From the Division of Endocrinology and Diabetes,
University Hospital Geneva, CH-1211 Geneva 14, Switzerland, the
§ Division of Genetics, Brigham & Women's Hospital,
Boston, Massachusetts 02115, and the ¶ Clinique de
Médecine II, Department of Medicine, University Hospital Geneva,
CH-1211 Geneva, Switzerland
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ABSTRACT |
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The peroxisome proliferator-activated
receptors (PPARs) are a subgroup of nuclear receptors activated by
fatty acids and eicosanoids. In addition, they are subject to
phosphorylation by insulin, resulting in the activation of PPAR The peroxisome proliferator-activated receptors
(PPARs)1 are nuclear hormone
receptors, the transcriptional activity of which is mostly thought to
be regulated by the binding of ligand, such as certain fatty acids and
eicosanoids (1, 2). However, the activity of some nuclear receptors is
also modulated by phosphorylation, usually resulting in transcriptional
enhancement, e.g. through the increased binding to ligand or
DNA (3-9). Similarly, PPARs were previously shown to be
phosphorylated, resulting in altered transcriptional activity depending
on the isoform and cellular systems examined (10-14). Whereas the
transcriptional capacity of the mostly adipocyte-specific PPAR The PPAR In the present study, we examined the hypothesis that PPAR Plasmid Constructs and Mutagenesis--
A fusion construct
linking the GAL4-DNA-binding domain (amino acids 1-147) upstream of
the amino-terminal region (amino acids 1-92) of hPPAR
Site-directed mutagenesis was performed by polymerase chain reaction
amplification of the entire pM-GAL4BD-hPPAR
The reporter plasmid pG5-chloramphenicol acetyltransferase (CAT)
(CLONTECH, Stehelin AG, Basel, Switzerland)
contains five consensus GAL4 binding sites (UASG 17-mer
(×5)). As a positive control, the plasmid pM3-VP16,
encoding a fusion protein of the GAL4 BD and the VP16 activation
domain, was used. The reporter plasmid pBL-1xPPRE-MEp-CAT8+, containing
the natural PPAR-response element from the malic enzyme promoter, was
described previously (17).
The pSV2-TR Transfection Experiments--
The human HepG2 hepatoma cell line
was cotransfected using the calcium phosphate method as described
previously with 4.2 µg of the expression plasmid for the parental
vector pM-GAL4BD or the fusion protein
pM-GAL4BD-hPPAR Phosphorylation Experiments--
HepG2 cells were transfected
with 4.6 µg of pM-GAL4BD or either wild-type or mutant
pM-GAL4BD-hPPAR
Immunoprecipitated products were analyzed by 10% SDS-polyacrylamide
gel electrophoresis.
Western Blotting--
As described above, HepG2 cells were
transfected with 4.6 µg of pM-GAL4BD or the appropriate
pM-GAL4BD-hPPAR The Amino Terminus of hPPAR
To examine whether the previously described stimulation of the basal
transcriptional activity of PPAR
Because we have shown recently that insulin rapidly stimulates the
phosphorylation of PPAR The Amino Terminus of hPPAR Serines 12 and 21, but Not 76/77, Mediate the Insulin Stimulation
of the AF-1 Domain of PPAR
These results are thus compatible with a model in which insulin
activates the AF-1 region of PPAR Serines 12 and 21 of hPPAR Expression of GAL4BD-PPAR Serines 12 and 21 Are Required for the Insulin-mediated Increase in
Transcriptional Activity of the Full-length PPAR Phosphorylation of the AF-1 Region of PPAR The human PPAR The concept that nuclear hormone receptors contain a ligand-independent
(AF-1) and a ligand-dependent (AF-2)
trans-activation domain has been well demonstrated for the
steroid and retinoid receptors (18, 20, 26). However, whereas the
activity of the AF-2 domain is regulated by the binding of ligand
resulting in the recruitment of coactivator proteins, the AF-1 region
is conventionally thought to be constitutively active by molecular mechanisms that are poorly understood (27). The presence of a strong
AF-1 region in the A/B domains of hPPAR In contrast to PPAR The phosphorylation sites in PPAR In summary, we have demonstrated that PPAR,
while inhibiting PPAR
under certain conditions. However, it was
hitherto unclear whether the stimulatory effect of insulin on PPAR
was direct and by which mechanism it occurs. We now demonstrate that
amino acids 1-92 of hPPAR
contain an activation function
(AF)-1-like domain, which is further activated by insulin through a
pathway involving the mitogen-activated protein kinases p42 and p44.
Further analysis of the amino-terminal region of PPAR
revealed that
the insulin-induced trans-activation occurs through the
phosphorylation of two mitogen-activated protein kinase sites at
positions 12 and 21, both of which are conserved across evolution. The
characterization of a strong AF-1 region in PPAR
, stimulating
transcription one-fourth as strongly as the viral protein VP16, is
compatible with the marked basal transcriptional activity of this
isoform in transfection experiments. However, it is intriguing that the
activity of this AF-1 region is modulated by the phosphorylation of two
serine residues, both of which must be phosphorylated in order to
activate transcription. This is in contrast to PPAR
2, which was
previously shown to be phosphorylated at a single site in a motif that
is not homologous to the sites now described in PPAR
. Although the
molecular details involved in the phosphorylation-dependent
enhancement of the transcriptional activity of PPAR
remain to be
elucidated, we demonstrate that the effect of insulin on the AF-1
region of PPAR
can be mimicked by the addition of triiodothyronine
receptor
1, a strong binder of corepressor proteins. In addition, a
triiodothyronine receptor
1 mutant deficient in interacting with
corepressors is unable to activate PPAR
. These observations suggest
that the AF-1 region of PPAR
is partially silenced by corepressor
proteins, which might interact in a phosphorylation-dependent manner.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
isoform is inhibited in certain cell types by the mitogen-activated
protein kinase (MAP-K)-dependent phosphorylation of a
serine residue in its amino terminus (Ser-112) (11, 14), we have shown
recently that PPAR
is phosphorylated in response to insulin,
resulting in an enhanced transcriptional response (10). However, it
remained unclear whether this represented a direct effect of insulin
and by which mechanism insulin stimulated the activity of PPAR
.
isoform is highly expressed in liver and brown adipose
tissue, both of which are target tissues for insulin action (15).2 PPAR
binds and is
activated by fibrates, certain fatty acids, arachidonic acid analogs
(e.g. 5,8,11,14-eicosatetraenoic acid), and leukotriene B4
(2, 15). However, in transient transfection systems, PPAR
exhibits
strong transcriptional activity even in the absence of exogenous
ligands (16, 17). Although this might be due to the presence of an
endogenous stimulator, this activity could also be mediated through a
constitutive trans-activating domain, similar to the
activation function (AF)-1 region present in some other members of the
nuclear receptor family, such as the retinoic acid and estrogen
receptors (18-20). Because we have previously demonstrated that
PPAR
is phosphorylated in response to insulin, resulting in the
stimulation of basal as well as ligand-dependent transcriptional activity, we hypothesized that PPAR
might contain a
phosphorylation-regulated trans-activation domain (10).
contains
a ligand-independent transcriptional activation domain that might be
subject to regulation by phosphorylation. We describe a novel AF-1
function within the A/B domain of PPAR
, which harbors two consensus
MAP-K sites, which are phosphorylated in response to insulin and both
of which are necessary to mediate the insulin-induced transcriptional
activation of PPAR
. Intriguingly, these MAP-K sites are distinct
from the inhibitory MAP-K site described for PPAR
. Moreover, our
results demonstrate that the transcriptional activation of PPAR
by
insulin may involve the dissociation from co-repressor proteins, which
may be related to the nuclear receptor co-repressor (NCoR) and the
silencing mediator for RAR and TR (SMRT) (21, 22). Hence, the
insulin-induced phosphorylation of PPAR
and
is mediated through
distinct phosphorylation motifs, resulting in transcriptional
activation or repression, respectively.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was
constructed by cloning the cDNA corresponding to the first 92 amino
acids of hPPAR
amplified by polymerase chain reaction using the
Pfu DNA polymerase (Life Technologies, Inc.) into the
BamHI and PstI sites of the
pM-GAL4-BD expression plasmid
(CLONTECH, Stehelin AG, Basel, Switzerland),
resulting in the vector pM-GAL4BD-hPPAR
. The expression
plasmid pSG5-hPPAR
was kindly provided by Dr. F. Gonzalez (NCI,
National Institutes of Health, Bethesda, MD) (16). The final construct
was verified by automated sequencing (ABI 373, Perkin-Elmer).
1-92 or
pSG5-hPPAR
plasmids with Pfu DNA using sense and
antisense primers containing the desired mutation: S77A mutation (AGC
GCC), S76A (TCG
GCG), S12A (TCC
GCC), S21A (AGC
GCC)
according to the manufacturer's protocol (QuickChange site-directed
mutagenesis, Stratagene, Basel, Switzerland). The presence of the
desired mutation and the absence of spurious mutations in the amplified
cDNA was assessed by automated sequencing.
1 expression vector encoding for the TR
1 was
described previously (23). The triple mutant
pcDNA/Amp-TR
1-
NCoR box (A223G/H224G/T227A) was generated by
site-directed mutagenesis. This mutant was shown previously to be
deficient in its interaction with corepressor proteins (21).
1-92, together with 0.42 µg of the
reporter plasmid pG5-CAT (17, 24). Twenty-four hours before cell
harvesting, cells were treated with 1 µM insulin and/or 30 µM of the MAP-K p42/p44 inhibitor PD98059 (Alexis
Corp., Läufelfingen, Switzerland) dissolved as described in the
manufacturer instructions. CAT activity was determined as described by
determining the incorporation of [14C]chloramphenicol
(Hartmann Analytic, Braunschweig, Germany) into acetyl-CoA as analyzed
by thin layer chromatography (24). The results were quantitated by a
PhosphorImager (Molecular Dynamics), and CAT activity was normalized to
the protein concentration as measured by the Bradford method
(Bio-Rad).
constructs. Twenty-four hours before
harvesting, cells were incubated in serum-free and phosphate-depleted
Dulbecco's modified Eagle's medium (Life Technologies, Inc., Basel,
Switzerland) followed by labeling with
[32P]orthophosphate for 2 h. Insulin (1 µM) was added for 20 min as described previously (10).
Cells were lysed in radioimmune precipitation buffer (0.15 M, NaCl, 10 mM sodium phosphate, pH 7.2, 2 mM EDTA, 1% Nonidet P-40, 1% deoxycholate, 50 mM NaF, CompleteTM protease inhibitors
(Boehringer Mannheim) and 5 mM orthovanadate), and the
fusion proteins were immunoprecipitated with a polyclonal anti-GAL4BD
antibody (1 µg) (Santa Cruz Biotechnology, Inc., Dr. Glaser AG,
Basel, Switzerland).
constructs. After 48 h, cells were
lysed in radioimmune precipitation buffer, and the fusion proteins were
immunoprecipitated by using 1 µg of rabbit polyclonal anti-GAL4BD
antibody. After electrophoresis, the proteins were electrotransferred
to a 0.45-µm nitrocellulose membrane (Bio-Rad) for 45 min at 100 V
(4 °C) in 25 mM Tris base, 190 mM glycine,
20% (v/v) methanol. Nonspecific binding was blocked by incubating the
membrane for 2 h in 5% powdered milk in TBST (20 mM
Tris, 150 mM NaCl, 0.1% Tween, pH 7.6). A monoclonal
anti-GAL4BD antibody (CLONTECH, Stehelin AG, Basel,
Switzerland) was added to a solution of 1% powdered milk/TBST for
12 h. The membrane was washed twice with TBST, followed by five
rinses of 15 min each in TBST. The second antibody (anti-mouse IgG
horseradish peroxidase (Santa-Cruz Biotechnology, Inc., Dr. Glaser AG,
Basel, Switzerland)) was diluted (1:1000 v/v) and was added for 2 h at 4 °C. After incubation, the membrane was washed as described
above. Immunolabeled proteins were revealed by chemiluminescence as
described by the manufacturer (Amersham Pharmacia Biotech, Zurich, Switzerland).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(amino acids 1-92) Contains a
Ligand-independent Activation Domain (AF-1), Which Is Regulated by
Phosphorylation--
In order to explore the possibility that the
previously described significant constitutive activity of the
full-length hPPAR
is due to an AF-1-like function in the amino
terminus of this protein, we created a fusion construct linking the
GAL4 DNA-binding domain to the first 92 amino acids of hPPAR
(pM-GAL4BD-hPPAR
). The transcriptional response of this construct
was assessed in transient transfection experiments in HepG2 cells using
a UAS-CAT reporter (pG5-CAT). Fig. 1
shows that the addition of the first 92 amino acids of hPPAR
to the
GAL4BD enhances the constitutive transcriptional activity over 20-fold,
as compared with an 80-fold stimulation by the known strong
transcriptional activator VP16.
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Fig. 1.
Transcriptional activity of the fusion
protein GAL4BD-hPPAR (1-92) on a GAL4 binding
site UASG 17 mers (×5) cloned upstream of a CAT reporter
gene. The effect of insulin and of the MAP-K p42/p44 inhibitor
PD98059 were tested. HepG2 cells were transiently transfected with
either pM-GAL4BD, pM-GAL4BD-hPPAR
(amino
acids 1-92), or the positive control plasmid pM3-VP16 together with
the pG5-CAT reporter construct. Twenty-four hours prior harvesting,
cells were either left untreated (treated with vehicle
(none)) or treated with insulin (1 µM),
PD98059 (30 µM), or both. Transcriptional activity is
expressed as relative CAT activity normalized to protein content
(percentage of GAL4BD-hPPAR
(1-92) activity with vehicle). Values
are the mean ± S.E. of four separate experiments, each performed
in triplicate.
by insulin might involve this novel
AF-1 domain, similar experiments were performed in the presence of 1 µM insulin for 24 h. As shown in Fig. 1, treatment with insulin enhances the activity of the amino terminus of PPAR
over 4-fold, whereas no effect was seen on the control constructs GAL4BD and VP16.
in adipose tissue and transfected cells, we
also assessed the effect of insulin on PPAR
in the presence of the
specific inhibitor of the p42/44 MAP-K pathway PD98059 (25). Compatible
with the hypothesis that the stimulation of the amino terminus of
PPAR
by insulin involves a MAP-K-mediated phosphorylation event,
PD95059 completely abolished this effect (Fig. 1).
Contains Several Putative MAP-K
Sites--
Because the results described above suggest that insulin
modulates the basal transcriptional activity of PPAR
through the phosphorylation of a MAP-K site in its amino terminus, we examined the
known sequences of PPAR
from various species for conserved MAP-K
motifs (PXnSP) (Fig.
2). Whereas several MAP-K motifs are
present within the first 92 amino acids of hPPAR
, three putative
sites were highly conserved across evolution: serine 12, serine 21, and
serine 77. Interestingly, the latter residue corresponds to a MAP-K
motif described in mPPAR
2, which has been shown to be phosphorylated
by MAP-K and results in an inhibition of transcription.
Hence, we created the corresponding mutants of the
pM-GAL4BD-hPPAR
construct for the functional analysis of
these sites (pM-GAL4BD-S12A, pM-GAL4BD-S21A, and
pMGAL4BD-S76A/S77A).
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Fig. 2.
Sequence alignment of the amino terminus of
PPAR across species. Putative MAP-K sites
are marked in boldface (h, human; r,
rat; m, mouse; cg, hamster; x,
Xenopus).
--
Using the various mutants of the
putative MAP-K sites within the amino terminus of PPAR
, we performed
transient transfection experiments in HepG2 cells to assess their
functional relevance. Fig. 3 shows that
the serine to alanine mutations at positions 12 and 21 were both able
to abolish the stimulatory effect of insulin, whereas similar mutations
of another putative MAP-K site (Ser-76/77) did not alter the fold
transcriptional stimulation by insulin. The lower transcriptional
activity of the S76A/S77A mutant correlates with its lower expression
as assessed in Western blot experiments (data not shown).
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Fig. 3.
Transcriptional activity of various mutants
of the putative MAP-K sites in the amino-terminal region of
PPAR . HepG2 cells were transiently
transfected with the pG5-CAT reporter gene together with either
wild-type pM-GAL4BD-hPPAR
(1-92) or the mutated
constructs containing the mutations S12A, S21A, or S76A/S77A. Cells
were treated with either vehicle or insulin (1 µM)
24 h prior harvesting. Transcriptional activity is expressed as
relative CAT activity, normalized to protein content. Values are the
mean ± S.E. of three separate experiments, each performed in
triplicate.
through the MAP-K-mediated phosphorylation of serines 12 and 21.
Are Phosphorylated in Response to
Insulin--
In order to test the hypothesis that insulin mediates the
phosphorylation of serine residues 12 and 21, we performed
phosphorylation experiments in HepG2 cells transfected with the
appropriate wild-type and mutant pM-GAL4BD-PPAR
constructs. Shown in Fig. 4 are the phosphorylated fusion proteins after precipitation with an anti-GAL4BD antibody, resulting in a main band at 39 kDa, corresponding to the
apparent molecular mass of the GAL4BD-PPAR
fusion protein (CLONTECH document 5399-1), as well as a smaller
band, which is likely to correspond to a proteolytic fragment. These
experiments demonstrated a 2.8-fold increased phosphorylation of
wild-type GAL4BD-PPAR
in response to insulin, which is
quantitatively comparable to the 4.5-fold enhancement in
trans-activation observed above. However, whereas the single
mutants S12A and S21A were still phosphorylated by insulin, the double
mutant S12A/S21A did not show an increase in its phosphorylation status
after treatment with insulin. These data demonstrate that serines 12 and 21 are both phosphorylated by insulin and that no other residues
contribute to the insulin-enhanced phosphorylation of the amino
terminus of PPAR
.
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Fig. 4.
Effect of insulin on the level of
phophorylation of the GAL4BD-hPPAR (1-92)
wild type (WT) and the mutated constructs S12A, S21A,
and S12A/S21A. HepG2 cells were transfected with
pM-GAL4BD, pM-GAL4BD-hPPAR
(1-92)
wild-type, or the appropriate mutants. Twenty-four hours before
harvesting, cells were placed in serum-free and phosphate-free medium,
followed by labeling with [32P]orthophosphate for 2 h, and stimulated with insulin (1 µM) for 20 min. After
cell lysis, fusion proteins were immunoprecipitated with anti-GAL4BD
antibodies and submitted to SDS-polyacrylamide gel electrophoresis.
Dried gels were autoradiographed and exposed on a PhosphorImager.
and Its Mutants at the Protein
Level--
In order to ascertain that the absent response of the
GAL4BD-PPAR
-S12A and S21A mutants was not due to the altered
expression of these proteins, we performed Western blot analyses with
an anti-GAL4BD antibody. As shown in Fig.
5, the S12A and S12A/S21A mutants were
expressed at similar levels as the wild-type protein, whereas the S21A
mutant was somewhat less abundant.
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Fig. 5.
Level of expression of the wild-type and
mutant GAL4BD-hPPAR constructs assessed by
Western blotting. HepG2 cells were transfected with
pM-GAL4BD, or the wild-type or mutant
pM-GAL4BD-hPPAR
(amino acids 1-92) constructs. After
harvesting and lysis, the fusion proteins were immunoprecipitated with
a polyclonal anti-GAL4BD antibody and submitted to SDS-polyacrylamide
gel electrophoresis. Proteins were electrotransferred to a
nitrocellulose membrane and blotted with a monoclonal anti-GAL4BD
antibodies and revealed with an anti-mouse IgG-horseradish peroxidase
antibody. Immunolabeled proteins were visualized by
chemiluminescence.
--
In order to
examine the role of the Ser-12 and Ser-21 residues in the context of
the intact hPPAR
, both serines were mutated to alanine
(pSG5-hPPAR
-S12A/S21A). As shown in Fig.
6, the wild-type intact hPPAR
was
activated by insulin in a dose-dependent manner (p = 0.03, one-way analysis of variance), whereas the
double-mutant S12A/S21A exhibited only a minimal and statistically not
significant response to insulin (p > 0.2, one-way
analysis of variance).
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Fig. 6.
Effect of insulin on the transcriptional
activity of intact wild-type hPPAR and the
mutated hPPAR
-S12A/S21A. HepG2 cells were
transfected with either pSG5-hPPAR
or pSG5-hPPAR
-S12A/S21A
together with the reporter construct pBL-1xPPRE-MEp-CAT8+. After
transfection, the cells were exposed to vehicle or insulin (2 or 10 µM) for 24 h. Transcriptional activity is expressed
as relative CAT activity, normalized to protein content. Values are the
mean ± S.E. (n = 3). The insulin dose-response
curves were analyzed by one-way analysis of variance. Whereas insulin
had a significant effect on the transcriptional activity of wild-type
PPAR
(p = 0.03), the response of the S12A/S21A
mutant was not statistically significant.
May Result in the
Dissociation of Co-repressor Proteins--
Multiple scenarios can
possibly explain how the phosphorylation of serines 12 and 21 activates
transcription, such as the recruitment or dissociation of putative
coactivator or corepressor proteins. Therefore, we hypothesized
specifically that the phosphorylation event might result in the
dissociation of a corepressor protein, such as NCoR or SMRT, thereby
resulting in transcriptional derepression and hence activation. The
testable prediction of this model is that the addition of a strong
binder of such co-repressor proteins should exert an insulin-like
stimulation on the AF-1 region of PPAR
. In order to examine this
hypothesis, we transfected HepG2 cells with GAL4BD-PPAR
(amino acids
1-92) either alone or in the presence of increasing amounts of TR
1,
which is known to strongly interact with corepressor proteins (21, 22).
Interestingly, the addition of TR
1 was able to increase the
transcriptional activity of the AF-1 domain of PPAR
in a
dose-dependent manner and to a level corresponding to that
observed with insulin (Fig. 7A). However, to exclude the
possibility that TR
1 might squelch other proteins nonspecifically, a
similar experiment was performed with a mutant TR
1, known to be
deficient in its interaction with corepressor proteins, such as NCoR
and SMRT (TR
1-
NCoR box). As shown in Fig. 7B, this
mutant has lost its capacity to activate PPAR
, compatible with the
model that TR
1 competes for corepressor proteins, resulting in a
derepression of the AF-1 domain of PPAR
.
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Fig. 7.
TR 1, but not
TR
1-
NcoR box, mimics
the effect of insulin on the transcriptional activity of
GAL4BD-hPPAR
(amino
acids 1-92). HepG2 cells were transiently transfected
with the pG5-CAT reporter gene and either pM-GAL4BD or
pM-GAL4BD-hPPAR
(1-92), with or without wild-type
TR
1 or TR
1-
NcoR box. Transcriptional activity is expressed as
relative CAT activity, normalized to protein content. Values are the
mean ± S.E. of a representative experiment performed in
triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was previously shown to exhibit significant
ligand-independent transcriptional activity, which can be modulated by
insulin (10). The observation that insulin also enhances the
phosphorylation of PPAR in adipocytes as well as in transfected cells
raised the possibility that insulin might directly phosphorylate PPAR
, thereby enhancing its transcriptional capacity. In the present
paper, (i) we demonstrate that the amino-terminal 92 amino acids of
PPAR
contain a AF-1-like trans-activation domain, which is further activated by insulin, (ii) we demonstrate that the effect of
insulin on the AF-1 region is dependent on MAP-K p42/44 and involves
the phosphorylation of two serine residues at positions 12 and 21, and
(iii) we present data indicating that the phosphorylation of the amino
terminus of PPAR
may result in the dissociation of corepressor
proteins, which may then result in a further transcriptional enhancement of this domain.
shown in the present paper
explains, at least in part, the basal activity observed with this PPAR
isoform in transient transfection experiments. Interestingly, the
present data demonstrate that this activity is subject to regulation
through phosphorylation of two MAP-K sites. Because we have previously
shown that PPAR
is a phosphoprotein in primary adipocyte cultures
stimulated with insulin, it is quite likely that the cross-talk between
these two signaling pathways is of functional relevance. This notion is
supported by our observation that the full-length PPAR
is also
activated by insulin and that this ligand-independent
trans-activation requires serines 12 and 21, compatible with
the results obtained with the GAL4-PPAR
1-92 fusion construct.
Although not statistically significant, the full-length
S12A/S21A-PPAR
retains a minimal residual response to insulin.
However, this small effect is not mediated by the amino-terminal 92 amino acids, as supported by experiments with a PPAR
deletion mutant
lacking the amino terminus (data not shown). This residual stimulation
by insulin may thus be mediated either through other regions of PPAR
or through insulin effects on, for example, co-activators or the
general transcriptional machinery. It is also of interest to note that
treatment of primary hepatocytes with insulin for 3 days decreases the
expression of the PPAR
gene, suggesting that short-term exposure to
insulin would increase trans-activation by PPAR
(via the
phosphorylation of PPAR
), whereas a longer exposition to this
hormone might result in the down-regulation of PPAR
activity in
liver (via the decreased expression of PPAR
mRNA) (28).
, the PPAR
2 isoform, which was recently shown
to be phosphorylated in its amino terminus in response to activators of
the MAP-K pathway, is inhibited after phosphorylation of a serine
residue at position 112 as assessed by its adipogenic potential (11).
Nevertheless, when examined in different experimental systems or in the
context of GAL4-fusion constructs, the amino terminus of PPAR
can
also be stimulated by phosphorylation. Hence, it appears that at least
in adipose tissue, which coexpresses the PPAR
and
isoforms at
high levels, the phosphorylation by insulin has opposite effects on
PPAR
and
, the former being activated, whereas the latter is
inhibited. This observation, together with their different ligand
binding spectrum and differences in DNA binding, provides a novel
mechanism for generating isoform-specific responses of PPAR (17).
are highly conserved across
species, and they are distinct from the homologous MAP-K site shown to
be phosphorylated in
2 (see Fig. 2). Hence, the functionally opposite effects of insulin on PPAR
and
2 are reflected by the presence of distinct phosphorylation sites. The mechanisms mediating phosphorylation-dependent alterations in transcription
rates are still poorly understood, as is the case for the PPARs.
Because the AF-1 domain as well as the insulin-stimulated
phosphorylation sites colocalize within the same domain of PPAR
, it
is tempting to speculate that the altered phosphorylation results in
the differential recruitment of coactivator and/or corepressor
proteins. However, the currently known coactivator proteins interact
preferentially in a ligand-dependent manner with the AF-2
domain of nuclear receptors, although some cooperation with AF-1 may
occur (29, 30). However, the corepressor proteins, such as NCoR and
SMRT, interact with different regions of the nuclear receptors in a
ligand-independent manner. Therefore, our finding that TR
1 can
activate the amino terminus of PPAR
is of interest, because the TRs
are known to strongly interact with NCoR and SMRT, thereby resulting in
a putative intranuclear sink for corepressor proteins. Because this
effect is abolished by a mutation in the NCoR box of TR
1, it is
tempting to speculate that the amino terminus of PPAR
interacts with
a NCoR-like corepressor protein that is able to partially silence the
activity of AF-1. However, whether the phosphorylation of serines 12 and 21 derepresses AF-1 by inducing the dissociation of corepressors is
a matter of conjecture. Although PPAR
was shown to interact with
NCoR in solution, this interaction involved the hinge region of PPAR,
rather than the amino terminus (31). Nevertheless, some recent data
demonstrate that an anti-NCoR antibody blocks the transcriptional
repression of PPAR
by insulin, suggesting at least indirectly a
functional interaction between the phosphorylated amino terminus of
PPAR
and NCoR (32). Preliminary experiments utilizing gel mobility
shift and glutathione S-transferase pull-down assays
examining the possibility of direct interactions of NCoR with the amino
terminus of PPAR
were
inconclusive.3 These findings
indicate that either the interactions between the AF-1 region of
PPAR
and NCoR are weak under the conditions used, or alternatively,
novel corepressor proteins are involved, which have the potential to
interact with the AF-1 region as well as the conventional NCoR box
motif of nuclear receptors.
contains a strong
ligand-independent AF-1 domain, which can be further activated through
the MAP-K-mediated phosphorylation of two serine residues, which are
distinct from the inhibitory phosphorylation site present in PPAR
2.
In addition, the AF-1 region of PPAR
can be equally well activated
by squelching corepressor proteins, suggesting that such proteins might
silence the amino terminus of PPAR
. It can thus be speculated that
the phosphorylation of the AF-1 domain of PPAR
might result in the
dissociation of such corepressor proteins, thereby resulting in
transcriptional activation.
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FOOTNOTES |
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* This project was supported by Grants 3231-51 957.97 and 3200-52 192.97 from the Swiss National Science Foundation (to C. A. M.) and 3200-037536.93 (to A. G. B.) and also in part through a grant-in-aid from Boehringer-Ingelheim International Inc. (to C. A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
41-22-372-9039; Fax: 41-22-372-9329; E-mail: cameier{at}genet.ch.
2 A. Gorla-Bajszczak and C. A. Meier, unpublished data.
3 C. Juge and C. A. Meier, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; hPPAR, human PPAR; TR, triiodothyronine receptor; NCoR, nuclear receptor co-repressor; SMRT, silencing mediator for RAR and TR; MAP-K, mitogen-activated protein kinase; CAT, chloramphenicol acetyltransferase; AF, activation function.
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REFERENCES |
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