Center for Biotechnology Department of Biosciences Karolinska Institute Novum, S-14157 Huddinge, Sweden
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ABSTRACT |
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INTRODUCTION |
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Within the nuclear receptor superfamily, PPARs have acquired unique
ligand-binding properties since they can bind to a broad range of
structurally diverse compounds including peroxisome proliferators
(i.e. clofibrates, WY-14, 643), antidiabetic
thiazolidinediones (i.e. BRL49653), eicosanoid metabolites
(i.e. prostaglandin PGJ2, leukotriene
LTB4), and naturally occurring fatty acids (i.e.
linoleic acid, oleic acid) (10, 11, 12, 13, 14, 15). In addition, since the PPAR/RXR
heterodimer is permissive also to RXR signaling, PPARs may be able to
integrate diverse ligand signals and to participate in the control of
such different biological events as lipid homeostasis, adipocyte
differentiation, inflammation, and carcinogenesis (16, 17). PPARs
comprise a subfamily with three different subtypes: PPAR and
exert major functions in tissues important for fatty acid metabolism
and lipid homeostasis such as liver or adipose tissue, whereas the
function of the ubiquitously expressed PPAR ß/
is unclear.
Most nuclear receptors, including PPARs, share a typical domain
structure: almost identical Zn-finger-type DNA-binding domains flanked
by nonconserved N-terminal regions usually exhibiting a constitutive
transcriptional activation function (AF-1) and conserved C termini
possessing a multifunctional ligand-binding domain (LBD) necessary for
ligand binding, heterodimerization, and ligand-dependent
transcriptional activation (AF-2). Central to AF-2 function appears to
be a highly conserved amphipathic -helix located at the C-terminal
end of the LBD (E region), termed AF-2 AD,
c, or Tau-4 (18, 19, 20, 21). The
contribution of both AF-1 and AF-2 activation domains to the
transcriptional activity of the entire receptor varies extremely
between receptors. In addition, many transcriptionally active nuclear
receptors do not possess a potent AF-1 function. Further, the
nonconservation of the N-terminal regions between different receptor
subtypes (compare, for example, PPAR
, ß, and
) argues against a
conserved mechanism for AF-1 function. For these reasons, the
recruitment of AF-2 coactivators is likely to represent a critical and
conserved step in ligand-dependent transcriptional activation by
nuclear receptors.
Although the AF-2 of nuclear receptors has been reported to interact directly with components of the basal transcriptional machinery, further experimental evidence has argued strongly for the existence of additional proteins acting as corepressors or coactivators (1, 22, 23). Receptors such as TR, RAR, or the orphan receptors RevErb and COUP, known to act as potent repressors in the absence of ligand, have been demonstrated to recruit the corepressors N-CoR/RIP13 and SMRT/TRAC (9, 24, 25, 26, 27, 28, 29, 30). Further, recent data suggest an additional involvement of corepressors in the regulation of antagonist-bound steroid receptors (31). Upon ligand binding, nuclear receptors undergo substantial conformational changes in their LBD, including a rearrangement of the activation domain helix that leads to the dissociation of corepressors and allows the association of coactivators as well as cofactors serving different functions. Protein-protein interaction screenings have provided several candidate proteins acting as cofactors for ligand-activated receptors (22, 23). Among these proteins, only the promiscuous coactivators, CBP/p300 (32, 33, 34) and members of the SRC-1 family of cofactors (SRC-1/N-CoA1, TIF-2/GRIP1/N-CoA2, p/CIP/ACTR), have been convincingly demonstrated to act as coactivators for many nuclear receptors (33, 35, 36, 37, 38, 39, 40, 41). The recent discovery of intrinsic histone acetyltransferase (HAT) activity in these coactivators functionally links nuclear receptor activation to histone acetylation and chromatin derepression (42, 43, 44).
In addition to SRC-1 coactivators (p160), biochemical studies using ligand-bound ER, RAR, and PPAR revealed the existence of a second group of predominant AF-2 cofactors with a molecular mass of approximately 140 kDa (9, 45, 46). As a major component of p140, nuclear factor RIP140 was originally identified in breast cancer cell lines and subsequently isolated by expression cloning using the ER AF-2 in the presence of estradiol (46, 47). Its ubiquitous expression and its ability to interact with the AF-2 of various nuclear receptors (48, 49) suggested that RIP140 might represent a common nuclear receptor cofactor. However, the coactivation effect of RIP140 on nuclear receptors in transient transfections was minimal and increasing amounts of RIP140 resulted in repression or down-regulation of receptor activity (32, 47). Also, microinjection experiments suggest that RIP140 cannot functionally substitute for coactivators of the SRC-1 family in vivo (50). These findings indicate alternative functions of RIP140 in nuclear receptor signaling.
To address this issue, we now report the characterization of RIP140
action with a focus on PPAR for several reasons. First, we identified
RIP140 multiple times in a yeast two-hybrid screen for liver proteins
interacting with the AF-2 domain of the rat PPAR. Second, recent
reports support the coactivator function of SRC-1 for PPAR (4, 41).
Although biochemical studies reveal predominant binding of p140
(RIP140) to PPAR in solution and as RXR heterodimer under certain
conditions (4), the relevance of these findings has not yet been
specifically addressed. Third, previous studies on RIP140 have mainly
focused on the estrogen receptor (47, 48, 51). Therefore, analyzing
RIP140 function in a different receptor context is of a certain
interest, considering the structural and regulatory differences between
steroid hormone receptor homodimers and RXR heterodimers with regard to
ligand action and cofactor binding. In addition to PPAR and RIP140, in
our study we included TR, a RXR heterodimer partner characteristic for
nonpermissive heterodimers, and SRC-1, an AF-2 cofactor with
established coactivator functions. Our results indicate that RIP140 has
properties of a negative coregulator of ligand-activated nuclear
receptor complexes that antagonizes coactivation mediated by SRC-1.
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RESULTS |
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Based on analogy to mutations in the AF-2 core described for several
other nuclear receptors, we substituted aa L459/L460 (mutation M3) and
E462 (mutation M2) of the rat PPAR with alanine (Fig. 4A
). In addition, to create a mutation
outside the conserved AF-2 core region, we substituted L434/V438
(mutation M1) with alanine. Both residues are part of the putative
heterodimerization helix and are believed to be important for the
structural stability of the LBD. To analyze the interaction of RIP140
and SRC-1 with the PPAR
mutations, we constructed the corresponding
GAL-PPAR LBD fusions and tested them in the two-hybrid system. From the
data presented in Fig. 4B
, we conclude that hydrophobic aa of the AF-2
core are the most important determinants for the interaction with both
RIP140 and SRC-1. We observed different effects of the E462
A
mutation (M2): whereas RIP140 still interacts to 30% compared with the
WT LBD, the mutation completely abolished the interaction with SRC-1.
Surprisingly, the mutation M1 uncovered further differences between the
two cofactors: the interaction with RIP140 is only slightly decreased
(consider the apparently lower expression of M1, Fig. 4C
), whereas the
SRC-1 interaction is definitely affected. These data imply overlapping,
but nonidentical, interaction surfaces of RIP140 and SRC-1 on PPAR
.
Alternatively, RIP140 and SRC-1 might bind with different affinities to
similar parts of the LBD.
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RIP140 Forms a Ternary Complex with RXR Heterodimers Bound to
DNA
To address the possibility that RIP140 might form a ternary
complex with the PPAR/RXR heterodimer on DNA, we performed
electrophoretic mobility shift assays (EMSA). We preincubated in
vitro-translated full-length receptor proteins with the
32P-labeled DR1 binding site derived from the rat acyl-CoA
oxidase (ACO) gene promoter as probe in the absence or presence of
the appropriate ligands and added purified recombinant HIS-tagged
RIP140 protein (aa 747-1158). To achieve a greater mobility of the
heterodimer complex and to be able to detect ternary complexes, both
RXR and RXR
C (see below) lack the first 102 aa of the N
terminus. Consistent with previous results, PPAR
and RXR
form a
DNA-bound complex in the absence of added ligand (Fig. 6A
, lane 1), and neither receptor alone
or in combination with RIP140 can bind to the ACO-peroxisome
proliferator response element (PPRE) (data not shown). Addition of
either PPAR or RXR ligands did not affect the DNA binding under our
conditions (lanes 24). When purified RIP140 C terminus was included
in the binding reaction, the complex was supershifted in the presence
of the RXR ligand 9-cis-RA, indicating the formation of a
ternary complex. Surprisingly, we failed to detect any RIP140 ternary
complex in the presence of the potent PPAR
ligand BRL49635. To
exclude subtype- or ligand-specific differences between PPARs, we
repeated the experiment with PPAR
and observed basically identical
results (data not shown): 9-cis-RA, but not the PPAR-ligand
(WY-14,643), induced the RIP140 ternary complex. Note that the in
vitro-translated PPARs interacted in a ligand-dependent manner
with GST-RIP140C in the pull-down assay (Fig. 5C
and data not shown).
Although we can not exclude differences between the sensitivity of the
two in vitro approaches, it is very likely that both DNA
binding and heterodimerization induce a different PPAR conformation
affecting the interaction with RIP140.
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The recruitment of AF-2 cofactors such as RIP140 to the PPAR/RXR
heterodimer in response to RXR ligands is in good agreement with the
active role of RXR as permissive partner in vivo (2, 3, 4, 6).
In contrast, in the TR/RXR heterodimer, RXR is believed to act as a
nonpermissive partner, unable to bind its ligand in vitro
and to activate transcription in vivo (7). Thus, we repeated
the EMSA experiment with the TR/RXR heterodimer bound to a labeled
synthetic DR4 binding site as probe. In contrast to the situation with
the PPAR/RXR heterodimer, the mobility of the TR/RXR heterodimer
containing the wild-type RXR (including the N terminus) was sufficient
to distinguish the heterodimer from the RIP140 ternary complex. As
demonstrated in Fig. 6B, addition of RIP140 and either
3,3,5-triiodothyroacetic acid (TRIAC), 9-cis-RA, or both
ligands in combination, induced the formation of the ternary complex
(lanes 46). The putative ternary complex is not formed after addition
of GST control protein (lanes 1 and 2). Note that the RXR-ligand
9-cis-RA appears to be more effective than the TR-ligand
TRIAC under the conditions of our bandshift assay (1 µM
ligands), supporting the idea that RIP140 might have a higher affinity
to the ligand-activated RXR subunit in different heterodimeric receptor
complexes. It is also interesting to note that the ternary complex with
TR/RXR bound to both ligands exhibited the same mobility as the ternary
complexes observed with only one ligand-bound receptor, indicating that
the number of RIP140 molecules recruited to the heterodimer was
identical. Although the stoichiometry of the ternary complex is
unknown, our results strongly suggest that heterodimeric receptors bind
RIP140 as a functional unit and not as independent subunits. This view
is further supported by the experiment shown in Fig. 6B
(lanes 712)
in which we used the C-terminal truncated RXR. Surprisingly, the RXR
ligand 9-cis-RA still induced the RIP140 ternary complex,
although the differential recruitment of RIP140 in response to the
individual ligands appears to be lost with the mutated RXR (compare
lanes 4/5 and 10/11). These results suggest strongly different modes of
interaction of RIP140 with PPAR/RXR or TR/RXR heterodimers,
respectively.
SRC-1, But Not RIP140, Functions as Potent Nuclear
Receptor Coactivator in Yeast
Having established that RIP140 interacts with the
ligand-activated AF-2/LBD of nuclear receptors in solution and
with heterodimers bound to DNA, we attempted to clarify the role of
RIP140 in transcriptional activation. Recent studies in yeast suggested
that RIP140 activates transcription when tethered to DNA and serves as
a nuclear receptor coactivator in yeast (48, 51). However, when fusing
the RIP140 WT protein to the GAL4 DNA-binding domain, we observed only
negligible transcriptional activity in our yeast system (data not
shown). Next, to monitor the ability of RIP140 to serve as a
coactivator for PPAR or TR in yeast, we established an in
vivo transactivation assay. In contrast to the two-hybrid
situation, RIP140 was now fused only to the SV40 nuclear localization
signal. As shown in Fig. 7, RIP140 WT (aa
11158) or C-terminus (aa 431-1158), and for comparison, the nuclear
receptor coactivator SRC-1 WT (aa 11061), were individually
coexpressed with PPAR or TR fused to the GAL4 DNA-binding domain and
assayed for activation of the lacZ reporter. Importantly,
none of the RIP140 fragments functions as potent coactivators in our
yeast system, in contrast to the strong coactivation seen with SRC-1.
Basically similar results were observed on plates (2 days growth) as
revealed from X-gal filter assays (data not shown).
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DISCUSSION |
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We and others have previously demonstrated that peroxisome
proliferators, such as WY-14,643, and natural fatty acids activate the
PPAR subtype in mammalian cells (2, 63). Here we show that WY-14,643
enhances the interaction of the rat PPAR
with RIP140 and SRC-1
in vitro, consistent with similar results recently reported
by Wahli and co-workers (13) using the Xenopus PPARs and
SRC-1 in pull-down assays. This suggests that at least some peroxisome
proliferators and endogenous fatty acids may act as PPAR
ligands
through direct binding to the receptor. In addition, binding of the
synthetic ligand BRL 49635 to PPAR
efficiently enhances the
interaction with both cofactors in vitro. However, several
PPAR activators and putative endogenous ligands (for instance,
PGJ2, LTB4, and ETYA) did not enhance the
RIP140 interaction in our assays. Such discrepancies may be partially
due to limitations of the pull-down approach using GST fusion proteins,
since Li et al. found that bacterially expressed receptor
LBDs could in some way be defective in proper folding and ligand
binding (64). This is important considering the difficulties observed
by us and others in obtaining soluble and functional PPAR protein of
mammalian origin (13). Further, it is not unlikely that
species-specific differences between the PPAR subtypes may complicate
the interpretation of these in vitro results.
With respect to cofactors relevant for ligand activation by PPARs, the
question remains whether, in addition to RIP140/SRC-1 proteins, other
perhaps specific cofactors contribute to the transcriptional regulation
in response to ligands. For example, the rat enzyme deoxyuridine
triphosphatase (dUTPase) was isolated as a PPAR-interacting protein
(65). However, the interaction domain of dUTPase seems to be specific
for rodents, arguing against the importance of dUTPase as a general
PPAR cofactor. One should also consider that p140/p160 proteins
identified in biochemical studies (4, 9, 45, 46) were purified mostly
from a limited number of cell lines (CV-1, HeLa, and breast cancer cell
lines), leaving the possibility open that other cell lines or tissues
might contain a different set of cofactors. For example, nothing is
known about relative expression levels of AF-2 cofactors in adipose
tissue, one of the major target tissues of PPAR action.
A Coactivator Function for RIP140?
Summarizing the results presented here and in previous studies,
RIP140 shares important features with coactivators of the SRC-1 family
(40, 46, 47, 48, 49). Biochemically, these proteins have been identified from
several cell extracts as the predominant nuclear receptor AF-2
cofactors in the presence, but not in the absence, of ligands. Using
different experimental approaches, RIP140 and SRC-1 have been
demonstrated to interact directly and ligand dependently with the AF-2.
Nuclear receptor modifications that abolish the AF-2 activity have been
shown also to abolish the interaction with both cofactors. Furthermore,
RIP140 and SRC-1 form ternary complexes with ligand-activated receptor
dimers bound to DNA. Also, RIP140 and SRC-1 family members seem to be
ubiquitously expressed and have been demonstrated individually to
colocalize with receptors to the nucleus (37, 47). In summary, both
cofactor classes fulfil important criteria crucial for coactivator
function.
However, a critical evaluation of the experimental data presented here and elsewhere might question such a role for RIP140, especially when compared with the evidence provided for SRC-1 and related coactivators. In fact, SRC-1/TIF-2 (31, 35, 37, 40, 41) and CBP/p300 (32, 33, 34, 36), but not RIP140, have been shown to significantly enhance the AF-2 activity of several nuclear receptors in mammalian cells, clearly indicating a role as nuclear receptor coactivators. The requirement for SRC-1 and CBP in ligand-regulated receptor activation has been further evaluated in inhibition experiments using microinjected antibodies (32, 33, 50). Coinjection of expression vectors for SRC-1 and TIF-2, but not for RIP140, could reverse the inhibitory effects of anti-SRC-1 IgG on ligand-dependent expression of a lacZ reporter gene, clearly demonstrating that RIP140 functionally cannot replace coactivators of the SRC-1 family (50). Further, far-Western analysis of cellular proteins binding mainly to ligand-activated ER suggested that different cell lines primarily contain relatively large amounts of p160, but apparently variable amounts of p140 (4, 9, 33, 45, 46). SRC-1 coactivators are now considered to act as histone acetyltransferases (HAT) and to function in concert with several other proteins exhibiting acetyltransferase activity, e.g. CBP/p300 and P/CAF (42, 43, 44, 66, 67, 68). Intriguingly, CBP/p300 as part of the RNA polymerase II holoenzyme might perhaps link nuclear receptors directly to the basal transcription machinery (69). Although RIP140 has been proposed to act as a bridging protein to the basal transcription machinery, there is no evidence for the involvement of RIP140 in such complexes in mammalian cells, consistent with the notion that RIP140 does not interact with TATA box binding protein (TBP), TFIIB, or CBP/p300 (33, 47). It should be noted that RIP140 coexpression in yeast only resulted in a minimal coactivation effect under nonsaturating ligand concentrations (51), whereas the coexpression of SRC-1 family members could efficiently restore the AF-2 activity of nuclear receptors (70, 71).
Differential Interaction of RIP140 with PPAR/RXR and TR/RXR
Heterodimers
Ligand-dependent ternary complex formation of cofactors with
receptor dimers on DNA is an important criterion for function. We
demonstrated in EMSA experiments that RIP140 supershifts both TR/RXR
and PPAR/RXR heterodimers under certain ligand conditions. Although
PPAR ligands increased RIP140 binding to PPAR in solution, they failed
to induce RIP140 ternary complexes with PPAR/RXR heterodimers on DNA.
The reason for this is unclear, especially since the in
vitro binding affinity of the BRL 49643 compound to PPAR is
comparable to that reported for TR or RXR ligands to their respective
receptors (14). However, RIP140 binds strongly to the PPAR/RXR
heterodimer in the presence of 9-cis-RA. Since this
interaction was dependent on the functional RXR AF-2 helix 12, RXR
might critically influence RIP140 binding to the PPAR/RXR heterodimer.
Previous studies suggested that in nonpermissive RXR heterodimers, RAR
and TR allosterically inhibit RXR from binding its ligand in
vitro (7, 8). This is contradicted by recent observations that, at
least in the case of RAR/RXR heterodimers, both partners can
independently bind their ligands (64). Our studies support the subunit
independency also for the DNA-bound TR/RXR heterodimer, since
9-cis-RA and TRIAC could independently induce the RIP140
ternary complex. These data do not conflict with the nonpermissivity
seen in vivo, since RXR ligands alone may not be sufficient
to dissociate dominant negative corepressors bound to the unliganded TR
or RAR subunit. Surprisingly and in contrast to the situation with
PPAR/RXR, we observed 9-cis-RA-dependent RIP140 ternary
complexes with TR/RXR also with an RXR lacking the AF-2 helix 12. It
remains to be shown whether this effect is due to a phantom ligand
effect (58) of 9-cis-RA on TR, or whether RIP140 binding to
RXR when heterodimerized with TR is not entirely dependent on helix 12.
The latter possibility is not unlikely since the putative interaction
surface for AF-2 cofactors is suggested to be complex and requires the
contribution of other LBD parts than helix 12 (such as helix 3/4).
Importantly, these differences between PPAR/RXR and TR/RXR heterodimers
with respect to RIP140 binding support the concept that RXR
heterodimers act as functional units with distinct specificities (30, 57, 58).
It is further interesting to note that in both the PPAR/RXR and the TR/RXR heterodimer, ligand-activated RXR appears to be very efficient in recruiting RIP140 to the complex. Independent far-Western studies aimed at detecting cofactors from CV-1 cell extracts using GST-RXR or the RAR/RXR heterodimer bound to DNA might support this notion since they revealed the predominant recruitment of p140 instead of p160, whereas in the absence of RXR ligands apparently either p140 or p160 can bind the heterodimer (4, 9). If true, the high affinity of RIP140 to the RXR subunit might have implications for the role of RIP140 in vivo, considering that RXR, as the ultimate heterodimerization partner for the majority of nuclear receptors, plays a central role in nuclear receptor activation. It remains to be determined how the recruitment of regulatory AF-2 cofactors, such as RIP140, through the activated RXR subunit contributes to the transcriptional activity of RXR heterodimers in vivo.
Competitive Binding of NR Box-Containing Cofactors to Nuclear
Receptors
During the completion of our work, interaction domain mapping
studies on TIF-1, RIP140, and SRC-1 led to the identification of a
short conserved peptide sequence LxxLL, serving as NR box or signature
motif in a variety of coactivators, including SRC-1, TIF-2, ACTR, CBP,
and p300, but also in other receptor cofactors, including RIP140,
TIF-1, and several TRIPs (50, 55, 56). However, since this motif is
found in many proteins not associated with receptor function, and since
AF-2 interacting proteins such as ARA70 (53) obviously lack the LxxLL
motif, it is likely that less-conserved motifs as well as additional
structural features determine the interaction of AF-2 cofactors with
receptors. For example, although the C-terminal RIP140 region (aa
981-1158) does not contain the consensus motif, we still detected an
appreciable interaction with PPAR. In addition, we noticed that RIP140
N and C termini exhibit different interaction characteristics to
nuclear receptors, although the interaction characteristics of NR box
peptides derived from these domains do not allow any prediction for
preferential binding (55). This suggests that the protein context and
structural influences of outside regions are important interaction
determinants of NR box-containing domains or proteins.
We have demonstrated that the C-terminal RIP140 interaction domain
could not only compete for binding of wild-type RIP140 but,
importantly, also for binding of wild-type SRC-1 to GST-PPAR in
pull-down assays. The competition observed in GST pull-down assays
indicates similar interaction sites of RIP140 and SRC-1 on the LBD, but
does not allow conclusions about the relative binding affinity of both
cofactors. However, preliminary data (not shown) suggest that
competition between RIP140 and SRC-1/TIF-2 also occurs in bandshift
assays with receptor heterodimers using equal amounts of cofactor
protein, suggesting that RIP140 and SRC-1/TIF-2 bind with similar
affinities to receptors. The importance of competition in
transcriptional activation may be illustrated, for example, by the
recent demonstration that competitive interaction occurs between
dTAFII 230 and the VP16 transcriptional activation domain
on the TBP surface (72). Similarly, competitive binding between the
adenovirus E1A protein and positive cofactors, such as the histone
acetyltransferase P/CAF to the same domain of CBP/p300, might
contribute to the negative effect of E1A on CBP/p300 signaling (67).
Interestingly, competition between RIP140 and SRC-1 might act at a
similar level considering the recent discovery of intrinsic HAT
activity in SRC-1 coactivators and functional evidence for the
existence of a complex between multiple HAT proteins. The capability of
RIP140 to compete for binding of SRC-1 coactivators in vitro
strongly suggests that the dominant negative in vivo effect
of transiently expressed RIP140 on nuclear receptor activation or on
AF-2-dependent synergism between nuclear receptors and transcription
factors such as Pit1 (73) is primarily due to competition for binding
and not to squelching or active repression mechanisms. Our finding
concerning competition between RIP140 and SRC-1 has potentially
important general implications for the mode of interaction of NR
box-containing cofactors with the LBD. Considering the different
interaction affinities of NR box peptides derived from several
cofactors, one could assume that not all NR box-containing proteins
bind with similar affinities to the LBD. For example, it remains to be
seen whether cofactors such as CBP/p300 bind competitively with RIP140
or SRC-1 to receptors. Competitive binding will undoubtedly favor a
limited number of cofactors with a relatively high binding affinity
over the majority of low-affinity cofactors. In consequence, the number
of biologically relevant cofactors might be smaller than anticipated
from the abundancy of cofactors described today.
A Regulatory Role for RIP140
Our study supports the concept that cofactors such as RIP140 may
function as regulatory proteins by critically influencing the
stoichiometry of individual components of a transcriptional activation
complex. Accordingly, a cell type expressing dominant levels of RIP140
would be predicted to exhibit a lower level of receptor activity
compared with one with dominant levels of p160 coactivators. With the
recent cloning of AIB1 (also known as p/CIP, ACTR), the third member of
the SRC-1 coactivator class, from chromosomal regions amplified in
certain breast and ovarian cancers (74), it became apparent how
overexpression of one critical cofactor might determine the activity of
the entire receptor-cofactor complex, resulting in deregulation of
normal cellular functions. Current models suggest a role of RIP140 in
regulating receptor activity directly as a coactivator and bridging
factor to the basal transcription complex. However, consistent with
previous suggestions for a role of RIP140 in receptor deactivation
(46), our findings indicate that RIP140 may alternatively regulate
nuclear receptor activity through competition with coactivators such as
SRC-1.
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MATERIALS AND METHODS |
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Yeast Expression Plasmids
All GAL4 DNA-binding domain (aa 1147) fusion constructs were derived
from the 2 µm plasmids pGBT9 or pAS2/21 (CLONTECH, Palo Alto, CA).
GAL-PPARLBD (aa 166468) served as bait for the two-hybrid
screening and was constructed by inserting a PCR fragment into
EcoRI-SalI linearized pGBT9. All GAL4-receptor
LBD fusion proteins (for details see figure legends) were expressed
from pAS21 after cloning the appropriate PCR fragments into the
EcoRI-SalI linearized vector. The GAD (aa
768881) fusion constructs were derived from the 2-µm plasmids
pGAD10, pGAD424, pGAD-GH, or pACT2 (CLONTECH). The GAD fusions to
hRIP140 (aa 431-1158) and rRXRß (aa 153451) were isolated in
two-hybrid screenings from pGAD10 cDNA libraries (CLONTECH) with
GAL4-rPPAR
as bait. GAD-SRC-1 was constructed by PCR amplification
of the SRC-1 coding sequence (aa 11061) from pBK-CMV-SRC-1 (gift from
B. W. OMalley) and cloning into BamHI-XhoI
linearized pACT2. GAD-RIP140 constructs were generated as follows:
GAD-RIP140 WT was constructed by PCR amplification of the RIP140 coding
sequence (aa 11158) from pEF-RIP140 (gift from M. G. Parker) and
cloning into BamHI-XhoI linearized pACT2. The
GAD-RIP140 fragments 36 were constructed by PCR amplification and
cloning into EcoRI-SalI linearized pGAD-GH.
GAD-RIP140 (aa 431-1158, fragment 2) was created by transferring hPIP32
as a BglII fragment from the pGAD10-construct to pACT2
cleaved with the same enzymes. GAD-RIP140 (aa 1472, fragment 7) was
derived from GAD-RIP140 (aa 11158) using partial digestion with
BamHI and BglII and religation. GAD-RIP140 (aa
1281, fragment 8) was derived from GAD-RIP140 (aa 11158) using
digestion with XhoI and religation. To create GAD-RIP140 (aa
431745, fragment 9), an EcoRI- SalI fragment
from GAD-hPIP32 (RIP140 aa 431-1158) was cloned into the corresponding
site of pACT2. The yeast expression vector pYEX-RIP140 C (aa 431-1158)
was constructed by internal deletion of an Asp
718-XhoI fragment (encoding the GAL4 activation domain of
pGAD10) from pGAD10-hPIP32. pYEX was made subsequently from pYEX-RIP140
C by internal deletion of the RIP140 BglII fragment. pYEX
RIP140WT and SRC-1WT fusions were made by inserting a BglII
fragment from the corresponding pACT2-constructs into the
BamHI site of pYEX.
Mammalian Expression Plasmids
Expression plasmids for RIP140 and SRC-1 were created by cloning of
BglII fragments from the corresponding pACT2-constructs into
pSG5 (Stratagene, La Jolla, CA). The PPRE luciferase reporter was
generated by inserting a double-stranded oligonucleotide derived from
the rat cytochrome P4504A6 gene (CYP4A6) (76) promoter
5'-AGCTTCTGAACTAGGGCAAAGTTGAG-3' into a thymidine kinase
(tk)-luciferase vector.
Plasmids for in Vitro Transcription/Translation
Proteins were synthesized in vitro using the T3 or T7 RNA
polymerase- based, rabbit reticulocyte lysate-coupled
transcription-translation kit (TNT, Promega, Madison, WI).
pBK-CMVHA-RIP140WT was constructed by PCR amplification of the RIP140
coding sequence (aa 11158) from pEF-RIP140 (gift from M. G.
Parker) and cloning into BglII-XhoI linearized
pBK-CMV (Stratagene) containing a hemagglutinin epitope (77).
pBK-CMVHA-RIP140 (aa 431-1158) was created by transferring hPIP32 as a
BglII fragment from the pGAD10 construct to the pBK-CMVHA
linearized with the same enzyme. The RIP140 fragments 36 (for details
see Fig. 7B) were constructed by cloning
EcoRI-SalI fragments from the corresponding GAD
yeast two-hybrid plasmids into pBK-CMVHA linearized with
EcoRI-XhoI. pBK-CMVHA-RIP140 N (aa 1472,
fragment 7) was made by cloning a BamHI-BglII
fragment from pBK-CMVHA-RIP140 WT into pBK-CMVHA linearized with
BglII.
rPPAR1 (aa 1475) was inserted after PCR amplification into a
NdeI-linearized derivative of pET19B (Novagen, Madison, WI).
pGEM3Z (Promega) containing rRXR
(aa 1467) has been described
previously. Plasmids expressing N- or C-terminal deletion variants of
rRXR
(RXR
N: aa 103467, RXR
C: aa 1457, RXR
N/C: aa
103467) were created by PCR amplification and subcloning into pGEM
vectors. pT7-hTRß (aa 1410) was a gift from Stefan Nilsson.
GST/HIS Fusion Constructs
To create pGEX fusion constructs to rPPAR (aa 166468), hTR
(aa
122410), rRXRß (aa 153451), and hTFIIB (aa 1316),
EcoRI-SalI fragments from the corresponding yeast
two-hybrid plasmids or pBK-CMV-derivatives were inserted into pGEX4T-1
(Pharmacia, Piscataway, NJ) linearized with
EcoRI-SalI. pGEX-rPPAR
(aa 175475) was
constructed by PCR amplification and insertion into pGEX4T-3 linearized
with BamHI-NotI. Similarly, GST-RIP140 N (aa
1281) and GST-RIP140 C (aa 747-1158) were derived from the
corresponding yeast two-hybrid plasmids. (His)10-tagged
RIP140 (aa 747-1158) was constructed by cloning a
BamHI-NotI fragment from pGEX-RIP140 C into pET19
(Novagen).
Yeast Two-Hybrid Interaction Screening
To identify PPAR-interacting proteins, a human liver cDNA
library (CLONTECH) in the activation domain vector pGAD10 was
introduced into the yeast reporter strain HF7c (MATa, ura-52, his
3200, lys 2801, ade 2101. trp 1901, leu 23, 112, gal4542,
gal80538, LYS::GAL1-HIS3, URA3::(GAL4 17
mers)3 -CYC1-lacZ) bearing pGBT9-PPAR
LBD. More than 3 x 106 transformants (as determined
from plating on SD media lacking leucine and tryptophan) were plated
onto selective synthetic medium (SD) lacking histidine, leucine, and
tryptophan and grown for 35 days at 30 C. From 89 HIS+
colonies, 69 remained positive after restreaking onto fresh selective
plates. When assayed for ß-galactosidase activity using an X-gal
filter assay, yeast from eight colonies turned blue. Library plasmid
DNA from all HIS+ colonies was isolated after
electroporation of total yeast DNA into Escherichia coli
strain HB101 and selection on synthetic M9 media lacking leucine,
followed by PCR analysis with GAD10-specific primers to detect
insert-containing library plasmids. After classification using PCR and
restriction analysis, 33 different cDNA inserts were sequenced using
the GAD10 5'-primer.
Yeast Two-Hybrid Interaction Assay/Coactivation Assay
The mating approach was used for both yeast two-hybrid and
coactivation assays. Briefly, pGAD or pYEX plasmids were introduced
into the reporter strain Y187 (MAT, ura352, his 3200, ade
2101. trp 1901, leu 23, 112, gal4
, met-, gal80
,
URA3::GAL1-lacZ) and mated with HF7c (MATa)
bearing various GAL4 constructs (pGBT9 or pAS2-derivatives) for 1216
h in liquid YPD (yeast-peptone-dextrose)-rich medium. Diploid strains
were selected for the presence of both Leu and Trp plasmids on plates
lacking tryptophan and leucine. Qualitative yeast growth assays,
quantitative liquid ß-galactosidase assays, and all standard yeast
manipulations were as essentially described.
Western Blotting
Yeast Two-Hybrid Assay
Yeast whole-cell extracts were prepared as described (78) and
fractionated by SDS/PAGE, and proteins were transferred onto a
nitrocellulose filter (Amersham, Arlington Heights, IL). Filters were
blocked with 5% milk powder in PBS-containing 0.5% Tween 80 and
incubated with a 1:1000 dilution of a mouse monoclonal antibody raised
against the GAL4 DNA binding domain (RK5C1, Santa Cruz Biotechnology,
Santa Cruz, CA) in PBS/Tween 80 for 60 min at room temperature. After
washing, the filters were incubated with horseradish
peroxidase-conjugated antimouse IgG antibody (Amersham) at a dilution
of 1:2000 in PBS/0.5% Tween 80 for 60 min. After washing, the GAL4
fusion proteins were visualized with x-ray film using an enhanced
chemiluminescense system (ECL, Amersham).
GST Pull-Down Competition Assay
Proteins in 1x SDS sample buffer were subjected to standard Western
analysis. GST-PPAR was detected with an rabbit polyclonal antibody
(PA3820, Affinity BioReagents, Golden, CO) recognizing the conserved
C terminus of all PPAR subtypes, and HIS-RIP140C was detected with a
mouse monoclonal antibody (dia 900, Dianova, Hamburg, Germany)
recognizing the HIS tag.
Expression and Purification of GST- and HIS-Tagged Proteins
Log-phase cultures of E. coli BL218(DE3) carrying the
appropriate fusion constructs were grown in LB medium containing 0.5%
casamino acids and 0.5% glucose at 30 C and were induced with 0.2
mM isopropyl ß-D-thiogalactoside for 23 h.
Cells were recovered by centrifugation and lysed in resuspension buffer
[1xPBS, 1 mM phenylmethylsulfonyl fluoride, 0.5 mg/ml
lysozyme, 10 mM MgCl2, 1 mM
MnCl2, 10 µg/ml deoxyribonuclease (DNase) I, 10 µg/ml
ribonuclease (RNase) A] for 30 min with rotation at 4 C. The lysates
were clarified by centrifugation at 10,000 x g for 30
min at 4 C and immediately used for the binding reaction (GST pull-down
assay). HIS-tagged RIP140 for gel-shift assays was purified on a TALON
affinity column after standard protocols (CLONTECH) and dialyzed
against the bandshift buffer (20% glycerol, 5 mM
dithiothreitol, 5 mM EDTA, 250 mM KCl, 100
mM HEPES, pH 7.5, 25 mM MgCl2,
0.05% Triton X-100). Protein concentration was determined by the
Bradford dye binding procedure (Bio-Rad Laboratories, Richmond, CA) The
stability of the proteins and concentration were confirmed by SDS-PAGE
analysis followed by Coomasie blue staining.
In Vitro Protein-Protein Interaction Assay (GST
Pull-Down)
Approximately 1 µg GST fusion protein bound to
glutathione-Sepharose-4B beads was incubated for 2 h with 4 µl
in vitro-translated [35S]methionine-labeled
protein in the presence of 1 µl ligand in dimethylsulfoxide (DMSO)
(final concentration between 1100 µM) or DMSO alone in
a total volume of 100 µl incubation buffer (50 mM KPi, pH
7.4; 100 mM NaCl; 1 mM MgCl2; 10%
glycerol; 0.1% Tween 20, 1.5% BSA) with rotation at 4 C. In the
competition assay, purified HIS-tagged RIP140 was included into the
binding reaction. Beads were collected by microfugation and washed
three times for 15 min with incubation buffer without BSA. Washed beads
were resuspended in 50 µl 1xSDS sample buffer, heated in boiling
water for 5 min, and pelleted in a microfuge, and 1015 µl of the
supernatant were subjected to SDS-PAGE. To control the stability of the
GST-fusion proteins and equal loading, gels were stained with Coomasie
blue before autoradiography. For quantification, autoradiographs were
analyzed using the GelPro Software (Media Cybernetics, Silver Spring,
MD).
EMSAs
Receptor proteins were synthesized in rabbit reticulocyte lysate
using the TNT coupled in vitro transcription/translation
system (Promega). Double-stranded oligonucleotides (1 µg) containing
either a PPRE corresponding to the rat ACO gene promoter (2)
5'-CTAGCGATATCATGACCTTTGTCCTAGGCCTC-3' or a synthetic DR4-TRE
5'-TCGATCAGGTCATTTCAGGTCAGAG-3' were end-labeled with
[-32P]ATP. Binding reactions (20 µl) included 1x
reaction buffer (5% glycerol, 5 mM dithiothreitol, 5
mM EDTA, 250 mM KCl, 100 mM HEPES,
pH 7.5, 1 µg poly(deoxyinosinic-deoxycytidylic)acid, 25
mM MgCl2, 1 mg/ml BSA, 0.05% Triton X-100),
protease inhibitors ('Complete', Boehringer Mannheim, Indianapolis,
IN), 0.5 ng labeled probe, 2 µl of each in vitro
translated receptor proteins, and, where indicated, 1 µl ligands in
DMSO. Purified GST or HIS-RIP140 protein (usually 200 ng/reaction) was
added last and binding was allowed to proceed for 20 min on ice.
Reactions were loaded on a 4% nondenaturing polyacrylamide gel
containing 5% glycerol and electrophoresed for 2 h in
0.5xTris-borate-EDTA at 4 C. Gels were dryed and autoradiographed.
Mammalian Cell Culture, DNA Transfections, and Luciferase
Assays
CV-1 cells were maintained in DMEM supplemented by 10% FCS
(GIBCO BRL, Gaithersburg, MD), 100 µl/ml penicillin, and 100 µl/ml
streptomycin (GIBCO BRL). Cells were transiently transfected by the
calcium phosphate method. Cotransfections were performed using 1 µg
PPRE-tk-luc per plate and indicated amounts (16 µg) of pSG-derived
expression vectors in the absence or presence of 5 µM
BRL49653. Lysated cells were mixed with luciferin reagent and ATP
reagent (Bio-Orbit) in the luminometer (Anthos Labtec Instruments,
Salzburg, Austria) according to the protocol of GENGlow-1000
(Bio-Orbit, Turku, Finland). Cells were harvested 20 h after
transfection. Diagnostic cotransfections with a control plasmid showed
the reproducibility of the transfections. Therefore, the luciferase
activities achieved did not have to be corrected; rather, the mean and
SD from independent triplicate experiments are
presented.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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E. T. is a recipient of a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft, and J. L. received a grant from the European Community (Marie Curie Fellowship Association). This work was supported by a grant from the Swedish Cancer Society.
Received for publication October 10, 1997. Revision received January 27, 1998. Accepted for publication February 18, 1998.
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REFERENCES |
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