ARIP3 (Androgen Receptor-Interacting Protein 3) and Other PIAS (Protein Inhibitor of Activated STAT) Proteins Differ in Their Ability to Modulate Steroid Receptor-Dependent Transcriptional Activation
Noora Kotaja,
Saara Aittomäki,
Olli Silvennoinen,
Jorma J. Palvimo and
Olli A. Jänne
Department of Physiology (N.K., J.J.P., O.A.J.) Institute of
Biomedicine University of Helsinki FIN-00014 Helsinki,
Finland
Department of Clinical Chemistry (O.A.J.)
University of Helsinki FIN-00290 Helsinki, Finland
Department of Medical Biochemistry (S.A., O.S.) University of
Tampere and Tampere University Hospital FIN-33014 Tampere,
Finland
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ABSTRACT
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Steroid receptors mediate their actions by using
various coregulatory proteins. We have recently characterized
ARIP3/PIASx
as an androgen receptor (AR)-interacting protein (ARIP)
that belongs to the PIAS [protein inhibitor of activated STAT
(signal transducer and activator of transcription)] protein family
implicated in the inhibition of cytokine signaling. We have analyzed
herein the roles that four different PIAS proteins (ARIP3/PIASx
,
Miz1/PIASxß, GBP/PIAS1, and PIAS3) play in the regulation of steroid
receptor- or STAT-mediated transcriptional activation. All PIAS
proteins are able to coactivate steroid receptor-dependent
transcription but to a differential degree, depending on the receptor,
the promoter, and the cell type. Miz1 and PIAS1 are more potent than
ARIP3 in activating AR function on minimal promoters. With the natural
probasin promoter, PIAS proteins influence AR function more
divergently, in that ARIP3 represses, but Miz1 and PIAS1 activate it.
Miz1 and PIAS1 possess inherent transcription activating function,
whereas ARIP3 and PIAS3 are devoid of this feature. ARIP3 enhances
glucocorticoid receptor-dependent transcription more efficiently than
Miz1 or PIAS1, and all PIAS proteins also activate estrogen receptor-
and progesterone receptor-dependent transcription but to a
dissimilar degree. The same amounts of PIAS proteins that modulate
steroid receptor-dependent transcription influence only marginally
transactivation mediated by various STAT proteins. It remains to be
established whether the PIAS proteins play a more significant
physiological role in steroid receptor than in cytokine signaling.
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INTRODUCTION
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The transcriptional activity of steroid receptors relies not only
on their ability to enter the nucleus and bind DNA but also on their
interactions with other transcription factors and a number of
coregulator protein complexes (1, 2, 3). Coregulators encompass
coactivators (e.g. Refs. 4, 5, 6, 7, 8, 9, 10, 11, 12), corepressors
(e.g. Refs. 13, 14, 15), cointegrators (e.g. Refs. 16, 17), and mediator protein complexes (e.g. Refs. 18, 19). In view of the central role of steroid receptors in the regulation
of cell growth, differentiation, and homeostasis, this large number of
coregulatory proteins is perhaps not surprising. Most of the auxiliary
proteins may interact with multiple signaling systems, and the same
coregulators can be used by diverse classes of signal-inducible
transcription factors.
ARIP3 (androgen receptor-interacting protein 3) is a steroid receptor
coregulator found in a yeast two-hybrid screen with the androgen
receptor (AR) zinc finger region (ZFR) as a bait (20). ARIP3 belongs to
a novel family of nuclear proteins that also includes Miz1
(Msx-interacting zinc finger), GBP (Gu/RNA helicase II-binding
protein), PIAS1 (protein inhibitor of activated Stat1) and PIAS3. These
proteins are reported to modulate functions of very different
transcription factors. Mouse Miz1 interacts with homeodomain-containing
Msx2 protein and may enhance its DNA binding (21). PIAS1 and PIAS3 bind
to Stat1 (signal transducer and activator of transcription 1) and
Stat3, respectively, and inhibit STAT-mediated signaling by perturbing
with DNA binding of Stat1 and Stat3 (22, 23). Human GBP, which is
almost identical to PIAS1, was isolated in a yeast two-hybrid screen
with Gu/RNA helicase II as a bait (24). Recently, Tan et al.
(25) identified PIAS1 as a steroid receptor coregulator through an
approach similar to the approach that we used for ARIP3. It is worth
pointing out in this context that, similar to ARIP3, the expression of
PIAS1 was mainly confined to the testis (25). Additional PIAS sequences
(PIASx
, PIASxß, and PIASy) were found in a cDNA library screen
with PIAS1 cDNA (22). Human PIASx
corresponds to rat ARIP3, and
PIASxß is the human counterpart of mouse Miz1.
Even though members of the PIAS protein family were identified through
interaction with very dissimilar signaling molecules, their high
sequence conservation predicts similar functions. In view of this, we
have compared the ability of different PIAS proteins to influence the
transactivation mediated by AR, glucocorticoid receptor (GR),
progesterone receptor (PR), and estrogen receptor
(ER
) and ß
(ERß). We report herein that the PIAS family members do indeed
interact with steroid receptors and modulate (i.e. activate
or repress) their function in a fashion that is dependent on the
promoter and the cell type. Under the experimental conditions used in
our studies, the effects of PIAS proteins on transcriptional activation
mediated by different STAT proteins were minor in comparison with those
on steroid receptors.
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RESULTS
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PIAS Proteins Modulate AR-Dependent Transactivation in a Distinct
Fashion
Sequence comparison of ARIP3 and PIAS family members examined in
this study is shown in Fig. 1A
. The
relative levels of FLAG-tagged proteins encoded by ARIP3 and PIAS
expression vectors were similar in HeLa and HepG2 cells (Fig. 1
, B and
C). The effects of ARIP3, Miz1, PIAS3, and PIAS1 on AR-dependent
transactivation were first studied in HeLa cells by cotransfecting a
constant amount of AR expression construct along with increasing
amounts (2, 10, and 20 ng) of expression vectors encoding the PIAS
proteins. When a reporter gene driven by two androgen response elements
(AREs) in front of E1b TATA sequence
(ARE2TATA-LUC) was used, low amounts of ARIP3
activated AR-dependent transcription up to approximately 3-fold, but
the effect vanished with increasing amounts of ARIP3 (Fig. 2A
). Even though ARIP3 and Miz1 differ
only in their very C-terminal 22 and 71 amino acids, respectively, the
two proteins displayed distinct actions on AR function. Whereas ARIP3
activated the ARE2TATA promoter approximately
3-fold, Miz1 enhanced AR-dependent transcription up to about 8-fold in
a dose-dependent fashion. The effect of PIAS1 on AR-mediated
transactivation was comparable to that of Miz1, and PIAS3 displayed a
dose-response curve similar to that of ARIP3. Comparable results were
obtained in COS-1 cells. ARIP3 or PIAS proteins did not influence the
reporter gene activity in the absence of hormone or alter the amount of
AR in transfected cells (data not shown).

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Figure 1. Sequence Comparison of the PIAS Family Members
A, Comparison of rat ARIP3 (PIASx ), mouse Miz1 (PIASxß),
mouse PIAS1, and mouse PIAS3 amino acid sequences. Gaps in the sequence
are shown by dashes. Black boxes and gray
shadings depict amino acids that are identical or conserved among
the sequences, respectively. B, Immunoblot analysis of proteins encoded
by the following expression vectors in HeLa cells: empty pFLAG-CMV2
(lane 1), pFLAG-ARIP3 (lane 2), pFLAG-Miz1 (lane 3), pFLAG-PIAS3 (lane
4), and pFLAG-PIAS1 (lane 5). HeLa cells were transfected by the FuGene
reagent with expression vectors (200 ng DNA/well, 12-well plate) and
cultured for 48 h. Whole-cell extracts were resolved by SDS-PAGE
and immunoblotted using monoclonal M2 antibody against the FLAG
epitope. C, Corresponding immunoblot analysis of proteins expressed in
HepG2 cells.
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Figure 2. PIAS Family Members Differ in Their Ability to
Potentiate AR-Dependent Transcription
A, Modulation of transactivation from minimal ARE2TATA
promoter. HeLa cells cultured on 12-well plates were transfected with
200 ng of pARE2TATA-LUC reporter, 20 ng of pSG5-rAR, 20 ng
of pCMVß, and increasing amounts (2 ng, 10 ng, and 20 ng) of
pFLAG-ARIP3, pFLAG-Miz1, pFLAG-PIAS3, or pFLAG-PIAS1 in the presence
(+) or absence (-) of 100 nM testosterone (T). Total
amount of DNA was kept constant by adding empty pFLAG-CMV2 as needed.
After normalization for transfection efficiency using ß-galactosidase
activity, reporter gene activities are expressed relative to those of
rAR + T without a coregulator (=1.0). B, Modulation of transactivation
from the natural probasin (PB) promoter. The experimental conditions
were the same as in panel A, except that pPB(-285/+32)-LUC reporter
was used. C and D, Effects of PIAS proteins on AR-dependent
transcriptional activation in HepG2 cells from the ARE2TATA
promoter and the probasin promoter, respectively. HepG2 cells were
cultured on 12-well plates and transfected with same amounts of
plasmids as described in panel A. The values represent means ±
SD from three to six independent experiments.
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Immunoblot analysis showed that ARIP3 is expressed to a level somewhat
higher than that of Miz1 or PIAS1 (Fig. 1B
). Since low amounts of ARIP3
activated AR function but higher levels were inhibitory (Fig. 2A
),
experiments were also performed with lower amounts of expression
vectors; 0.5 ng of ARIP3 plasmid enhanced AR-dependent transcription by
1.5-fold, 1 ng of ARIP3 led to an approximately 2.5-fold increase, and
5 ng of ARIP3 attenuated the maximal activation. In the case of Miz1,
0.5 and 1 ng of Miz1 plasmid exhibited marginal effects, and 5 ng
stimulated AR-dependent transcription by about 4-fold (data not
shown).
With the more complex probasin promoter, cotransfection with 10 ng and
20 ng of ARIP3 repressed AR-dependent transactivation, whereas Miz1 and
PIAS1 enhanced it by approximately 2.5- to 3-fold (Fig. 2B
). PIAS3
behaved in a fashion similar to that of ARIP3, in that it repressed the
transcription at the highest dose. ARIP3 or PIAS proteins did not
influence probasin promoter activity in the absence of hormone (data
not shown).
The effects of PIAS proteins on AR-dependent transactivation were also
studied in HepG2 cells (Fig. 2C
). Interestingly, the PIAS proteins
activated AR function on the minimal ARE2TATA
promoter to a similar degree; maximal induction was 4- to 5-fold by
ARIP3, Miz1, and PIAS1, and about 7-fold by PIAS3. In contrast to HeLa
or COS-1 cells, ARIP3 or other PIAS proteins failed to repress the
probasin promoter in HepG2 cells; rather, they all activated
AR-dependent transcription and PIAS3 was the most potent activator
(
6-fold activation) (Fig. 2D
). As shown in Fig. 1
, relative
expression levels of the PIAS proteins in HepG2 cells were comparable
to those in HeLa cells, and therefore, cell line-dependent differences
in their activities are not due to different protein levels. In sum,
the PIAS proteins modulate AR-dependent transcription in a cell line-
and promoter-dependent fashion.
Miz1 and PIAS1 Possess Intrinsic Transcription-Activating
Functions
To examine whether the ability of PIAS proteins to enhance
AR-dependent transcription is explainable by differences in their
intrinsic transcription-activating functions, ARIP3, Miz1, PIAS3, and
PIAS1 were fused to Gal4 DNA-binding domain (Gal4) and transfected to
HeLa cells with a reporter construct driven by five Gal4-binding sites
(G5-LUC) (Fig. 3A
). Gal4-Miz1 activated
the reporter gene by 23-fold and Gal4-PIAS1 by 10-fold [compared with
the activity of Gal4 DNA-binding domain (DBD) alone], indicating the
presence of transcription-activating regions in these proteins. If
anything, Gal4-ARIP3 and Gal4-PIAS3 fusion proteins repressed the
promoter activity. In HepG2 cells, the relative activities of Gal4-Miz1
and Gal4-PIAS1 were lower than in HeLa cells, and Gal4-ARIP3 and
Gal4-PIAS3 again repressed Gal4 DBD activity by 40% and 70%,
respectively (Fig. 3B
). The use of lower or higher amounts (100250
ng) of expression plasmids yielded essentially identical results, in
that only PIAS1 and Miz1 exhibited intrinsic transcription-activating
function (data not shown). This presence of transcription activation
regions in PIAS1 and Miz1 may, at least in part, explain their
differential ability to stimulate AR-dependent transcription in HeLa
cells, but it does not apply to HepG2 cells.

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Figure 3. Miz1 and PIAS1 Contain Intrinsic
Transcription-Activating Functions
A, Activation of transcription by Gal4 DBD fusion proteins in HeLa
cells. Cells were transfected with 200 ng of pG5-LUC reporter
(containing five Gal4-binding sites in front of the minimal TATA box),
20 ng of pCMVß, and 150 ng of Gal4 DBD (Gal4) fusion constructs
Gal4-ARIP3, Gal4-Miz1, Gal4-PIAS3, or Gal4-PIAS1. After normalization
for transfection efficiency using ß-galactosidase activity, reporter
gene activities are expressed relative to that of Gal4 DBD alone
(=1.0). B, Activation of transcription by Gal4 DBD fusion proteins in
HepG2 cells. The experimental conditions were the same as those
described in panel A. The values are means ± SD
from three independent experiments.
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PIAS Proteins Activate and Repress GR Function in a
Promoter-Dependent Fashion
HeLa and HepG2 cells were used to study the influence of PIAS
proteins on GR-dependent transcription. Hormone response elements in
the ARE2TATA promoter also mediate GR-dependent
signaling (26). Coexpression of ARIP3 with GR enhanced GR-dependent
transcription
30-fold from the minimal promoter in HeLa cells (Fig. 4A
), which is 10 times more than that
with AR. On the other hand, Miz1 or PIAS1 stimulated transcriptional
activity of GR to the same extent as that of AR. In contrast to AR,
PIAS3 was as efficient as PIAS1 or Miz1 in coactivating the function of
GR. Interestingly, when GR-dependent transcription from the minimal
promoter was examined in HepG2 cells, the differences between PIAS
proteins diminished, and they all activated GR function by 5- to
6-fold, which is comparable to that of AR in the same cells (c.f.,
Figs. 2C
and 4B
). Thus, PIAS proteins possess steroid receptor
selectivity in their coregulatory properties, but this selectivity is
dependent on the cell context.

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Figure 4. Modulation of GR-Dependent Transcription by PIAS
Proteins
A and B, Effects of cotransfection of PIAS proteins with GR (pSG5-hGR)
on the ARE2TATA promoter activity in HeLa cells (A) and in
HepG2 cells (B). Experimental conditions were same as those in Fig. 2A , except that GR-dependent transcription was activated by exposure to 100
nM dexamethasone (DEX). C, The same experiment was
performed in HeLa cells using pHH-LUC reporter, which contains region
-203/+105 of the mouse mammary tumor virus promoter in front of the
LUC gene. The values represent means ± SD from
three to six independent experiments.
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When HH-LUC containing the mouse mammary tumor virus promoter was used
as the reporter, both ARIP3 and PIAS3 caused a dose-dependent
repression of GR-dependent transactivation in HeLa cells (Fig. 4C
).
These findings resemble those of ARIP3 and PIAS3 on AR with the
probasin promoter. By contrast, Miz1 and PIAS1 did not modulate
GR-dependent transcription from HH-LUC. The effect of GR on this latter
promoter in another cell type, CV-1 cells, has been reported to be
activated 2-fold by coexpression of PIAS1 (25).
ARIP3 was originally identified by an interaction screen using AR ZFR
(AR DBD plus one-third of the hinge region) as the bait (20). Since the
influence of PIAS proteins differed on AR- and GR-dependent
transcription, it was pertinent to determine whether the DBDs were
mainly responsible for their dissimilar responses. To study this
possibility, receptor chimeras GAG (GR DBD is replaced with AR DBD) and
AGA (AR containing GR DBD) (27) were examined in cotransfections with
ARIP3 and Miz1 along with ARE2TATA-LUC reporter.
As shown in Fig. 5
, ARIP3 and Miz1
influenced chimeric GAG receptor function in a fashion identical with
that of wild-type GR. Likewise, AR and AGA responded to coexpressed
PIAS proteins in a comparable manner. Thus, the receptor selectivity in
the action of these two PIAS proteins appears to require receptor
regions outside the DBDs.

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Figure 5. Effects of ARIP3 and Miz1 on Chimeric Forms of AR
and GR
A, Cotransfections of ARIP3 and Miz1 with rGR. B, Cotransfections with
the GAG chimera (rGR DBD is replaced with that of AR). C,
Cotransfections of ARIP3 and Miz1 with mAR. D, Cotransfections with the
AGA chimera (=mAR containing GR DBD). HeLa cells were transfected with
200 ng of pARE2TATA-LUC, 20 ng of pCMV5-rGR, pCMV5-GAG,
pCMV5-mAR, or pCMV5-AGA (panels AD, respectively), 20 ng of pCMVß,
and increasing amounts (2 ng, 10 ng, and 20 ng) of pFLAG-ARIP3 or
pFLAG-Miz1, in the presence (+) or absence (-) of 100
nM testosterone (T) or dexamethasone (DEX) under the
experimental conditions described in Fig. 2 . Mean ±
SD values from three independent experiments are
shown.
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PIAS Proteins Modulate PR-, ER
-, and ERß-Dependent
Transactivation
To study whether PIAS proteins modulate steroid receptor function
more generally, PR, ER
, and ERß were coexpressed with PIAS
proteins in HeLa cells, and the activities of their cognate minimal
promoters were monitored. All PIAS family members were able to enhance
ligand-dependent transactivation by PR, but they modulated PR function
in a manner clearly different from that of GR; ARIP3 and Miz1 activated
PR function to a similar degree (
6-fold increase) that exceeded the
effect of PIAS1 or PIAS3 (max.
3-fold stimulation) (Fig. 6
). It is of note that with a more
complex promoter (the mouse mammary tumor virus promoter), Tan et
al. (25) found PIAS1 to repress PR-dependent transactivation. In
the case of ER
, the activities of PIAS proteins were similar, and
they all elicited 2- to 3-fold maximal stimulation of transcription,
whereas with ERß, Miz1 activity exceeded that of other PIAS proteins
(Fig. 6
, B and C). Modulatory effects of PIAS proteins on thyroid
hormone receptor-dependent transcription were minor in comparison to
those of the five steroid receptors examined in this study (data not
shown).
PIAS Proteins Interact with Steroid Receptors in
Vitro and in Vivo
Physical interaction of steroid receptors with PIAS proteins was
examined by glutathione S-transferase (GST) pull-down
experiments. GST-ARIP3 and GST-Miz1 bound to glutathione sepharose were
incubated with [35S]methionine-labeled
receptors synthesized by translation in vitro. All receptors
studied were capable of interacting with GST-Miz1 and GST-ARIP3
in vitro (Fig. 7
, A and B, and
data not shown). The interactions were specific, as the receptors
failed to adhere markedly to GST alone, and no binding of a control
protein, luciferase, was observed under the conditions used. The
interactions were largely hormone independent, as comparable amounts of
proteins were bound without the cognate ligand, as illustrated for
rAR-Miz1 and ER
-Miz1 interactions in Fig. 7C
. Other steroid
receptors behaved the same way with Miz1 and ARIP3 (data not shown).
This was in contrast to the interaction of AR and ER
with amino acid
residues 5631,121 of glucocorticoid receptor interacting protein
1 (GRIP1) (GRIP1b) fused to GST, which was clearly
ligand-enhanced under the same in vitro conditions (Fig. 7C
). Overall, the interactions of ARIP3 and Miz1 with the five steroid
receptors were quite similar; ER
and ERß bound somewhat more
efficiently to GST-Miz1 and GST-ARIP3 than the other receptors. In any
event, receptor selectivity of the PIAS proteins in transactivation
assays does not seem to be caused by their markedly dissimilar in
vitro binding affinities for PIAS proteins.

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Figure 7. Miz1 and ARIP3 Interact with Steroid Receptors
in Vitro
A, AR, GR, PR, ER , and ERß were labeled with
[35S]methionine by translation in vitro
and incubated with Glutathione Sepharose-bound GST or GST-Miz1. Bound
proteins were eluted with the SDS sample buffer, resolved by SDS PAGE,
and visualized by fluorography. Lane 1 of each panel represents 5% of
the labeled protein incubated with the matrix. Testosterone,
dexamethasone, progesterone, or estradiol (each 1
µM) was included in binding reactions with AR, GR,
PR, or ER, respectively. B, The corresponding pull-down experiments
were performed by using GST-ARIP3 fusion protein except that hPR was
not included. C, [35S]Methionine-labeled AR and ER
were synthesized by translation in vitro and incubated
with Glutathione Sepharose-bound GST, GST-Miz1, or GST-GRIP1b with (+)
or without (-) 1 µM testosterone (T) or estradiol
(E) as indicated. The conditions were otherwise identical with those in
panel A. Lane 1 represents 5% of the input.
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Interactions of the PIAS proteins with AR were also compared by using a
mammalian two-hybrid system in HeLa cells (Fig. 8
). Full-length ARIP3 and PIAS proteins
fused to VP16 activation domain (VP16) were cotransfected with
expression vector encoding Gal4 DBD-AR (Gal4-AR) and G5-LUC reporter.
All PIAS proteins interacted with AR in this assay, in that they
increased reporter gene activity over that of Gal4-AR and polyoma virus
coat protein fused to VP16 (VP16-CP). ARIP3 and Miz1 were more active
than PIAS3 and PIAS1. Direct comparison of their potencies, however, is
hampered by dissimilar expression levels of the VP16 fusion proteins;
VP16-PIAS1 and VP16-PIAS3 were expressed to levels lower than those of
VP16-ARIP3 and VP16-Miz1 fusions (data not shown). ARIP3-AR interaction
was detectable already in the absence of hormone, but the
presence of androgen increased it markedly (Fig. 8
). Miz1-apo-AR
interaction was minor compared with that of ARIP3 with apo-AR. It was,
however, greatly enhanced by the hormone (
30-fold induction), whereas
the effect of androgen was intermediate on other PIAS proteins. Thus,
in contrast to cell-free conditions, the interaction of AR with PIAS
proteins in intact cells is highly hormone dependent.

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Figure 8. Interaction of AR with PIAS Proteins in Mammalian
Cells
Interaction of AR with ARIP3 and other PIAS proteins was examined by a
two-hybrid system in HeLa cells. The cells were transfected using the
FuGene reagent with 200 ng of pG5-LUC, 20 ng of pCMVß, and 100 ng of
Gal4 DBD fusion of AR (Gal4-AR) together with VP16 AD fusion of ARIP3
(VP16-ARIP3), Miz1 (VP16-Miz1), PIAS3 (VP16-PIAS3), and PIAS1
(VP16-PIAS1), or polyoma virus coat protein (VP16-CP) as indicated.
Twenty hours after transfection, the medium was changed to one
containing charcoal-stripped 2% FBS with (+) or without (-) 100
nM testosterone, and the cells were incubated for an
additional 28 h. After normalization for transfection efficiency
using ß-galactosidase activity, the reporter gene activities are
expressed relative to that of Gal4 DBD alone (=1.0). Mean ±
SD values from three independent experiments are
shown.
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Effects of PIAS Proteins on Transcriptional Activation Mediated by
STATs
PIAS1 and PIAS3 are reported to function as specific inhibitors of
Stat1 and Stat3 signaling, respectively (22, 23). To study whether
ARIP3/PIAS proteins are involved in the regulation of STATs and
cytokine signaling more generally, we examined the effects of ARIP3,
PIAS1, and PIAS3 on STAT-dependent transcriptional activation by using
the same or higher amounts of PIAS expression vectors than those in
experiments on steroid receptor-dependent signaling.
The three PIAS proteins were first tested for their ability to regulate
interferon-
(IFN-
)-activated Stat1. HeLa cells were transfected
with GAS-LUC reporter containing a Stat1-binding site from the IRF-1
promoter in front of the minimal tk promoter (28) and increasing
amounts (2 ng, 10 ng, and 20 ng) of ARIP3, PIAS1, or PIAS3 expression
vectors. The cells were treated with IFN-
or left untreated. Ectopic
expression of ARIP3, PIAS1, or PIAS3, in the amounts used in the
preceding studies on steroid receptor function, minimally influenced
IFN-
-induced activation of GAS-LUC in HeLa cells (Table 1
). However, when higher amounts of PIAS1
expression plasmid (3090 ng) were used in HepG2 cells, a 2030%
decrease in IFN-
-induced activation of GAS-LUC reporter was observed
(Fig. 9A
). By contrast, even the higher
amounts of ectopically expressed PIAS1 failed to perturb with the
function of endogenous Stat1 in HeLa cells (Fig. 9B
).
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Table 1. Influence of ARIP3, PIAS1, and PIAS3 on
Transcriptional Activation Mediated by Stat1, Stat5, and Stat6 in HeLa
Cells
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Figure 9. Influence of Ectopic Expression of PIAS1 on Stat1-
and Stat5-Dependent Transcriptional Activation in HeLa and HepG2 Cells
HeLa and HepG2 cells were transfected using the FuGene reagent on
12-well plates with 200 ng of the indicated LUC reporter, 20 ng of
pCMVß, and 20 ng of Stat5 expression vector together with the amounts
of PIAS1 expression plasmid shown (in nanograms). With GAS-LUC, no
exogenous Stat1 was transfected, and 30 ng of EpoR expression vector
were cotransfected in Stat5 experiments. Twenty-four hours after
transfection, the cells were cultured in the absence (-) or presence
(+) of cytokines (10 ng/ml IFN- or 4 U/ml Epo) for 24 h.
Reporter gene activities were normalized by the ß-galactosidase
activity and are expressed relative to that of the corresponding
cytokine alone (=100, black bars). The values are
means ± SD from three separate experiments.
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We also tested whether ARIP3, PIAS1, and PIAS3 affect
transcriptional activation brought about by ectopically expressed Stat5
or Stat6. HeLa cells were cotransfected with a Stat5-responsive Spi-LUC
reporter driven by six repeats of a Stat5-binding site from the serine
protease inhibitor 2.1 gene (29) and expression vectors encoding
erythropoietin receptor (EpoR) and Stat5, as well as ARIP3, PIAS1, or
PIAS3, and cells were treated with erythropoietin (Epo) or left
untreated. Alternatively, HeLa cells were transfected with a
Stat6-responsive fN
N4-LUC reporter containing four repeats of a
Stat6-binding site in front of the c-fos minimal promoter
(30) and expression vectors for Stat6, as well as ARIP3, PIAS1, or
PIAS3, and cells were exposed to interleukin-4 (IL-4) or left
untreated. PIAS1 or PIAS3 had negligible effects on Epo- or
IL-4-induced transcriptional activation mediated by Stat5 and Stat6,
respectively (Table 1
). ARIP3, on the other hand, up-regulated
Stat6-mediated transactivation to a modest degree. Ectopic Miz1
expression with different STAT-responsive reporters did not influence
STAT activities in HeLa cells (data not shown). Likewise, higher
amounts (30 and 90 ng) of PIAS1 did not alter Stat5-dependent
transactivation in HeLa or HepG2 cells (Fig. 9
, C and D).
There is cross-talk between cytokine and glucocorticoid signaling (31),
as exemplified by the synergistic activation of the Spi promoter by
glucocorticoids and Epo (32). Even though ARIP3 is a powerful
coactivator of GR function on glucocorticoid-dependent promoters (Fig. 4
) and interacts with GR in vitro (Fig. 7
), ectopically
expressed ARIP3 did not influence significantly the synergism between
Stat5 and GR in the activation of Spi-LUC reporter in HeLa cells (S.
Aittomäki and O. Silvennoinen, unpublished).
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DISCUSSION
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The receptors for androgens, glucocorticoids, mineralocorticoids,
and progesterone may recognize the same or similar DNA response
elements and yet they regulate distinct target genes in vivo
(33). In addition to the obvious ways to achieve steroid-specific gene
activation, including cell-specific expression of the receptor protein
and differential availability of the ligand, coregulatory proteins
provide an additional level of control to ensure that appropriate
responses to hormones are achieved. Although most coregulatory proteins
identified to date are recognized by the conserved activation
function-2 (AF-2) located in the C terminus of the ligand-binding
domain (1, 2, 3, 4) and, in the case of AR, by the less well conserved AF-1
in the N-terminal region (34, 35), recent data indicate that also the
DBD and hinge region of nuclear receptors present important interaction
interfaces for coregulatory proteins. SNURF (36), Ubc9 (37), ANPK (38),
TLS/FUS (39), HET/SAF-B (40), PCAF (11), N-CoR (15), SMRT (14), and
also POU domain-containing proteins Oct-1/2 and Brn-3a/3b (41, 42) are
among the proteins that can interact with the ZFRs and/or hinge regions
of nuclear receptors. All of them are not necessarily involved in
direct transcriptional control; instead, they may mediate other
processes, such as nuclear targeting and intranuclear
compartmentalization. In this regard, GR ZFR is shown to be required
for the interaction with nuclear matrix (43), and SNURF is capable of
modulating nuclear trafficking of AR (44).
ARIP3, a rat counterpart of human PIASx
, is among the proteins that
interact with the AR ZFR/hinge region in vitro and in
vivo and modulate AR-dependent transactivation in intact cells
(20). It is predominantly expressed in testis, albeit lower ARIP3 mRNA
levels are found in other tissues as well. Likewise, another family
member, PIAS1, which has recently been reported to activate AR
function, shows the highest expression in testis (25). In contrast to
ARIP3 and PIAS1, PIAS3 is reported to be ubiquitously expressed (23).
The PIAS proteins are relatively well conserved, as
Drosophila genome contains a gene termed zimp
that encodes a homolog of the PIAS family (45). Zimp is an
essential gene for Drosophila development. It is expressed
as three alternatively spliced forms, two of which are detected only in
adult flies. The zimp transcripts encode proteins of 544 and
522 amino acids that share an N-terminal 515-amino acid region and
differ in their C termini. The splice variants of zimp
resemble ARIP3/PIASx
and Miz1/PIASxß, in that residues 1550 of
these latter proteins are identical, and they differ only in their C
termini (ARIP3 residues 551572, Miz1 residues 551621). The presence
of a PIAS homolog, but not relatives of the p160 gene family of nuclear
receptor coactivators in the Drosophila genome (46),
suggests that the PIAS proteins serve a function different from, and
perhaps more ancient than, that of the p160 coactivators in steroid
receptor signaling.
Since the PIAS family members are highly homologous, with regions
exhibiting amino acid sequence identities of 6080% (Ref. 23
and Fig. 1
), it was pertinent to compare their ability to
modulate steroid receptor-dependent transcription. Each PIAS family
member activated steroid receptor function from simple promoters, and
none of them influenced basal transcription in the absence of ligand.
However, the proteins behaved in a receptor-selective fashion, in that
their ability to modulate transcription mediated by different steroid
receptors varied substantially. There were also interesting differences
in their cell line-specific functions. On more complex promoters,
such as probasin and mouse mammary tumor virus promoters, PIAS3 and
ARIP3 acted predominantly as corepressors of AR and GR function in HeLa
and COS-1 cells, whereas in HepG2 cells, all PIAS proteins activated
AR-dependent transcription. Moreover, the differences among the PIAS
protein activities on the minimal promoter were diminished in HepG2
cells, in that they all activated the function of GR and AR to a
similar degree.
Miz1 and PIAS1 exhibited intrinsic transcription-activating
function in both HeLa and HepG2 cells when fused to Gal4 DBD,
whereas ARIP3 and PIAS3 were devoid of this feature. This intrinsic
transcription-activating function of PIAS1 and Miz1 was in line with
their more robust activity on AR in HeLa cells but, surprisingly, not
in HepG2 cells or with other steroid receptors in either HeLa or HepG2
cells. Since Miz1 and ARIP3 differ merely in their very C-terminal 71
and 22 amino acids, respectively, it is likely that the Ser/Thr-rich
extension in the Miz1 C terminus contributes to the activating
function. In this regard, ARIP3 and Miz1 resemble the C-terminal SRC-1
variants, SRC-1a and SRC-1e, which differ in their ability to
potentiate transcription by ER in a promoter context-dependent fashion,
and their functional differences relate to an activation domain present
only in the SRC-1e isoform (47).
Inhibition of STAT-DNA interaction is the postulated mechanism
underlying the down-regulation of STAT signaling by PIAS1 and PIAS3
(22, 23). The contrasting effects of PIAS proteins on steroid receptor
function in different cell lines imply that their action on steroid
receptors is hardly based on the interference with receptor-DNA
interaction, i.e. a mechanism suggested for Stat1 and Stat3.
Experiments with chimeric AR and GR forms also showed that the
receptor-selective effects of PIAS proteins are dependent on regions
other than AR or GR DBD. Moreover, our previous work indicated that
ARIP3 does not influence significantly the interaction of AR with ARE
(20). Differences in steroid receptor-PIAS interactions also failed to
provide a mechanistic explanation for the dissimilar effects of PIAS
proteins on steroid receptor function. It is likely that PIAS proteins
form complexes with other coregulatory proteins, perhaps simultaneously
with steroid receptors. Dissimilar amounts of these
yet-to-be-identified PIAS-interacting proteins might form the basis for
the cell- and promoter-specific actions of PIAS proteins on steroid
receptor-dependent transcriptional activation.
ARIP3 contains two LXXLL motifs starting at residues 18 and 304
that are conserved in mammalian PIAS proteins, but not in Zimp.
However, these putative nuclear receptor boxes do not seem to play an
important role in the ability of the PIAS proteins to modulate steroid
receptor function (N. Kotaja, O. A. Jänne, and J. J.
Palvimo, in preparation). Mammalian PIAS proteins, Zimp, and the
predicted proteins in Caenorhabditis elegans (Ce 1523698),
and Saccharomyces cerevisiae Nfi-1 (2104683), all share a
well conserved region comprising one His and five Cys residues that may
form a zinc-binding motif. The possibility that this region serves as
an interaction interface for steroid receptors will be addressed in our
future experiments. Interestingly, this region is not essential
for the interaction of PIAS1 with Stat1 (48).
The ability of PIAS proteins to interact with steroid receptors and,
depending on the promoter and cell type context, to play both positive
and negative regulatory roles is intriguingly similar to the behavior
of Zac1b (49). Like ARIP3, Zac1 (zinc finger protein that regulates
apoptosis and cell cycle arrest) is a member of a larger protein
family, the PLAG (pleomorphic adenoma gene) family (50). In addition to
nuclear receptors, Zac1b may also bind to the C-terminal activation
domain of GRIP1 and interact with CREB-binding protein (CBP) and p300
(49). Similar to ARIP3 and Miz1, Zac1 also interacts with nuclear
receptors in a hormone-independent manner in vitro. Altered
Zac1 expression has been associated with cancer, and its expression is
repressed in ovarian cancer cell lines (51, 52). In this regard, it is
of interest that PIAS1 expression is severely repressed in
HRAS-transformed fibroblasts and the repression is blocked by a
mitogen-activated protein (MAP) kinase inhibitor (53).
Taken together, the PIAS proteins modulate transcriptional
activity of steroid receptors and, depending on the cell and promoter
context, they either activate or repress transcription dependent on
steroid receptors. The biological functions of this protein family are
obviously not restricted to the inhibition of STAT signaling. It is
currently unknown which of the functions of the PIAS proteins,
i.e. the modulation of steroid receptor action or the
inhibition of STAT-mediated signaling, is biologically more important.
These actions do not have to be mutually exclusive, and they may well
be dependent on the concentration of individual PIAS proteins and their
interaction partners in a given cell type. ARIP3 and PIAS1 are
predominantly expressed in the testis (20, 25) which is a target for
cytokine regulation through STAT proteins (54, 55) and for steroid
hormone action. Testis may thus represent a tissue where the cross-talk
between steroids and cytokines is governed by the function of PIAS
proteins, such as ARIP3 and PIAS1.
 |
MATERIALS AND METHODS
|
---|
Materials
pARE2TATA-LUC reporter containing two AREs
(from the first intron of the rat C3 gene) in front of minimal TATA
sequence and pPB(-285/+32)-LUC containing nucleotides -285 to +32 of
the rat probasin promoter driving luciferase coding region have been
described (36, 56). ERE2TATA-LUC was constructed
in the same way except that the inserted 45-bp oligomer contained two
estrogen response elements in lieu of AREs (57). Mouse mammary tumor
virus promoter LUC construct (pHH-LUC, containing region -203/+105 of
the promoter) was obtained from American Type Culture Collection (ATCC, Manassas, VA). pG5-LUC has five
Gal4-binding sites in front of the minimal TATA box sequence driving
the LUC gene (Promega Corp., Madison, WI). pSG5-rAR
expression vector was constructed as previously described (58). pM-rAR
was also described previously (36).
pSG5-hPR1 was gift from Dr. Pierre Chambon. pSG5-hGR was created as
described previously (36). pCMV5-hER
and pCMV5-hERß were from Drs.
Benita S. Katzenellenbogen and Jan-Åke Gustafsson, respectively.
pSG5-ER
was created by ligating ER
digested with EcoRI
and BamHI into the pSG5 (Stratagene, La Jolla,
CA). pSG5-hERß was constructed by first inserting ERß C terminus as
an EcoRI/BamHI fragment into pSG5 and by
subsequently cloning the N terminus as an EcoRI fragment.
pCMV5-AGA derived from the full-length mouse AR by swapping its DBD
(amino acid residues 575634) for that of the rat GR, the
corresponding pCMV5-GAG derived from full-length rat GR, and their
wild-type counterparts pCMV5-mAR and pCMV5-rGR were gifts from Dr.
Diane M. Robins (27). PIAS3 and PIAS1 cDNAs were from Dr. K. Shuai, and
Miz1 cDNA was a gift from Dr. Rob Maxon. The following mammalian
two-hybrid vectors were used (from CLONTECH Laboratories, Inc., Palo Alto, CA): pM for expressing the DBD of the
Saccharomyces cerevisiae Gal4 protein (residues 1147),
pVP16 for expressing the transcriptional activation domain (VP16 AD) of
the herpes simplex virus VP16 protein (amino acid residues 411456),
and VP16-CP for expressing a fusion of VP16 AD to the polyoma virus
coat protein. The ß-galactosidase expression plasmid pCMVß was
purchased from CLONTECH Laboratories, Inc. Luciferase
reporter constructs GAS-LUC, Spi-LUC, and fN
N4-LUC and plasmids
encoding EpoR, Stat1, Stat5, and Stat6 have been described previously
(28, 29, 30, 32). Testosterone was from Makor Chemicals (Jerusalem,
Israel), progesterone, estradiol, and dexamethasone were from
Sigma (St. Louis, MO), and IFN-
was from Immugenex (Los
Angeles, CA). Luciferase assay reagent was purchased from Promega Corp.. Restriction endonucleases, DNA-modifying enzymes, and
[35S]methionine were purchased from
Amersham Pharmacia Biotech (Arlington Heights, IL).
Plasmid Construction
pFLAG-ARIP3 was constructed by cloning PCR-generated cDNA
fragments into pFLAG-CMV2 (Kodak IBI,
Rochester, NY) as described previously (20). Full-length Miz1 was
constructed by digesting the ARIP3 N terminus from pFLAG-ARIP3 with
KpnI and SpeI and ligating it to the Miz1 C
terminus digested from pBluescript IIKS-Miz1. pM2-ARIP3 was cloned by
digesting full-length ARIP3 from pFLAG-ARIP3 with EcoRI and
ligating the insert into pM2 vector. To construct pM2-Miz1,
EcoRI and XbaI were used to digest Miz1 cDNA from
pFLAG-Miz1, and the insert was then ligated into pM2 vector.
pVP16-ARIP3 was created by first cloning the N-terminal PCR-generated
EcoRI/BamHI fragment to pVP16 vector and then
inserting the C-terminal BamHI fragment downstream of the
BamHI site. To construct pVP16-Miz1, Miz1 C terminus was
digested from pFLAG-Miz1 with BamHI and then inserted to
pVP16-ARIP3(1103) cut with the same enzyme. pM-PIAS1 and pVP16-PIAS1
were generated by inserting full-length PIAS1 cleaved from
pCMV5-FLAG-PIAS1 with BglII and HindIII into pM
or pVP16 vectors digested with BamHI and HindIII.
pM-PIAS3 and pVP16-PIAS3 were constructed by cloning the full-length
PIAS3 from pFLAG-PIAS3 to pM and pVP16 with SalI and
HindIII. pGEX4T3-ARIP3 was obtained by transferring
full-length ARIP3 from pFLAG-ARIP3 to the pGEX-4T3 vector as an
EcoRI fragment. To create pGEX-5X1-Miz1, Miz1 was cleaved
with EcoRV and XhoI from pFLAG-Miz1 and inserted
into the SmaI/XhoI sites of pGEX-5X1.
pGEX-5X1-GRIP1b (amino acids 5631,121) was constructed by digesting
pM-GRIP1(5631,121) (a gift from Dr. Michael Stallcup) with
EcoRI and SalI and transferring the insert into
the corresponding site of pGEX-5X1 vector.
Cell Culture and Transfections
HeLa (American Type Culture Collection) cells were
maintained in DMEM containing penicillin (25 U/ml), streptomycin (25
U/ml), 10% (vol/vol) FBS, and nonessential amino acids. HepG2 cells
were maintained in DMEM containing penicillin, streptomycin, 10% FBS,
and sodium pyruvate. Cells were seeded onto 12-well plates and
transfected 24 h later by FuGene transfection method (Roche Molecular Biochemicals, Indianapolis, IN). In brief, each well
received 200 ng of the luciferase reporter plasmid, 20 ng of
ß-galactosidase (pCMVß) internal control plasmid, and 20 ng of
different steroid receptor or STAT expression vectors, and indicated
amounts of ARIP3/PIAS expression vectors. Four hours before
transfection, the medium was changed to one containing 10%
charcoal-stripped FBS. Twenty hours after transfection, the cells
received fresh medium containing 2% charcoal-stripped FBS with or
without 100 nM steroid hormone or, for STAT experiments,
with 10 ng/ml IFN-
, 4 U/ml Epo, or 10 ng/ml IL-4. Forty-eight hours
after transfection, the cells were harvested, lysed in Reporter Lysis
Buffer (Promega Corp., Madison, WI) and the cleared
supernatants were used for luciferase measurements with reagents from
Promega Corp. using a Luminoskan RT reader (Labsystems,
Helsinki, Finland) and for ß-galactosidase assays as described
previously (34, 59). Independent transfection experiments were
conducted using triplicate dishes three to six times, and at least two
different plasmid batches were used for each set of experiments.
Immunoblotting
Whole-cell extracts from HeLa and HepG2 cells were resolved by
electrophoresis on 12% polyacrylamide gels (PAGE) under denaturing
conditions. Proteins were electroblotted onto Hybond ECL membrane
(Amersham Pharmacia Biotech, Arlington Heights, IL).
Membranes were incubated with M2 monoclonal antibody against FLAG
epitope (Kodak, Rochester, NY) and horseradish
peroxidase-conjugated goat antimouse IgG antibody (Zymed Laboratories, Inc., South San Francisco, CA), and
immunocomplexes were visualized using ECL Western blotting detection
reagents from Amersham Pharmacia Biotech according to the
manufacturers instructions.
Protein-Protein Interaction in Vitro
GST-ARIP3, GST-Miz1, and GST-GRIP1b were produced in
Epicurian coli BL21-CodonPlus bacteria
(Stratagene, La Jolla, CA) and purified with Glutathione
Sepharose 4B (Amersham Pharmacia Biotech) as previously
described (60). Lysis buffer containing 50 mM
Tris-HCl (pH 7.8), 150 mM KCl, 0.1% Nonidet
P-40, 0.1% Triton-X 100, 0.5 mM EDTA, 10%
glycerol, 5 mM MgCl2, and
1:200 protease inhibitor cocktail (Sigma, St. Louis, MO)
was used. AR, GR, PR, ER
, and ERß were translated in
vitro using the TNT-coupled transcription/translation system
(Promega Corp.) in the presence of
[35S]methionine. Protein-protein affinity
chromatography with purified GST fusion proteins bound to Glutathione
Sepharose and 10 µl of
[35S]methionine-labeled in vitro
translated protein was carried out at 4 C for 2 h, with or without
the cognate hormone (1 µM), in binding buffer
containing 4 mM Tris-HCl (pH 8.0), 40
mM NaCl, 10% glycerol, 0.5
mM EDTA, 0.4% Nonidet P-40, 0.1% Triton-X 100,
5 mM MgCl2, 50
µM ZnCl2, 20 µg/ml BSA,
and 1:200 protease inhibitor cocktail in a total volume of 500 µl.
The resin was washed four times with 1 ml of binding buffer. Bound
proteins were released by boiling in SDS-PAGE sample buffer. After
electrophoresis, the gels were fixed in methanol (45%)-acetic acid
(10%), treated with Amplify (Amersham Pharmacia Biotech)
and dried, and radioactive proteins were visualized by
fluorography.
 |
ACKNOWLEDGMENTS
|
---|
The excellent technical assistance of Kati Saastamoinen, Leena
Pietilä, and Seija Mäki is gratefully acknowledged. We
thank Pierre Chambon, Jan-Åke Gustafsson, Benita Katzenellenbogen, Rob
Maxon, Diane Robins, Laura Seikku, Ke Shuai, and Michael Stallcup for
plasmids.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Olli A. Jänne, M.D., Ph.D., Institute of Biomedicine, Department of Physiology, University of Helsinki, P.O. Box 9 (Siltavuorenpenger 20 J), FIN-00014 Helsinki, FINLAND. E-mail olli.janne{at}helsinki.fi
This work was supported by grants from the Academy of Finland, the
Finnish Foundation for Cancer Research, the Sigrid Jusélius
Foundation, Biocentrum Helsinki, Helsinki University Central Hospital,
and CaP CURE.
Received for publication August 2, 2000.
Revision received September 6, 2000.
Accepted for publication September 7, 2000.
 |
REFERENCES
|
---|
-
McKenna NJ, Lanz RB, OMalley BW 1999 Nuclear receptor
coregulators: cellular and molecular biology. Endocr Rev 20:321344[Abstract/Free Full Text]
-
Glass CK, Rosenfeld MG 2000 The coregulator exchange in
transcriptional functions of nuclear receptors. Genes Dev 14:121141[Free Full Text]
-
Torchia J, Glass C, Rosenfeld MG 1998 Co-activators and
co-repressors in the integration of transcriptional responses. Curr
Opin Cell Biol 10:373383[CrossRef][Medline]
-
Onate SA, Tsai SY, Tsai M-J, OMalley BW 1995 Sequence and
characterization of a co-activator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional
co-activator in yeast for the hormone binding domains of steroid
receptors. Proc Natl Acad Sci USA 93:49484952[Abstract/Free Full Text]
-
Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK,
Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and
mediates nuclear-receptor function. Nature 387:677684[CrossRef][Medline]
-
Anzick SL, Kononen J, Walker RL, Azorsa DO, Tanner MM, Guan
XY, Sauter G, Kallioniemi OP, Trent JM, Melzer PS 1997 AIB1, a steroid
receptor co-activator amplified in breast and ovarian cancer. Nature 277:965968
-
Chen H, Lin RJ, Schiltz RL, Chkravarti D, Nash A, Nagy L,
Privalsky ML, Nakatani Y, Evans RM 1997 Nuclear receptor co-activator
ACTR is a novel histone acetyltransferase and forms a multimeric
activation complex with p/CAF and CBP/p300. Cell 90:56980[Medline]
-
Li H, Gomes PJ, Chen JD 1997 RAC3, a steroid/nuclear
receptor-associated co-activator that is related to SRC-1 and TIF2.
Proc Natl Acad Sci USA 94:84798484[Abstract/Free Full Text]
-
Takeshita A, Cardona GR, Koibuchi N, Suen CS, Chin WW 1997 TRAM-1, a novel 160-kDa thyroid hormone receptor activator molecule,
exhibits distinct properties from steroid receptor co-activator-1.
J Biol Chem 272:2762927634[Abstract/Free Full Text]
-
Blanco JCG, Minucci S, Lu JM, Yang XJ, Walker KK, Chen HW,
Evans RM, Nakatani Y, Ozato K 1998 The histone acetylase PCAF is a
nuclear receptor coactivator. Genes Dev 12:16381651[Abstract/Free Full Text]
-
Korzus E, Torchia J, Rose DW, Xu L, Kurokawa R, McInerney EM,
Mullen TM, Glass CK, Rosenfeld MG 1998 Transcription factor-specific
requirements for coactivators and their acetyltransferase functions.
Science 279:703707[Abstract/Free Full Text]
-
Chen JD, Evans RM 1995 A transcriptional co-repressor that
interacts with nuclear hormone receptors. Nature 377:454457[CrossRef][Medline]
-
Hörlein AJ, Näär AM, Heinzel T, Torchia J,
Gloss B, Kurokawa R, Ryan A, Kamei Y, Söderström M, Glass
CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid
hormone receptor mediated by a nuclear receptor co-repressor. Nature 377:397404[CrossRef][Medline]
-
Perissi V, Staszewski LM, McInerney EM, Kurokawa R, Krones A,
Rose DW, Lambert MH, Milburn MV, Glass CK, Rosenfeld MG 205 1999 Molecular determinants of nuclear receptorcorepressor interaction.
Genes Dev 13:319831[Abstract/Free Full Text]
-
Kamei Y, Xu L, Heinzel T, Torchia J, Kurokawa R, Gloss B, Lin
SC, Heyman RA, Rose DW, Glass CK, Rosenfeld MG 1996 A CBP integrator
complex mediates transcriptional activation and AP-1 inhibition by
nuclear receptors. Cell 85:403414[Medline]
-
Chakravarti D, LaMorte VJ, Nelson MC, Nakajima T, Schulman IG,
Juguilon H, Montminy M, Evans RM 1996 Role of CBP/p300 in nuclear
receptor signalling. Nature 383:99103[CrossRef][Medline]
-
Rachez C, Lemon BD, Suldan Z, Bromleigh V, Gamble M, Naar AM,
Erdjument-Bromage H, Tempst P, Freedman LP 1999 Ligand-dependent
transcription activation by nuclear receptors requires the DRIP
complex. Nature 398:824828[CrossRef][Medline]
-
Ito M, Yuan CX, Malik S, Gu W, Fondell JD, Yamamura S, Fu ZY,
Zhang X, Qin J, Roeder RG 1999 Identity between TRAP and SMCC complexes
indicates novel pathways for the function of nuclear receptor and
diverse mammalian activators. Mol Cell 3:361370[Medline]
-
Moilanen A-M, Karvonen U, Poukka H, Yan W, Toppari J,
Jänne OA, Palvimo JJ 1999 A testis-specific androgen receptor
coregulator that belongs to a novel family of nuclear proteins. J
Biol Chem 274:37003704[Abstract/Free Full Text]
-
Wu L, Wu H, Ma L, Sangiorgi F, Wu N, Bell JR, Lyons GE, Maxson
R 1997 Miz1, a novel zinc finger transcription factor that interacts
with Msx2 and enhances its affinity for DNA. Mech Dev 65:317[CrossRef][Medline]
-
Liu B, Liao J, Xiaoping R, Kushner SA, Chung CD, Chang DD,
Shuai K 1998 Inhibition of Stat1-mediated gene activation by PIAS1.
Proc Natl Acad Sci USA 95:1062610631[Abstract/Free Full Text]
-
Chung CD, Liao J, Liu B, Rao X, Jay P, Berta P, Shuai K 1997 Specific inhibition of Stat3 signal transduction by PIAS3. Science 278:18031805[Abstract/Free Full Text]
-
Valdez BC, Henning D, Perlaky L, Busch RK, Busch H 1997 Cloning and characterization of Gu/RH-II binding protein. Biochem
Biophys Res Commun 234:33540[CrossRef][Medline]
-
Tan J, Hall SH, Hamil KG, Grossman G, Petrusz P, Liao J, Shuai
K, French FS 2000 Protein inhibitor of activated STAT-1 (signal
transducer and activator of transcription-1) is a nuclear receptor
coregulator expressed in human testis. Mol Endocrinol 14:1426[Abstract/Free Full Text]
-
Schoenmakers E, Alen P, Verrijdt G, Peeters B, Verhoeven G,
Rombauts W, Claessens F 1999 Differential DNA binding by the androgen
and glucocorticoid receptors involves the second Zn-finger and a
C-terminal extension of the DNA-binding domains. Biochem J 341:515521[CrossRef][Medline]
-
Scheller A, Hughes E, Golden KL, Robins DM 1998 Multiple
receptor domains interact to permit, or restrict, androgen-specific
gene activation. J Biol Chem 273:2421624222[Abstract/Free Full Text]
-
Pine R, Canova A, Schindler C 1994 Tyrosine phosphorylated p91
binds to a single element in the ISGF2/IRF-1 promoter to mediate
induction by IFN
and IFN
, and is likely to autoregulate the p91
gene. EMBO J 13:15867[Abstract]
-
Wood TJ, Sliva D, Lobie PE, Goullieux F, Mui AL, Groner B,
Norstedt G, Haldosen LA 1997 Specificity of transcription enhancement
via the STAT responsive element in the serine protease inhibitor 2.1
promoter. Mol Cell Endocrinol 20:6981
-
Pesu M, Takaluoma K, Aittomäki S, Lagerstedt A, Saksela
K, Kovanen PE, Silvennoinen O 2000 Interleukin-4-induced
transcriptional activation by Stat6 involves multiple serine/threonine
kinase pathways and serine phosphorylation of Stat6. Blood 95:494502[Abstract/Free Full Text]
-
Cella N, Groner B, Hynes NE 1998 Characterization of Stat5a
and Stat5b homodimers and heterodimers and their association with the
glucocorticoid receptor in mammary cells. Mol Cell Biol 18:17831792[Abstract/Free Full Text]
-
Aittomäki S, Pesu M, Groner B, Jänne OA, Palvimo
JJ, Silvennoinen O 2000 Cooperation among Stat1, glucocorticoid
receptor, and PU.1 in transcriptional activation of the high-affinity
Fc
receptor I in monocytes. J Immunol 164:56895697[Abstract/Free Full Text]
-
Beato M, Herrlich P, Schütz G 1995 Steroid hormone
receptors: many actors in search for a plot. Cell 83:851857[Medline]
-
Ikonen T, Palvimo JJ, Jänne OA 1997 Interaction between
amino- and carboxyl-terminal regions of rat androgen receptor modulates
transcriptional activity and is influenced by nuclear receptor
coactivators. J Biol Chem 272:2982129828[Abstract/Free Full Text]
-
Alen P, Claessens F, Verhoeven G, Rombauts W, Peeters B 1999 The androgen receptor amino-terminal domain plays a key role in p160
coactivator-stimulated gene transcription. Mol Cell Biol 19:60856097[Abstract/Free Full Text]
-
Moilanen A-M, Poukka H, Karvonen U, Häkli M, Jänne
OA, Palvimo JJ 1998 Identification of a novel RING finger protein as a
coregulator in steroid receptor-mediated gene transcription. Mol Cell
Biol 18:51285139[Abstract/Free Full Text]
-
Poukka H, Aarnisalo P, Karvonen U, Palvimo JJ, Jänne OA 1999 Ubc9 interacts with the androgen receptor and activates
receptor-dependent transcription. J Biol Chem 274:1944119446[Abstract/Free Full Text]
-
Moilanen A-M, Karvonen U, Poukka H, Jänne OA, Palvimo JJ 1998 Activation of androgen receptor function by a novel nuclear
protein kinase. Mol Biol Cell 9:25272543[Abstract/Free Full Text]
-
Powers CA, Mathur M, Raaka BM, Ron D, Samuels HH 1998 TLS
(translocated-in-liposarcoma) is a high-affinity interactor for
steroid, thyroid hormone, and retinoid receptors. Mol Endocrinol 12:418[Abstract/Free Full Text]
-
Oesterreich S, Zhang Q, Hopp T, Fuqua SAW, Michaelis M, Zhao
HH, Davie JR, Osborne CK, Lee AV 2000 Tamoxifen-bound estrogen receptor
(ER) interacts with the nuclear matrix protein HET/SAF-B, a novel
inhibitor of ER-mediated transactivation. Mol Endocrinol 14:369381[Abstract/Free Full Text]
-
Prefontaine GG, Lemieux ME, Giffin W, Schild-Poulter C, Pope
L, LaCasse E, Walker P, Hache RJ 1998 Recruitment of octamer
transcription factors to DNA by glucocorticoid receptor. Mol Cell
Biol 18:34163430[Abstract/Free Full Text]
-
Budhram-Mahadeo V, Parker M, Latchman DS 1998 POU
transcription factors Brn-3a and Brn-3b interact with the estrogen
receptor and differentially regulate trans-criptional activity via an
estrogen response element. Mol Cell Biol 18:10291041[Abstract/Free Full Text]
-
Tang Y, Getzenberg RH, Vietmeier BN, Stallcup MR, Eggert M,
Renkawitz R, DeFranco DB 1998 The DNA-binding and tau2 transactivation
domains of the rat glucocorticoid receptor constitute a nuclear
matrix-targeting signal. Mol Endocrinol 12:14201431[Abstract/Free Full Text]
-
Poukka H, Karvonen U, Yoshikawa N, Tanaka H, Palvimo JJ,
Jänne OA 2000 The RING finger protein SNURF modulates nuclear
trafficking of the androgen receptor. J Cell Sci 113:29913001[Abstract/Free Full Text]
-
Mohr SE, Boswell RE 1999 Zimp encodes a homologue of mouse
Miz1 and PIAS3 and is an essential gene in Drosophila
melanogaster. Gene 18:109116
-
Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD,
Amanatides PG, et al 2000 The genome sequence of
Drosophila melanogaster. Science 287:21852195[Abstract/Free Full Text]
-
Kalkhoven E, Valentine JE, Heery DM, Parker MG 1998 Isoforms
of steroid receptor co-activator 1 differ in their ability to
potentiate transcription by the oestrogen receptor. EMBO J 17:232243[Abstract/Free Full Text]
-
Liao J, Fu Y, Shuai K 2000 Distinct roles of the
NH2- and COOH-terminal domains of the protein
inhibitor of activated signal transducer and activator of transcription
(STAT) 1 (PIAS1) in cytokine-induced PIAS1-Stat1 interaction. Proc Natl
Acad Sci USA 97:52675272[Abstract/Free Full Text]
-
Huang SM, Stallcup MR 2000 Mouse Zac1, a transcriptional
coactivator and repressor for nuclear receptors. Mol Cell Biol 20:18551867[Abstract/Free Full Text]
-
Kas K, Voz ML, Hensen K, Meyen E, Van de Ven WJM 1998 Transcriptional activation capacity of novel PLAG family of zinc finger
proteins. J Biol Chem 36:2302623032[CrossRef]
-
Abdollahi A, Roberts D, Godwin AK, Schultz DC, Sonoda G, Testa
JR, Hamilton TC 1997 Identification of a zinc-finger gene at 6q25: a
chromosomal region implicated in development of many solid tumors.
Oncogene 14:19731979[CrossRef][Medline]
-
Abdollahi A, Godwin AK, Miller PD, Getts LA, Schultz DC,
Taguchi T, Testa JR, Hamilton TC 1997 Identification of a gene
containing zinc-finger motifs based on lost expression in malignantly
transformed rat ovarian surface epithelial cells. Cancer Res 57:20292034[Abstract]
-
Zuber J, Tchernitsa OI, Hinzmann B, Schmitz A-C, Grips M,
Hellriegel M, Sers C, Rosenthal A, Schäfer R 2000 A genome-wide
survey of RAS transformation targets. Nat Genet 24:144152[CrossRef][Medline]
-
Jenab S, Morris P 1997 Transcriptional regulation of
Sertoli cell immediate early genes by interleukin-6 and
interferon-
is mediated through phosphorylation of STAT-3 and
STAT-1 proteins. Endocrinology 138:27402746[Abstract/Free Full Text]
-
Jenab S, Morris P 1998 Testicular leukemia inhibitory factor
(LIF) and LIF receptor mediate phosphorylation of signal transducers
and activators of transcription (STAT)-3 and STAT-1 and induce
c-fos transcription and activator protein-1 activation in
rat Sertoli but not germ cells. Endocrinology 139:18831890[Abstract/Free Full Text]
-
Palvimo JJ, Reinikainen P, Ikonen T, Kallio PJ, Moilanen A,
Jänne OA 1996 Mutual transcriptional interference between RelA
and androgen receptor. J Biol Chem 271:2415124156[Abstract/Free Full Text]
-
Karvonen U, Kallio PJ, Jänne OA, Palvimo JJ 1997 Interaction of androgen receptors with androgen response element in
intact cells: roles of amino- and carboxyl-terminal regions and the
ligand. J Biol Chem 272:1597315979[Abstract/Free Full Text]
-
Palvimo JJ, Kallio PJ, Ikonen T, Mehto M, Jänne OA 1993 Dominant negative regulation of trans-activation by the rat androgen
receptor: roles of the N-terminal domain and heterodimer formation. Mol
Endocrinol 7:13991407[Abstract]
-
Rosenthal N 1987 Identification of regulatory elements of
cloned genes with functional assays. Methods Enzymol 152:704720[Medline]
-
Kallio PJ, Poukka H, Moilanen A, Jänne OA, Palvimo JJ 1995 Androgen receptor-mediated transcriptional regulation in the
absence of direct interaction with a specific DNA element. Mol
Endocrinol 9:10171028[Abstract]