From the Department of Physiology and Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, Michigan 48109
Received for publication, November 8, 2000, and in revised form, February 23, 2001
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ABSTRACT |
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Growth hormone (GH) regulates transcription
factors associated with c-fos, including C/EBP Growth hormone (GH)1
exhibits a variety of effects on somatic growth, differentiation, and
metabolism (1, 2), which often involve changes in gene expression.
Analysis of the regulation of gene transcription by GH has provided
additional insight into signaling mechanisms between the GH receptor
(GHR) and nuclear events regulating transcription (3, 4). Regulation of
the expression of the proto-oncogene c-fos has served as a
useful model for studying regulation of gene expression by GH (5, 6),
and has demonstrated diverse effects of GH on the phosphorylation of
transcription factors. For example, GH-stimulated tyrosine phosphorylation of STATs and serine phosphorylation of Elk-1 are required for these transcription factors to activate transcription of
c-fos in response to GH (7-13).
The CCAAT/Enhancer-Binding Proteins (C/EBPs) have recently been shown
to participate in GH-regulated transcription of c-fos (14).
C/EBPs, which include C/EBP GH increases the binding of C/EBP Materials--
3T3-F442A fibroblasts were provided by Dr. H. Green (Harvard University) and Dr. M. Sonenberg (Sloan-Kettering).
Chinese hamster ovary cells expressing rat GHR containing the
N-terminal half of the cytoplasmic domain (CHO-GHR) were provided by G. Norstedt (Karolinska Institute, Stockholm, Sweden) and N. Billestrup (Hagedorn Laboratory, Gentofte, Denmark) (26). Human
embryonic kidney 293T cells were provided by Dr. M. Lazar (University
of Pennsylvania). Recombinant human GH was provided by Eli Lilly.
Culture media were purchased from Irvine Scientific. Calf serum and
fetal calf serum were purchased from Life Technologies, Inc., albumin
(BSA, CRG7) from Intergen. Wortmannin was purchased from Calbiochem. LY294002, lithium chloride, and sodium orthovanadate were purchased from Sigma Chemical Co., and alkaline phosphatase was purchased from
Roche Molecular Biochemicals. The MEK inhibitor PD098059 and the
GSK-3 peptide substrates were gifts from Dr. A. Saltiel (Pfizer, Ann
Arbor, MI). Recombinant GSK-3 was purchased from New England BioLabs.
Aprotinin, leupeptin, and Complete® protease inhibitor mixture
(EDTA-free) were purchased from Roche Molecular Biochemicals and used
according to the supplier's instructions. [ Cell Culture and Hormone Treatment--
3T3-F442A preadipocytes
and 293T cells were grown in Dulbecco's modified Eagle's medium
containing 4.5 g/liter glucose and 8% calf serum in an atmosphere of
10% CO2/90% air at 37 °C. CHO-GHR cells were grown in
Ham's F-12 medium containing 10% fetal calf serum and 0.5 mg/ml G418
in an atmosphere of 5% CO2/95% air at 37 °C. All media
were supplemented with 1 mM L-glutamine, 100 units/ml penicillin, 100 mg/µl streptomycin, and 0.25 µg/ml
amphotericin. Prior to treatment, cells were incubated overnight in the
appropriate medium containing 1% BSA instead of serum, then cells were
incubated with or without GH at 500 ng/ml (22 nM) as indicated.
Plasmids and Antibodies--
Plasmids encoding LAP or LIP driven
by the CMV promoter (CMV-LAP and CMV-LIP) were gifts from Dr. U. Schibler (University of Geneva) courtesy of L. Sealy (Vanderbilt
University). The plasmid RSV-
Specific rabbit polyclonal antibodies against a peptide corresponding
to amino acids 278-295 at the C terminus of C/EBP Immunoblotting--
Cell lysis and immunoblotting for C/EBP GSK-3 Activity--
3T3-F442A cells were washed with
phosphate-buffered saline and lysed in GSK-3 extraction buffer (50 mM Hepes (pH 7.4), 1 mM EGTA, 1 mM
EDTA, 10 mM C/EBP Electrophoretic Mobility Shift Assay (EMSA)--
Confluent
3T3-F442A cells were deprived of serum overnight and incubated for the
indicated times with hormone or vehicle as described above. Nuclear
extracts were prepared as described previously (14). Binding reactions
proceeded for 30 min at room temperature with labeled oligonucleotides
containing wild-type c-fos C/EBP site and flanking SRE (wt
C/EBP-SRE) or the C/EBP binding site from the 422/aP2 gene
(aP2-C/EBP) that were previously described (14). Complexes formed by
LAP and/or LIP were separated by nondenaturing 7% PAGE followed by
autoradiography. For analysis of LAP or LIP, 293T cells were
transfected using calcium phosphate coprecipitation (28) with plasmids
CMV-LAP (1 µg) or CMV-LIP (l µg) with or without CMV-GSK-3 S9A (8 µg). 48 h later, cell lysates enriched in nuclear proteins were
prepared using high salt buffer (420 mM NaCl, 20 mM Hepes (pH 7.9), 1 mM EDTA, 1 mM
EGTA, 50% glycerol, protease inhibitor mixture) and were stored at
Gene Expression Assay--
CHO-GHR (1 × 105
cells/35-mm well) were transiently transfected by calcium phosphate
coprecipitation (28) with wt-fos-Luc (0.4 µg) and the RSV
GH-induced Dephosphorylation of LAP and LIP Is Blocked by PI3K
Inhibitors--
GH causes a rapid and transient dephosphorylation of
C/EBP
Because activation of PI3K is known to lead to phosphorylation and
inactivation of GSK-3, involvement of GSK-3 in GH-induced dephosphorylation of C/EBP GH Activates Akt--
Akt is a downstream effector of PI3K
(36-38). The ability of GH to stimulate phosphorylation of Akt was
examined by immunoblotting, using an antibody that specifically
recognizes a peptide of Akt phosphorylated on Ser-473 (anti-P-Akt). GH
was found to increase the amount of Ser-phosphorylated Akt within 5 min
(Fig. 2, A, B, and
C, upper panels, lanes 1,
2). The stimulation of Akt by GH subsided from 15 to 60 min
(Fig. 2, A, B, and C, upper
panels, lanes 3-6). The lower panels show total Akt.
The PI3K inhibitor wortmannin completely blocked the GH-stimulated
activation of Akt (Fig. 2A, lanes 7-12),
consistent with GH-mediated activation of Akt being dependent on PI3K.
When 3T3-F442A cells were pretreated with PD098059, the appearance of
phosphorylated Akt in response to GH was not altered (Fig.
2B, lanes 7-12), suggesting that the MEK-ERK
pathway is not involved in GH activation of Akt. Lithium, a GSK-3
inhibitor, also failed to alter Akt phosphorylation in response to GH
(Fig. 2C, lanes 7-12), as expected, because
GSK-3 lies downstream of Akt. Taken together, these results indicate that, in 3T3-F442A cells, GH promotes phosphorylation and activation of
Akt downstream of PI3K.
GH Inhibits GSK-3--
GSK-3 activity is inhibited by growth
factors that activate PI3K and Akt in several cell types (39-41).
GSK-3 is inactivated when it is phosphorylated downstream of Akt (39).
Hence, it would be predicted that activation of Akt by GH would be
associated with inhibition of GSK-3. The activity of GSK-3 was measured
by its ability to phosphorylate a synthetic peptide containing a GSK-3
consensus sequence in the presence of [
Akt-dependent phosphorylation of the GSK-3 LAP and LIP Are Substrates of GSK-3 in Vitro--
Because
inhibition of GSK-3 by GH might contribute to the ability of GH to
promote dephosphorylation of LAP and LIP, and because both LAP and LIP
contain a putative consensus sequence for GSK-3, the possibility that
LAP and/or LIP are GSK-3 substrate(s) was tested. LAP and LIP were
immunoprecipitated from 3T3-F442A cells and incubated with
[ Dephosphorylation of LAP Increases DNA Binding--
The effect of
dephosphorylation of LAP and LIP on their DNA binding capacity was
investigated for insight into the functional importance of the
phosphorylation state of C/EBP
To examine the effect of dephosphorylation on binding to the
c-fos C/EBP site, extracts enriched in LAP were incubated
with alkaline phosphatase (AP) and subjected to EMSA. The resulting dephosphorylation of LAP caused a dramatic increase in binding of LAP
homodimers (Fig. 5B, lane 2 versus
lane 1). The increase in binding by alkaline phosphatase was
prevented by the simultaneous addition of the phosphatase inhibitor
orthovanadate to the incubation medium (data not shown). The
dephosphorylation of LAP by alkaline phosphatase was verified by
immunoblotting (Fig. 5C), showing that alkaline phosphatase
treatment caused LAP to migrate more rapidly (lane 2 versus lane 1), an effect that was prevented by orthovanadate (data not shown). Interestingly, alkaline phosphatase treatment of extracts enriched with LIP led to a decrease in binding of
LIP homodimer to the C/EBP site (Fig. 5B, lane 4 versus lane 3). Alkaline phosphatase treatment
caused LIP to shift to a faster mobility form on immunoblots (Fig.
5C, lane 4 versus lane 3)
consistent with LIP dephosphorylation. Thus, dephosphorylation has
opposite effects on the ability of LAP and LIP to bind as homodimers to the C/EBP site of the c-fos promoter, increasing binding of
LAP homodimers and decreasing binding of LIP homodimers.
When alkaline phosphatase-treated extracts enriched in LAP and LIP were
combined, dephosphorylation increased binding of LAP homodimers (Fig.
5B, lane 6 versus lane 5)
and decreased binding of LIP homodimers (detectable in longer
autoradiographic exposures, not shown) similar to the changes observed
when LAP and LIP were tested alone. Interestingly, binding of LAP/LIP
heterodimers was increased by dephosphorylation, suggesting that LAP
may recruit LIP for heterodimer formation. Taken together, these
results suggest that the phosphorylation status of LAP and LIP plays a
key role in their ability to bind as homo- and/or heterodimers to the
C/EBP site of the c-fos promoter.
To test further the effect of dephosphorylation on the DNA binding
capacity of LAP and LIP complexes, a probe based on the C/EBP site from
the 422/aP2 gene, which is not associated with the SRE, was
tested. Alkaline phosphatase treatment of LAP-enriched extracts
increased binding of LAP homodimers (Fig. 5D, lane
2 versus lane 1), indicating that the
increase in binding with dephosphorylation of LAP is likely to be
independent of the presence of the SRE sequence. However,
dephosphorylation of LIP did not alter binding of LIP homodimers to the
aP2-C/EBP probe (Fig. 5D, lane 4 versus lane 3), in contrast to the decrease
obtained with the C/EBP-SRE probe (lane 7 versus
8). When alkaline phosphatase-treated extracts enriched in LAP and LIP
were combined, dephosphorylation increased binding of LAP homodimers
(Fig. 5D, lane 6 versus lane
5, upper band). The binding of LIP homodimers
(bottom band) was not altered even in longer
autoradiographic exposures (not shown), whereas the binding of LAP/LIP
dimers (middle band) was slightly decreased.
Taken together, these results indicate that in vitro
dephosphorylation of LAP facilitates its binding to DNA in the absence as well as the presence of the SRE sequence. In contrast, in
vitro dephosphorylation of LIP decreases binding of LIP homodimers
to the c-fos C/EBP site but does not modify its binding to
the C/EBP site in the aP2 probe, suggesting that dephosphorylation of
LIP may have different consequences in the regulation of LIP DNA
binding capacity.
Dephosphorylation Facilitates Binding of Endogenous LAP to the
c-fos C/EBP Site--
To evaluate whether the binding of endogenous
LAP and LIP is also altered by dephosphorylation, nuclear extracts from
3T3-F442A cells were incubated with alkaline phosphatase for 1 h
and analyzed by EMSA. The resulting dephosphorylation of endogenous LAP
and LIP by alkaline phosphatase increased binding of LAP/LAP and
LAP/LIP dimers (Fig. 6, lane 2 versus lane 1). GH increased the binding of
endogenous LAP homodimers and LAP/LIP heterodimers, as reported previously (14). The extent of the increase with GH was comparable to
the increase promoted by alkaline phosphatase-mediated
dephosphorylation of LAP and LIP in the same experiment (Fig. 6,
lane 2 versus lane 3). The comparable
increases with alkaline phosphatase and GH support the idea that
GH-promoted dephosphorylation of endogenous LAP contributes to the
ability of GH to increase binding of LAP complexes. The binding of LIP
homodimers was not detectable in 3T3-F442A cells under any condition
tested. The inability to detect endogenous LIP homodimers may be
related to apparent recruitment of LIP to LAP/LIP heterodimers.
Dephosphorylation of LAP and LIP was confirmed by immunoblot (data not
shown). These data suggest that dephosphorylation of endogenous LAP, as
promoted by GH, can increase the binding of LAP homodimers and LAP/LIP
heterodimers to the c-fos promoter.
GSK-3 Restrains LAP Binding to the c-fos C/EBP Site--
If
dephosphorylation increases binding of LAP complexes to DNA, then it
would be predicted that phosphorylation, as mediated by GSK-3, might
reduce such binding. To examine whether GSK-3 alters the ability of LAP
and/or LIP to bind DNA, LAP or LIP was overexpressed in 293T cells in
the presence or absence of constitutively active GSK-3 (GSK-3 S9A).
Co-expression of LAP and GSK-3 S9A reduced the binding of LAP
homodimers to DNA by almost half (Fig. 7,
lane 2 versus lane 1). The decrease in
LAP binding was also evident when extracts from cells overexpressing
LAP or LIP in the presence of GSK-3 S9A were combined; binding of
LAP/LAP homodimers, and to a lesser extent of LAP/LIP heterodimers, was
reduced in the presence of GSK-3 S9A (Fig. 7, lane 6 versus lane 5). Co-expression of LIP and GSK-3
S9A slightly decreased the binding of LIP homodimers (Fig. 7,
lane 3 versus lane 4). These data
suggest that phosphorylation, as mediated by GSK-3, decreases the
ability of LAP and LIP to bind as homo- or heterodimers to the C/EBP
site of the c-fos promoter.
Role of GSK-3 on GH-stimulated c-fos Promoter
Activity--
To examine whether GSK-3 modulates GH-regulated
c-fos promoter expression, constitutively active GSK-3
(GSK-3 S9A) was expressed in combination with a luciferase reporter
driven by wild type c-fos promoter (wt-fos-Luc)
in GH-responsive CHO-GHR cells. Expression of GSK-3 S9A reduces the
basal level of c-fos promoter activity by 50% (Fig.
8A, open bars).
Moreover, expression of GSK-3 S9A reduces the ability of GH to
stimulate reporter expression via the c-fos promoter
(Fig. 8A, hatched bars). The c-fos
promoter is capable of being stimulated by 10% calf serum to the same
level in the presence (5.5- ± 0.6-fold) or absence (5.1- ± 0.5-fold) of GSK-3 S9A. These data indicate that GSK-3 activity restrains c-fos expression and specifically interferes with
stimulation of c-fos by GH.
Overexpression of LAP elevates basal c-fos promoter
activity with respect to that observed in vector-transfected cells
(Fig. 8B versus 8A, leftmost
open bars), as reported previously (14). Stimulation of
c-fos promoter activity by GH is ~2-fold relative to
untreated control in the presence or absence of overexpressed LAP (Fig.
8B versus Fig. 8A, leftmost
hatched bars), as reported previously (14). To determine
whether LAP could overcome the ability of GSK-3 to interfere with
GH-induced c-fos promoter activity, GSK-3 S9A was
co-expressed with LAP and wt-fos-Luc. Basal c-fos promoter activity was reduced in the presence of GSK-3 S9A despite the
presence of LAP (Fig. 8B, open bars).
Furthermore, GSK-3 S9A interfered with GH-stimulated c-fos
promoter activity in the presence of LAP (Fig. 8B,
hatched bars) as well as the absence of LAP (Fig. 8A, hatched bars). Taken together, these data
indicate that GSK-3 restrains c-fos expression-stimulated by
GH and suggest that transient inhibition of GSK-3 by GH plays a role in
the GH-stimulated expression of c-fos. These data also
suggest that GSK-3 activity interferes with the ability of LAP to
stimulate the c-fos promoter in the absence or presence of
GH; such interference could be due in part to the restraining effect of
GSK-3 on the binding of LAP dimers to the c-fos promoter.
GH-stimulated PI3K Signaling Regulates C/EBP GH Activates Akt and Inhibits GSK-3--
A link between PI3K and
regulation of C/EBP C/EBP Dephosphorylation of C/EBP
In contrast to the increase in DNA binding that accompanies LAP
dephosphorylation, the dephosphorylation of LIP leads to a decrease in
its binding to the c-fos promoter. This supports the possibility that the reciprocal changes in binding of LAP and LIP with
dephosphorylation lead directly to changes in transcription, because
LAP can increase and LIP can inhibit transcription mediated by the
c-fos promoter (14). The differences in DNA binding observed between dephosphorylated LAP and LIP suggest that phosphorylation sites
that are not shared by LAP and LIP may be determinants of their
abilities to bind to c-fos and to regulate transcription. It
is not yet known which (if any) of these unshared sites is critical.
Although dephosphorylation of LIP led to a decrease in its binding to
the c-fos C/EBP site, LIP binding to the aP2 C/EBP site was not decreased by dephosphorylation. This suggests that
the consequences of dephosphorylation, at least for LIP, may be
specific for the gene to which the protein binds.
In addition to the phosphorylation state, other determinants such as
participation in nucleoprotein complexes may also modulate the ability
of LAP and LIP to bind to c-fos and to regulate
transcription. Because mutation of the SRF binding site abolished the
binding of C/EBP complexes (7) or of LAP and LIP dimers (data not
shown) to the c-fos promoter, the integrity of
SRF binding may contribute to the ability of C/EBP Regulation of C/EBP
Inhibition of GSK-3 may be critical for GH-stimulated c-fos
promoter activity, because expression of constitutively active GSK-3
S9A interfered with GH-stimulated transcription via the c-fos promoter in the absence and presence of LAP. It has
been demonstrated that GSK-3-mediated phosphorylation of c-Jun not only
inhibits c-Jun binding to AP-1 sites but also expression of
AP-1-sensitive reporter constructs (54, 60-61). It is possible that GSK-3 can modulate binding of complexes to the AP-1 site as well
as to the C/EBP site in c-fos, thereby reducing
GH-stimulated transcription. The decrease in binding of LAP that
accompanies its phosphorylation by GSK-3 may therefore be functionally
important for the ability of LAP to activate transcription.
In summary, these studies implicate a PI3K pathway in mediating
signaling between the GH receptor and the nucleus. GH stimulates PI3K
and Akt and inhibits GSK-3, which is implicated in regulating the
phosphorylation of C/EBP. Two
forms of C/EBP
, liver-activating protein (LAP) and liver inhibitory
protein (LIP), are dephosphorylated in GH-treated 3T3-F442A
fibroblasts. GH-induced dephosphorylation of LAP and LIP is reduced
when cells are preincubated with phosphatidylinositol 3'-kinase
(PI3K) inhibitors. GH activates Akt and inhibits glycogen synthase kinase-3 (GSK-3). Lithium, a GSK-3 inhibitor, increases GH-dependent dephosphorylation of LAP and LIP. Both are
in vitro substrates of GSK-3, suggesting that GSK-3
inactivation contributes to GH-promoted dephosphorylation of C/EBP
.
Alkaline phosphatase increases binding of LAP homodimers and decreases
binding of LIP homodimers to c-fos, suggesting that
dephosphorylation of C/EBP
modifies their ability to bind DNA. Both
alkaline phosphatase- and GH-mediated dephosphorylation comparably
increase binding of endogenous LAP in 3T3-F442A cells. In cells
overexpressing LAP and GSK-3, LAP binding decreases, suggesting that
GSK-3-mediated phosphorylation interferes with LAP binding. Expression
of constitutively active GSK-3 reduced GH-stimulated
c-fos promoter activity. These studies indicate that
PI3K/Akt/GSK-3 mediates signaling between GH receptor and the nucleus,
promoting dephosphorylation of C/EBP
. Dephosphorylation increases
binding of LAP complexes to the c-fos promoter and may
contribute to the participation of C/EBP
in GH-stimulated
c-fos expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, C/EBP
, C/EBP
, C/EBP
, and
C/EBP
/CHOP, belong to the bZIP family of transcription factors characterized by a C-terminal dimerization domain (leucine zipper) adjacent to a basic DNA binding domain. The N-terminal region of C/EBPs
contains the transcription activation and inhibitory domains. C/EBP
,
the prominent GH-regulated form (14) is present in cells as three
alternate translation products. The 32- and 35-kDa forms of C/EBP
,
also known as liver-activating proteins (LAP), are potent
transactivators. The 20-kDa form of C/EBP
, known as liver
inhibitory protein (LIP), possesses a truncated transactivation
domain and inhibits transcription (15). LIP inhibits the
transactivating potential of LAP, even at relatively low molar ratios
(15). It has also been demonstrated that overexpression of LIP in
hepatocytes overcomes LAP-mediated cell cycle arrest (16). In the
adipocyte differentiation program, C/EBP
appears to play an
important role in the transition between cell cycle progression and
terminal differentiation (17-19).
and C/EBP
to the
c-fos C/EBP site, which lies
295 to
303 bp relative to
the transcription start site, and overlaps the c-fos serum
response element (SRE) (14). GH promotes a rapid and transient
dephosphorylation of LAP and LIP in 3T3-F442A cells, in addition to
increasing binding of C/EBP
to the C/EBP site in c-fos
(14). It is well established that modulation of target gene expression
can be achieved through regulation of the phosphorylation state of
transcription factors, which can positively or negatively alter their
DNA binding affinity and/or their capacity to activate gene
transcription (20-25). The focus of the present study was to identify
signaling events by which GH regulates the dephosphorylation of LIP and
LAP and to determine whether dephosphorylation of LAP and LIP modulates
their ability to bind to DNA. These studies implicate PI3K/Akt/GSK-3 signaling in the regulation of the dephosphorylation of C/EBP
by GH,
thereby identifying PI3K as a signaling intermediate between the GH
receptor and the nucleus. Furthermore, these studies demonstrate that
GH-induced dephosphorylation of C/EBP
dramatically increases binding
of LAP complexes to the c-fos promoter and may thereby modulate transcriptional activation of c-fos by C/EBP
in
response to GH.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP
and [
-32P]ATP were from PerkinElmer Life Sciences. The
ECL detection system was purchased from Amersham Pharmacia Biotech.
Luciferin was purchased from Promega and
-galactosidase
chemiluminescence reagents were from Tropix.
galactosidase (RSV-
-gal) was
provided by Dr. M. Uhler (University of Michigan). CMV-GSK-3 S9A was
provided by P. J. Roach (Indiana University), and pcDNA3.1 was
purchased from CLONTECH. The plasmid wt-fos-Luc, provided by Dr. W. Wharton (University of South
Florida) (27) contains 379 bp of the mouse c-fos promoter
immediately 5' of the transcription start site cloned upstream of the
luciferase gene.
were purchased
from Santa Cruz Biotechnology, Inc. Polyclonal antibodies that
recognize phosphorylated Ser-473 of Akt (anti-P-Akt), and polyclonal
antibodies made against the Akt peptide 466-479 (anti-Akt) were
purchased from New England BioLabs. Polyclonal antibodies against an
oligopeptide corresponding to amino acids 16-26 of GSK-3
phosphorylated on Ser-21 (anti-P-GSK-3) and to a peptide corresponding
to amino acids 203-219 of GSK-3 (anti-GSK-3) were purchased from
Upstate Biotechnology Inc.
were performed as previously described (14) and for Akt and GSK-3 as
follows: confluent 3T3-F442A cells on 100-mm plates were washed with
phosphate-buffered saline with vanadate (10 mM Tris (pH
7.4), 150 mM sodium phosphate, 1 mM sodium
vanadate) and scraped in 0.3 ml of L-RIPA lysis buffer (50 mM Hepes, pH 7.0, 250 mM NaCl, 0.5% Triton
X-100) containing 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of
aprotinin and leupeptin. Whole cell lysates (35-50 µg) were analyzed
by immunoblotting using antibodies against phospho-Akt or
phospho-GSK-3. Blots were reprobed as previously described (8) using
the corresponding antibody against Akt or GSK-3. The apparent
Mr are based on prestained molecular weight
standards (Life Technologies, Inc.).
-glycerophosphate, 5 mM
Na2PO4, 100 mM KCl, 0.5% Triton
X-100, 1 mM dithiothreitol, 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin,
and 0.5 mM sodium vanadate). Samples were centrifuged at
15,000 × g for 10 min. GSK-3 activity was measured in
the supernatant (8 µg of protein) in a final volume of 25 µl of 50 mM Hepes (pH 7.4), 10 mM MgCl2, 1 mM dithiothreitol, 0.5 mg/ml synthetic peptide (RRAAEELDSRAG(p)SPQL) or negative control peptide (RRAAEELDSRAGAPQL), and 100 µM [
-32P]ATP (1000 cpm/pmol).
After incubation for 15 min at 30 °C, the reactions were terminated
by the addition of (5 µl) 100 mM EDTA, 5 mM
ATP, and tubes were placed on ice. An aliquot (20 µl) of the reaction
mixture was spotted onto Whatman p81 phosphocellulose paper, washed in
three changes of 175 mM phosphoric acid for a total of 20 min, air-dried, and 32P incorporation was measured by
liquid scintillation counting. 32P incorporation into the
negative control peptide was subtracted from values obtained using the
GSK-3 peptide substrate. Results are expressed as nanomoles of
phosphate incorporated per min per mg of protein.
Immunoprecipitation and in Vitro GSK-3 Phosphorylation
Assay--
3T3-F442A cells were washed and scraped in RIPA 0.5% SDS
buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM EGTA, 0.5% SDS). C/EBP
was immunoprecipitated with
an antibody against the C-terminal domain of C/EBP
(5 µl) (or an
equivalent volume of rabbit non-immune serum) for 2 h at 4 °C.
After centrifugation, pellets were washed three times with RIPA 0.5%
SDS buffer and resuspended in 50 µl of GSK-3 buffer (50 mM Hepes (pH 7.4), 10 mM MgCl2, 1 mM dithiothreitol). Recombinant GSK-3 (15 units) and
[
-32P]ATP (500 µCi/µmol) were added. After 40 min
of incubation at 30 °C, the reaction was stopped by boiling the
samples in the presence of sample buffer. The proteins were separated
by 15% SDS-PAGE as previously described (14), and phosphorylation was determined by autoradiography. An aliquot of the sample was subjected to immunoblot analysis to identify C/EBP
in the immunoprecipitate.
80 °C. In some experiments, the enriched lysates were incubated
with or without 200 units of alkaline phosphatase, in the presence or
absence of orthovanadate (30 mM), for 1 h at 37 °C
prior to EMSA or immunoblotting.
-galactosidase plasmid (0.1 µg), with or without a plasmid
encoding a constitutively active GSK-3 (GSK-3 S9A 0.8 µg), in the
presence or absence of CMV-LAP DNA (1 ng) or corresponding amounts of
pcDNA3.1 vector per 35-mm well. Twenty-four hours after
transfection, cells were deprived of serum by incubation in medium
containing 1% BSA for 18 h prior to treatment as indicated. Cell
lysates were prepared in reporter lysis buffer (100 mM
potassium phosphate, 0.2% Triton X-100, 1 mM
dithiothreitol), and luciferase or
-galactosidase activity was
measured using an Opticomp luminometer. The luciferase values were
normalized to
-galactosidase activity. Each condition was tested in
duplicate or triplicate in each experiment. Analysis of variance with
factorial Scheffe F test was used to analyze data as indicated.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, as indicated by an increased mobility on SDS-PAGE (14). GH is
known to activate PI3K and MAPKs ERK1 and ERK2 in several cell types,
including 3T3-F442A fibroblasts (29-33). To evaluate whether either of
these signaling pathways mediates GH-regulated dephosphorylation of LAP
and LIP, we tested the effect of PI3K or MEK inhibitors on the ability
of GH to promote dephosphorylation of LAP and LIP. Treatment of the
cells with the PI3K inhibitor wortmannin (30 min) prior to treatment
with GH (60 min) markedly impaired the GH-promoted dephosphorylation of
LIP (Fig. 1A, band
b, lane 4 versus lane 2) and LAP
(not shown). Similar inhibition of GH-promoted dephosphorylation was
obtained when cells were treated with LY294002, another PI3K inhibitor,
prior to GH (Fig. 1A, band b, lane 8 versus lane 6). Taken together these results
suggest a role for PI3K in the GH-promoted dephosphorylation of
C/EBP
. Treatment with the MEK inhibitor PD098059 (40 µM, 30 min) prior to GH did not interfere with the
GH-promoted dephosphorylation of LIP and LAP (not shown), indicating
that the MEK-ERK pathway is unlikely to be a major contributor to this
response to GH. Similar changes in phosphorylation in response to GH
were observed for LAP and LIP throughout these studies.
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Fig. 1.
Inhibition of PI3K reduces GH-induced
dephosphorylation of LIP. A, 3T3-F442A fibroblasts were
incubated with wortmannin (W, 200 nM)
(lanes 3 and 4), LY294002 (LY, 10 µM), or respective vehicles (lanes 1-2 and
5-6) for 30 min prior to treatment without ( ) or with (+)
GH for an additional 60 min. The cells were lysed and used for
immunoblotting with anti-C/EBP
(1:1000). Bands representing LIP (20 kDa) are shown, and the slower (a) and faster (b)
migrating forms of LIP are indicated. Data are representative of four
independent experiments. Similar changes were observed for LAP (not
shown) in each experiment. B, 3T3-F442A cells were incubated
with LiCl (Li, 25 mM) for 30 min prior to
treatment without (
) or with (+) GH for additional 60 min. The cells
were lysed and analyzed by immunoblotting as indicated in part
A. Data are representative of four independent experiments.
Similar results were observed for LAP (not shown) in each
experiment.
was examined. Cells were pretreated with
LiCl, a GSK-3 inhibitor (34, 35), which slightly increased the
intensity of the rapidly migrating LIP band in control cells (Fig.
1B, band b, lane 3). Notably, the
GH-dependent shift to the rapidly migrating form of LIP was
further enhanced in the presence of LiCl (Fig. 1B,
band b, lane 4 versus lane
2), suggesting that inhibition of GSK-3 favors the presence of the
dephosphorylated form of C/EBP
. Treatment with LiCl increased
dephosphorylation of LAP similarly to the increase observed for LIP in
GH-treated and untreated cells. In contrast, neither wortmannin nor
LiCl altered the pattern of phosphorylation of ERK-1 or -2 in the
absence or presence of GH (data not shown), indicating that wortmannin and LiCl do not affect the MAPK pathway under the conditions of these
experiments. These data are consistent with GH utilizing a PI3K pathway
to modulate dephosphorylation of C/EBP
and suggest that GSK-3 may be
involved downstream of PI3K.
View larger version (52K):
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Fig. 2.
GH transiently activates Akt. 3T3-F442A
cells were treated without or with (A) wortmannin (200 nM), (B) PD098059 (40 µM), or
(C) LiCl (25 mM) for 30 min. Then GH
(lanes 2-6, 8-12) or vehicle (lanes
1, 7) were added for the times indicated. Cells lysates
were immunoblotted with anti-P-Akt (1:1000) (upper panels).
Blots were stripped and reprobed with anti-Akt (1:1000) (lower
panels). Similar findings were obtained in two independent
experiments.
-32P]ATP
(42). Treatment of 3T3-F442A cells with GH resulted in a 30-40%
inhibition of GSK-3 activity within 15 min (Fig.
3A). The GH-mediated
inhibition of GSK-3 was transient, and GSK-3 activity returned to
baseline within 30 min.
View larger version (34K):
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Fig. 3.
GH transiently inhibits GSK-3 activity.
A, 3T3-F442A cells, treated with or without GH (500 ng/ml)
for the indicated times, were lysed, and GSK-3 activity was measured
using a synthetic peptide and [ -32P]ATP. Data shown
are the average ± S.E. of three independent experiments.
B, 3T3-F442A cells were treated without or with wortmannin
(200 nM) for 30 min prior to incubation with GH for the
times indicated. Cells were lysed and analyzed by immunoblotting with
anti-P-GSK-3 (1:1000). The blots were stripped and reprobed with
anti-GSK-3 (1:1000). The immunoblots are representative of three
independent experiments.
isoform on
Ser-21 or of GSK-3
on Ser-9 results in partial inactivation of the kinase (43). Consistent with GH-mediated inactivation of GSK-3, GH was
found to increase the amount of serine-phosphorylated GSK-3, as
detected by immunoblotting in lysates from 3T3-F442A cells with an
antibody that specifically recognizes phosphorylated Ser-21 of
GSK-3
. The increase was evident within 5 min of GH treatment (Fig.
3B, lane 2), and subsided progressively (15-90
min)(Fig. 3B, lanes 3-6). As expected,
pretreatment with wortmannin reduced the GH-induced phosphorylation of
GSK-3 (Fig. 3B, lanes 7-12 versus 1-6). These results indicate that GH stimulates
phosphorylation and inhibition of GSK-3 most likely by a mechanism
involving PI3K.
-32P]ATP in the presence or absence of recombinant
GSK-3. When anti-C/EBP
immunoprecipitates were incubated with GSK-3
in the presence of [
-32P]ATP, autoradiography revealed
prominent phosphorylated proteins (Fig.
4A, lane 4) with
the sizes appropriate for LAP and LIP (Fig. 5B, lane 6). No
phosphoproteins were detected when the incubation was performed in the
absence of GSK-3 (Fig. 4A, lane 2) indicating that other kinase activity did not co-precipitate with C/EBP
. When
non-immune serum was used instead of anti-C/EBP
, labeled proteins
were not detected in the absence or presence of GSK-3 (lanes
1 and 3). These results indicate that in
vitro LAP and LIP are substrates of GSK-3.
View larger version (25K):
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Fig. 4.
LAP and LIP are in vitro
substrates of GSK-3. A, lysates of 3T3-F442A
fibroblasts were immunoprecipitated with anti-C/EBP or treated with
non-immune serum (NI). Immune complexes were incubated with
[
-32P]ATP (ATP*) in the absence
(lanes 1, 2) or presence of GSK-3 (lanes
3, 4). B, aliquots of the same samples as in
A were incubated with non-immune serum (lane 5)
or anti-C/EBP
(lane 6) and immunoblotted with
anti-C/EBP
(1:1000). Data are representative of two independent
experiments.
View larger version (50K):
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Fig. 5.
Dephosphorylation of LAP, but not LIP,
increases DNA binding. A, extracts from 293T cells
overexpressing LAP or LIP were mixed and incubated in the absence
(lane 1) or presence (lane 2) of antiserum
specific for C/EBP (anti-
), and complexes containing
LAP and/or LIP were identified by EMSA using wt C/EBP-SRE as probe.
Migration of complexes representing LAP homodimers
(LAP/LAP), LIP homodimers (LIP/LIP), and LAP/LIP
heterodimers (LAP/LIP) is shown (left margin).
The arrow (lane 2) marks supershift with
anti-C/EBP
. The faint upper band in lane 1 represents endogenous SRF. B, extracts from 293T cells
overexpressing LAP or LIP were incubated without (lanes 1,
3, 5) or with alkaline phosphatase
(AP, 200 units) (lanes 2, 4,
6). The extracts were incubated separately (lanes
1-4) or in combination (lanes 5-6) with the wt
C/EBP-SRE probe for EMSA. LAP- or LIP-containing complexes are
indicated as in A. Similar results were obtained in 10 independent experiments. C, extracts used in part
B containing LAP (lanes 1, 2) or LIP
(lanes 3, 4) were analyzed by immunoblotting with
antibody specific for C/EBP
after treatment with (lanes
2, 4) or without (lanes 1, 3)
alkaline phosphatase. Slowly (phosphorylated) and rapidly
(dephosphorylated) migrating forms of LAP and LIP are marked by
upper and lower bars, respectively, in the
right margin. D, extracts enriched in LAP
(lanes 1, 2) or LIP (lanes 3,
4, 7, 8), with or without alkaline
phosphatase treatment, were incubated alone (lanes 1-4) or
in combination (lanes 5, 6) with a probe
containing the C/EBP site of the aP2/422 gene for EMSA.
Binding of the same preparation of LIP to the c-fos
C/EBP-SRE probe, with and without alkaline phosphatase treatment, is
compared in the same experiment (lanes 7 and 8).
Migration of LAP- and LIP-containing complexes is indicated as in
A. Similar results were obtained in three independent
experiments.
. Extracts from 293T cells (which lack
detectable C/EBP
) overexpressing LAP or LIP were subjected to EMSA
to detect LAP and LIP complexes bound to the c-fos C/EBP
site and overlapping SRE (wtC/EBP-SRE) as probe. When a combination of
extracts each enriched in LAP or LIP was subjected to EMSA, homodimers
of LAP (upper complex) and LIP (bottom complex),
and heterodimers of LAP and LIP (middle band) were
detected (Fig. 5A, lane 1). The addition of
antibodies specific for C/EBP
caused a supershift of these complexes
(Fig. 5A, lane 2, arrow). A faint band
representing endogenous SRF in 293T cells was also observed (Fig.
5A, lane 1, top band).
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Fig. 6.
Dephosphorylation of endogenous LAP increases
its binding to DNA. Nuclear extracts from 3T3-F442A cells were
incubated without (lane 1) or with alkaline phosphatase
(AP, 200 units) (lane 2) prior to EMSA with the
wt C/EBP-SRE probe. Nuclear extracts from cells incubated with GH for
60 min (lane 3) were also analyzed by EMSA. Migration of
complexes containing LAP and/or LIP is shown to the left and
was determined by comparison to LAP- or LIP-enriched extracts from 293T
cells on same gel. Similar data were obtained in two independent
experiments.
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Fig. 7.
GSK-3 impairs the DNA binding capacity of
LAP. LAP or LIP were expressed in 293T cells in the presence or
absence of constitutively active GSK-3 (GSK-3 S9A). The
amount of expressed protein in the extracts was quantified by prior
immunoblotting and equivalent amounts of LAP (lanes 1,
2) or LIP (lanes 3, 4) were tested
individually (lanes 1-4) or in combination (lanes
5, 6) by EMSA with wt C/EBP-SRE. LAP- and
LIP-containing complexes are indicated as in Fig. 5. Similar results
were obtained in four independent experiments.
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Fig. 8.
A, Expression of GSK-3 reduces
GH-stimulated c-fos promoter activity. CHO-GHR
cells were transiently transfected with CMV-GSK-3 S9A (+GSK-3
S9A) or empty vector, with wt-c-fos-luc plasmid and
RSV- -gal. After 48 h, cells were treated with GH (hatched
bars) or vehicle (open bars) for 4 h, and
luciferase activity was measured and normalized to
-galactosidase
activity. Each bar represents the mean ± S.E. for six
independent experiments. Luciferase activity was significantly
different (p < 0.001) between control and GH-treated
cells in the absence of GSK-3 S9A and between controls in the absence
and presence of GSK-3 S9A. There was not a significant difference
between control and GH-treated cells in the presence of GSK-3 S9A.
B, LAP does not overcome GSK-3 restraint of basal or
GH-stimulated c-fos promoter activity. CHO-GHR cells were
transiently transfected with plasmids for c-fos-luc,
RSV-
-gal, CMV-LAP, and CMV-GSK-3 S9A or empty vector and were
treated and analyzed as in part A. Each bar
represents the mean ± S.E. for four independent experiments. The
response to GH is significant (p < 0.04) in the
absence of GSK-3 S9A.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in the
Nucleus--
The present studies are consistent with GH utilizing a
PI3K-mediated pathway to alter the phosphorylation state of C/EBP
in
the nucleus, by a mechanism involving activation of Akt and inhibition
of GSK-3. It has been known for some time that GH can initiate PI3K
signaling by stimulating tyrosine phosphorylation of insulin receptor
substrate family proteins, and the association of insulin receptor
substrates 1 and 2 with PI3K (44-47). This study demonstrates that
C/EBP
is a likely nuclear end point for a GH-stimulated
PI3K-mediated pathway, based on reversal of LAP and LIP
dephosphorylation by PI3K inhibitors. Thus for the first time, C/EBP
is identified as a nuclear target for a GH-stimulated PI3K pathway and
shows that PI3K may be a factor that mediates signaling between GHR and
the nucleus.
by GH is strengthened by the additional
observations that GH stimulates the downstream PI3K target Akt and that
GH induces phosphorylation and inhibition of GSK-3. Many growth factors
have been shown to activate Akt (48). Here GH stimulates Akt
phosphorylation on Ser-473. Phosphorylation of Ser-473 and Thr-308 are
required for the full activation of Akt (49). It is likely that an
enzyme such as PDK1/PDK2 mediates activation of Akt in response to GH,
as reported for insulin (49). Autophosphorylation could also contribute
to Akt activation, as reported for IGF-1(50). The activation of Akt
results in phosphorylation and inhibition of GSK-3 (43). This study
shows that GH causes a partial inhibition of GSK-3 most likely by
promoting GSK-3 phosphorylation, as shown for GSK-3
. PDGF produced a
decrease in GSK-3 activity similar to that produced by GH in the same
experiment; the inhibition was comparable to that reported for PDGF in
L6 myotubes, nerve growth factor in PC12 cells, and insulin in 3T3-L1
fibroblasts (39, 40, 42, 51). Dependence of GSK-3 phosphorylation on
GH-stimulated PI3K is supported by inhibition of GH-promoted GSK-3
phosphorylation by wortmannin. Wortmannin also increased basal GSK-3
activity,2 suggesting that
PI3K may contribute to a tonic restraint of GSK-3 in resting cells.
Furthermore, GH was unable to reduce GSK-3 activity below basal levels
in wortmannin-treated cells, reinforcing the idea that inhibition of
GSK-3 by GH is mediated by PI3K.
Is a GSK-3 Substrate--
GSK-3 substrates include
bZIP transcription factors, including C/EBP
, CREB, and c-Jun, as
well other transcription factors such as c-Myc (52-54). C/EBP
contains a putative consensus sequence site for GSK-3 phosphorylation
at Ser-184, adjacent to a MAPK site at Thr-188, that is shared by LAP
and LIP. In the present studies, both LAP and LIP were phosphorylated
by GSK-3 in vitro. One can speculate that the GH-mediated
inhibition of GSK-3 activity contributes to the dephosphorylation of
C/EBP
. Lithium, which can inhibit GSK3 activity, favored
dephosphorylation of LAP and LIP in the presence of GH, supporting a
role for GSK-3 in regulation of C/EBP
phosphorylation by GH.
Similarly, insulin stimulates dephosphorylation of C/EBP
through
inhibition of GSK-3 in 3T3-L1 adipocytes (52). Insulin and PDGF promote
GSK-3 inhibition in 3T3-L1 fibroblasts, resulting in activation of
glycogen synthase (42). However, in 3T3-L1 adipocytes, protein
phosphatase-1 activation rather than GSK-3 inactivation appears to be
the major mechanism by which insulin mediates glycogen synthase
dephosphorylation and activation (42). It will be of great interest to
determine whether GH can also activate a phosphatase that contributes
to C/EBP
dephosphorylation in 3T3-F442A cells.
Results in Functionally Important and
Distinct Changes in Binding of LAP and LIP to DNA--
Regulation of
the phosphorylation state of transcription factors is an important
mechanism for regulation of gene expression (20). The dephosphorylation
of LAP leads to a dramatic increase in its ability to bind to C/EBP
sites in the c-fos and aP2 promoters. Binding of LAP/LAP
homodimers and LAP/LIP heterodimers increased with dephosphorylation of
both endogenous and overexpressed LAP. The increased binding associated
with dephosphorylation corresponded with the increased LAP binding
induced by GH treatment, suggesting that GH-promoted dephosphorylation
enhances LAP binding. C/EBP
contains multiple phosphorylation sites,
including sites for Ras-MAPK (Thr-235),
calcium/calmodulin-dependent protein kinase (Ser-276), protein kinase C (Ser-105), and protein kinase A (Ser-105, Ser-173, Ser-233, Ser-299) (21, 24, 55-57). However, several reports indicate
that phosphorylation of C/EBP
by PKA and/or PKC attenuates site-selective DNA binding (57), whereas others suggest that phosphorylation may increase binding (58). It remains to be determined
which site(s) in LAP are dephosphorylated by GH treatment to increase
DNA binding capacity of LAP.
to bind to
c-fos. Furthermore, SRF and C/EBP
have been shown to
interact in vivo through the DNA binding domain of SRF and
the C terminus of C/EBP
(59). LAP may thus be recruited to the
c-fos promoter not only by binding to DNA, but also by
protein·protein interactions with SRF, which are reported to be
stimulated by activated Ras (59). Mutation of the C/EBP site abolished
the binding of LAP and LIP dimers to the c-fos promoter,
suggesting that they do not bind to the SRE. Interestingly, mutation of
the C/EBP site in the c-fos upstream regulatory region
enhanced the ability of GH to stimulate c-fos expression in
3T3-F442A cells stably expressing SRE luc (14), raising the possibility
that proteins bound to the C/EBP site might restrain the ability of GH
to stimulate c-fos promoter activity. It remains to be
determined whether interaction(s) of C/EBP
with other protein(s)
bound to the c-fos promoter, and/or the recruitment of other
factors to an enhanceosome on c-fos may participate in the
GH-mediated regulation of c-fos expression.
by GH--
Phosphorylation of LAP and LIP,
secondary to overexpression of constitutively active GSK-3, reduces the
binding of LAP and LIP. Similarly for c-Jun, gel shift assays using
nuclear extracts of cells overexpressing GSK-3 show decreased binding
of c-Jun to AP1-sites. c-Jun is phosphorylated by GSK-3 on residues
clustered near the DNA binding domain (54). Such results with c-Jun and GSK-3 are similar to those observed here for LAP and LIP binding to the
c-fos C/EBP site when GSK-3 is co-expressed with LAP or LIP.
The proximity of the consensus sequence for GSK-3 phosphorylation of
LAP and LIP to the DNA binding domain raises the possibility that, as
suggested for c-Jun, charge repulsion via the phosphate groups on amino
acids close to the DNA binding domain may interfere with the
interaction of this region of the protein with DNA, thereby decreasing
LAP and LIP binding in the presence of GSK-3. Future mapping of the
phosphorylation sites of GSK-3 in C/EBP
and mutation of the site(s)
of phosphorylation will provide insight into the mechanism of
GH-mediated regulation of C/EBP
phosphorylation and function.
. Changes in the phosphorylation state of
LAP and LIP are functionally important in modulating their ability to
bind DNA and regulate transcription. Further studies on C/EBP
phosphorylation will provide more insight into the complex mechanisms
by which GH regulates gene expression.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Alan Saltiel for the gift of GSK-3 peptide substrate. We also thank Drs. L. Argetsinger and J. Herrington for critical review of this manuscript, and S. Reoma, A. Bates, and B. Hawkins for assistance in the preparation of the figures and manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health (NIH) Grants DK46072 (to J. S.) and DK51563 (to O. 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.
Supported by Medical Scientist Training Program Grant GM07863 from NIH.
§ Supported by the Natural Sciences and Engineering Research Council of Canada.
¶ To whom correspondence should be addressed: Dept. of Physiology, University of Michigan Medical School, Ann Arbor, MI 48109-0622. Tel.: 734-647-2124; Fax: 734-647-9523; E-mail: jeschwar@umich.edu.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.M010193200
2 G. Piwien-Pilipuk and J. Schwartz, unpublished.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GH, growth hormone; GHR, growth hormone receptor; STAT, signal transducers and activators of transcription; C/EBP, CCAAT/enhancer-binding proteins; LAP, liver-activating protein; LIP, liver inhibitory protein; bp, base pair(s); SRE, serum response element; PI3K, phosphatidylinositol 3-kinase; CHO, Chinese hamster ovary; BSA, bovine serum albumin; MAPK, mitogen-activate protein kinase; AP, alkaline phosphatase; ERK, extracellular signal-regulated kinase; MEK, MAPK/ERK kinase; GSK-3, glycogen synthase kinase-3; CMV, cytomegalovirus; RSV, Rous sarcoma virus; PAGE, polyacrylamide gel electrophoresis; wt, wild-type; PDGF, platelet-derived growth factor; CREB, cAMP-response element-binding protein; AP-1, activator protein-1; SRF, serum response factor..
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