Regulation of Glucocorticoid Receptor Activity by 143-3-Dependent Intracellular Relocalization of the Corepressor RIP140
Johanna Zilliacus,
Elin Holter,
Hideki Wakui1,
Hiroshi Tazawa,
Eckardt Treuter and
Jan-Åke Gustafsson
Department of Medical Nutrition (J.Z., H.W., H.T., J.-A.G.) and
Department of Biosciences (E.H., E.T., J.-A.G.) Karolinska
Institutet Novum, S-141 86 Huddinge, Sweden
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ABSTRACT
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Proteins belonging to the 143-3 family interact
with various regulatory proteins involved in cellular signaling, cell
cycle regulation, or apoptosis. 143-3 proteins have been suggested to
act by regulating the cytoplasmic/nuclear localization of their target
proteins or by acting as molecular scaffolds or chaperones. We have
previously shown that overexpression of 143-3 enhances the
transcriptional activity of the glucocorticoid receptor (GR), which is
a member of the nuclear receptor family. In this study, we show that
143-3 interacts with the nuclear receptor corepressor RIP140. In
transfection assays, RIP140 antagonizes 143-3- enhanced GR
transactivation. Using colocalization studies we demonstrate that
143-3 can export RIP140 out of the nucleus and, interestingly, can
also change its intranuclear localization. Moreover, we also observed
that 143-3 can bind various other nuclear receptors and cofactors. In
summary, our findings suggest that 143-3-mediated intracellular
relocalization of the GR corepressor RIP140 might be a novel mechanism
to enhance glucocorticoid responsiveness of target genes. They
furthermore indicate a more general role for 143-3 protein by
influencing the nuclear availability of nuclear receptor-associated
cofactors.
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INTRODUCTION
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Proteins belonging to the 143-3 family have recently been shown
to interact with many proteins involved in cellular signaling, such as
Raf-1, protein kinase C, Bcr, MEK kinase, insulin-like growth factor I
(IGF I) receptor, and IRS 1 (1, 2, 3). 143-3 proteins also bind
proteins regulating the cell cycle (Cdk 25 and Wee1) and proteins
involved in apoptosis (A20 and Bad) (1, 4, 5, 6). The functional role of
the interactions has, in many cases, been unclear. In the case of Raf-1
it has been shown that 143-3 is permissive for recruitment of Raf-1
to the cell membrane and for Raf-1 activation (7). Interaction between
143-3 and Cdc25 retains Cdc25 in the cytoplasm, thus suppressing its
ability to induce entry into mitosis (8, 9). Furthermore, 143-3
counteracts the apoptotic activity of BAD by disrupting the
heterodimerization between BAD and Bcl2 (6). These effects could be
explained by 143-3 stabilizing a specific structure of the
heterodimerizing partner. 143-3 is a dimer and can also bind two
different proteins simultaneously and thus function as a scaffold that
connects two protein signals (10). Recently it has been demonstrated
that 143-3 proteins can regulate the subcellular localization of a
number of interacting proteins. Cdc25 has been shown to interact with
143-3 and be retained in the cytoplasm, either by a 143-3-mediated
export from the nucleus due to a putative nuclear export signal present
in 143-3 or by inhibition of nuclear import due to 143-3 binding
next to a nuclear localization sequence on Cdc25 (8, 9, 11, 12, 13).
143-3 has also been shown to retain Forkhead transcription factor
FKHRL1 (14), the yeast nutrient-regulated transcription factors MSN2
and MSN4 (15), protein kinase U-
(16), as well as histone
deacetylase 4 and 5 (17, 18) in the cytoplasm. In contrast, 143-3 can
enhance nuclear localization of telomerase (19) and the homeodomain
Tlx-2 transcription factor (20). Many 143-3 interacting proteins
contain the consensus 143-3 interacting sequence
R(S)X1,2pSX(P) where pS is a phosphoserine
(1). Other 143-3 interacting sequences have also been reported, and
in many cases these include a serine residue (21).
We have recently demonstrated an interaction between the 143-3
protein and the ligand-activated glucocorticoid receptor (GR) (22).
Overexpression of the 143-3 protein enhances the transcriptional
activity of GR in transient transfection experiments (22). GR mediates
the effects of glucocorticoid hormones and is a member of the nuclear
receptor family (23). The nuclear receptors have three major functional
domains, an N-terminal transactivation domain, a central DNA-binding
domain, and a C-terminal ligand-binding domain (LBD) (24). The LBD is,
in addition, involved in transcriptional activation and interaction
with several cofactor proteins (25, 26, 27). In the absence of ligand, GR
is found in a cytoplasmic complex with heat shock protein 90 (hsp90)
and other associated proteins (28). The hsp90 multiprotein complex
stabilizes a structure of GR that is competent of ligand binding. The
ligand-activated receptor regulates transcription either by binding to
glucocorticoid response elements located close to target genes or by
interacting with other transcription factors (23). The nuclear
receptors interact with many transcriptional cofactors that modulate
the activity of receptors, including RIP140, TRAP220, CBP, and members
of the p160/SRC-1 coactivator family (e.g. SRC-1, TIF2,
ACTR) (25, 26, 27). The p160/SRC-1 coactivators and CBP have been
demonstrated to enhance the transcriptional activity of nuclear
receptors, at least partly by modulating chromatin structure by histone
acetylation (29), and TRAP220 has been suggested to function by
connecting the receptor with the transcriptional machinery (26). RIP140
was originally identified as a coactivator protein for estrogen
receptor (ER) that stimulated the transcriptional activity of the
receptor (30), and it has also been shown to stimulate the activity of
androgen receptor (AR) and aryl hydrocarbon receptor (AhR) (31, 32). In
contrast, RIP140 does not act as a coactivator for other nuclear
receptors tested such as peroxisome proliferator-activated receptor
(PPAR), TR2, retinoic acid receptor (RAR), and thyroid hormone receptor
(TR) (33, 34, 35). This led to the hypothesis that RIP140 might function in
regulation of nuclear receptor activity by competition for nuclear
receptor coactivators such as SRC-1 for binding to the receptor (34).
Recently, the interaction between RIP140 and histone deacetylase 1 and
3 was reported, suggesting that RIP140 also may act as corepressor by
negatively modulating chromatin structure and gene regulation (36). In
the case of GR, RIP140 shows a strong inhibition of GR function both in
transactivation and transrepression (37, 38), indicating that it
generally inhibits glucocorticoid-dependent gene regulation.
To further elucidate the function of 143-3 proteins in the regulation
of GR activity, we were interested in determining whether any nuclear
receptor cofactors could directly interact with the 143-3 protein. In
this paper we describe a functional interaction between 143-3 and
RIP140 that could influence the transcriptional activity of GR.
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RESULTS
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Interaction of RIP140 with 143-3
To determine whether the GR interacting protein 143-3 could bind
directly to any GR cofactor proteins, we performed glutathione
S-transferase (GST) pull-down experiments. The results
showed that the in vitro translated corepressor RIP140 did
interact specifically with GST-143-3 (Fig. 1A
, lane 4). The in vitro
translated RIP140 protein also bound specifically to the GST-GR LBD in
the presence of ligand (Fig. 1A
, lane 3), in accordance with the
previously described ligand-dependent interaction between RIP140 and GR
(38). We next analyzed whether the same domains of RIP140 were involved
in the interactions with 143-3 and GR. Analysis of the interaction of
in vitro translated RIP140 domains with GST-143-3 and
GST-GR LBD showed that N- and C-terminal domains of RIP140 bound both
143-3 and GR LBD (Fig. 1B
, lanes 5, 6, 11, and 12), whereas the
middle domain did not interact with either protein (Fig. 1B
, lanes 8
and 9).

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Figure 1. Interaction of RIP140 with GR LBD and 143-3 and
Mapping of the Binding Sites on RIP140: GST Pull-Down Assays
A, In vitro translated 35S-labeled RIP140
was incubated with GST (lane 2), GST-GR LBD (lane 3), or GST-143-3
(lane 4) bound to glutathione agarose beads in PDB with 0.01% Igepal
CA-630. GST-GR LBD bound to glutathione agarose beads was incubated
with 1 µM Dex for 2 h before mixing with translated
protein. The eluted samples from washed beads were analyzed by SDS-PAGE
and visualized by autoradiography. The input (lane 1) represents 15%
of the amount of labeled protein incubated with GST-protein-bound
beads. B, In vitro translated 35S-labeled
RIP140 N (aa 1472) (lanes 1 and 46), RIP140 M (aa
431745) (lanes 2 and 79), and RIP140 C (aa 747-1,158) (lanes 3 and
1012) were incubated with GST (lanes 4, 7, and 10), GST-GR LBD (lanes
5, 8, and 11), or GST-143-3 (lanes 6, 9, and 12) bound to glutathione
agarose beads in PDB with 0.01% Igepal CA-630. GST-GR LBD bound to
glutathione agarose beads was incubated with 1 µM Dex for
2 h before mixing with translated protein. The input (lanes 13)
represents 15% of the amount of labeled protein incubated with
GST-protein-bound beads.
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Role of Protein Phosphorylation for 143-3 Interaction
143-3 has been shown in many cases to interact with
phosphorylated forms of proteins (1), and phosphatase treatment of the
143-3 interacting protein can result in reduced binding (45, 46, 47). To
test whether protein phosphorylation is involved in 143-3 binding to
RIP140 we studied the binding of in vitro translated 143-3
with bacterially expressed GST-RIP140 domains, since proteins expressed
in bacteria are not phosphorylated. The experiment showed that in
vitro translated 143-3 did not bind GST-RIP140 N or C proteins
(Fig. 2A
, lanes 3 and 4). Control
reactions showing interaction between in vitro translated GR
and GST-RIP140 N and C (Fig. 2A
, lanes 7 and 8) proved that there was
no general defect in the bacterially expressed proteins. The role of
RIP140 phosphorylation for 143-3 binding was further studied by
analyzing the interaction of in vitro translated and
alkaline phosphatase-treated RIP140 with GST-143-3. The results
showed that alkaline phosphatase treatment of RIP140 decreased the
interaction with 143-3 (Fig. 2B
, compare lanes 3 and 6).

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Figure 2. Role of Protein Phosphorylation for 143-3
Interaction: GST Pull-Down Assays
A, In vitro translated 35S-labeled 143-3
(lanes 14) and GR (lanes 58) were incubated with GST (lanes 2 and
6), GST-RIP140 N (aa 1281) (lanes 3 and 7), or GST-RIP140 C (aa
747-1,158) (lanes 4 and 8) bound to glutathione agarose beads in PDB
with 0.01% Igepal CA-630. In vitro translated GR was
diluted into PDB and incubated with 1 µM Dex for 2 h
at 4 C before mixing with the GST protein. The eluted samples from
washed beads were analyzed by SDS-PAGE and visualized by
autoradiography. The input (lanes 1 and 5) represents 15% of the
amount of labeled protein incubated with GST-protein-bound beads. B,
In vitro translated 35S-labeled RIP140 was
incubated in the presence or absence of alkaline phosphatase (AP) and
mixed with GST (lanes 2 and 5) or GST-143-3 (lanes 3 and 6) bound to
glutathione agarose beads in PDB with 0.03% Igepal CA-630. The input
(lanes 1 and 4) represents 15% of the amount of labeled protein
incubated with GST-protein-bound beads. The relative binding of
alkaline phosphatase-treated RIP140 to GST-143-3 compared with
untreated, was 0.22 ± 0.14 (mean ± SD, n =
4) as determined by PhosphoImager analysis.
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RIP140 Antagonizes the 143-3-Enhanced GR Transactivation
To analyze the functional role of RIP140 interaction with 143-3,
we performed transient transfection experiments in COS-7 cells. As
expected, 143-3 enhanced the GR- induced transactivation (Fig. 3
). The results of coexpression of RIP140
and 143-3 on GR-induced transactivation showed that
RIP140-antagonized 143-3 enhanced GR transactivation (Fig. 3
). In
cells transfected with 5 ng of RIP140 expression plasmid, no
143-3-enhanced GR transactivation could be detected (Fig. 3
).
RIP140 also reduced the GR transactivation in the absence of
coexpressed 143-3. RIP140 only worked via ligand- activated GR since
no effect of RIP140 on basal transactivation levels in the absence of
dexamethasone (Dex) was seen (Fig. 3
).

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Figure 3. RIP140 Antagonizes the 143-3 Enhanced GR
Transactivation
COS-7 cells were transiently transfected with 100 ng of p19-tk-luc and
5 ng of pCMV-ß gal reporter plasmids and 2.5 ng of pCMV4-hGR
expression plasmid, as well as indicated amounts of pSG5-RIP140 and
pBKCMV-143-3 expression plasmids or the corresponding empty plasmids.
The total amount of each kind of plasmid was kept constant. The cells
were treated with or without 1 µM Dex and harvested for
luciferase and ß-galactosidase assays. The GR-regulated luciferase
reporter gene activity was related to the control ß-galactosidase
reporter gene activity. The results shown represent mean values and
SD obtained from three independent experiments.
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Noncompetitive Interactions between RIP140, 143-3, and GR
The negative effect of RIP140 on GR transactivation has previously
been suggested to be due to RIP140 competing with positively acting
coactivator proteins for binding to GR (34). We have previously
described a ligand-dependent interaction between 143-3 and GR (22).
To analyze whether the mechanism for the antagonizing effect of RIP140
on 143-3-enhanced GR transactivation was due to RIP140 competing with
143-3 for binding to GR, we performed GST pull-down competition
experiments. Unlabeled in vitro translated RIP140 or
bacterially expressed His-RIP140 C were added to binding reaction to
determine the effect on interaction between GST-143-3 and
in vitro translated GR in the presence of ligand. The
results showed that neither of the RIP140 proteins could disrupt the
binding of GR to 143-3 (Fig. 4A
, compare lanes 3 and 5, and 8 and 10, respectively). To determine
whether the positive effect of 143-3 on GR transactivation was due to
143-3 competing with RIP140 for binding to GR, unlabeled in
vitro translated 143-3 was added to the binding reaction for
GST-GR LBD and in vitro translated RIP140 and to the binding
reaction for GST-RIP140 C and in vitro translated GR (Fig. 4B
). In neither case did 143-3 inhibit the interaction between GR and
RIP140 (Fig. 4B
, compare lanes 3 and 5, and 8 and 10,
respectively).

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Figure 4. Noncompetitive Interactions between RIP140,
143-3, and GR: GST Pull-Down Assays
A, RIP140 does not disrupt the interaction between GR and 143-3.
In vitro translated 35S-labeled GR (7.5
µl) was incubated in the presence of 1 µM Dex with 10
µg of GST (lanes 2, 4, 7, and 9) or GST-143-3 (lanes 3, 5, 8, and
10) bound to glutathione agarose beads in the absence or presence of 30
µl of in vitro translated unlabeled RIP140 (lanes
25) or 10 µg of bacterially expressed His-RIP140 C (aa 7471,158)
(lanes 710) in PDB with 0.03% Igepal CA-630. The ratio of in
vitro translated RIP140/GR was 3-fold and the ratio of
His-RIP140/GR was more than 100-fold. GR was diluted into PDB
and incubated with 1 µM Dex for 2 h at 4 C before
mixing with the GST protein. The eluted samples from washed beads were
analyzed by SDS-PAGE and visualized by autoradiography. The input
(lanes 1 and 6) represents 10% of the amount of labeled protein
incubated with GST-protein-bound beads. The relative binding of GR to
GST-143-3 in the presence of added in vitro translated
RIP140 or His-RIP140 C compared with in the absence, was 1.5 ±
0.7 (mean ± SD, n = 3) and 1.2 ± 0.5
(mean ± SD, n = 4), respectively, as determined
by densitometric scanning of films. B, 143-3 does not disrupt the
interaction between RIP140 and GR. Five microliters of in
vitro translated 35S-labeled RIP140 (lanes 15)
and GR (lanes 610) were incubated in the presence of 1
µM Dex with 10 µg of GST (lanes 2, 4, 7, and 9), GST-GR
LBD (lanes 3 and 5), or GST-RIP140 C (aa 7471,158) (lanes 8 and 10)
bound to glutathione agarose beads in the absence or presence of 25
µl of in vitro translated unlabeled 143-3 in PDB with 0.01% Igepal
CA-630. The ratio of in vitro translated 143-3/RIP140
and 143-3/GR was 5-fold. GR was diluted into PDB and incubated
with 1 µM Dex for 2 h at 4 C before mixing with the
GST protein. The input (lanes 1 and 6) represents 15% of the amount of
labeled protein incubated with GST-protein bound beads. C, RIP140,
143-3, and GR can form a tertiary complex. Bacterially expressed
His-RIP140 C was incubated in the presence of 1 µM Dex
with GST (lanes 2 and 4) or GST-143-3 (lanes 3 and 5) bound to
glutathione agarose beads in the absence or presence of in
vitro translated unlabeled GR. GR was diluted into PDB and
incubated with 1 µM Dex for 2 h at 4 C before mixing
with the GST protein. The eluted samples from washed beads were
analyzed by SDS-PAGE and visualized by Western blotting using a His
tag-specific antibody.
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We have demonstrated here that all three proteins, GR, RIP140, and
143-3, can interact pairwise with each other. To determine whether
all three proteins could form a tertiary complex, we used the inability
of bacterially expressed protein to bind to 143-3. The binding of
bacterially expressed His-RIP140 C to GST-143-3 was analyzed in the
presence or absence of in vitro translated GR (Fig. 4C
). In
the absence of GR, His-RIP140 C did not bind GST-143-3 (Fig. 4C
, lane
3). However, in the presence of GR, His-RIP140 C interacted with
GST-143-3 (Fig. 4C
, lane 5), indicating that GR could function as a
bridging protein between RIP140 and 143-3 and that all three proteins
were present in the same complex.
143-3 Regulates the Subcellular Localization of RIP140
143-3 proteins have been shown to regulate the subcellular
localization of some interacting proteins (8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). To determine
whether this mechanism was involved in the case of 143-3 binding to
RIP140 we studied the effect of 143-3 on the subcellular localization
of green fluorescent protein (GFP)-fused RIP140 in transfected COS-7
cells. GFP-RIP140 was detected only in the nucleus, in a punctate
pattern as previously described (Fig. 5A
, panel a) (30). In contrast, in cells cotransfected with an expression
plasmid for 143-3, the majority of GFP-RIP140 was relocalized into
the cytoplasm (Fig. 5A
, panel b). Interestingly, remaining nuclear
GFP-RIP140 had lost its punctate expression pattern (Fig. 5A
, panel b),
suggesting a function for 143-3 in relocalization of RIP140 also
within the nucleus. To study the expression of both 143-3 and RIP140
in the same cell, GFP-fused 143-3 and hemagglutinin (HA)-tagged
RIP140 that could be detected using indirect immunofluorescence were
used. In cells cotransfected with HA-RIP140 and GFP, HA-RIP140 was
present only in the nucleus in a similar punctate pattern to GFP-RIP140
(Fig. 5B
, panel a). As expected, the control GFP protein was detected
both in the cytoplasm and the nucleus (Fig. 5B
, panel b). In cells
coexpressing HA-RIP140 and GFP-143-3, HA-RIP140 was found in the
cytoplasm, confirming the 143-3-induced redistribution observed using
GFP-RIP140 (Fig. 5B
, panel c). In these cells GFP-143-3 could be
detected both in the cytoplasm and the nucleus (Fig. 5B
, panel d). In
most cells, the coexpression of HA-RIP140 did not affect the
localization of GFP-143-3 (data not shown). Apparently, the export
function of 143-3 seems to dominate over the import function of
RIP140.

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Figure 5. Relocalization of RIP140 from the Nucleus to the
Cytoplasm by 143-3: Confocal Microscopy Analysis
A, COS-7 cells were transfected with 0.5 µg of pEGFP-C2-RIP140 and
0.5 µg of pcDNA3-FLAG (panel a) or with 0.5 µg of pEGFP-C2-RIP140
and 0.5 µg of pcDNA3-FLAG-143-3 (panel b). The localization of
GFP-RIP140 was visualized using a TCS SP Multiband Confocal Imaging
System (Leica Corp.). B, COS-7 cells were transfected with
0.5 µg of pSG5-HA-hRIP140 and pEGFP-C2 (panels a and b) or with 0.5
µg of pSG5-HA-hRIP140 and pEGFP-C214-33 (panels c and d). The
localization of HA-RIP140 and GFP or GFP-143-3 in the same cells was
visualized using indirect immunofluorescence and a Leica TCS SP
Multiband Confocal Imaging System.
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Interaction of 143-3 with Nuclear Receptors and Nuclear Receptor
Cofactors
To determine whether 143-3 could interact with any other nuclear
receptors, in addition to GR, we performed GST pull-down experiments.
GST-143-3 interacted with in vitro translated GR, AR,
ER
, ERß, and TR
, but not with retinoid X receptor
(RXR
)
or PPAR
(Fig. 6A
, lanes 5, 10, 15, 20,
25, 30, and 35). The interaction between GR and 143-3 was inhibited
by molybdate, which stabilizes the GR-hsp90 complex that is formed in
the absence of ligand (Fig. 6A
, lane 3) (22). However, molybdate did
not inhibit the interaction between 143-3 and the other steroid
receptors, AR, ER
, or ERß (Fig. 6A
, lanes 8, 13, and 18). The
interaction between 143-3 and TR
was also ligand independent (Fig. 6A
, lane 23).

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Figure 6. Interaction of 143-3 with Nuclear Receptors and
Nuclear Receptor Cofactors: GST Pull-Down Assays
A, In vitro translated 35S-labeled GR (lanes
15), AR (lanes 610), ER (lanes 1115), ERß (lanes 1620),
TR (lanes 2125), RXR (lanes 2630), and PPAR (lanes 3135)
were incubated with GST or GST-143-3 bound to glutathione agarose
beads in PDB with 0.01% Igepal CA-630. The interaction of GR, AR,
ER , and ERß was studied in the presence of 20 mM
molybdate (Mo) or 1 µM of the respective ligand,
i.e. Dex, testosterone (Test), and 17ß-estradiol
(E2) as indicated. The interaction of TR , RXR , and
PPAR was studied in the absence (-) or presence of 10
µM of their respective ligands, i.e.
T3, 9-cis retinoic acid (9-cis), and
WY-14,643 (WY) as indicated. The translated receptor proteins were
diluted into PDB and incubated in the presence or absence of the
corresponding ligand or molybdate for 2 h at 4 C before mixing
with the GST protein. The eluted samples from washed beads were
analyzed by SDS-PAGE and visualized by autoradiography. The input
represents 10% of the amount of labeled protein incubated with
GST-protein-bound beads. B, In vitro translated
35S-labeled TRAP220 (lanes 1 and 68), CBP (lanes 2 and
911), ACTR (lanes 3 and 1214), SRC-1 (lanes 4 and 1517), and TIF2
(lanes 5 and 1820) were incubated with GST, GST-GR LBD, or
GST-143-3 bound to glutathione agarose beads in PDB with 0.01%
Igepal CA-630. GST-GR LBD bound to glutathione agarose beads was
incubated with 1 µM Dex for 2 h before mixing with
translated protein. The input (lanes 15) represents 15% of the
amount of labeled protein incubated with GST-protein-bound beads.
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Using the yeast two-hybrid method we tested whether nuclear receptor
cofactors previously identified in a two-hybrid screening for PPAR
interacting proteins could also bind to GR LBD (34, 44, 48). The
results showed that, in the presence of ligand, GR LBD interacted not
only with RIP140 [amino acids (aa) 4311,158], but also with TRAP220
(aa 503754) and TIF2 (aa 3201,119) (data not shown). To further
study the interaction of GR LBD with cofactor proteins, we performed
GST pull-down experiments. GST-GR LBD interacted in the presence of
ligand with in vitro translated full-length TRAP220 and
ACTR, and weakly with SRC-1 and TIF2 (Fig. 6B
, lanes 7, 13, 16, and
19). No interaction was detected with in vitro translated
full-length CBP (Fig. 6B
, lane 10). To determine whether any of the GR
cofactor proteins, in addition to RIP140, could interact with 143-3,
we used GST-143-3 in the GST pull-down experiments. The results
showed that the in vitro translated full-length TRAP220 and
ACTR interacted specifically with GST-143-3 (Fig. 6B
, lanes 8 and
14), whereas CBP, SRC-1, and TIF2 did not interact or interacted weakly
(Fig. 6B
, lanes 11, 17, and 20).
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DISCUSSION
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We show in this paper that the 143-3 protein can interact with
the nuclear receptor corepressor RIP140 and change its subcellular
localization. Therefore, we suggest that the positive effect of 143-3
on GR transactivation is due to 143-3 binding to RIP140 and removing
the negatively acting RIP140 from the nucleus, allowing positively
acting coactivator proteins to interact with the receptor. Recently it
has been reported that 143-3 may act by regulating the subcellular
localization of interacting proteins (8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20). We show here that
coexpression of 143-3 can relocalize RIP140 from the nucleus to the
cytoplasm, as well as change the punctate pattern of remaining nuclear
RIP140 to a homogeneous nuclear distribution. Therefore it is tempting
to speculate that the 143-3- induced disappearance of the punctate
pattern could be connected with functional changes of RIP140 within the
nucleus. Further, it is interesting that agonist-, but not antagonist-,
bound GR is also localized to intranuclear dot structures (49). 143-3
has been suggested to act by exporting proteins from the nucleus using
a nuclear export sequence that is present in 143-3. Alternatively,
143-3 may inhibit nuclear import of interacting proteins by binding
to a site on the interacting protein overlapping with a nuclear
localization sequence. The mechanism for the redistribution of RIP140
into the cytoplasm by 143-3 is at present not known. However, the
observed 143-3-induced intranuclear redistribution of RIP140 suggests
that 143-3 is binding to RIP140 in the nucleus and not only acting by
sequestering RIP140 in the cytoplasm.
RIP140 has previously been shown to compete with the coactivator TIF2
for binding to GR (37). We show here that overexpression of RIP140
inhibits the 143-3-enhanced GR transactivation, which can be due to
an increased level of RIP140 that is not bound to 143-3 and can stay
in the nucleus and compete with coactivators for binding to GR. Since
RIP140 and 143-3 interact noncompetitively with GR, it is unlikely
that the antagonistic effect of RIP140 on 143-3- enhanced GR
transactivation is due to RIP140 disrupting the 143-3/GR complex or
that the positive effect of 143-3 on GR transactivation is due to
143-3 disrupting the RIP140/GR. Both RIP140 and 143-3 interact with
GR LBD. RIP140 has been shown to interact with the activation function
2 (AF2) domain in nuclear receptor LBDs (30) whereas the exact
interaction surface for 143-3 on GR LBD has not been determined. The
lack of binding competition between RIP140 and 143-3 suggests that
143-3 does not interact with the AF2 domain in GR LBD. It is possible
that all three proteins can form a complex, but that this tertiary
complex is inhibitory in contrast to the heterodimer between GR and
143-3.
Many 143-3 target proteins are phosphorylated on serine residues, and
phosphatase treatment of the proteins has been shown to reduce the
interaction with 143-3 (1, 45, 46, 47). RIP140 is a serine-rich protein,
and we show here that phosphorylation of RIP140 may also be important
for 143-3 recognition, since phosphatase-treated in vitro
translated RIP140 or nonphosphorylated bacterially expressed RIP140 did
not interact efficiently with 143-3. The effect of 143-3 of
subcellular localization of interacting proteins has been demonstrated
to be regulated by phosphorylation of the target protein and subsequent
association with 143-3 (8, 14, 15). It would be interesting to
determine whether a regulated serine phosphorylation of RIP140
contributes to the function of the protein and the subcellular
localization.
Interestingly, the same domains of RIP140 are involved in both GR and
143-3 interaction. RIP140 contains many nuclear receptor boxes (NR
boxes) (LXXLL motifs) that have been shown to be important for nuclear
receptor interaction (34, 44, 50, 51, 52). The GR-interacting domains
RIP140 N (aa 1472) and RIP140 C (aa 7471,158) contain five and
three NR boxes, respectively. However, the middle domain RIP140 M (aa
431745), which does not interact with GR, still contains two NR
boxes. Possibly, sequences surrounding the LXXLL core of these NR boxes
account for the low affinity to GR (44). The C-terminal part of RIP140
contains a consensus 143-3 binding motif (RTFSYP), whereas the
N-terminal and middle parts of RIP140 only contain sequences with weak
similarity to the consensus motif. However, in addition to the
consensus motif, other less well characterized 143-3 binding
sequences have been identified, and possibly such sequences are also
involved in the 143-3/RIP140 interaction.
Our results indicate that in addition to RIP140, 143-3 can interact
with the nuclear receptor coactivators TRAP220 and ACTR. Although the
importance of these coactivators for GR signaling is not yet clear,
143-3 could be involved in the regulation of these cofactors as well.
Nuclear receptor cofactors have been suggested to function within
larger coactivator complexes such as the p160 complex and the TRAP220
complex (26). Since 143-3 has been suggested to act as a scaffold to
bring together different proteins, it is interesting to speculate that
143-3, which potentially interacts with proteins in both complexes,
may play a role in the formation or regulation of the coactivator
complexes needed for receptor function. In addition, our results
suggest that 143-3 can interact with other nuclear receptors. The
binding of 143-3 to these receptors was ligand independent and not
inhibited by molybdate, which is consistent with a weaker complex
formation between these receptors and hsp90 (28). Since several nuclear
receptors can interact with 143-3, the interaction between 143-3
and RIP140 that we describe here might be involved in the regulatory
function of RIP140 on other receptors in addition to GR.
 |
MATERIALS AND METHODS
|
---|
Plasmids
GST fusion proteins were expressed from pGEX plasmids
(Amersham Pharmacia Biotech, Arlington Heights, IL). The
plasmids expressing GST-143-3
, GST-RIP140 N (aa 1281), and
GST-RIP140 C (aa 747-1,158) have been described previously (22, 34).
The plasmid expressing GST-LBD (aa 485777) was constructed by
subcloning of a BamHI fragment from pEG202/GR LBD into
pGEX-5X-3 (22). The His-tagged RIP140 C (aa 7471,158) was expressed
from plasmid pET19 (Novagen Inc., Madison, WI) (34). pSG5-HA-hRIP140
expressing an HA-tagged human RIP140 (aa 11,158) has been described
previously (34). GFP-RIP140 was made by subcloning the corresponding
fragment from the pBK-CMV-based expression plasmid into pEGFP-C2
(CLONTECH Laboratories, Inc., Palo Alto, CA).
GFP-143-3
was generated by subcloning an
EcoRIXhoI fragment from pBK-CMV-143-3
into
pEGFP-C2 (CLONTECH Laboratories, Inc.). FLAG-143-3
was made by cloning the same fragment into pcDNA3-FLAG (a gift from A.
Ström). Plasmids for in vitro
transcription/translation of hGR, 143-3
, RIP140, RIP140 N (aa
1472), RIP140 M (aa 431745), RIP 140 C (aa 747-1,158), hAR, hER
,
hERß, hTR
, rRXR
, rPPAR
, mTRAP220, hSRC1, mTIF2, hACTR, and
mCBP were used (22, 34, 39, 40, 41, 42, 43, 44). Mammalian expression plasmids
pCMV4-hGR and pBKCMV-143-3
and reporter plasmids p19-tk-luc and
pCMV-ß gal were described previously (22, 34).
Protein Expression and Purification
GST- and His-tagged proteins were expressed in the
Escherichia coli strain BL21(DE3)pLysS. Bacteria were grown
to an OD600 of 0.5, and the expression of fusion
protein was induced for 2 h by adding isopropyl
1-thio-ß-D-galactopyranoside to a final
concentration of 0.5 mM. Bacteria expressing GST
and GST-143-3 were harvested by centrifugation and resuspended in 10
mM phosphate buffer, pH 7.4, 140
mM NaCl, 3 mM KCl, 1
mM dithiothreitol (DTT), 1
mM phenylmethylsulfonyl fluoride (PMSF), 1
µg/ml leupeptin, 0.1% Igepal CA-630. The suspension was treated with
0.1 mg/ml lysozyme, 20 µg/ml DNaseI, 10 µg/ml RNaseA and cleared by
centrifugation. The resulting soluble protein lysate was incubated with
glutathione-agarose beads (Sigma, St. Louis, MO) for
2 h at 4 C. The beads were washed with 10 mM
phosphate buffer, pH 7.4, 140 mM NaCl, 3
mM KCl. Fusion proteins were eluted with 20
mM HEPES-KOH, pH 7.9, 10% (vol/vol) glycerol,
100 mM KCl, 5 mM
MgCl2, 0.2 mM EDTA, 1
mM DTT, 0.2 mM PMSF, 0.01%
Igepal CA-630, 20 mM glutathione, and dialyzed
using the same buffer without glutathione. GST-RIP140 proteins and
GST-GR LBD, which were insoluble under normal purification conditions,
were solubilized in sodium-N-lauroylsarcosinate, and the
extracts were used directly in GST pull-down experiments. Bacteria
expressing GST-RIP140 and GST-GR LBD were harvested by centrifugation
and resuspended in 10 mM Tris-HCl, pH 8.0, 150
mM NaCl, 1 mM EDTA, 1
mM DTT, 1 mM PMSF, 1
µg/ml leupeptin. The suspension was treated with 0.1 mg/ml lysozyme,
20 µg/ml DNaseI, 10 µg/ml RNaseA, and 1.5% sodium-N-
lauroylsarcosinate and cleared by centrifugation. The cleared
extract was used directly in GST pull-down experiments. Bacteria
expressing His-RIP140 C were harvested by centrifugation and
resuspended in 20 mM Tris-HCl, pH 8.0, 100
mM NaCl, 1 mM PMSF, 1
µg/ml leupeptin. The suspension was treated with 0.1 mg/ml lysozyme,
20 µg/ml DNaseI, 10 µg/ml RnaseA, and 0.9%
sodium-N-lauroylsarcosinate and cleared by centrifugation.
The resulting soluble protein lysate was incubated with a TALON
affinity resin (CLONTECH Laboratories, Inc.) for 30 min at
room temperature. The resin was washed with 20 mM
Tris-HCl, pH 8.0, 100 mM NaCl. His-RIP140 C was
eluted with 20 mM Tris-HCl, pH 8.0, 100
mM NaCl, 1 mM PMSF, 1
µg/ml leupeptin, 1% Tween 20, and 50 mM
imidazole and dialyzed using the same buffer without Tween 20 or
imidazole. [35S]Methionine-labeled proteins
were synthesized in vitro using TnT-coupled reticulocyte
lysate (Promega Corp., Madison, WI).
Alkaline Phosphatase Treatment
To dephosphorylate the in vitro translated RIP140
protein, 10 µl of translation reaction were incubated in the presence
or absence of 5 U of alkaline phosphatase (Roche Molecular Biochemicals, Indianapolis, IN) in 50 mM
Tris-HCl, pH 8.5, 0.1 mM EDTA at 37 C for 1
h. The reactions were subsequently used in GST pull-down
experiments.
GST Pull-Down Experiments
GST-fusion protein (5 or 10 µg) bound to 30 µl glutathione
beads was incubated with 510 µl of in vitro translated
protein in 200 µl pull-down buffer (PDB) [20
mM HEPES-KOH, pH 7.9, 10% (vol/vol) glycerol,
100 mM KCl, 5 mM
MgCl2, 0.2 mM EDTA, 1
mM DTT, 0.2 mM PMSF, 1
mg/ml BSA and 0.010.03% Igepal CA-630] overnight at 4 C. The beads
were recovered by centrifugation and washed four times with PDB without
BSA, and bound proteins were eluted by SDS-PAGE sample buffer, analyzed
on SDS-PAGE, and visualized by autoradiography. Dex, 1
µM, was used in all GST pull-down experiments
using in vitro translated GR or GST-GR LBD. The translated
receptor proteins were diluted into PDB and incubated in the presence
or absence of the corresponding ligand or molybdate for 2 h at 4 C
before mixing with the GST protein. In experiments using GST-GR LBD,
the fusion protein bound to glutathione beads was incubated with ligand
for 2 h before mixing with translated proteins. In competition GST
pull-down experiments, His-RIP140 C or unlabeled in vitro
translated protein was included in the binding reaction. For
quantification, gels were analyzed using PhosphoImager (BAS-2000,
Fuji Photo Film Co., Ltd., Tokyo, Japan) or films
were analyzed using Image Scanner (Amersham Pharmacia Biotech, Arlington Heights, IL) and the relative amount
of protein input was calculated by subtracting unspecific binding (GST)
from specific binding (GST-fusion protein).
Western Blotting
Proteins were separated on SDS-PAGE and transferred to Hybond-C
extra nitrocellulose filters (Amersham Pharmacia Biotech).
Filters were blocked with 5% milk powder in PBS containing 0.05%
Tween 20. For detection of His-RIP140 C the filter was incubated with a
1:500 dilution of a rabbit polyclonal antibody recognizing the His tag
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in
PBS/Tween 20 for 2 h at room temperature. After washing, the
filters were incubated with horseradish peroxidase-linked antirabbit
antibody (Amersham Pharmacia Biotech) at a dilution of
1:8,000 in PBS/Tween 20 for 1 h at room temperature. After
washing, the proteins were visualized using the Supersignal West Pico
chemiluminescent substrate (Pierce Chemical Co.).
Reporter Gene Assays
COS-7 cells were seeded in 24-well plates and transfected with
plasmids using the Fugene 6 transfection agent (Roche Molecular Biochemicals). After transfection, cells were maintained in the
presence of 1 µM Dex or ethanol vehicle for 24 h and
then harvested for luciferase and ß-galactosidase assay. The
luciferase activity was determined using the Gen-Glow-1000 kit
(Bioorbit, Turku, Finland) and ß-galactosidase using the
Galacto-Light Plus kit (Tropix, Inc., Bedford, MA) in the luminometer
Anthos Lucy I (Anthos Labtec-Instruments, Salzburg,
Austria).
Confocal Microscopy
COS-7 cells were plated on glass cover slips in six-well plates
and grown for 24 h. The cells were transfected for 8 h with
0.5 µg of each plasmid (total amount 1 µg) using Lipofectin
(Life Technologies, Inc., Gaithersburg, MD). Then fresh
medium was added, and the cells were further grown for 20 h before
fixing with 3% paraformaldehyde in 5% sucrose/PBS, 20 min at room
temperature. For indirect immunofluorescence, fixed cells were rinsed
three times with PBS before being permeabilized with PBS/Tween
(0.1 %), three times for 5 min at room temperature. The cells
were blocked by 5% goat serum (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in PBS/Tween (1 h at room
temperature) before incubating with mouse monoclonal antibody, HA.1
(Babco, Richmond, CA) for 1 h (diluted 1:200 in PBS/Tween). After
removal of the HA.1 antibody by washing three times 5 min with
PBS/Tween, cells were treated with tetramethyl rhodamine
isothiocyanate (TRITC)-conjugated goat antimouse IgG (H+L)
(Jackson ImmunoResearch Laboratories, Inc.) for 1 h
at room temperature (diluted 1:100 in PBS/Tween). Cells were washed
five times for 5 min in PBS/Tween before being fixed to slides using
antiphotobleaching fluorosave (Calbiochem, La Jolla, CA).
Subcellular localization was determined using a TCS SP Multiband
Confocal Imaging System. (Leica Corp., Deerfield, IL).
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. R. M. Evans, G. Kuiper, J. Leers, B.W.
OMalley, M. G. Parker, K. Pettersson, A. Ström, and S.
Nilsson for generously providing plasmids and Sanna Hulkko for
technical assistance during the work.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Johanna Zilliacus, Department of Medical Nutrition, Karolinska Institutet, Novum, S-141 86 Huddinge, Sweden.
This work was supported by grants from The Swedish Medical Research
Council (12557), Åke Wiberg Foundation, Lars Hierta Foundation, and
Emil and Wera Cornell Foundation. Dr. Zilliacus was supported by a
junior research position from the Swedish Medical Research Council. Dr.
Holter was supported by a grant from Nordic Academy of Advanced Study
(99.30.1650).
1 Present address: Third Department of Internal Medicine, Akita
University School of Medicine, 11-1 Hondo, Akita-shi, Akita 010-8543,
Japan. 
Received for publication May 22, 2000.
Revision received December 19, 2000.
Accepted for publication January 5, 2001.
 |
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