBD, were constructed by inserting the
corresponding cDNA fragments of the human GR
LBD or GR
LBD into
pLexA (Clontech) in an in-frame fashion, respectively.
pGAD424-14-3-3
-(1270) and -(106270), and
pB42AD-14-3-3
-(1244), -(141244), -(190244),
-(210244), -(110210), and -(1110), which respectively
express the GAL4 or LexA activation domain (AD) fusions of the indicated
14-3--3 fragments, were constructed by inserting cDNA fragments of the
indicated regions of 14-3-3
or 14-3-3
into pGAD424 (Clontech) or
pB42AD (Clontech), respectively. p8OP-LacZ was purchased from Clontech.
Cell Cultures and TransfectionsHuman colon cancer-derived
HCT116 wild type (WT) and 14-3-3 knock-out (KO) cells were kindly
provided by Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD)
(31). These cells are
defective in functional GR
(data not shown). They were maintained in
McCoy's 5A medium supplemented with 10% fetal bovine serum, 100 units/ml of
penicillin, and 1 µg/ml of streptomycin. They were transfected using
LipofectinTM with 1 µg/well of pF25-hGR
and/or 0.3 µg/well
of 14-3-3
-expressing plasmids for the study of GR
subcellular
localization, as described previously
(32). For reporter assays, 0.5
µg/well of pRShGR
and 0.3 µg/well of 14-3-3
-expressing
plasmids together with 1.0 µg/well of pMMTV-Luc and 0.3 µg/well of
pSV40-
-Gal were used.
Detection of Subcellular Localization of GFP-fused GR
and 14-3-3
Cells were plated on 25-mm dishes
and were transfected as described above. 24 h after transfection, the medium
was replaced with McCoy's 5A medium containing 10% charcoal/dextran-treated
fetal bovine serum with antibiotics. 48 h after transfection, the cells were
analyzed with an inverted fluorescence microscope (Leica DM IRB, Wetzlar,
Germany) as described previously
(13,
33). 12-Bit black-and-white
images were captured using a digital CCD camera (Hamamatsu Photonics K.K.,
Hamamatsu, Japan). Image analysis and presentation was performed using the
Openlab software (Improvision, Boston, MA). To examine the subcellular
distribution of GFP-GR
, numbers of cells exhibiting five different
distribution patterns from complete cytoplasmic distribution (C) to nuclear
localization (N) were counted and percentages of each fraction to the total
transfected cell number were calculated. For the experiments examining the
nuclear export of GFPGR
, cells were exposed to
106 M dexamethasone 48 h after the
transfection. After culturing for 1 h, the cells were washed with PBS two
times and placed in McCoy's 5A medium containing 10% charcoal/dextran-treated
fetal bovine serum and antibiotics. Eight hours after replacement of the
medium, numbers of cells that contained GFP-GR
mainly in the cytoplasm
(subgroups corresponding to "C" and "N < C") were
counted, and the nuclear export of GR
was expressed as percentages of
these numbers to the total transfected cell number.
Reporter AssaysCells were plated on six-well plates and
transfected as described above. 24 h after transfection, the cells were
exposed to the indicated amounts of dexamethasone. 48 h after transfection,
the cell lysis buffer (Promega) was placed in each well, and the resulting
cell lysates were harvested. Luciferase and -galactosidase activities
were determined as described previously
(32). All measurements of
reporter gene activity were conducted in triplicate and all experiments were
repeated at least three times.
Yeast Two-hybrid AssayYeast strain EGY48 (Clontech) was
transformed with the lacZ reporter plasmid p8OP-LacZ,
pLexA-GRLBD or -GR
LBD, and the indicated pGAD424-14-3-3
-
or pB42AD-14-3-3
-related plasmids
(34). The cells were grown in
a selective medium to the early stationary phase, permeabilized with
CHCl3-SDS treatment, and
-galactosidase activity was measured
in the cell suspension using GalactolightTM PLUS (Tropix, Bedford, MA),
as described previously
(33).
Statistical AnalysesStatistical analysis was carried out by analysis of variance, followed by Student t test with Bonferroni correction for multiple comparisons.
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RESULTS |
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Endogenous 14-3-3 Suppresses the Transcriptional
Activity of GR
We then examined the transactivation
activity of GR
stimulated with increasing concentrations of
dexamethasone in WT and KO cells (Fig.
2). GR
stimulated the MMTV promoter in response to
106 M dexamethasone by about 80- and
300-fold in WT cells and KO cells, respectively. The dexamethasone titration
curve was shifted upward in the latter cells. Transfection of wild type
14-3-3
partially reversed this change in KO cells. The EC50
(mean ± S.E.: in mM) was 4.01 ± 0.33 and 6.46
± 1.04 in WT and KO cells, respectively (p > 0.10), whereas
the Bmax (mean ± S.E.: x
102 relative luminescence unit) was 9.11 ±
0.89 and 28.01 ± 1.19, respectively (p < 0.001).
Transfection of 14-3-3
partially reversed this change in KO cells.
EC50 values were similar in transfected and KO cells (p
> 0.30), while the Bmax in the transfected cells was
significantly lower than in KO cells (p < 0.01). These results
indicated that endogenous 14-3-3
functions as a negative regulator of
GR
-induced transactivation.
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The C-terminal Half of 14-3-3 Interacts with the
GR
LBD in a Yeast Two-hybrid AssayWe next examined
the interaction of GR
and 14-3-3
in a yeast two-hybrid assay
(Fig. 3A).
Administration of dexamethasone stimulated the LexA-DBDGR
LBD-induced,
but not LexA-GR
LBD-induced,
-galactosidase activity by about
3-fold in the EGY48 yeast strain. Co-expression of GAL4-AD fusions of the
full-length or the COOH-terminal half of 14-3-3
enhanced
-galactosidase activity induced by LexA-DBD-GR
LBD in a partially
dexamethasone-dependent fashion, whereas it did not affect the activity of
LexA-DBD-GR
LBD. These results indicated that GR
LBD interacted
with the COOH-terminal half of 14-3-3
in a partially ligand-dependent
fashion, while GR
LBD did not. No increase of
-galactosidase
activity was observed in the transformed yeast cells, when they were cultured
in galactose-deficient medium that did not support the expression of bait
proteins (data not shown). This result indicated that expression of
GAL4-AD-fused 14-3-3
s did not influence basal promoter activity. We
obtained similar results using a plasmid expressing the LexA-DBD fusions of
the full-length GR
(data not shown).
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Our results showed that 14-3-3 interacts with GR
in the
absence of dexamethasone as well as in its presence. In our system,
14-3-3
interacted with the GR
LBD in the absence of dexamethasone
and the interaction was enhanced in its presence
(Fig. 3B). 14-3-3
fragments, which contained the region from 210 to 240 that corresponds to the
ninth
-helix, supported the binding to GR
LBD.
Destruction of 14-3-3 NES Diminishes the Ability of
This Protein to Promote Cytoplasmic Retention/Nuclear Export of
GR
and Suppression of GR
Transactivation14-3-3 proteins may help translocate their
partner proteins into the cytoplasm via their classic NES located in their
ninth
-helix (16,
26). Thus, we examined the
contribution of 14-3-3
NES on the cytoplasmic retention of GR
,
by constructing a plasmid expressing a 14-3-3
mutant (14-3-3
NES
Mut), in which the NES was destroyed by clustered mutations, as a fusion with
EGFP (26). The EGFP-fused wild
type 14-3-3
was mainly located in the cytoplasm, while a small fraction
of this fusion protein was also observed in the nucleus
(Fig. 4A). The
EGFP-14-3-3
NES Mut, on the other hand, was distributed more in the
nucleus, indicating that the introduced mutations inactivated the NES.
Co-expression of 14-3-3
NES Mut did not change the distribution of
unliganded GFP-GR
, in contrast to the wild type 14-3-3
(Fig. 4B). We next
examined the effect of this mutant on the nuclear export of GFP-GR
after withdrawal of dexamethasone. Supplementation of the wild type
14-3-3
brought GFP-GR
into the cytoplasm in 23% of KO cells,
while expression of 14-3-3
NES Mut did not change the nuclear export of
this receptor (Fig.
4C). In a functional reporter assay, 14-3-3
NES Mut
did not suppress GR
-induced transactivation of the MMTV promoter in a
dexamethasone titration curve (Fig.
4D). These results suggest that 14-3-3
retained
the ligand-free GFP-GR
in the cytoplasm and helped it through its NES
to redistribute in the cytoplasm after the withdrawal of dexamethasone. Since
the destruction of NES also abolished the suppressive effect of 14-3-3
on GR
transactivation, it is possible that 14-3-3
suppressed
GR
-induced transcriptional activity by segregating GR
away from
the nucleus.
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The Phosphopeptide Binding Activity of 14-3-3 May Not
Be Necessary for Cytoplasmic Retention of GFP-GR
and
Suppression of GR
-dependent TransactivationSince a
previous publication indicated that 14-3-3 forms a complex with GR
together with its partner protein Raf-1, we employed a 14-3-3
E182K
mutant, to examine whether 14-3-3 partner proteins might contribute to the
action of 14-3-3
on the activity of GR
(29). This mutation
corresponds to the replacement of a glutamic acid at 180 by a lysine in
Drosophila 14-3-3
that inactivates the binding activity of this
protein to Raf-1 because of destruction of the phosphopeptide-binding pocket
(26). 14-3-3
E182K was
distributed mainly in the cytoplasm similarly to the wild type 14-3-3
(Fig. 5A).
14-3-3
E182K preserved property of the wild type 14-3-3
on the
subcellular distribution and transactivation activity of GR
(Fig. 5, B and
C), suggesting that association of 14-3-3
to
partner proteins, such as Raf-1, may not be necessary for its effect on these
GR
activities.
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DISCUSSION |
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We also demonstrated that endogenous 14-3-3 functioned as a negative
regulator of GR
-induced transactivation. This activity correlated with
the ability of 14-3-3
to localize unliganded GFP-GR
in the
cytoplasm, in the experiment employing the 14-3-3
mutants NES Mut and
E182K. Therefore, it is likely that 14-3-3
suppresses GR
-induced
transactivation by shifting intracellular circulation of GR
toward the
cytoplasm, possibly by reducing the chance of ligand-bound GR
to
interact with GREs, steroid hormone receptor co-activators, and other related
specific or general transcription factors in the nucleus. For instance,
GR
dynamically interacts with GREs in living cells, binding to and
dissociating from them in the order of seconds
(35,
36). In this situation, the
drive created by 14-3-3
, which shifts GR
toward the cytoplasm,
may reduce the probability of GR
binding to GREs and, hence, may reduce
its transcriptional activity. A similar effect was also observed in a recent
report, which showed that c-Jun NH2-terminal kinase suppressed
GR
-induced transactivation by phosphorylating serine 216 and
facilitating its nuclear export
(9).
A previous report demonstrated that overexpressed 14-3-3 enhanced
GR
-induced transactivation of a synthetic GRE-containing heterologous
promoter in African monkey kidney-derived COS7 cells, while we showed that
endogenous 14-3-3
suppressed GR
transactivation in HCT116 cells
(30). This discrepancy may
result from differences in the experimental systems employed, including the
type of cell lines, the different 14-3-3 isoforms and overexpression
versus normal expression versus knock-out of 14-3-3. Indeed,
overexpression of a protein may sometimes cause artificial effects
(37). We examined 14-3-3
"loss of function" by employing the KO cells and showed that the
physiologic activity of 14-3-3
is that of a negative regulator of the
glucocorticoid signaling pathway.
We demonstrated that the GR LBD interacts with 14-3-3
as well
as
in a partially ligand-dependent fashion. A previous report indicated
that this domain of GR
interacted with 14-3-3
in an absolutely
ligand-dependent fashion in the same LexA yeast two-hybrid system
(30). Differences in the
GR
fragments employed or yeast strains used in the assay might have led
to the different results. Since a recent report also demonstrated partial
ligand-dependent interaction between GR
and 14-3-3 in a
semiquantitative coimmunoprecipitation assay, it is likely that GR
and
14-3-3 proteins associate with each other in the absence of ligand. In
contrast to the GR
LBD, the GR
"LBD" did not interact
with 14-3-3
at all. Since GR
is constitutively located in the
nucleus, the inability of GR
to interact with 14-3-3
might, to
some extent, contribute to its constitutive nuclear localization
(13,
3840).
Our results employing 14-3-3E182K suggest that the association of
known partner proteins with 14-3-3
may not be necessary for this
molecule to influence the subcellular localization of GR
and the
suppression of its transactivation. However, an E180K mutation in
Drosophila 14-3-3
, which corresponds to the E182K mutation in
human 14-3-3
, abolishes the interaction of this protein with Raf-1 and
BAD, but preserves that with IRS-1, indicating that the E182K mutation in
14-3-3
might not completely exclude the interaction of this 14-3-3
subtype with all partner proteins
(26,
41). In agreement with the
above-indicated evidence, about half of the 14-3-3-partner proteins use a
different phosphopeptide-binding motif to interact with 14-3-3, suggesting
that a single surface of 14-3-3
in the phosphopeptide-binding pocket,
which the E182K mutation destroys, may not support its association generically
with all partner proteins. Further experiments are required to address this
issue.
In summary, endogenous 14-3-3 functions as a negative regulator of
GR
-induced transactivation, most likely by shifting the subcellular
distribution and circulation of GR
toward the cytoplasm. These results
indicate that change in the intracellular concentration as well as the
subcellular distribution of 14-3-3
may contribute to the altered
sensitivity of tissues to glucocorticoids seen in several physiologic and
pathologic conditions (1).
Since 14-3-3 proteins are involved in a broad array of cellular activities,
such as cell cycle progression, growth, differentiation, and apoptosis, these
activities might indirectly influence the transcriptional activity of
GR
, by changing the availability of 14-3-3 and/or altering partner
proteins associated with 14-3-3. On the other hand, the opposite may be true.
Ligand-bound GR
may influence these cellular processes by segregating
and/or influencing 14-3-3 and partner molecules.
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FOOTNOTES |
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To whom correspondence should be addressed: Pediatric and Reproductive
Endocrinology Branch, NICHD, NIH, Bldg. 10, Rm. 9D42, 10 Center Dr., MSC 1583,
Bethesda, MD 20892-1583. Tel.: 301-496-6417; Fax: 301-480-2024; E-mail:
kinot{at}mail.nih.gov.
1 The abbreviations used are: GR, glucocorticoid receptor; GRE,
glucocorticoid response element; NES, nuclear export signal; DBD, DNA-binding
domain; LBD, ligand-binding domain; GFP, green fluorescence protein; EGFP,
enhanced GFP; MMTV, mouse mammary tumor virus;AD, activation domain; WT, wild
type; KO, knock-out; C, cytoplasmic distribution; N, nuclear localization.
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ACKNOWLEDGMENTS |
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REFERENCES |
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