(Received for publication, December 10, 1996, and in revised form, January 29, 1997)
From the Department of Biosciences, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden
The glucocorticoid receptor (GR) is a
ligand-activated transcription factor. In this study, we used the yeast
two-hybrid system to isolate cDNAs encoding proteins that interact
with the human GR ligand-binding domain (LBD) in a
ligand-dependent manner. One isolated cDNA from a HeLa
cell library encoded the COOH-terminal portion of the -isoform of
the 14-3-3 protein (residues 187-246). Glucocorticoid agonists,
triamcinolone acetonide and dexamethasone, induced the GR LBD/14-3-3
protein fragment interaction, but an antagonist, RU486, did not.
Glutathione S-transferase pull-down experiments in
vitro showed that full-length 14-3-3
protein also interacted
with the activated GR. Transient transfection studies using COS-7 cells
revealed a stimulatory effect of 14-3-3
protein on transcriptional
activation by the GR. The 14-3-3 family members have recently been
found to associate with a number of important signaling proteins, such
as protein kinase C and Raf-1, as functional modulators. Our findings
suggest a novel regulatory role of 14-3-3
protein in GR-mediated
signaling pathways and also point to a mechanism whereby GR may
cross-talk with other signal transduction systems.
The human glucocorticoid receptor
(hGR),1 a member of the nuclear receptor
superfamily, mediates the effects of glucocorticoids by regulating the
transcription of target genes (for review, see Ref. 1). The GR contains
three major structural domains. The 1 transactivation
domain in the NH2-terminal portion is important for gene
activation by DNA-bound GR and interacts with components of the
transcriptional machinery (2). The DNA-binding domain (DBD), in the
central part of the receptor protein, associates with specific
glucocorticoid response elements (GREs) within the target genes
(reviewed in Ref. 3). The COOH-terminal ligand-binding domain (LBD)
contains overlapping functional domains responsible for ligand binding
(4), nuclear translocation (5), dimerization (6), transactivation
(e.g. the
2 domain) (7), and binding of
90-kDa heat-shock protein (HSP90) (8). The unliganded GR in the
cytoplasm forms a complex with HSP chaperones to keep the GR in an
inactive, yet ligand-activable state. Upon ligand binding, HSP90
dissociates from the activated GR, which can enter the nucleus and act
as a transcription factor (see Refs. 1 and 9 and references
therein).
Using advanced screening methods for the protein-protein interaction, recent studies have shown that the transcriptional activity of nuclear hormone receptors can be modulated by the interaction with other proteins, such as coactivators or corepressors of transcription and cointegrators of diverse signal transduction pathways (for reviews, see Refs. 10 and 11).
In our yeast two-hybrid screening, the COOH-terminal portion of the
human -isoform of the 14-3-3 protein family was found to interact
with the hGR-LBD in a ligand-dependent manner. The interaction between full-length 14-3-3
protein and the activated GR
was also confirmed in in vitro experiments. Moreover, the
expression of 14-3-3
protein in mammalian cells showed a stimulatory
effect on transcriptional activation by GR. 14-3-3 proteins have
recently been the subjects of considerable attention, since they can
interact with a wide variety of signaling proteins, including those in the Ca2+ signaling and mitogen-activated protein kinase
cascades (for review, see Ref. 12). Our observations indicate that
14-3-3
protein may also play a role in GR-mediated signaling
pathways.
A LexA-based yeast two-hybrid screening was performed according to protocols from the Brent Laboratory, Massachusetts General Hospital, Boston, MA (13). The following plasmids were used: pEG202 (HIS3+) expressing LexA-fused baits, pRFHM1 (HIS3+) expressing a LexA-fused bicoid protein, pSH18-34 (URA+) expressing a lacZ reporter gene with LexA-binding sites, and pJG4-5 (TRP1+) expressing B42 transactivation domain fused to HeLa cell cDNA library-encoded proteins. The LexA-fused baits were expressed from plasmids, pEG202/GR LBD (residues 485-777) and pEG202/GR DBD (418-503).
Yeast strain EGY188 (MAT, his3,
trp1, ura3, 2LexAop-LEU2) was
transformed with pSH18-34, pEG202/GR LBD, and a HeLa cell cDNA library cloned in pJG4-5, and transformants were selected on
galactose-Ura
His
Trp
Leu
plates containing 50 µM triamcinolone acetonide (TA).
LEU2+ colonies were further tested for LacZ activity on
Ura
His
Trp
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside plates
containing 0.5 µM TA. The library plasmids from LEU2+ and
LacZ+ colonies were recovered, and sequences of nonidentical clones
were determined.
To verify the specificity, the empty pJG4-5 plasmid or isolated pJG4-5
cDNA plasmid was introduced either into the original yeast strain
expressing the LexA-GR LBD bait or strains expressing LexA-fused GR DBD
or bicoid protein. The LEU2+ and LacZ+ activities of these strains were
tested on plates and by -galactosidase assays (14) in the absence or
presence of various steroids.
A cDNA
encoding full-length 14-3-3 protein was isolated from human liver
cDNA library (Clontech, Palo Alto, CA), using specific polymerase
chain reaction primers, and inserted into the
EcoRI/XhoI sites of the pBKCMV expression vector
(Stratagene, La Jolla, CA).
[35S]Methionine-labeled
full-length hGR and 14-3-3 proteins were translated in
vitro using an in vitro translation kit (Promega, Madison, WI) at 30 °C for 90 min.
The EcoRI/XhoI fragment from pBKCMV-14-3-3 was
subcloned into the glutathione S-transferase (GST) fusion
vector pGEX-4T-1 (Pharmacia Biotech Inc., Uppsala, Sweden). The
original and modified pGEX vectors were transformed into
Escherichia coli, strain BL21(DE3)pLysS. The expression
of GST and GST-fused proteins was induced by adding isopropyl
1-thio-
-D-galactopyranoside to a final concentration of
0.1 mM into the bacterial cultures. The induced proteins
were purified from the bacterial extracts using glutathione-agarose beads (Sigma) by standard procedures. GST-fused hGR
1 domain was a gift from Dr. Jacqueline Ford (Karolinska
Institute, Sweden).2
Purified GST and GST fusion proteins were bound to glutathione beads at an approximate concentration of 1 mg of protein/ml of beads.
Immediately after the in vitro translation, 10 µl of the
35S-labeled GR translation mix was diluted either into 400 µl of ice-cold GST pull-down buffer (20 mM Hepes-KOH, pH
7.9, 10% (v/v) glycerol, 100 mM KCl, 5 mM
MgCl2, 0.2 mM EDTA, 0.01% (v/v) Nonidet P-40, 1 mg/ml bovine serum albumin (BSA), 1 mM dithiothreitol,
0.2 mM phenylmethylsulfonyl fluoride) containing 0.2 µM TA, 1 µM dexamethasone (Dex), or 1 µM RU486, and incubated at 4 °C for 2 h. To
stabilize the GR·HSP90 complex in the translation mixture, the buffer
containing 20 mM sodium molybdate (15) was also used. 200 µl of each sample was then mixed either with 25 µl of GST- or
GST/14-3-3-glutathione beads at 4 °C overnight. The beads were
recovered by centrifugation. After washing the beads, bound proteins
were eluted by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) sample
buffer, analyzed on SDS-PAGE, and visualized by autoradiography.
In experiments using in vitro translated 14-3-3 protein,
25 µl of the 35S-labeled 14-3-3
protein translation
mix was diluted either into 600 µl of the GST pull-down buffer or the
same buffer without Nonidet P-40 and containing 0.1 mg/ml BSA. 200 µl
of the sample was then mixed either with 25 µl of GST-,
GST/14-3-3
-, or GST/GR
1 domain-glutathione beads at
4 °C overnight.
The essential
procedures for cell culture and transfections of monkey kidney COS-7
cells were described previously (16). Cells were transiently
transfected with 5 µg of p19LUC-TK reporter construct containing two
GREs upstream of the reporter gene, 0.2 µg of pCMV4-hGR expression
vector, and various equimolar amounts of pBKCMV or pBKCMV-14-3-3
expression vectors. The total amount of DNA was kept constant using
plasmid pGEM 3Zf(+) (Promega). Following transfections, cells were
maintained in the absence or presence of Dex (10 pM to 1 µM) for 24 h and then harvested for luciferase
assays using enhanced luciferase assay kits (Bio-Orbit, Turku,
Finland).
Using a LexA-based yeast two-hybrid screening, we isolated
cDNAs from a HeLa cell library encoding proteins that interact with
the hGR LBD in a ligand-dependent manner. Sequence analysis revealed that one of the clones encoded the COOH-terminal portion of
human 14-3-3 protein (residues 187-246) (17).
We next verified the interaction of the COOH-terminal 14-3-3 protein
fragment with the ligand-activated hGR LBD. The plasmid expressing the
truncated 14-3-3
protein fused to the B42 transactivation domain or
the control plasmid expressing only the transactivation domain was
re-transformed either into the original yeast strain expressing
LexA-fused GR LBD or other strains expressing LexA-fused GR DBD or the
bicoid protein. The 14-3-3
protein fragment interacted specifically
with GR LBD, since only the strain coexpressing LexA-GR LBD and the
14-3-3
protein fragment grew on LEU
plates (not
shown). Furthermore, the interaction was ligand-dependent, since the cell growth was only observed on plates containing a GR
ligand, TA (not shown). The interaction was studied in more detail by
determining the levels of the lacZ reporter expression in
extracts prepared from the yeast strains using
-galactosidase assays. The strain coexpressing the 14-3-3
protein fragment and LexA-fused GR LBD showed a strong
-galactosidase expression in the
presence of TA (Fig. 1A). In accordance with
the results on LEU
plates, the 14-3-3
protein fragment
did not interact with GR DBD or bicoid protein (Fig. 1A). We
also tested the 14-3-3
protein fragment/GR LBD interaction in the
presence of various steroids. The results showed that the two GR
agonists TA and Dex induced the protein-protein interaction, but the
antagonist RU486 did not (Fig. 1B).
We next isolated a full-length cDNA of human 14-3-3 protein and
constructed a plasmid for expression of a full-length 14-3-3
protein. However, the expression of this protein in the EGY188 yeast
strain resulted in a strong toxic effect on cell growth, and thus we
could not examine the GR LBD/full-length 14-3-3
protein interaction
in the yeast two-hybrid system. Similar toxic effects were also
observed in yeast strains overexpressing the yeast 14-3-3 gene
(18).
To determine whether the interaction between 14-3-3 protein and GR
LBD could also occur in vitro, as well as in vivo
in yeast cells, we performed GST pull-down experiments. In these
experiments, full-length proteins of both 14-3-3
and GR were used.
It is known that the in vitro translated GR forms a complex
with HSP90 from the reticulocyte lysate and that this GR·HSP90
complex can be stabilized by molybdate (15). In vitro
translated and molybdate-treated GR showed no significant binding to
GST protein or GST-fused 14-3-3
protein (Fig.
2A, lanes 2 and 3). On the other
hand, the GR treated with the GR agonists TA and Dex preferentially
bound to GST-fused 14-3-3
protein (Fig. 2A, lanes 5 and
7), compared with GST protein alone (Fig. 2A, lanes
4 and 6). In addition, treatment with the antagonist,
RU486, resulted in a lower level of receptor interaction with the
14-3-3
protein (Fig. 2A, lane 9). Since molybdate and RU486 are known to stabilize the GR·HSP90 complex (15, 19), these
results suggest that prior dissociation of the GR from HSP90 is
necessary for the interaction with 14-3-3
protein in
vitro.
14-3-3 protein isoforms have a molecular mass of around 30 kDa and can
form homo- and heterodimers (20). Crystallization of the proteins
revealed that the dimeric molecule has a large negatively charged
groove (21, 22). This groove is likely to represent the binding surface
of 14-3-3 proteins with their target proteins, and consistent with our
findings, it is largely constructed from the COOH-terminal part of
14-3-3 proteins. In our experiments, in vitro translated
14-3-3 protein could bind to GST-fused 14-3-3
protein (Fig.
2B, lane 3), indicating that our expressed proteins dimerize
and that they could interact with the GR as dimers.
Recently, 14-3-3 protein-binding motifs in two target proteins, Raf-1
and the platelet adhesion receptor, have been identified as
(RSXSXP) and
(XXXSXXSXXXSXXSX),
respectively (23, 24). Furthermore, the (RSXSXP)
motif has been found in many other known 14-3-3 protein-binding
molecules (23), but some evidence suggests that there are alternative
protein contacts with 14-3-3 proteins in the case of A20, an inhibitor
of tumor necrosis factor-induced apoptosis (25). This is also true for
the GR LBD protein, which does not contain either motif but interacts
strongly with the 14-3-3 protein fragment in the yeast two-hybrid
system. However, the (RSXSXP) motif is found in
the
1 region of the GR NH2-terminal domain
(23). To examine whether the 14-3-3
protein could interact with the
hGR
1 domain, we used GST-fused GR
1
domain. In vitro translated 14-3-3
protein did not bind
to GST-fused GR
1 domain under normal stringency
conditions (Fig. 2B, lane 4), but a weak interaction was
seen at lower stringency (Fig. 2B, lane 6). Since our
results showed that the interaction between 14-3-3
protein and the
GR
1 domain is relatively weak, we suggest that the
interaction we observe with the intact GR in vitro is mainly
via the GR LBD.
To determine whether the interaction between the ligand-activated GR
and 14-3-3 protein had any effect on transcriptional activation by
the GR, we performed transient transfections of COS-7 cells. In
cotransfection experiments, 14-3-3
protein enhanced the response to
Dex in a dose-dependent manner. At the highest levels of
14-3-3
tested, Dex-induced transactivation was enhanced by 7-fold
(Fig. 3A, left panel). Although the levels of
transactivation were also increased in the absence of Dex (Fig.
3A, left panel), this was largely dependent on expression of
the GR, because no significant increase was seen in control cells
transfected with the empty expression vector (Fig. 3A, right
panel). It is possible that small amounts of hormone might have
been present in media components or that GR overexpression resulted in
a small proportion of GR that was active in the absence of ligand. Even
if these effects are disregarded, Dex-induced transactivation was still increased from 16-fold in the absence of 14-3-3
to 28-fold in its
presence (35 µmol; Fig. 3A, left panel). To determine
whether 14-3-3
protein affected steroid sensitivity, a dose-response curve was performed (Fig. 3B). In cells cotransfected with
the 14-3-3
expression vector, the amount of Dex needed to induce half maximal activity was marginally reduced. This indicates that 14-3-3
protein might facilitate receptor activation by hormone in
addition to any effects at other levels, such as transactivation capacity.
Highly conserved 14-3-3 protein family members have been identified in mammalian cells (at least seven isoforms) and nonmammalian eukaryotic cells. These proteins have been found to associate with a number of key signaling proteins and cell cycle regulators (see Ref. 12 and references therein and Refs. 24 and 26), such as protein kinase C, Raf-1, phosphatidylinositol 3-kinase, polyomavirus middle tumor antigen, Bcr and Bcr-Abl, platelet adhesion receptor, and Cdc25 phosphatase. Although the functional role of 14-3-3 proteins in these interactions is still poorly defined, 14-3-3 proteins apparently modulate the functions of protein kinase C and Raf-1. Recently, a model for the Raf-1 activation by 14-3-3 proteins has been proposed, suggesting that 14-3-3 protein dimers mediate Raf-1 oligomerization and that this is an essential step in the activation of Raf-1 (27).
It has been shown that 14-3-3 protein dimers can serve as adaptors, bringing together different target proteins (25, 28). 14-3-3 proteins are also known to have chaperone functions in mitochondrial protein import (29) and in solubilizing A20 (25), and they act as a solubility cofactor for keratins in a cell cycle-dependent manner (30). Moreover, nonmammalian 14-3-3 protein family members have been shown to associate with transcriptional regulatory elements (31) and to be required for DNA checkpoints during the cell cycle (32).
Considering the characteristic structure and known features of 14-3-3 proteins, several possible mechanisms could account for the role of
14-3-3 in stimulating the transactivation capacity of the GR. An
attractive model would envisage a role as a chaperone that accepts the
monomeric GR released by the HSP90 complex and facilitates its
dimerization and/or translocation to the nucleus. This would be
consistent with the recent proposal that 14-3-3 proteins mediate
dimerization of the Raf-1 protein (27) and with a previous report of a
6 S complex that potentiates DNA binding by the GR (33). Other models,
including roles as a transcriptional coactivator or as a mediator of
cross-talk mechanisms with other signal transduction pathways, are also
possible.
We thank Drs. Roger Brent and Jenö
Gyuris, Massachusetts General Hospital, for providing the plasmids,
yeast strains, and HeLa cell cDNA library. We also thank Dr.
Jacqueline Ford, Karolinska Institute, for providing GST-fused hGR
1 domain.