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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. 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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). 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 (100–250
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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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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).
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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-, and ERß-Dependent
Transactivation, 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).
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, 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 563–1,121 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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). 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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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 (30–90 ng) were used in HepG2 cells, a 20–30%
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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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 1–550 of
these latter proteins are identical, and they differ only in their C
termini (ARIP3 residues 551–572, Miz1 residues 551–621). 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 60–80% (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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 575–634) 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 1–147),
pVP16 for expressing the transcriptional activation domain (VP16 AD) of
the herpes simplex virus VP16 protein (amino acid residues 411–456),
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 fNN4-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(1–103) 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 563–1,121) was constructed by digesting
pM-GRIP1(563–1,121) (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
manufacturer’s 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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M. Kobayashi, S. Kishida, A. Fukui, T. Michiue, Y. Miyamoto, T. Okamoto, Y. Yoneda, M. Asashima, and A. Kikuchi Nuclear Localization of Duplin, a beta -Catenin-binding Protein, Is Essential for Its Inhibitory Activity on the Wnt Signaling Pathway J. Biol. Chem., February 15, 2002; 277(8): 5816 - 5822. [Abstract] [Full Text] [PDF]
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B. Saville, H. Poukka, M. Wormke, O. A. Janne, J. J. Palvimo, M. Stoner, I. Samudio, and S. Safe Cooperative Coactivation of Estrogen Receptor alpha in ZR-75 Human Breast Cancer Cells by SNURF and TATA-binding Protein J. Biol. Chem., January 18, 2002; 277(4): 2485 - 2497. [Abstract] [Full Text] [PDF]
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H.-J. Ting, S. Yeh, K. Nishimura, and C. Chang Supervillin associates with androgen receptor and modulates its transcriptional activity PNAS, January 22, 2002; 99(2): 661 - 666. [Abstract] [Full Text] [PDF]
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D. Schmidt and S. Muller Members of the PIAS family act as SUMO ligases for c-Jun and p53 and repress p53 activity PNAS, March 5, 2002; 99(5): 2872 - 2877. [Abstract] [Full Text] [PDF]
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