From the Department of Biochemistry and Molecular Biology, Faculty of Medicine, Sir Charles Tupper Medical Bldg., Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada
Received for publication, November 26, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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Mitogen-activated protein kinase (MAPK) is
required for cell growth and cell differentiation. In adipogenesis,
MAPK activation opposes the differentiation process. The regulatory
mechanisms or the cellular factors that regulate the switch between
growth and differentiation in the adipogenic lineage have been largely unelucidated. We show here that AEBP1, a transcriptional repressor that
is down-regulated during adipogenesis, complexes and protects MAPK from
its specific phosphatase in mammalian cells. We further show evidence
that the modulation of MAPK activation by AEBP1 is a biologically
relevant process in adipogenesis. Our results suggest that modulation
of MAPK activation by the protective effect of AEBP1 may constitute a
critical part in the determination between cell growth and
differentiation in the adipogenic lineage. The proposed mode of action
by which a transcription factor regulates MAPK activation is novel.
Adipogenesis is a complex process regulated by a variety of
hormones, growth factors, and cytokines that act via specific receptors
to transduce external growth and differentiation signals through a
cascade of intracellular events. The initiation of differentiation of
preadipocytic cell lines, such as 3T3-L1, into mature adipocytes is
induced by continuous exposure to pharmacological doses of insulin or
physiological doses of
IGF-1.1 In addition, cAMP and
glucocorticoids are generally considered necessary for the induction of
differentiation. However, the mechanistic actions of insulin and other
ligands on differentiation are not clearly defined (1). The
determination that the activation of the IGF-1 receptor is a potent
inducer of the differentiation process implies that a
tyrosine-kinase-mediated signaling pathway is involved in adipocyte
differentiation. Furthermore, constitutive expression of Ras or Raf-1
(two components of receptor tyrosine kinase pathways) can induce
preadipocyte differentiation in the absence of insulin or IGF-1 (2, 3).
However, Raf-1 was able to induce only partial differentiation,
indicating that Raf-independent pathways downstream of Ras may be
involved in adipocyte differentiation. Moreover, the signals generated
by Raf-1 did not activate MAPK or a 90-kDa S6 kinase, suggesting
a functional dissociation between Raf-1 and MAPK/S6 kinase activation
in Ras signaling pathways leading to 3T3-L1 differentiation (4).
Moreover, it was demonstrated that MAPK activation is not required for,
but rather antagonizes, the differentiation process (5). In sharp
contrast, studies by Sale et al. (6) showed that MAPK is a
key element in adipogenesis, with MAPK activation being required for
signaling initiated by insulin and serum stimulation, for activation of
DNA synthesis, and for the induction of differentiation in 3T3-L1 cells.
MAPK is activated by phosphorylation at specific threonine and tyrosine
residues on the MAPK molecule by the upstream kinase, MAPK kinase
(MAPKK, or MEK (7- 9)), and if this activation is sustained, MAPK
translocates to the nucleus. However, if this activation is transient,
MAPK is prevented from translocating (10). MAPK phosphorylation
(activation) is a reversible process, in which protein phosphatases
play a crucial role in controlling cellular activities. Because MEK is
known to be transiently activated by phosphorylation, the regulation of
prolonged or transient MAPK activation may be through phosphatases (10,
11). An emerging class of dual-specificity phosphatases that directly
and specifically regulate MAPK family members has been characterized.
Among them is the dual-specificity phosphatase PYST1 (also called MKP3
or rVH6) that is selective for inactivation of the
extracellular-signal-regulated kinase (ERK) family MAPKs (ERK1 and
ERK2) (12-14).
We have previously identified a novel transcriptional repressor (termed
AEBP1) with carboxypeptidase (CP) activity (15) that regulates an
adipocyte-specific gene (aP2); furthermore, we showed
that its CP activity is vital for the transcriptional repression
function (16). In this paper, we use coimmunoprecipitation experiments
to demonstrate that AEBP1 complexes with ERK in vivo and
in vitro. We also use ectopically expressed AEBP1, along
with the MAPK-specific phosphatase PYST1/MKP3/rVH6, to demonstrate that
AEBP1 attenuates MAPK inactivation, resulting in increased MAPK
activity. Finally, we show evidence that the modulation of MAPK
activation by AEBP1, which is independent of its transcriptional function, is a biologically relevant process in adipogenesis. This
modulation of MAPK activation by the protective effect of AEBP1
illustrates a potential new mode of regulation in adipogenesis. The
notion that a transcription factor can also function as a regulator of
MAPK activation is unexpected and unprecedented.
Cell Culture, Transfection, and Plasmids--
COS-7, 3T3-L1, and
NIH-3T3 cells were cultured in 90-mm dishes and transfected at 80%
confluence by the polybrene procedure as described previously (17).
Detailed descriptions of plasmid constructions and the generation of
the overexpressing and the antisense cell lines are available from the
authors on request. The HA-tagged expression plasmids pJ3H-AEBP1,
pJ3H-AEBP1( Immunoprecipitation and Kinase Assay--
Cell extracts were
prepared in cold radioimmune precipitation buffer containing 1 mM phenylmethylsulfonyl fluoride, 10 µg aprotinin/ml, 10 µg leupeptin/ml, 5 mM EDTA, and 5 mM EGTA.
Cell extracts were incubated with protein A-agarose (Santa Cruz
Biotechnology) for 1 h, after which the beads were discarded and
the supernatants were incubated with specific antibodies (2 µg/ml
anti-HA, anti-ERK1, anti-MEK1, or anti-IGFR, or 2.5 µg/ml of either
affinity-purified anti-AEBP1 antibodies or IgG from preimmune serum)
for 1 h and then overnight with protein A-agarose. Samples were
collected and washed four times with radioimmune precipitation buffer.
The precipitated samples were resolved by SDS-PAGE and analyzed by Amersham Pharmacia Biotech's ECL blotting system with specific antibodies (200 ng/ml). For the immunocomplex kinase assay, MAPK was
immunoprecipitated from cell lysates with specific antibodies overnight. The pelleted agarose beads were washed three times with
radioimmune precipitation buffer and equilibrated three times in kinase
buffer (30 mM Tris-HCl (pH 7.4), 10 mM
MgCl2, 0.1 mM EGTA, 0.1 mM sodium
orthovanadate, 1 mM dithiothreitol), then 15 µCi of
[ Phosphatase Protection Assay--
For the in vivo
assay, COS-7 cells transfected with plasmids expressing AEBP1, PYST1,
and HA-ERK1 were starved for 2 h in Dulbecco's modified Eagle's
medium (DMEM) containing 0.1% calf serum, then stimulated for 15 min
with 10 nM of EGF (Sigma). Cell lysates in cold radioimmune
precipitation buffer were immunoprecipitated with anti-HA antibody (2 µg/ml) for 1 h and incubated with protein A-agarose for 30 min.
The assay for MAPK activity was performed with MBP as above.
Alternatively, phosphorylated ERK was detected by immunoblotting with
Phospho-p44/42 MAPK monoclonal antibody (New England BioLabs). For the
in vitro assay, recombinant phospho-ERK2 protein (2.5 ng,
New England BioLabs) was incubated with 100 ng of recombinant AEBP1 (or
its mutant derivatives) protein for 1 h on ice. The mixture was
treated with the recombinant MAPK phosphatase rVH6 (13) for 30 min at
30 °C. The treated kinases were analyzed by immunoblotting with
Phospho-p44/42 MAPK monoclonal and p44/42 MAPK antibodies, respectively.
CAT Assay--
Transfection of NIH-3T3 cells was performed with
5 µg of paP2(3AE-1/-120)CAT and 10 µg of the fusion HA-AEBP1 (or
its mutant derivatives) expression plasmid pJ3H-AEBP1 (or the control
plasmid pJ3H-AEBP1( Adipocyte Differentiation and Lipid Staining--
Stable cell
lines were maintained in DMEM containing 10% calf serum and 200 ng of
G418/ml (Life Technologies, Inc.). At confluence, cells were treated
with DMEM containing 10% cosmic calf serum (HyClone Laboratories), 0.5 µM dexamethasone, 5 µg insulin/ml, 0.5 mM
1-methyl-3-isobutylxanthine for 2 days then maintained in DMEM
containing 10% cosmic calf serum and 5 µg insulin/ml for 6 days.
Cells were washed three times with phosphate-buffered saline, fixed by
incubating in 4% paraformaldehyde for 10 min, and stained with a
saturated oil Red O in 60% triethyl phosphate solution (BDH).
AEBP1 Is Phosphorylated by, and Associated with, MAPK--
AEBP1,
besides being a novel carboxypeptidase with transcriptional repression
function, has a serine, threonine, proline (STP)-rich region with a few
possible MAPK proline-directed phosphorylation sites in the C terminus.
Indeed, the recombinant AEBP1 protein can be phosphorylated by
immunoprecipitated MAPK, and the phosphorylation site(s) may be located
at the C terminus, because no phosphorylation was detected with a
truncated mutant form of AEBP1 (AEBP1
Immunoprecipitation and immunoblot experiments with antibodies toward
AEBP1 and phosphorylated tyrosine have indicated that AEBP1 was not
phosphorylated by a tyrosine kinase. Interestingly, two phosphoproteins
with the approximate molecular weights of the MAPKs ERK1 and ERK2 were
detected when AEBP1 immunoprecipitates were immunoblotted with
anti-phosphotyrosine antibody (data not shown). These observations
prompted us to examine the possibility that MAPK can be coprecipitated
with AEBP1. To verify this intriguing possibility, we utilized cell
extracts from different stages of adipocyte differentiation for
coimmunoprecipitation, because AEBP1 expression is abolished in
adipocytes (16). Fig. 1A
(top) shows the immunoprecipitation analysis of AEBP1
abundance during 7 days after exposure to differentiation conditions.
Expression of AEBP1 was decreased on day 1 and persisted until 4 days
after exposure to differentiation medium, then decreased on day 5 and
was subsequently abolished at the terminal stage of adipocyte
differentiation on days 6 and 7. Because AEBP1 is undetectable on days
6 and 7, we would not expect to observe any coprecipitation of MAPK on
these days, and MAPK was not detected in the AEBP1 immunoprecipitates from days 6 and 7 (Fig. 1A, middle).
Immunoblotting of the cell lysates showed no significant change in the
content of MAPK during adipocyte differentiation (Fig. 1A,
bottom).
To further assess the interaction between AEBP1 and MAPK we carried out
reciprocal coimmunoprecipitation experiments. Cell extracts were
immunoprecipitated with either anti-AEBP1 or anti-ERK1 antibodies, and
then the post-precipitated cell extracts were further
immunoprecipitated with the opposite antibodies. The precipitated samples were then analyzed by immunoblotting with anti-AEBP1 and anti-ERK1 antibodies, respectively. Fig. 1B shows that the
amount of AEBP1 that remains unbound (top, lane
2) is approximately similar to the amount of AEBP1 associated with
ERKs (top, lane 1). Conversely, the amount of
MAPK that remains unbound (bottom, lane 4) is
approximately similar to the amount of MAPK associated with AEBP1
(bottom, lane 3). These results indicate that
about 50% of AEBP1 and MAPK molecules interact with each other in
mammalian cells.
In light of the above studies, it would be interesting to determine
whether AEBP1 interacts with the phosphorylated, dephosphorylated, or
both forms of MAPK to determine whether the AEBP1-MAPK interaction was
further involved in regulation of MAPK. To determine the
phosphorylation state of the MAPK molecules that complex with AEBP1, we
used cell extracts from quiescent and from serum-stimulated 3T3-L1
cells. Activation (phosphorylation) of MAPK, which can be detected by the slower migration of the phosphorylated form in SDS-polyacrylamide gels, was induced by serum stimulation (Fig. 1C,
top). AEBP1 was then isolated from cell extracts by
immunoprecipitation with anti-AEBP1 antibody and MAPK was detected by
immunoblot analysis. Fig. 1C (bottom) shows that
ERKs are detected in the AEBP1 immunoprecipitates prepared from both
quiescent (lane 3) and the serum-stimulated cells
(lane 4). Thus, AEBP1 associates with both forms of
MAPK.
MAPK Directly Interacts with the N-terminal Domain of
AEBP1--
To further confirm that AEBP1 and MAPK interact in
vivo, extracts from cells transfected with an expression plasmid
(pJ3H-AEBP1) encoding an HA-tagged version of AEBP1 (HA-AEBP1) were
subjected to immunoprecipitation with anti-HA antisera, and the
immunoprecipitates were probed with anti-ERK1 antisera. Fig.
2A (bottom) shows
that ERK1 was detected in the HA immunoprecipitates from cells
transfected with pJ3H-AEBP1 (lane 1) but not from cells
transfected with the control plasmid (lane 4). Reciprocal
immunoprecipitation with anti-ERK1 antisera and immunoblotting with
anti-HA antisera (top) demonstrated that HA-AEBP1 is
detected from pJ3H-AEBP1-transfected cells (lane 2) but not
from cells transfected with the control plasmid (lane 5).
Similar coimmunoprecipitation experiments with an upstream component of
the MAPK signaling module showed that AEBP1 does not interact with MEK1
(data not shown). However, we cannot rule out the possibility that
AEBP1 interacts with other upstream components of the MAPK pathway.
To locate a domain(s) of AEBP1 responsible for MAPK interaction, we
generated constructs encoding mutant derivatives of the HA-tagged AEBP1
protein (Fig. 2B); these constructs were then transfected
individually and analyzed for the ability of the encoded AEBP1
polypeptides to interact with MAPK by coimmunoprecipitation experiments. Fig. 2C (bottom) shows that ERK1 was
coprecipitated from cells transfected with the wild-type (lane
1) and most mutant derivatives (lanes 3-5) but not
from cells transfected with either the mutant derivative
pJ3H-AEBP1
We developed an in vitro coimmunoprecipitation system for
assessing the interaction to determine whether AEBP1 interacts with MAPK directly or via intermediates. The wild-type and mutant
derivatives of the His-tagged recombinant AEBP1 proteins (16) were
mixed individually with the recombinant ERK2 protein, then the mixture was immunoprecipitated with anti-ERK or anti-AEBP1 antisera. The precipitates were then analyzed by immunoblotting with anti-AEBP1 or
anti-ERK antisera. As shown in Fig. 2D, the wild-type
(lane 3) and all the mutant derivatives of His-AEBP1
(lanes 5-7), except the mutant Modulation of MAPK Activation by AEBP1--
Because AEBP1
associates specifically with MAPK, AEBP1 may participate in the MAPK
signaling pathway by modulating MAPK activation. We asked whether MAPK
activity could be modulated by altering the abundance of AEBP1 in
3T3-L1 cells. The pooled stable 3T3-L1 cell lines were generated with a
retrovirus directing expression of either wild-type (AEBP1/Neo-P) or
the mutant derivatives (AEBP1
The above results suggest that AEBP1 can modulate MAPK activation.
However, because the conclusions are based on results obtained with
AEBP1-overexpressing cell lines, it cannot be concluded whether AEBP1
operates similarly at physiological concentrations. It would thus be
important to examine whether suppression of endogenous AEBP1 by
antisense RNA expression can lead to decreased MAPK activity. Cells
were transfected with an expression vector (pAS/Neo) that constitutively expresses an antisense RNA corresponding to the 5'
700-bp region of the AEBP1 transcript. We established two stable cell
lines (AS/Neo-7 and -11), which showed decreased levels of AEBP1 in
comparison to the level in the control cell line (Neo-12) established
with the empty vector. Of the two clones, AS/Neo-11 exhibited a lower
level of AEBP1 expression (Fig. 3B, top row). As
predicted, AS/Neo-11 exhibited lowest MAPK activity (24% of the
control level) (Fig. 3B, second row), but MEK
activity (detected by phospho-MEK1 antibody) was similar among these
cell lines (Fig. 3B, fourth row). These
results suggest that AEBP1 specifically modulates MAPK activation, and
the modulation may be physiologically relevant.
AEBP1 Specifically Modulates MAPK Activation by Protecting from Its
Phosphatase--
A possible mechanism of MAPK modulation by AEBP1 is
through protection of MAPK from its phosphatase. Because AEBP1 was
observed at similar levels in the nuclear and post-nuclear soluble
fractions (25), we predict that AEBP1 may protect MAPK from the
cytosolic MAPK-specific phosphatase PYST1/MKP3/rVH6. To test this
hypothesis we used ectopically expressed AEBP1, along with ERK1 and
PYST1, to demonstrate that AEBP1 attenuates ERK1 inactivation. To
establish a system that allows assessment of MAPK regulation by AEBP1,
we expressed PYST1 and measured mitogen-stimulated ERK1 activity in
transfected COS-7 cells. Fig.
4A (top) shows that
PYST1 expressed in COS-7 cells blocked heterologously expressed ERK1
(pECE/HA-ERK1) activation following stimulation with epidermal growth
factor (EGF), and this inhibition increased when cells were transfected with increasing amounts of PYST1 plasmid (pSG5/PYST1-Myc (12)). Next,
we tested whether expressing AEBP1 restored the EGF-stimulated ERK1
activation under conditions where the PYST1-mediated inhibition would
be maximal. Fig. 4B (top) shows that
EGF-stimulated ERK1 activation was recovered in a
dose-dependent manner when COS-7 cells were transfected
with the plasmid expressing AEBP1 (lanes 4-6) but not with
the control plasmid (lanes 1-3). The PYST1 inhibition is
unlikely mediated by AEBP1 binding to the phosphatase, because coimmunoprecipitation experiments did not reveal such interaction (data
not shown). To rule out the possibility that the transcriptional repression activity of AEBP1 is involved in the modulation of MAPK
activation, we repeated the protection experiments with mutant derivatives of AEBP1 defective in transcriptional repression activity but still able to interact with MAPK. We predicted that the mutants
To further substantiate the conclusion that AEBP1 modulates MAPK
activation by a protective interaction, we utilized another mutant
derivative (DLD) that consists of only the interaction domain (Fig. 2)
as a dominant-negative mutant. We would predict that this mutant
competes away MAPK from the wild-type and attenuates the protective
activity exhibited by the wild-type in a dominant-negative fashion.
Indeed, DLD expressed in COS-7 cells attenuated the protective activity
of AEBP1 in a dose-dependent manner (Fig. 4E,
top). Taken together, these results strongly suggest that
AEBP1 modulates MAPK activation through protective interaction.
To test whether AEBP1 alone is sufficient for blocking
dephosphorylation of activated MAPK by a MAPK phosphatase, an in
vitro phosphatase assay was designed. Recombinant phospho-ERK2
protein was treated with the recombinant rVH6, a MAPK phosphatase,
after incubation with either wild-type or mutant derivatives of the recombinant AEBP1 protein. The treated MAPK was then analyzed by
immunoblotting with the phospho-MAPK antibody, and we found that the
recombinant AEBP1 protein alone is not sufficient for the protection
(data not shown). These results suggest that one or more accessory
proteins are required, along with AEBP1, for stabilizing the
phosphorylation status of MAPK and making it immune to dephosphorylation.
The MAPK-protective Activity of AEBP1 Modulates the Adipogenic
Process--
MAPK maintained in its active phosphorylated form by
protection from phosphatases may cause overstimulation of MAPK
pathways. Several recent reports showed that activation of the MAPK
pathway phosphorylates a nuclear hormone receptor, PPAR
The stable cell lines that overexpressed either wild-type or mutant
derivatives of AEBP1 (Fig. 3A) were induced to
differentiate, and these cultures were monitored for the differentiated
phenotype. Examination of these cultures indicated striking differences
in the ability of the cells to accumulate lipid, a feature of mature adipocytes. It was clear that the stable cell line AEBP1/Neo-P, generated by a virus harboring AEBP1 and showing MAPK protection activity (Fig. 4D), was unable to convert to the adipocyte
phenotype (Fig. 5A).
Importantly, AEBP1
Because expression of the dominant-negative mutant DLD caused an
attenuation of the MAPK protection activity of AEBP1 (Fig. 4E), we tested whether the anti-adipogenic effect in the
AEBP1/Neo-P cell line can be attenuated by ectopic expression of the
dominant-negative mutant DLD. The stable cell line AEBP1/DLD(+), which
was derived from the AEBP1/Neo-P cell line by infection with the virus
expressing DLD (AEBP1DLD/Puro), was able to differentiate, albeit less
efficiently than the Neo-P cells (Fig. 5B). The control
stable cell line AEBP1/DLD( MAPK localizes to the cytoplasm by binding to the cytoplasmic
anchor MAPKK under quiescent conditions. After stimulation, MAPK
becomes phosphorylated by MAPKK and dissociates from MAPKK. The
dissociated MAPK then enters the nucleus by either passive diffusion or
active transport mechanisms (30). It has been postulated that the
nuclear accumulation of MAPK may require a putative nuclear anchor(s)
(31). AEBP1 may be a mammalian nuclear anchor that is responsible for
the nuclear accumulation of MAPK. The translocation is also dependent
on the protection of ERK from MKP3, a cytosolic phosphatase.
Protein-protein interaction between AEBP1 and ERK may protect ERK from
MKP3 in the cytoplasm, thus allowing ERK to be translocated to the
nucleus. It has been demonstrated that MKP3 is activated by direct
binding to regions localized within the C-terminal structural lobe of
ERK that includes regions believed to be important for substrate
binding (32-34). Thus, the protection by AEBP1 may be mediated by a
simple competitive interaction between MKP3 for ERK and through rapid
nuclear translocation.
The persistent activation of MAPK as a result of the protective effect
of AEBP1 would allow phosphorylation of different sets of proteins in
comparison to the situation for transient activation. The activation of
different subsets of transcription factors may thus determine which
genes are turned on, thereby determining cell fate. It has been
demonstrated that, in fibroblasts, sustained activation of MAPK is
associated with proliferation, not differentiation (9, 35, 36).
Sustained activation of MAPK also leads to the proliferation of 3T3-L1
cells.2 In other cell types,
the converse may be true. For example, in PC12 cells the sustained
activation of MAPK by fibroblast growth factor or nerve growth factor
induces differentiation, whereas the transient activation by EGF
induces proliferation (37). These differences in the duration of MAPK
activation appear not to be the result of MEK activation but rather of
a MEK-independent pathway in which phosphatases that inactivate MAPK
both in the cytoplasm and nucleus are regulated (10, 11, 38). Our
results further add to this, in that AEBP1 may regulate the duration of MAPK activation through its protective effect from specific phosphatases.
It has been well documented that adipose differentiation is inhibited
by a large number of mitogens and growth factors (39-41). Recently, it
has been demonstrated that this inhibition results, in part, from
MAPK-mediated phosphorylation of PPAR The apparent conflicting roles of MAPK, in which MAPK activation in
preadipocytes appears to be required for differentiation (6, 45), but
MAPK is also capable of repressing PPAR Our studies provide new information that the modulation of MAPK
activation could constitute a critical part of the molecular mechanism
of adipogenesis. The finding that the MAPK signal-transduction pathway
is stimulated in response to factors that initiate cell differentiation, and the finding that MAPK physically interacts with
AEBP1, strongly implies that this interaction is important in
adipogenesis. AEBP1 may function to block inappropriate signals by
balancing the level of activated MAPK in conjunction with regulation by
specific phosphatases, thus maintaining the preadipocyte phenotype. When the cells are stimulated to differentiate, the protective effect
of AEBP1 on MAPK activation may be attenuated, thus allowing the signal
to begin the differentiation process. Our results demonstrate a novel
function of AEBP1 as an important mediator, through its protective
effect on MAPK, in the adipogenic signal-transduction pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), pJ3H-AEBP1
DLD, pJ3H-AEBP1DLD, pJ3H-AEBP1
Hic, and
pJ3H-AEBP1
Sty were derived from pJ3H (18). The retrovirus plasmids
pAEBP1/Neo, pAEBP1
Hic/Neo, pAEBP1
Sty/Neo, and the antisense
plasmid pAS/Neo were derived from pWZLNeo (19). The retrovirus plasmids
pAEBP1DLD/Puro and pAEBP1DLD(
)/Puro were derived from pBabe-puro
(20).
-32P]ATP and specific substrate (AEBP1 or the
myelin basic protein (MBP)) were added and the mixture was
incubated for 30 min at 37 °C. Reactions were stopped by adding
SDS-PAGE loading buffer. Samples were resolved by either 8.5% (AEBP1),
or 15% (MBP) SDS-PAGE and transferred to nitrocellulose membranes.
Bands were detected and quantitated by the Molecular Imager system
(Bio-Rad Laboratories).
)) along with 1 µg of pHermes-lacZ, a plasmid
that expresses the lacZ gene under the control of the CMV
promoter.
-Galactosidase activity was assayed 48 h after
transfection to normalize transfection efficiency, and CAT activity was
assayed as described previously (16).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Sty (16)), which lacks the
C-terminal domain (data not shown). However, it is possible that
conformational changes caused by the truncation in this mutant may mask
potential phosphorylation sites in other regions of AEBP1.
Nevertheless, these results have suggested that one kinase responsible
for AEBP1 phosphorylation is MAPK.
View larger version (34K):
[in a new window]
Fig. 1.
AEBP1 associates with MAPK in mammalian
cells. A, confluent 3T3-L1 cells (day 0) were subjected
to the differentiation protocol, and total cell extracts were prepared
at the times indicated during differentiation. The extracts were
immunoprecipitated with anti-AEBP1 antibody and then immunoblotted with
either anti-AEBP1 (top) or anti-ERK1 antibodies
(middle). Bottom, Western blot analysis of the cell extracts
with anti-ERK1 antibody (Santa Cruz Biotechnology) that also
cross-reacts with ERK2. B, total protein (1 mg) from 3T3-L1
cell extracts was subjected to immunoprecipitation with either
anti-AEBP1 or anti-ERK1 antibodies (1st), and the
post-immunoprecipitation extracts were further immunoprecipitated with
the opposite antibodies (2nd). Both sets of precipitated
samples were analyzed by immunoblotting with anti-AEBP1
(top) and anti-ERK1 (bottom) antibodies,
respectively. As a control, the same amount of total protein was
immunoprecipitated with either anti-IGFR antibody (lane 5)
or pre-immune IgG (lane 6). C, top,
3T3-L1 cells were starved in DMEM containing 0.1% calf serum for
24 h, then treated with DMEM containing 15% fetal bovine serum
for various lengths of time. Five micrograms of total cell lysate
protein was resolved by SDS-PAGE. Both unphosphorylated and
phosphorylated forms of ERK1 and -2 were detected by immunoblotting
with anti-ERK1 antibody (200 ng/ml). Bottom, cell extract
from quiescent (lanes 1, 3) or serum-stimulated
(lanes 2, 4) cells was immunoprecipitated with
either anti-ERK1 (protein A-conjugated anti-ERK1 antibody, Santa Cruz
Biotechnology) (lanes 1, 2) or anti-AEBP1
(lanes 3, 4) antibodies, and then analyzed by
immunoblotting with anti-ERK1 antibody.
View larger version (32K):
[in a new window]
Fig. 2.
AEBP1 directly interacts with MAPK through
its N-terminal domain. A, equal amounts of COS-7 cell
extracts, transfected with 10 µg of pJ3H-AEBP1 (lanes
1-3) or the control plasmid pJ3H-AEBP1( ) (lanes
4-6), were used in immunoprecipitation studies with anti-HA
antibody (lanes 1, 4), anti-ERK1 antibody
(lanes 2, 5), or anti-IGFR antibody (lanes
3, 6). The immunoprecipitated samples were
immunoblotted with either anti-HA antibody (top) or
anti-ERK1 antibody (bottom). B, schematic
representations of only AEBP1 coding regions of HA-AEBP1 fusion-protein
derivatives. Small black boxes represent sequences created
by frameshift mutations. The numbers indicate amino acid
residues of AEBP1. The presence (+) and absence (
) of interaction
with MAPK is shown. C, COS-7 cells were individually
transfected with 10 µg of pJ3H-AEBP1 (lane 1) and its
mutant derivatives as indicated (lanes 2-5). Lane
6 contains material from cells transfected with the empty vector
pJ3H. Cell extracts from transfected cells were analyzed by
immunoblotting with anti-HA antibody (top). The
asterisk indicates HA-AEBP1 and its mutant derivatives. Cell
extracts were also immunoprecipitated with anti-HA antibody and then
analyzed by immunoblotting with anti-ERK1 antibody (bottom).
D, 300 ng of the His-tagged recombinant AEBP1 (lanes
1-3) and its mutant derivative (lanes 4-7) proteins
was individually mixed with (lanes 3-7) or without
(lanes 1 and 2) the recombinant ERK2 protein (300 ng). The mixtures were immunoprecipitated either with preimmune IgG
(lane 1) or p44/42 MAPK antibody (lanes 2-7).
The precipitates were immunoblotted with anti-AEBP1 antibody. The
asterisk indicates His-AEBP1 and its mutant
derivatives.
DLD (lane 2), which expresses an HA-tagged
AEBP1 that lacks the N-terminal 233 amino acids (Fig. 2B) or
the empty vector pJ3H (lane 6). Interaction with MAPK was abolished neither by deletion in the carboxypeptidase domain
(pJ3H-AEBP1
Hic, lane 4) nor by truncation at the C
terminus (pJ3H-AEBP1
Sty, lane 5). Significantly, ERK1 was
coprecipitated from cells transfected with the mutant derivative
pJ3H-AEBP1DLD (lane 3), which expresses only the N-terminal
204 amino acids (residues 32-235) of AEBP1 (Fig. 2B).
Therefore, these results indicate that the domain of AEBP1 responsible
for MAPK interaction is located at the N terminus, which contains
sequences that were previously termed discoidin-like domain (DLD (16)),
and suggest that phosphorylation is not required for AEBP1 to interact
with MAPK.
DLD with amino-terminal
deletion (lane 4), were detected in the immunoblot.
Specifically, a mutant derivative, which consists of only the
N-terminal 204 amino acids (DLD), was able to coprecipitate with the
recombinant ERK2 protein (lane 5). The apparent inability of
the N-terminal deletion mutant to interact with MAPK is due neither to
its inability to be recognized by the anti-AEBP1 antisera nor to its
prevention of immunoprecipitation by anti-ERK antisera (data not
shown). Taken together these results suggest that MAPK directly
interacts with AEBP1 at the N terminus. Examination of the sequences in
the N-terminal domain of AEBP1 did not reveal any MAPK docking sites
described previously (21-23). However, a putative MAPK-docking site,
which is characterized by a cluster of positively charged amino acids
(24), is present in the N-terminal domain of AEBP1.
Hic/Neo-P and AEBP1
Sty/Neo-P) of
AEBP1. Fig. 3A (second
row) shows the amount of phosphorylated ERK in the stable cells.
The AEBP1/Neo-P cells contained the highest level among the stable cell
lines, whereas the AEBP1
Sty/Neo-P cells contained the lowest,
similar to the level in the control cell line (Neo-P) generated by the empty retroviral vector. Significantly, the amount of phosphorylated ERK in the AEBP1
Hic/Neo-P cells was much higher than the level in
the control cells, but lower than the level in the AEBP1/Neo-P cells.
These results indicate that overexpression of AEBP1 increases MAPK
activity and that the ability to interact with MAPK by the mutant
derivative AEBP1
Sty is not sufficient for the modulation of MAPK
activation. To rule out the possibility that AEBP1 overexpression could
be eliciting expression of a growth factor that is activating the MAPK
pathway, the activity of MEK1 was determined, and we found no
difference in the activity among these cell lines (data not shown).
Furthermore, in vitro kinase assay showed that
phosphorylation of the recombinant ERK2 protein by immunoprecipitated
MEK1 was not stimulated by addition of recombinant AEBP1 protein (data not shown). These results suggest that the modulation of MAPK activation by AEBP1 overexpression may not be mediated by stimulation of MEK1 activity.
View larger version (47K):
[in a new window]
Fig. 3.
Modulation of MAPK activation by AEBP1.
A, total protein (20 µg) from the stable cells was
analyzed by immunoblotting with AEBP1 (top), Phospho-p44/42
MAPK monoclonal (upper middle), and p44/42 MAPK (lower
middle) antibodies. Cell extracts were also analyzed by
immunoblotting with anti-PPAR antibody (Santa Cruz Biotechnology) at
7 days after the confluent cells were subjected to the differentiation
protocol (bottom). B, AEBP1 protein level in each
antisense stable cell line was determined by Western blot analysis
(top). ERKs were immunoprecipitated with anti-ERK1 antibody
and the MAPK activity analysis was carried out by the immunocomplex
assay using MBP (second row) as described under
"Experimental Procedures." The radiolabeled MBP bands were
quantitated by Molecular Imager (Bio-Rad), and the percent activity was
estimated for each sample. Phosphorylated MEK1 (fourth row)
and total MEK1 (fifth row) were detected by immunoblotting
of the cell extracts with Phospho-MEK1/2 and MEK1 antibodies (New
England BioLabs), respectively.
Hic and
Sty are defective in transcriptional function, because
Sty lacks the C-terminal DNA-binding domain whereas
Hic lacks the
CP activity that is required for transcriptional function (16). Indeed,
both mutants were defective in transcriptional repression activity
(Fig. 4C). Importantly, the EGF-stimulated ERK1 activation
(detected by phospho-ERK1 antibody), under conditions where inhibition
by PYST1 is maximal, was recovered in a dose-dependent manner when COS-7 cells were transfected with the plasmid expressing the mutant derivative
Hic (Fig. 4D, top,
lanes 4-6). These results are in agreement with the MAPK
modulation activity retained by the mutant derivative
Hic (Fig.
3A). In agreement with the results in Fig. 3A,
the protection by
Hic was not as strong as that conferred by
wild-type AEBP1 (Fig. 4D, top, lanes
7-9), perhaps due to the large deletion in the
Hic protein
(Fig. 2B). We did not observe any protection effect with the
Sty mutant (Fig. 4D, top, lanes 1-3), which suggests that the protection domain is located at the
C terminus that is missing in the
Sty protein (Fig. 2B). These results are consistent with the data showing lack of MAPK modulation activity in cells overexpressing the
Sty mutant (Fig. 3A). The EGF-stimulated MEK1 activation (detected by
Phospho-MEK1/2 antibody) was not affected in any of the transfected
cells (Fig. 4D, second row). Also, the
decreased protection activity of
Hic and
Sty are not due to
defects in their expression, because the expression level of each
mutant was similar to the wild-type level (Fig. 4D,
third row). These results indicate that AEBP1 can protect MAPK from a MAPK-specific phosphatase and suggest that the protection is most likely mediated by protective interaction not by
transcriptional function.
View larger version (58K):
[in a new window]
Fig. 4.
MAPK-protection activity of AEBP1.
A, COS-7 cells were cotransfected with pECE/HA-ERK1 (1 µg)
together with pSG5/PYST1-Myc in varying amounts as indicated. Following
48 h of growth and 2 h of serum starvation, cells were
stimulated for 15 min with 10 nM of EGF. ERK1 was
immunoprecipitated with anti-HA antibody, and the immunocomplex assay
(top) using MBP and immunoblotting with anti-HA antibody
(bottom) was carried out. The radiolabeled MBP bands were
quantitated by Molecular Imager (Bio-Rad), and the percent activity was
estimated for each sample. B, COS-7 cells were cotransfected
with pECE/HA-ERK1 (1 µg) and pSG5/PYST1-Myc (5 µg) along with
either pSVAEBP1 (lanes 4-6) or pSVAEBP1( ) (lanes
1-3) in varying amounts as indicated. The MAPK activity
(top) and immunoblotting (bottom) analyses were
as in A. C, cells were transiently transfected
with the reporter construct paP2(3AE-1/-120)CAT (17) along with an
expression vector encoding either wild-type or mutant derivatives of
HA-AEBP1 proteins. The control plasmid pJ3H-AEBP1(
) contains the
AEBP1 cDNA in the opposite orientation. The transcription activity
was estimated as absolute levels of CAT activity (% conversion of
chloramphenicol to acetylated chloramphenicol) expressed from the
aP2 promoter. The values shown represent three separate
transfection experiments. D, COS-7 cells were cotransfected
with pECE/HA-ERK1 (1 µg) and pSG5/PYST1-Myc (5 µg) along with
either pJ3HAEBP1
Sty (lanes 1-3), pJ3HAEBP1
Hic
(lanes 4-6), or pJ3HAEBP1 (lanes 7-9) in
varying amount as indicated. Cells were then stimulated with EGF at
48 h post-transfection, and cell lysates were immunoprecipitated
as described in A. The immunoprecipitated samples were
immunoblotted with either anti-phospho-p44/42 MAPK (top
row), anti-phospho-MEK1/2 (second row),
anti-HA (third and fourth rows), or
anti-Myc (bottom row) antibodies. E, COS-7 cells
were cotransfected with pECE/HA-ERK1 (1 µg), pSG5/PYST1-Myc (5 µg),
and pJ3HAEBP1 (10 µg) along with pJ3HAEBP1DLD in varying amount as
indicated. The cell lysates were analyzed as in D.
, and
inhibits adipogenesis (26-29). Thus, we tested the hypothesis that the
modulation of MAPK activity by AEBP1 is a biologically relevant process
in adipogenesis.
Sty/Neo-P cells, which did not show any MAPK
modulation (Fig. 3A) and protection (Fig. 4D)
activities, were able to differentiate as efficiently as the control
cell line Neo-P, in which more than 90% of the cells accumulated
lipid. In contrast, the stable cell line AEBP1
Hic/Neo-P, generated
by the virus harboring the mutant derivative
Hic and showing MAPK protection activity (Fig. 4D), was not able to differentiate
efficiently. As predicted from the differentiated phenotype, expression
of the differentiation-specific gene PPAR
in the AEBP1
Hic/Neo-P cells was much lower than the level in the control cells following the
differentiation protocol (Fig. 3A, bottom). The
amount of PPAR
in the AEBP1
Sty/Neo-P cells was similar to the
level in the control cells, but the level in the AEBP1/Neo-P cells was much lower. Because AEBP1
Hic is defective in transcription function (Fig. 4C) the reduced level of PPAR
in the
AEBP1
Hic/Neo-P and AEBP1/Neo-P cells is most unlikely due to
transcriptional repression of PPAR
expression by overexpression of
AEBP1. These results suggest that the MAPK-protection activity is
responsible for the anti-adipogenic effect.
View larger version (47K):
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Fig. 5.
Modulation of adipogenesis by the
MAPK-protective activity of AEBP1. A, the
differentiated phenotype was monitored by microscopic
examination either before (top) or after (bottom)
staining with oil Red O at 7 days after the confluent cells were
subjected to the differentiation protocol described under
"Experimental Procedures." Magnification, × 300. B,
microscopic analysis of the differentiation phenotype of the stable
cell lines was as in A. C, total protein (20 µg) from the stable cells was analyzed by immunoblotting with AEBP1
(top row), phospho-p44/42 MAPK monoclonal (second
row), and p44/42 MAPK (third row) antibodies. Cell
extracts were also analyzed by immunoblotting with anti-PPAR
antibody at 7 days after the confluent cells were subjected to the
differentiation protocol (bottom row).
), which was generated with the control
virus (AEBP1DLD(
)/Puro), was unable to differentiate as in the case
of the parental cell line AEBP1/Neo-P. As predicted, the amount of the
differentiation-specific gene PPAR
was increased in the AEBP1/DLD(+)
cells following the differentiation protocol (Fig. 5C,
bottom). The amount of phosphorylated ERK in AEBP1/DLD(+)
but not in AEBP1/DLD(
) cells was significantly decreased from the
level in the parental AEBP1/Neo-P cells (Fig. 5C,
second row). However, the MEK activity was not affected by ectopic expression of DLD (data not shown). These results strongly suggest that adipogenesis is regulated by the MAPK-protection activity of AEBP1.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, a potent transcriptional
activator of adipogenesis (42, 43), which results in reduction of its
transcriptional activation function (29, 31). More recently, Lazar and
colleagues (44) have observed that modification of the phosphorylation
site, by phosphorylation or replacement with an acidic amino acid,
lowers the affinity of the holoreceptor for ligand. These results
suggest that the anti-adipogenic effect exhibited by some growth
factors might act through the MAPK pathway that phosphorylates PPAR
.
However, insulin treatment, which stimulates adipocyte differentiation, also results in activation of MAPK (45). This study also showed that
insulin stimulates the phosphorylation of PPAR
in an
MAPK-dependent manner and showed that MAPK is an important
mediator of cross-talk between insulin signal-transduction pathways and
PPAR
activation. This apparent discrepancy requires further studies.
function (26, 28) and
opposes cell differentiation in 3T3-L1 cells (5), may be resolved by
examining the differences in stimulation of MAPK, because MAPK will
translocate to the nucleus upon prolonged activation, or by examining
the activation of MAPK by different receptor types that utilize similar
signaling pathways but cause drastic differences in cellular outcomes
(37). Adipogenic agents such as insulin may cause transient MAPK
activation, whereas mitogenic agents such as growth factors may cause
prolonged MAPK activation. In support of this proposal, overexpression
of the IRS-1, the major substrate of the insulin receptor
tyrosine kinase (46), causes the activation of MAPK and induces cell
proliferation in fibroblasts (47). Insulin normally causes these cells
to differentiate, but prolonged activation of the IRS-1 component of
the insulin signaling pathway induces cell proliferation, again
indicating an activation-dependent function of MAPK in adipogenesis.
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ACKNOWLEDGEMENTS |
---|
We thank R. A. Singer and N. D. Ridgway for discussion and comments on the manuscript; G.-P. He for help in the generation of stable cell lines; S. O. Freytag and B. M. Spiegelman for providing the retroviral vectors pWZLneo and pBabe-puro, respectively; S. Keyse for plasmid pSG5/Pyst1-Myc; A. Brunet for pECE/HA-ERK1; J. Chernoff for the epitope-tag vector pJ3H; J. E. Dixon for the bacterial expression plasmid pT7-rVH6(His)6; and J. Shi for technical assistance.
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FOOTNOTES |
---|
* This work was supported by grants from the Canadian Diabetes Association, the Heart and Stroke Foundation of Canada, and the National Sciences and Engineering Research Council of Canada (to H.-S. R.).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: Dept. of Biochemistry
and Molecular Biology, Tupper Medical Bldg., Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada. Tel.: 902-494-2367; Fax: 902-494-1355; E-mail: hsro@is.dal.ca.
Published, JBC Papers in Press, January 4, 2001, DOI 10.1074/jbc.M010640200
2 S.-W. Kim, and H.-S. Ro, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
IGF-1, insulin-like
growth factor-1;
AEBP1, adipocyte enhancer binding protein 1;
MAPK, mitogen-activated protein kinase;
MKP, MAPK phosphatase;
ERK, extracellular signal-regulating kinase;
MEK, MAPK/ERK kinase (or MAPKK);
EGF, epidermal growth factor;
CAT, chloramphenicol
acetyltransferase;
PPAR, peroxisome-proliferator-activated receptor
;
CP, carboxypeptidase;
HA, hemagglutinin;
PAGE, polyacrylamide gel
electrophoresis;
MBP, myelin basic protein;
DMEM, Dulbecco's modified
Eagle's medium;
DLD, discoidin-like domain.
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