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J Biol Chem, Vol. 273, Issue 47, 31327-31336, November 20, 1998
From the Growth hormone (GH), a major regulator of normal
body growth and metabolism, regulates cellular gene expression. The
transcription factors Elk-1 and Serum Response Factor are necessary for
GH-stimulated transcription of c-fos through the Serum
Response Element (SRE). GH stimulates the serine phosphorylation of
Elk-1, thereby enabling Elk-1 to mediate transcriptional activation.
The contribution of the Ras/mitogen-activated protein kinase kinase
(MEK)/extracellular signal-regulated kinase (ERK) pathway to
Elk-1-mediated transcriptional activation of the c-fos SRE
in response to GH was examined. The MEK inhibitor PD098059 attenuated
GH-induced expression of the endogenous SRE-regulated genes
c-fos, egr-1, and junB as well as
transcriptional activation mediated by the c-fos promoter. The MEK inhibitor blocked GH-stimulated activation of MEK,
phosphorylation of ERK1/ERK2, and MAP kinase activity in 3T3-F442A
cells. Blocking MEK activation prevented GH-induced phosphorylation of
Elk-1, as well as the ability of Elk-1 to mediate transcriptional
activation in response to GH. Overexpression of dominant-negative Ras
or the ERK-specific phosphatase, mitogen-activated protein kinase phosphatase-1, blocked the Ras/MEK/ERK pathway and abrogated
GH-induced phosphorylation of Elk-1. GH failed to stimulate
phosphorylation or activation of Jun N-terminal kinase under the
conditions used. GH slightly increased p38-mediated mitogen-activated
protein kinase-activated protein (MAPKAP) kinase-2 activity, but the
p38 inhibitor SB203580 did not attenuate GH-promoted Elk-1
phosphorylation. Wortmannin, which inhibited GH-induced ERK
phosphorylation, also attenuated transcriptional activation of
c-fos by GH. Taken together, these data suggest that
GH-dependent activation of the Ras/MEK/ERK pathway and subsequent serine phosphorylation of Elk-1 contribute to
GH-stimulated c-fos expression through the SRE.
Expression of the c-fos proto-oncogene is rapidly
induced by growth hormone
(GH)1 (1, 2). The Serum
Response Element (SRE), an enhancer sequence upstream of the
c-fos gene, can mediate transcriptional activation in
response to GH (3, 4). Transcriptional activation via the SRE in
response to GH requires the presence of the transcription factors
Ternary Complex Factor (TCF) and Serum Response Factor (SRF) at the SRE
(4).
Elk-1 is a member of the TCF family of transcription factors, which
also includes SAP-1 and SAP-2/ERP/Net (5, 6). GH was recently found to
stimulate serine phosphorylation of Elk-1 and to stimulate
Elk-1-dependent transcriptional activation (4). Phosphorylation and activation of Elk-1 in response to agents such as
serum and ultraviolet light are reported to be mediated by
mitogen-activated protein kinase (MAPK) pathways, including extracellular signal-regulated kinases (ERKs) 1 and 2, as well as the
stress-induced kinases Jun N-terminal kinase (JNK) and p38 (7-12).
The binding of GH to its receptor results in the activation of JAK2
(13), initiating several signaling cascades, including one in which a
SHC-Grb2-SOS complex activates Ras (14). These events correlate in time
with GH-induced activation of Raf, MAPK/ERK kinase (MEK), and ERK1 and
-2 (14-17). Activation of the ERK pathway by GH raises the possibility
that a MAPK pathway may be involved in the regulation of GH-promoted
Elk-1 activity. GH has also been documented to stimulate tyrosine
phosphorylation of insulin receptor substrate-1 and -2 and concomitant
association of both insulin receptor substrate proteins with the 85-kDa
regulatory subunit of phosphatidylinositol (PI) 3-kinase (18-21).
Studies in which the PI 3-kinase inhibitor, wortmannin, interferes with
ERK activation suggest a link between PI 3-kinase activation and
activation of ERKs (22-25). Thus, signaling pathways involving PI
3-kinase may modulate the ERK pathway. This study examines the
involvement of Ras, MEK, and ERK1 and -2 in Elk-1-mediated
transcriptional activation of c-fos in response to GH and
possible sensitivity of this pathway to the PI 3-kinase inhibitor
wortmannin. Potential roles for JNK and p38 in mediating
GH-induced Elk-1 phosphorylation were also investigated.
Materials--
Stocks of 3T3-F442A cells were provided by Dr. H. Green (Harvard University) and Dr. M. Sonenberg (Sloan-Kettering, New
York, NY). Chinese hamster ovary (CHO) cells expressing full-length rat
GH receptor (GHR-(1-638)) or GHR lacking the C-terminal half of the
cytoplasmic domain (GHR-(1-454)) were provided by Drs. Gunnar Norstedt
(Karolinska, Stockholm, Sweden) and Nils Billestrup (Hagedorn
Laboratory, Gentofte, Denmark) (26). Recombinant human GH was provided
by Lilly. The MEK inhibitor PD098059 was a gift from Dr. A. Saltiel
(Parke Davis, Ann Arbor, MI). Wortmannin, the p38 inhibitor SB203580,
and anisomycin were purchased from Calbiochem. Sorbitol and
phenylmethylsulfonyl fluoride were from Sigma. Leupeptin, aprotinin,
and pepstatin were purchased from Boehringer Mannheim, bovine serum
albumin (CRG7) from Intergen, and radioisotopes from NEN Life Science
Products. Lipofectamine was purchased from Life Technologies, Inc., and
luciferin was from Promega. The ECL detection system and RediprimeTM
kit were purchased from Amersham Pharmacia Biotech.
Cell Culture and Treatment--
3T3-F442A fibroblasts were grown
to confluence in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose and 8-9% calf serum in an atmosphere of 10%
CO2/90% air at 37 °C. CHO-GHR cells were grown in
Ham's-F12 medium containing 0.5 mg/ml G418 and 10% fetal calf serum
in an atmosphere of 5% CO2, 95% air. All media were
supplemented with 1 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml
amphotericin. Prior to treatment, cells were deprived of serum for
16-18 h in Dulbecco's modified Eagle's medium containing 4.5 g/liter
glucose or Ham's F-12 medium containing 1% bovine serum albumin.
Cells were preincubated with inhibitor or vehicle (Me2SO,
0.2% final concentration, or ethanol) for 1 h prior to treatment
with GH or other agents. Unless otherwise indicated, the final
concentration of the MEK inhibitor was 100 µM, and GH was
used at a concentration of 500 ng/ml (22 nM). This
concentration of GH is within the physiological range for circulating
rodent GH (27, 28).
Northern Blot Analysis--
Total RNA was isolated as described
(29) and subjected to Northern blot analysis using ExpressHyb
(CLONTECH). Prehybridization was performed at
55 °C, and hybridization was performed at 68 °C. The membranes
were probed with cDNAs for mouse c-fos (2), egr-1 (from Dr. L. Lau, University of Illinois, Chicago, IL
(30)), junB (from Dr. D. Nathans, John Hopkins, Baltimore MD
(31)), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, from Dr. M. Alexander-Bridges, Harvard University), which were labeled with
32P using a Rediprime kit from Amersham Pharmacia Biotech.
Membranes were subjected to autoradiography and/or phosphor imaging,
and quantification was performed using Multi-Analyst/PC Molecular Imager software (Bio-Rad).
Luciferase Assay--
3T3-F442A cells were stably transfected
with a GH-responsive luciferase reporter plasmid driven by the region
of the c-fos promoter from Elk-1-mediated Reporter Activation--
An expression-reporter
system was used to measure Elk-1-mediated transcriptional activation.
The expression plasmid Gal4/ElkC (34) encodes a fusion protein
containing the Elk-1 transactivation domain fused to the Gal4 DNA
binding domain. The control plasmid Gal4 (1-147) (35, 36) encodes
amino acids 1-147 of the Gal4 DNA binding domain without an activation
domain. The reporter plasmid, 5× Gal/Luc (37) contains five copies of
the Gal4 binding site upstream of luciferase cDNA. The plasmids
Gal4/ElkC and 5× Gal/Luc were provided by Dr. C. Der (University
of North Carolina), and Gal4 (1-147) was received from Dr. M. Ptashne
(Harvard University). CHO cells expressing GHR-(1-454) were plated on
35-mm plates and transiently co-transfected using calcium phosphate
with 1 µg of Gal4/ElkC or Gal4(1-147) DNA, 1 µg of Gal/Luc DNA,
and 0.1 µg of RSV Elk-1 Phosphorylation--
The cDNA for Elk-1 (from Dr. R. Treisman, Imperial Cancer Research Fund, London) was inserted into the
vector pcDNA3 downstream of a CMV promoter to generate CMV-Elk-1
(38). In experiments evaluating Elk-1 phosphorylation, CMV-Elk-1 or
pcDNA3 was transiently transfected into CHO cells expressing
GHR-(1-454) using calcium phosphate. Other plasmids were
co-transfected with CMV-Elk-1 as follows. The plasmids pZIP-ras(WT),
pZIP-ras(15A), and the vector pZIP-NeoSV(x)1 (39, 40) provided by Dr.
C. Der (University of North Carolina) were each used at 30 µg/100-mm
plate. The plasmid encoding MAP kinase phosphatase-1 (MKP-1) (41) was
from Dr. J. Pessin (University of Iowa). The CLDN vector, used as a
control, was recovered by excising the
EcoRI/BamHI fragment containing the MKP-1
sequence. Klenow was then used to generate blunt ends, which were
ligated with T4 ligase. Both MKP-1 and CLDN were used at 5 µg/100-mm
plate. 26 h after transfection, the cells were incubated in medium
containing 0.5% fetal calf serum for 16 h and were then incubated
in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose
and 1% bovine serum albumin for 5 h prior to treatment as
indicated. Cells were lysed and immunoblotted as described.
Antibodies--
Polyclonal antibodies against a peptide
corresponding to residues 379-392 of Elk-1 phosphorylated on Ser-383
( Immunoblotting--
Following treatment, cells were washed three
times with PBSV buffer (10 mM Tris, 150 mM
sodium phosphate, 100 µM Na3VO4,
pH 7.4). For analysis of ERKs and Elk-1, cells were scraped in 0.5 ml
of 50 mM Hepes, pH 7.0, containing 250 mM NaCl,
0.1% Nonidet P-40, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each
aprotinin and leupeptin. In assays for JNK phosphorylation, cells were
lysed in 50 mM Hepes, pH 7.5 containing 150 mM
NaCl, 1.5 mM EGTA, 10% glycerol, and 1% Nonidet P-40, 1 mM Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 10 µg each of aprotinin and leupeptin,
100 mM NaF, and 0.5 mM
Na4P2O7. Cell lysates were analyzed on 7.5% polyacrylamide gels for Elk-1, 12% polyacrylamide gels for
JNK, and 5-12% gradient polyacrylamide gels for ERK1 and -2. Western
blot analysis was performed as described (43) with the indicated
antibodies. Enhanced chemiluminescence was used for detection. The
apparent Mr indicated are based on the migration of prestained molecular weight standards (Life Technologies, Inc.). Membranes were stripped by submerging them in 100 mM
Kinase Assays--
MAP kinase assays were performed with myelin
basic protein as substrate as described previously (16). MEK activity
was measured as described previously (14). Briefly, cell lysates were
incubated for 2 h with an antibody recognizing both MEK1 and -2 (1:1000 dilution). Immune complexes were collected on protein A-agarose during a 1-h incubation with rotation at 4 °C; washed three times with 50 mM Tris, pH 7.5, 0.1% Triton X-100, 137 mM NaCl, and 2 mM EGTA; and washed once with 25 mM HEPES, pH 8.0, 0.5 mM EDTA, 0.25%
Inhibition of the MEK/ERK Pathway Impairs GH-stimulated
Transcriptional Activation Mediated by the c-fos Promoter--
GH has
been shown to promote c-fos gene expression (2, 45) as well
as activation of Ras, Raf, MEK, and ERK1 and -2 in 3T3-F442A
fibroblasts (14-16). To determine whether the MEK/ERK pathway
contributes to GH-stimulated c-fos expression, the MEK inhibitor PD098059 was used to block activation of the MEK/ERK pathway.
PD098059 suppresses MEK activation by binding inactive MEK and
preventing its phosphorylation by upstream kinases (46).
The GH-promoted increase in endogenous c-fos expression
detected after 30 min (Fig.
1A, lane
2, top) was progressively inhibited by increasing
concentrations of MEK inhibitor (Fig. 1A, lanes 3-5). When normalized to GAPDH expression, which is
unaffected by GH or PD098059 treatment, expression of c-fos
was attenuated an average of 31, 69, and 84% with 25, 50, and 100 µM PD098059, respectively (n = 2).
To determine whether blocking MEK activation decreases GH-induced
activation of the c-fos promoter, PD098050 was added to 3T3-F442A fibroblasts stably transfected with a plasmid (mC/E) containing a GH-responsive c-fos promoter fragment
(
The SRE, which mediates GH-promoted expression of c-fos (3,
4) is also present in the promoters of the GH-responsive genes
egr-1 (29) and junB (47). To assess whether MEK
is involved in the expression of SRE-regulated genes other than
c-fos, the effect of PD098059 on the expression of
egr-1 and junB was examined (Fig.
2). In 3T3-F442A cells, GH stimulated
expression of both egr-1 and junB (Fig. 2,
A and B, top, lane
2). In two experiments in which mRNA levels were
normalized to GAPDH mRNA levels, expression of egr-1 and
junB was inhibited by pretreatment with PD098059 (Fig. 2,
lanes 3-5). Pretreatment of cells with 50 µM PD098059 decreased GH-promoted expression of
egr-1 by 35% and junB by 43%, and 100 µM PD098059 decreased the expression of egr-1
by 61% and junB by 55%.
To verify that PD098059 effectively blocks GH-stimulated activation of
MEK and ERK, 3T3-F442A fibroblasts were preincubated with PD098059 for
1 h prior to GH addition (Fig. 3).
The activity of ERK1 and -2 was assessed by Western blot analysis with
an antibody (
To provide additional evidence that the MEK inhibitor attenuates
GH-promoted MAP kinase activity, MAP kinase activity was directly
examined in 3T3-F442A lysates using an in vitro kinase assay
with myelin basic protein as substrate. GH increased MAP kinase
activity almost 2-fold (Fig. 3B). PD098059 reduced
GH-stimulated MAP kinase activity at concentrations (25, 50, and 100 µM) that inhibit GH-induced phosphorylation of ERK1 and
ERK2 (Fig. 3B). PD098059 decreased basal MAP kinase activity
slightly (8%, data not shown). MEK activity was measured using MEK
that was immunoprecipitated from 3T3-F442A cell lysates with antibodies
that recognize MEK1 and MEK2 (Fig. 3C). MEK
immunoprecipitates were used in an in vitro kinase assay
containing a glutathione S-transferase-ERK fusion protein
(14) and myelin basic protein. GH stimulated an 11-fold increase in MEK
activity, which was reduced approximately 75% when cells were
pretreated with 50 or 100 µM MEK inhibitor (Fig.
3C). In Fig. 3C, the MEK inhibitor failed to
abrogate MEK activity completely. It seemed possible that MEK2 activity
might account for the residual activity, although 10-fold higher
concentrations of PD098059 are needed to inhibit MEK2 than MEK1 (46).
However, results similar to those shown in Fig. 3C were
obtained using antibodies specific for MEK1, suggesting that the
residual MEK activity is not due to MEK2.
Wortmannin Impairs GH-stimulated Expression and Promoter Activation
of c-fos--
Wortmannin, a potent PI 3-kinase inhibitor, has been
demonstrated to interfere with GH-induced ERK activation, presumably via a mechanism involving PI 3-kinase (25, 48). If ERKs mediate GH-promoted c-fos expression, then wortmannin would also be
expected to inhibit GH-induced c-fos expression. In
3T3-F442A cells, expression of c-fos mRNA (normalized to
GAPDH mRNA levels) was reduced by 41% with 100 nM
wortmannin and by 84% with 500 nM wortmannin (Fig. 4A, lanes
3 and 4 versus lane
2). Wortmannin inhibited GH-induced c-fos
promoter-mediated reporter expression by 64% (250 nM) and 86% (500 nM) (Fig. 4B). Concentrations of
wortmannin of 100 and 500 nM inhibited GH-induced ERK
phosphorylation (Fig. 4C, top, lanes
4 and 5 versus lane
2) in the absence of a change in ERK expression. Thus,
wortmannin effectively inhibits ERK phosphorylation and also decreases
expression of c-fos mRNA and c-fos promoter activity in response to GH. Taken together, the inhibition by wortmannin is consistent with ERKs mediating GH-dependent
activation of c-fos. Furthermore, a wortmannin-sensitive
molecule, possibly PI 3-kinase, may participate in
ERK-dependent activation of gene expression in response to
GH.
Inhibition of the MEK/ERK Pathway Blocks GH-stimulated Activation
of Elk-1--
Previous studies (4) demonstrated that GH stimulates
Elk-1-mediated transcriptional activation of a luciferase reporter gene
in CHO cells stably expressing full-length GHR-(1-638) or truncated
GHR-(1-454). GH induces c-fos mRNA and stimulates
transcriptional activation via the SRE to comparable extents in both
cell lines (29). Unless otherwise indicated, CHO cells expressing
GHR-(1-454) (referred to hereafter as CHO-GHR cells) were used in the
present study. To examine whether blocking ERK activity alters the
ability of Elk-1 to mediate transcriptional activation in response to GH, CHO-GHR cells transfected with plasmids encoding a
Gal-Elk/Gal-luciferase expression-reporter system (see "Experimental
Procedures") were treated with PD098059 prior to GH (Fig.
5, left panel). GH
alone stimulated Elk-1-mediated transcriptional activation in CHO-GHR cells, as expected. Incubation with PD098059 prior to GH treatment blocked GH-stimulated luciferase activity (Fig. 5, left
panel), indicating that GH-stimulated
Elk-1-dependent gene expression requires MEK activation.
Similar results were observed in CHO cells stably expressing
full-length GHR-(1-638) (data not shown). In control experiments, GH
failed to stimulate Elk-1-mediated reporter expression in cells
transfected with a plasmid (Gal4 (1-147)) encoding the Gal4 DNA
binding domain lacking an activation domain (Fig. 5, right
panel), indicating that the Elk-1 transcriptional activation
domain is required for GH-stimulated reporter expression.
GH-stimulated Phosphorylation of Elk-1 Is Blocked by the MEK
Inhibitor, Dominant Negative Ras, and MAPK Phosphatase-1--
Since
phosphorylation of Elk-1 is thought to be important in Elk-1
activation, the ability of the MEK inhibitor to block GH-stimulated Elk-1 phosphorylation was examined. CHO-GHR cells were transiently transfected with a plasmid (CMV-Elk-1) encoding Elk-1, and
phosphorylated Elk-1 was detected by immunoblotting with antibodies
against a synthetic Elk-1 peptide phosphorylated on serine 383 (Fig.
6). The stimulation of Elk-1
phosphorylation by GH was concentration-dependent from 5 to
500 ng/ml (0.22-22 nM). The appearance of multiple bands with decreased mobility in the lysates of GH-treated cells is consistent with Elk-1 being phosphorylated on multiple sites
(upper panel). Pretreatment of cells with
PD098059 (100 µM) obliterated all bands recognized by the
To demonstrate further the role of the signaling pathway involving Ras,
MEK, and ERK in GH-stimulated Elk-1 phosphorylation, additional steps
of this MAPK pathway were modulated. Interfering with MAPK activation
by co-expressing MKP-1 with CMV-Elk-1 prevented the appearance of the
bands recognized by JNK and p38 Kinase Do Not Mediate GH-stimulated Phosphorylation and
Activation of Elk-1--
Although ERK1 and ERK2 appear to be the major
Elk-1 kinases regulated by growth factors (10, 49), JNK and p38 kinase
are also capable of activating Elk-1 in response to cellular stresses such as ultraviolet light and interleukin-1 (8, 11). To determine whether GH regulates JNK and as a step toward determining whether JNK
contributes to GH-stimulated transcriptional activation via Elk-1, JNK
activity was assayed in 3T3-F442A cells treated with GH. JNK
phosphorylation was measured using an antibody (
To investigate the possible contribution of p38 kinase activity to
GH-stimulated Elk-1 phosphorylation and activation, CHO-GHR cells
transfected with CMV-Elk-1 were pretreated with the specific p38
inhibitor, SB203580. The p38 inhibitor (10 µM) failed to
reduce GH-stimulated Elk-1 phosphorylation when compared with
vehicle-treated cells and may have increased it (Fig.
9B, lane
6 versus lane 2). In the
same experiment, PD098059 blocked GH-promoted Elk-1 phosphorylation completely (lane 4). Furthermore, the p38
inhibitor failed to block the ability of Elk-1 to mediate
transcriptional activation in response to GH (data not shown). To
determine whether GH regulates p38, the activity of the p38 substrate,
MAPKAP kinase-2, was measured in 3T3-F442A cells (Fig. 9B).
GH induced a modest 0.6-fold increase in MAPKAP kinase-2 activity,
compared with the more robust (4-fold) stimulation by anisomycin (data
not shown), which is known to stimulate p38 kinase activation. The
modest increase in GH-induced p38 kinase activity was blocked
completely when cells were preincubated with 10 µM of the
p38 inhibitor SB203580 (Fig. 9B). In contrast, the MEK
inhibitor did not reduce the slight GH-promoted increase in MAPKAP
kinase-2 activity in two independent experiments (data not shown).
Thus, GH stimulates a modest increase in p38-dependent MAPKAP kinase-2 activity, and this activity is not sensitive to MEK
inhibition. Although the p38 inhibitor blocked the modest GH-induced
increase in p38 activity, it failed to attenuate GH-induced phosphorylation of Elk-1, suggesting that GH-promoted p38 activity is
not likely to play a role in GH-stimulated Elk-1 phosphorylation. These
data are consistent with the hypothesis that MEK-activated ERK1 and -2 are the key MAPKs that mediate GH-induced Elk-1 phosphorylation and
activation.
Activation of Ras, MEK, and ERK1 and -2 Mediates GH-stimulated
Phosphorylation and Activation of Elk-1--
This study demonstrates
that GH-stimulated phosphorylation of Elk-1 and
Elk-1-dependent gene expression are mediated by a pathway
involving activation of Ras, MEK, and ERK1 and -2. Overexpression of a
dominant negative Ras mutant reduces GH-stimulated Elk-1 phosphorylation, suggesting that Ras plays a role in Elk-1
phosphorylation. Conversely, overexpression of wild-type Ras elevates
basal and GH-induced phosphorylation of Elk-1. Since activation of Ras
leads to activation of the MEK/ERK pathway (50, 51), these data are
consistent with the idea that Ras acts upstream of MEK and ERK to
participate in GH-stimulated phosphorylation of Elk-1. However,
treatment with GH increased the phosphorylation of Elk-1 in cells
overexpressing wild-type Ras, suggesting that other signaling molecules
may also contribute to activation of a MEK/ERK pathway in response to GH.
Inhibition of MEK or ERK activity blocks GH-promoted Elk-1
phosphorylation and activation completely. The MEK inhibitor PD098059 was used as one tool for examining the role of MEK and ERK in GH
signaling, because it specifically inhibits the phosphorylation of
inactive MEK by upstream kinases while failing to inhibit directly the
activation of other signaling molecules (46) such as p38, JNK, ERK2,
MAPKAP kinases 1 and 2, Raf (an activator of MEK), MKK4 (a JNK kinase),
and protein kinase C (implicated in GH-stimulated c-fos
expression) (2, 47, 52). GH-stimulated phosphorylation and activation
of Elk-1 was blocked effectively by the MEK inhibitor (100 µM). Since the MEK inhibitor also abrogated GH-induced
ERK activity, these data support the involvement of both MEK and ERKs in the phosphorylation and activation of Elk-1 in response to GH.
Furthermore, dephosphorylation of ERKs with the ERK-specific phosphatase, MKP-1, also successfully blocks Elk-1 phosphorylation, substantiating the role of ERKs in the phosphorylation of Elk-1. Together, these data support a model in which activation by GH of a
Ras/MEK/ERK cascade leads to ERK-mediated phosphorylation and
activation of Elk-1.
Recent evidence suggests that the EGFR may contribute to JAK2-mediated
activation of the MEK/ERK pathway in response to GH (53). The present
study does not address specifically the mechanisms upstream of Ras by
which GH stimulates activation of the MEK/ERK pathway. Rather, the data
presented here clearly show that the MEK/ERK pathway is essential for
the activation of downstream end points such as Elk-1 phosphorylation
and transcriptional activation and expression of early response genes
in response to GH. However, results presented here indicate that the
effects of GH on Elk-1 phosphorylation and c-fos expression
can occur independently of EGFR-mediated signaling, because CHO-GHR
cells, in which these responses were studied, lack the EGFR. In
3T3-F442A cells, which have both endogenous GHR and EGFR, it is
possible that EGFR-mediated signaling contributes to GH-stimulated
activation of the MEK/ERK pathway. Future investigation of the varying
contributions of GHR and EGFR signaling to GH-stimulated activation of
the MEK/ERK pathway as well as downstream gene expression may yield
insights into how cross-talk occurs between various signaling pathways.
JNK and p38 Kinase Do Not Mediate GH-stimulated Phosphorylation and
Activation of Elk-1--
Among the MAPKs, both JNK and p38 MAPKs are
also reported to phosphorylate Elk-1 in several cell types (7, 9, 11, 12). JNK and p38 are thought to be activated primarily in response to
cellular stresses such as inflammatory cytokines, heat and chemical
shock, bacterial endotoxin, and ischemia (reviewed in Ref. 54).
However, activation and integration of MAP kinase signaling pathways
utilizing JNK, p38, and ERK are complex and dependent on many factors
such as cell type and the nature of the stimulus (54). In 3T3-F442A
cells, GH failed to stimulate phosphorylation of JNK or JNK activity as
measured by the phosphorylation of its substrate, Jun. Although GH
produced a slight (0.6-fold) increase in p38-dependent
MAPKAP kinase-2 activity, the inability of the p38 inhibitor SB203580
to attenuate GH-promoted Elk-1 phosphorylation or Elk-1-mediated
transcriptional activation makes it highly unlikely that p38
contributes to GH-stimulated Elk-1 activation. These data do not
preclude possible involvement of p38 in other GH-dependent events. Together, the present data are consistent with the view that
JNK and p38 do not contribute significantly to GH-promoted phosphorylation and activation of Elk-1. Further, because the MEK
inhibitor PD098059 does not inhibit JNK or p38 activity directly in vitro, the complete block of GH-induced Elk-1
phosphorylation and activation observed with PD098059 suggests that
ERK1 and -2 are the primary, and perhaps the sole, Elk-1 kinases
activated in response to GH.
ERK1 and -2 Mediate Activation of SRE-regulated Genes in Response
to GH--
The present data suggest that GH-stimulated
c-fos expression is mediated by ERK activation of
transcription factors bound to the c-fos SRE. The
c-fos SRE includes a CArG box, which binds SRF, and an Ets
motif, which binds TCF family members (55). In GH-treated cells, Elk-1
and SRF are both present in the complex bound to the c-fos
SRE (4). Binding of SRF to the CArG box is thought to recruit TCF
family members, such as Elk-1, to the Ets motif sequence (55). Thus, GH
fails to stimulate transcriptional activation via a c-fos
promoter fragment or the SRE alone when either the SRF or TCF binding
sites are mutated (4, 56). Other TCF family members such as SAP-1a and
SAP-2/ERP/NET can bind to the Ets motif (6). Both of these TCFs are
reported to mediate transcriptional activation when activated by ERK2, although SAP-1a has also been shown to be activated by the
stress-induced MAPKs, JNK and p38 (6, 57, 58). Future investigation of the effects of GH and other agents on the binding, phosphorylation, and
activation of SAP-1a and SAP-2/ERP/NET may provide additional insight
into how TCF proteins regulate gene expression in a stimulus-specific manner and whether they play a role in transcriptional activation via
the SRE in response to GH .
Activation of ERK1 and -2 by GH may facilitate binding of SRF to the
SRE. SRF can be phosphorylated by an ERK-dependent
mechanism involving p90rsk (59-63); SRF phosphorylation is
thought to facilitate binding of SRF to the SRE (60, 61). GH stimulates
activation of p90rsk in 3T3-F442A cells (64), but it is not
currently known whether GH promotes phosphorylation of SRF. Although
Elk-1 and SRF are constitutively bound to the SRE in 3T3-F442A cells,
GH rapidly and transiently increases further binding of both proteins
(4). More work is needed to determine whether ERK-dependent
binding of SRF is necessary for transcriptional activation through the SRE in response to GH.
The model of GH-stimulated activation of SRE-containing genes mediated
by ERK1 and -2 is supported by the observation that attenuation of ERK
activation with the MEK inhibitor also impaired GH-stimulated
expression of egr-1 and junB. Two of the four SRE sequences upstream of egr-1 mediate transcriptional
activation in response to human granulocyte-macrophage
colony-stimulating factor, mouse interleukin-3, urea, and antigen
receptor cross-linking (65-67). The induction of egr-1
transcription by these agents requires the Ets sequences adjacent to
the CArG boxes (66, 67). Moreover, the expression of egr-1
is thought to be ERK-dependent in response to urea and
insulin (68, 69) and to involve activation of JAK2 and Ras in the case
of human granulocyte-macrophage colony-stimulating factor (65). The
junB enhancer regions also contain two SREs that mediate
transcriptional activation in response to serum (70, 71). One of the
junB SREs is similar to the c-fos SRE (70, 71),
but it is not yet clear whether Ets sites play a role in transcriptional activation through the junB SREs. Further,
Ets-independent transcriptional activation through the c-fos
SRE can be stimulated by treatment with lysophosphatidic acid (72),
indicating that multiple mechanisms exist for stimulation of
c-fos transcription via the SRE. It will be of interest to
determine whether ERK-mediated Elk-1 activation of transcription
through the SRE is the only pathway by which GH induces expression of
SRE-regulated genes.
The MEK inhibitor may block GH-stimulated transcriptional activation of
c-fos, egr-1, and junB in part by
inhibiting the ERK-dependent activation of transcriptional
complexes other than the SRE in the promoters of these genes. For
example, previous work has shown that GH induces tyrosyl
phosphorylation and binding of STATs 1 and 3 to the Sis-inducible
element in the c-fos promoter (73-75). Serine
phosphorylation, possibly mediated by ERKs, is thought to contribute to
STAT-mediated transcriptional activation (76-79). While these studies
suggest that STAT-mediated transcriptional activation may be
susceptible to MEK inhibition, the role of STATs in GH-stimulated
activation of c-fos is still being explored.
Role of PI 3-Kinase in GH-stimulated ERK
Activation--
GH-induced signaling through the Ras/MEK/ERK cascade
may be modulated by the activity of other signaling molecules, such as those sensitive to wortmannin. This study demonstrates that wortmannin interferes with GH-stimulated ERK activation as well as downstream transcriptional activation of c-fos. GH promotes
phosphorylation of Insulin Receptor Substrate-1 and -2 and association
of these proteins with the Src homology 2 domain of the 85-kDa
regulatory subunit of PI 3-kinase, resulting in activation of PI
3-kinase (18, 19, 21). The PI 3-kinase inhibitor wortmannin has been used to implicate PI 3-kinase in GH-stimulated lipid synthesis (25) and
activation of p70S6K (80). In addition, GH-promoted ERK
activation is inhibited in wortmannin-treated cells (23, 48, 80), as
observed here. The data here demonstrate that GH-stimulated expression
of c-fos and c-fos promoter function is blocked
by wortmannin. Such inhibition is consistent with a requirement for ERK
activation in GH-promoted transcription of c-fos and raises
the possibility that a member of the PI 3-kinase family may participate
in GH-induced c-fos expression.
In summary, the data presented support a model in which activation by
GH of the Ras/MEK/ERK cascade promotes Elk-1 phosphorylation and
Elk-1-mediated transcriptional activation of gene expression. When the
Ras/MEK/ERK pathway was blocked, GH failed to stimulate phosphorylation
and activation of Elk-1. Since GH did not activate JNK and produced
only a slight increase in p38 activity, which did not contribute
significantly to GH-stimulated Elk-1 phosphorylation or activation, JNK
and p38 are not likely to activate Elk-1 in response to GH. The
Ras/MEK/ERK cascade was found to mediate GH-induced expression of
several SRE-regulated genes, including c-fos,
egr-1, and junB. A wortmannin-sensitive molecule,
possibly PI 3-kinase, may regulate ERK activation and subsequent
transcriptional activation of c-fos in response to GH.
Knowledge of the mechanisms by which GH promotes changes in gene
expression provides a foundation for exploring the many functions of
this major regulator of growth and differentiation.
We thank Drs. L. Argetsinger, J. Bragado, D. Gumucio, T. Kerppola, L. Mathews, D. Meyer, C. Schaefer, and J. VanderKuur for helpful discussions; D. Meyer also for help with assays
for JNK activity; P. Du for technical assistance with CHO cells; and
Drs. L. Argetsinger, J. Herrington, and O. MacDougald for critical comments on the manuscript.
*
This work was supported by National Science Foundation (NSF)
Grant IBN 9221667 (to J. S.) and National Institutes of Health Grants
DK 46072 (to J. S.), DK34171 (to C. C. S.), and GM51586 (to
K. L. G.).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 a NSF predoctoral fellowship and by a Merit Fellowship
from the University of Michigan.
§§
To whom all 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{at}umich.edu.
The abbreviations used are:
GH, growth hormone; GHR, GH receptor; SRE, Serum Response Element; SRF, Serum Response
Factor; TCF, Ternary Complex Factor; MAPK, mitogen-activated protein
kinase; ERK, extracellular signal-regulated kinase; JNK, Jun N-terminal
kinase; MKP-1, MAPK phosphatase-1; MAPKAP kinase, MAPK-activated
protein kinase; PI, phosphatidylinositol; SAP, SRE-associated protein; STAT, signal transducer and activator of transcription; EGFR, epidermal
growth factor receptor; CHO, Chinese hamster ovary; RSV, Rous sarcoma
virus; CMV, cytomegalovirus; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Growth Hormone Stimulates Phosphorylation and Activation of
Elk-1 and Expression of c-fos, egr-1, and
junB through Activation of Extracellular Signal-regulated
Kinases 1 and 2*
§,
,,¶, and¶§§
Program in Cellular and Molecular Biology
and Departments of ¶ Physiology,
Biological Chemistry, and ** Microbiology
and Immunology, University of Michigan Medical School,
Ann Arbor, Michigan 48109-0622
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
379 to +1 with a mutation in
the CCAAT/enhancer-binding protein binding site (mC/E) (32) (provided
by Dr. W. Walton, Moffitt Cancer Center, Tampa, FL) and RSV-neo (from
Dr. Nils Billestrup, Hagedorn Laboratory). Pooled clones were generated
and maintained as described previously (4). Cells were deprived of
serum overnight and incubated with GH for 4 h. Cell lysates were
prepared in reporter lysis buffer (100 mM potassium
phosphate, pH 7.8, 0.2% Triton X-100, 1 mM
dithiothreitol), and luciferase (Promega) or 
-galactosidase (Tropix)
activity was measured using a Wallac-Berthold Autolumat luminometer or
an MGM Opticomp luminometer. Analysis of variance with factorial
Scheffe F-test was used to analyze data. A value of p < 0.05 was considered significant. For control experiments, the
plasmids Spi tkluc and RSV--gal were transiently co-transfected into
CHO cells expressing GHR-(1-454), and luciferase values were measured
as above. Spi tkluc containing eight copies of the spi 2.1 gene (147/102) upstream of a luciferase reporter gene was constructed as follows: the CAT gene was excised from Spi tkCATAn (from
S. Berry, University of Minnesota, Minneapolis, MN (33)) with
XhoI/SssT1, leaving the Spi tk backbone. A
2.7-kilobase pair XhoI/SstI fragment containing
the luciferase gene was excised from tkluc and then ligated into the
Spi tk vector backbone to create Spi tkluc.
-galactosidase DNA (provided by Dr. M. Uhler,
University of Michigan). At 44-48 h after transfection, cells were
deprived of serum for 16-18 h and treated as indicated. Luciferase
assays were performed as described above, and luciferase values from experiments evaluating transcriptional activation were normalized to
-galactosidase activity.
-P-Elk-1), and polyclonal antibodies against the corresponding
nonphosphorylated Elk-1 peptide (Elk-1) were purchased from New
England Biolabs. Antibodies to dually phosphorylated ERK1 and ERK2
(-P-ERK), and dually phosphorylated JNK (-P-JNK) were purchased
from Promega. According to the manufacturer, the -P-JNK antibodies
cross-react with phosphorylated ERK1 and -2. Antibodies to JNK and p38
were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Antibodies recognizing MEK1 and MAPKAP kinase-2 were purchased from
Upstate Biotechnology, Inc. (Lake Placid, NY). Antibodies to
nonphosphorylated ERK1/ERK2 and to MEK1/MEK2 were made as described
(42).
-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7 at
50 °C for 30 min with shaking.
-mercaptoethanol. The immobilized MEK preparation was then incubated
with 0.3 µg of ERK/sample in kinase buffer (90 mM HEPES, pH 7.5, 5 mM magnesium acetate, and 250 µM
ATP) for 30 min at 30 °C. Triplicate aliquots (10 µl) of each
sample were incubated with 30 µl of kinase buffer containing 20 µg
of myelin basic protein and 2 µCi of [
-32P]ATP for
30 min. Samples were spotted on p81 Whatman filter paper, washed with
1% phosphoric acid, and counted. Assays for p38 utilized its substrate
MAPKAP kinase-2. Antibody to MAPKAP kinase-2 (5 µg/sample) was used
for immunoprecipitation, and MAPKAP kinase-2 activity was measured in
an in vitro kinase assay using MAPKAP kinase-2 substrate
peptide (250 µmol/sample) as described (44). JNK activity was assayed
using lysates from 3T3-F442A cells that were incubated with glutathione
S-transferase-Jun (from B. Margolis, University of Michigan)
complexed to glutathione beads for 3 h. Beads were washed twice
with wash buffer (140 mM NaCl, 50 mM Tris, pH
8.0, 5 mM EDTA) and 1% Triton-100 and twice with wash
buffer without Triton X-100. Beads were incubated for 20 min at
30 °C with 10 µCi/sample [-32P]ATP in 50 mM Tris, pH 7.4, 10 mM MgCl, 1 mM
EDTA, 40 µM ATP. They were then washed and boiled, and
eluted proteins were analyzed by SDS-polyacrylamide gel electrophoresis
using a 12% polyacrylamide gel.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
View larger version (29K):
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Fig. 1.
The MEK inhibitor PD098059 attenuates
GH-stimulated expression and promoter activation of
c-fos. A, 3T3-F442A fibroblasts were
incubated for 1 h with the indicated concentrations of PD098059 in
Me2SO (MI, lanes 3-6) or
Me2SO alone (lanes 1 and
2). Then GH (lanes 2-5) or vehicle
(lanes 1 and 6) were added for an
additional 30 min. Total RNA was isolated from cells and assayed for
endogenous c-fos (top) and GAPDH mRNA levels
(bottom) by Northern blot analysis. Similar results were
obtained in three independent experiments. B,
left, 3T3-F442A fibroblasts stably transfected with a
plasmid (mC/E) containing a c-fos promoter region upstream
of a luciferase reporter gene were treated with PD098059 (100 µM) or Me2SO. GH (hatched
bars) or vehicle (C, solid
bars) was added for 5 h. Luciferase activity in cell
lysates is normalized to the activity in untreated control cells
(control = 1). Results show the mean + S.E. for three
independent experiments, each assayed in triplicate. Luciferase
activity was significantly different (p < 0.005)
between control and GH-treated cells and between GH-treated cells in
the presence and absence of PD098059. There was not a significant
difference between vehicle-treated control cells and PD098059-treated
control or GH-treated cells. c-fos promoter-mediated
luciferase values from control and GH-treated cells incubated with
PD098059 were not significantly different from those from
Me2SO-treated control cells. Right, CHO cells
stably expressing GHR-(1-454) were transiently co-transfected with a
spi 2.1 luciferase reporter and RSV- gal. The results are
representative of two independent experiments.
379/+1) upstream of a luciferase reporter gene (Fig. 1B).
GH produced a greater than 4-fold stimulation of reporter expression
(Fig. 1B). Pretreatment of cells with 100 µM
MEK inhibitor resulted in an 80% reduction in GH-promoted
transcriptional activation through the c-fos promoter (Fig.
1B, left). This degree of reduction corresponds
well with the 84% decrease in endogenous c-fos expression
observed with 100 µM PD098059 (Fig. 1A). In
contrast, PD098059 did not reduce reporter expression mediated by eight
tandem copies of the GH-responsive promoter of the spi 2.1 gene (Fig. 1B, right), indicating some specificity of the MEK inhibitor for the pathway leading to expression of the c-fos promoter.
View larger version (66K):
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Fig. 2.
The MEK inhibitor PD098059 attenuates
GH-induced expression of egr-1 and junB.
3T3-F442A fibroblasts were incubated for 1 h with
Me2SO (lanes 1 and 2) or
the indicated concentrations of PD098059 (MI,
lanes 3-6) and then with GH (lanes
2-5) or vehicle (lanes 1 and
6) for an additional 30 min. Total RNA was assayed by
Northern blot analysis for endogenous GAPDH expression
(lower panels) and either endogenous
egr-1 expression (A) or endogenous
junB expression (B) (upper
panels). Similar results were obtained in two
experiments.
-P-ERK) specific for dually phosphorylated, activated
ERK1 and ERK2. GH treatment for 5 min increased levels of
phosphorylated ERK1 and -2 (Fig. 3A, top,
lanes 4 and 5 versus
lanes 2 and 3). The addition of
increasing concentrations of PD098059 progressively lowered levels of
dually phosphorylated ERK1 and ERK2 in GH-treated cells (Fig.
3A, top, lanes 6-11). In
five separate experiments, ERK phosphorylation was inhibited 47% with
25 µM, 77% with 50 µM, and 101% with 100 µM PD098059. Levels of nonphosphorylated ERK were not
affected by treatment with PD098059 (lane 1 versus lane 2, bottom).
View larger version (26K):
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Fig. 3.
The MEK inhibitor PD098059 attenuates
GH-induced activation of ERK1 and ERK2. A, 3T3-F442A
fibroblasts were preincubated for 1 h with the indicated
concentrations of PD098059 (lanes 1 and
6-11) or Me2SO alone (lanes
2-5). Cells were then treated with GH (lanes
4-11) or vehicle (lanes 1-3) for an
additional 5 min. Duplicate aliquots of whole cell lysates were
immunoblotted with -P-ERK (1:20,000, top). Blots were
stripped and reprobed with -ERK (1:2000, bottom). When
aliquots of the same lysates were initially blotted with -ERK,
constant levels of ERK protein were observed in all conditions (data
not shown). Apparent Mr values × 1000 are
shown on the left in this and subsequent figures. Similar
results were obtained in five independent experiments. B,
3T3-F442A fibroblasts were preincubated with the indicated
concentrations of PD098059 or Me2SO for 1 h, and
vehicle (solid bar) or GH (hatched bars) was
added for an additional 5 min. Extracts were prepared and assayed for
MAPK activity. Bars represent the means + S.E. from
three independent experiments, each assayed in triplicate. Stimulation
of MAPK activity by GH was significant (p < 0.005).
GH-stimulated MAPK activity in the presence of 50 or 100 µM PD098059 was significantly different from that with GH
alone (p < 0.02) and was not different from untreated
control (p > 0.05). C, 3T3-F442A
fibroblasts were preincubated with the indicated concentrations of
PD098059 or Me2SO and treated with GH (hatched
bars) or vehicle (solid bar) for 5 min. Extracts were
prepared and assayed in triplicate for MEK activity. Similar results
were obtained in two experiments.
View larger version (25K):
[in a new window]
Fig. 4.
Wortmannin impairs GH-stimulated c-fos
expression and promoter activation of c-fos.
A, 3T3-F442A fibroblasts were preincubated for 1 h with
the indicated concentrations of wortmannin in Me2SO
(WT, lanes 3 and 4) or
Me2SO alone (lanes 1 and
2) and treated with GH (lanes 2-4) or
vehicle (lane 1) for an additional 30 min. Total
RNA from cells was assayed by Northern blot analysis for
c-fos expression (upper panel) or
GAPDH expression (lower panel). Similar results
were obtained in three experiments. B, 3T3-F442A fibroblasts
stably transfected with a plasmid (mC/E) containing the
c-fos promoter upstream of a luciferase reporter gene were
pretreated with Me2SO or the indicated concentrations of
wortmannin and then treated with GH (hatched bars) or
vehicle (C, solid bars) for an additional 5 h. Luciferase activity in cell lysates is normalized to the activity in
untreated control cells (control = 1). Bars represent
the means + S.E. of triplicate determinations from one experiment.
Similar results were obtained in three experiments. C,
3T3-F442A cells were preincubated for 1 h with wortmannin
(lanes 3-5) or Me2SO
(lanes 1 and 2) prior to the addition
of GH for 15 min. ERK phosphorylation was measured in whole cell
lysates by immunoblotting as described for Fig. 3.
View larger version (13K):
[in a new window]
Fig. 5.
The MEK inhibitor PD098059 attenuates
GH-induced Elk-1-mediated transcriptional activation.
Left, CHO-GHR cells were transiently co-transfected with
plasmids Gal4/ElkC, 5× Gal/Luc, and RSV- gal. Cells were treated
with or without MEK inhibitor (100 µM) for 1 h and
subsequently incubated with GH (hatched bars) or
vehicle (C, solid bars) for an additional 4 h. Luciferase activity, normalized for -galactosidase activity, is
expressed relative to that in control cells treated with
Me2SO (control = 1). Bars represent the
means + S.E. of three independent experiments, each measured in
triplicate. Stimulation of Gal4/ElkC-mediated luciferase activity by GH
is significant (p < 0.005). The values from cells
treated with GH and PD098059 are statistically different from those in
cells treated with GH in the absence of PD098059 (p < 0.005). CHO-GHR cells were transfected with the plasmids for Gal4 DNA
binding domain alone, 5× Gal/Luc, and RSV-gal (Gal4
DBD, right panel). Cells were treated
with vehicle (solid bars) or GH (hatched
bars) as above. Bars represent the means + S.E.
for two independent experiments, each measured in triplicate.
-P-Elk-1 antibodies at all concentrations of GH (Fig. 6,
lanes 5-8), consistent with a role for ERKs in GH-stimulated Elk-1 phosphorylation. Nonphosphorylated Elk-1 protein levels were comparable under all conditions (Fig. 6, lower
panel).
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Fig. 6.
The MEK inhibitor PD098059 blocks
GH-stimulated Elk-1 phosphorylation. CHO-GHR cells were
transiently transfected with the plasmid CMV-Elk-1. 36 h after
transfection, cells were deprived of serum for 6 h, and PD098059
(MI, 100 µM, lanes 5-8)
or Me2SO (lanes 1-4) was added
during the final hour of deprivation. Cells were then treated with the
indicated concentrations of GH or vehicle for 30 min. Whole cell
lysates were analyzed by immunoblotting with -P-Elk-1 (1:1,000;
upper panel) or Elk-1 (1:1,000;
lower panel). Data are representative of three
experiments.
-P-Elk-1 in response to GH (Fig.
7A, top,
lane 4), whereas GH clearly induced bands representing phosphorylated Elk-1 in the absence of MKP-1 (Fig. 7A, top, lane 2). Dominant
negative Ras (15A) also decreased GH-stimulated Elk-1 phosphorylation
compared with vector (pZIP-Neo) alone (Fig. 7B,
top, lane 4 versus
lane 2). Conversely, overexpression of wild-type
Ras elevated levels of protein recognized by -P-Elk-1 in both GH-
and vehicle-treated cells. (Fig. 7B, upper
panel, lanes 5 and 6).
Elk-1 protein was present in all conditions (Fig. 7, A and
B, lower panels). In lysates from
GH-treated cells analyzed with Elk-1, a slower migrating band is
observed that comigrates with the band recognized by -P-Elk-1 (Fig.
7, A and B, lane 2, bottom versus top). This slower
migrating band is likely to represent Elk-1 phosphorylated on serine
383 and is absent in lysates from cells transfected with MKP-1 or
dominant negative Ras. These data are consistent with observations with
PD098059 and together support a role for Ras and MAP kinase activation
in GH-stimulated Elk-1 phosphorylation.
View larger version (32K):
[in a new window]
Fig. 7.
GH-stimulated Elk-1 phosphorylation is
altered by MAP kinase phosphatase and Ras. CHO-GHR cells were
transiently co-transfected with CMV-Elk-1 (all
lanes) and one of the following. A, irrelevent
vector, pcDNA3 (Control, lanes 1 and 2) or CMV-MKP-1 (MKP-1, lanes
3 and 4); B, pZIP-NeoSV(x)1
(Neo, lanes 1 and 2),
dominant negative Ras, pZIP-ras(15A) (D/N Ras,
lanes 3 and 4), or wild-type Ras,
pZIP-ras(WT) (wt Ras, lanes 5 and
6). 36 h after transfection, cells were deprived of
serum for 5 h, and GH (A, lanes 2 and 4; B, lanes 2,
4, and 6) or vehicle (A,
lanes 1 and 3; B,
lanes 1, 3, and 5) were
added for 30 min. Whole cell lysates were analyzed as described for
Fig. 6. Lanes 1-4 were exposed to x-ray film for
a longer period of time than lanes 5 and
6. Similar results were observed in three experiments.
-P-JNK) that
recognizes dually phosphorylated, activated p54 and p46 JNK. In
3T3-F442A fibroblasts, GH treatment for 10, 20, or 30 min failed to
stimulate phosphorylation of p54 or p46 JNK (Fig.
8A, top, lanes 4-6). GH also failed to induce JNK
phosphorylation in CHO-GHR cells (data not shown). The capability of
-P-JNK to detect phosphorylation of JNK was verified using
anisomycin, which stimulated phosphorylation of both p46 and p54 JNK in
3T3-F442A cells (Fig. 8A, lane 7). Due
to cross-reactivity of the -P-JNK antibody with phosphorylated ERKs,
bands that co-migrate with phosphorylated ERK1 and -2 (data not shown)
were observed, most notably in lysates from cells treated with GH for
10 min (Fig. 8A, lane 4). To confirm
that GH did not stimulate JNK activity in 3T3-F442A cells, lysates from
GH-treated cells were also tested for their ability to phosphorylate
glutathione S-transferase-Jun. Clearly, GH failed to induce
JNK activity (Fig. 8B, lanes 4-6),
while anisomycin (lane 7) was effective. The
failure of GH to stimulate JNK activity suggests that JNK is unlikely to play a role in GH-promoted Elk-1-mediated gene expression.
View larger version (51K):
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Fig. 8.
GH fails to induce JNK phosphorylation in
3T3-F442A fibroblasts. 3T3-F442A fibroblasts were treated with
vehicle (C, lanes 1-3) or 500 ng/ml
GH (lanes 4-6) for the times indicated or for 20 min with 10 µg/ml anisomycin (A, lane
7) or with ethanol vehicle (C, lane
8). A, whole cell lysates were analyzed by
immunoblotting with -P-JNK (1:5000; upper
panel) or JNK (1:1,000; lower
panel). The migrations of p54 JNK and p46 JNK are indicated
by arrows. The comigration of the bands seen in
lanes 4-6 with ERK1 and -2 was determined by
stripping the membrane and blotting with ERK (data not shown).
B, glutathione S-transferase-Jun complexed to
glutathione beads was incubated with cell lysates and used subsequently
in a kinase assay. A representative experiment is shown.
View larger version (30K):
[in a new window]
Fig. 9.
GH-induced p38 activity does not contribute
to GH-stimulated Elk-1 phosphorylation. A, CHO-GHR
cells were transiently transfected with the plasmid CMV-Elk-1
(all lanes). 36 h later, cells were deprived
of serum for 6 h. Me2SO (lanes 1 and 2), MEK inhibitor PD098059 (MI, 100 µM, lanes 3 and 4), or
p38 inhibitor SB203580 (10 µM, lanes
5 and 6) was added for the final hour. Cells were
treated with GH or vehicle for an additional 30 min. Whole cell lysates
were analyzed for the presence of phospho-Elk-1 or Elk-1 by
immunoblotting as described for Fig. 6. Similar results were observed
in two experiments. B, 3T3-F442A cells were preincubated
with Me2SO or a 10 µM concentration of the
p38 inhibitor SB203580 for 1 h and then treated for 20 min with
vehicle (C, solid bars) or GH
(hatched bars). Cell lysates were then harvested and assayed
for MAPKAP kinase-2 activity. MAPKAP kinase-2 activity is expressed
relative to that in control cells (control = 1, solid
bar). Bars show the means + S.E. for three
separate experiments. The stimulation of MAPKAP kinase-2 activity by GH
is significant (p < 0.01), and the activity observed
with the p38 inhibitor in the absence and presence of GH did not differ
significantly from that in control Me2SO-treated
cells.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of NIH Postdoctoral Fellowship DK 09293.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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 Y. Zhang, M. Xu, S. Zhang, L. Yan, C. Yang, W. Lu, Y. Li, and H. Cheng The role of G protein-coupled receptor 40 in lipoapoptosis in mouse {beta}-cell line NIT-1 J. Mol. Endocrinol., June 1, 2007; 38(6): 651 - 661. [Abstract] [Full Text] [PDF]
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 S. Simo, L. Pujadas, M. F. Segura, A. L. Torre, J. A. Del Rio, J. M. Urena, J. X. Comella, and E. Soriano Reelin Induces the Detachment of Postnatal Subventricular Zone Cells and the Expression of the Egr-1 through Erk1/2 Activation Cereb Cortex, February 1, 2007; 17(2): 294 - 303. [Abstract] [Full Text] [PDF]
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T. I. Cesena, J.-R. Cardinaux, R. Kwok, and J. Schwartz CCAAT/Enhancer-binding Protein (C/EBP) beta Is Acetylated at Multiple Lysines: ACETYLATION OF C/EBPbeta AT LYSINE 39 MODULATES ITS ABILITY TO ACTIVATE TRANSCRIPTION J. Biol. Chem., January 12, 2007; 282(2): 956 - 967. [Abstract] [Full Text] [PDF]
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 M. Adriani, C. Garbi, G. Amodio, I. Russo, M. Giovannini, S. Amorosi, E. Matrecano, E. Cosentini, F. Candotti, and C. Pignata Functional Interaction of Common {gamma}-Chain and Growth Hormone Receptor Signaling Apparatus J. Immunol., November 15, 2006; 177(10): 6889 - 6895. [Abstract] [Full Text] [PDF]
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S. Cottet, L. Michaut, G. Boisset, U. Schlecht, W. Gehring, and D. F. Schorderet Biological characterization of gene response in Rpe65-/- mouse model of Leber's congenital amaurosis during progression of the disease FASEB J, October 1, 2006; 20(12): 2036 - 2049. [Abstract] [Full Text] [PDF]
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J. Xu, Z. Liu, T. L. Clemens, and J. L. Messina Insulin Reverses Growth Hormone-induced Homologous Desensitization J. Biol. Chem., August 4, 2006; 281(31): 21594 - 21606. [Abstract] [Full Text] [PDF]
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 Y. Chang, H.-H. Lee, Y.-T. Chen, J. Lu, S.-Y. Wu, C.-W. Chen, K. Takada, and C.-H. Tsai Induction of the early growth response 1 gene by epstein-barr virus lytic transactivator zta. J. Virol., August 1, 2006; 80(15): 7748 - 7755. [Abstract] [Full Text] [PDF]
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 T. Ahmed, G. Yumet, M. Shumate, C. H. Lang, P. Rotwein, and R. N. Cooney Tumor necrosis factor inhibits growth hormone-mediated gene expression in hepatocytes Am J Physiol Gastrointest Liver Physiol, July 1, 2006; 291(1): G35 - G44. [Abstract] [Full Text] [PDF]
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 A. M. Mazurkiewicz-Munoz, L. S. Argetsinger, J.-L. K. Kouadio, A. Stensballe, O. N. Jensen, J. M. Cline, and C. Carter-Su Phosphorylation of JAK2 at Serine 523: a Negative Regulator of JAK2 That Is Stimulated by Growth Hormone and Epidermal Growth Factor. Mol. Cell. Biol., June 1, 2006; 26(11): 4052 - 4062. [Abstract] [Full Text] [PDF]
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J. Xu, A. B. Keeton, J. L. Franklin, X. Li, D. Y. Venable, S. J. Frank, and J. L. Messina Insulin Enhances Growth Hormone Induction of the MEK/ERK Signaling Pathway J. Biol. Chem., January 13, 2006; 281(2): 982 - 992. [Abstract] [Full Text] [PDF]
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T. X. Cui, G. Piwien-Pilipuk, J. S. Huo, J. Kaplani, R. Kwok, and J. Schwartz Endogenous CCAAT/Enhancer Binding Protein {beta} and p300 Are Both Regulated by Growth Hormone to Mediate Transcriptional Activation Mol. Endocrinol., August 1, 2005; 19(8): 2175 - 2186. [Abstract] [Full Text] [PDF]
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M. L. Shumate, G. Yumet, T. A. Ahmed, and R. N. Cooney Interleukin-1 inhibits the induction of insulin-like growth factor-I by growth hormone in CWSV-1 hepatocytes Am J Physiol Gastrointest Liver Physiol, August 1, 2005; 289(2): G227 - G239. [Abstract] [Full Text] [PDF]
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S. Katsuma, N. Hatae, T. Yano, Y. Ruike, M. Kimura, A. Hirasawa, and G. Tsujimoto Free Fatty Acids Inhibit Serum Deprivation-induced Apoptosis through GPR120 in a Murine Enteroendocrine Cell Line STC-1 J. Biol. Chem., May 20, 2005; 280(20): 19507 - 19515. [Abstract] [Full Text] [PDF]
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C. N. Hancock, S. Dangi, and P. Shapiro Protein Phosphatase 2A Activity Associated with Golgi Membranes during the G2/M Phase May Regulate Phosphorylation of ERK2 J. Biol. Chem., March 25, 2005; 280(12): 11590 - 11598. [Abstract] [Full Text] [PDF]
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K. Narayanan, R. Srinivas, M. C. Peterson, A. Ramachandran, J. Hao, B. Thimmapaya, P. E. Scherer, and A. George Transcriptional Regulation of Dentin Matrix Protein 1 by JunB and p300 during Osteoblast Differentiation J. Biol. Chem., October 22, 2004; 279(43): 44294 - 44302. [Abstract] [Full Text] [PDF]
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 Y.-M. Wu, D. R. Robinson, and H.-J. Kung Signal Pathways in Up-regulation of Chemokines by Tyrosine Kinase MER/NYK in Prostate Cancer Cells Cancer Res., October 15, 2004; 64(20): 7311 - 7320. [Abstract] [Full Text] [PDF]
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 C.-J. Tai, S.-J. Chang, P. C. K. Leung, and C.-R. Tzeng Adenosine 5'-Triphosphate Activates Nuclear Translocation of Mitogen-Activated Protein Kinases Leading to the Induction of Early Growth Response 1 and Raf Expression in Human Granulosa-Luteal Cells J. Clin. Endocrinol. Metab., October 1, 2004; 89(10): 5189 - 5195. [Abstract] [Full Text] [PDF]
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 K.-C. Leung, G. Johannsson, G. M. Leong, and K. K. Y. Ho Estrogen Regulation of Growth Hormone Action Endocr. Rev., October 1, 2004; 25(5): 693 - 721. [Abstract] [Full Text] [PDF]
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S. Fujioka, J. Niu, C. Schmidt, G. M. Sclabas, B. Peng, T. Uwagawa, Z. Li, D. B. Evans, J. L. Abbruzzese, and P. J. Chiao NF-{kappa}B and AP-1 Connection: Mechanism of NF-{kappa}B-Dependent Regulation of AP-1 Activity Mol. Cell. Biol., September 1, 2004; 24(17): 7806 - 7819. [Abstract] [Full Text] [PDF]
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Y. Huang, Y. Chang, X. Wang, J. Jiang, and S. J. Frank Growth Hormone Alters Epidermal Growth Factor Receptor Binding Affinity via Activation of Extracellular Signal-Regulated Kinases in 3T3-F442A Cells Endocrinology, July 1, 2004; 145(7): 3297 - 3306. [Abstract] [Full Text] [PDF]
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M. Maggiolini, A. Vivacqua, G. Fasanella, A. G. Recchia, D. Sisci, V. Pezzi, D. Montanaro, A. M. Musti, D. Picard, and S. Ando The G Protein-coupled Receptor GPR30 Mediates c-fos Up-regulation by 17{beta}-Estradiol and Phytoestrogens in Breast Cancer Cells J. Biol. Chem., June 25, 2004; 279(26): 27008 - 27016. [Abstract] [Full Text] [PDF]
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Y. Huang, S.-O. Kim, N. Yang, J. Jiang, and S. J. Frank Physical and Functional Interaction of Growth Hormone and Insulin-Like Growth Factor-I Signaling Elements Mol. Endocrinol., June 1, 2004; 18(6): 1471 - 1485. [Abstract] [Full Text] [PDF]
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N. Yang, Y. Huang, J. Jiang, and S. J. Frank Caveolar and Lipid Raft Localization of the Growth Hormone Receptor and Its Signaling Elements: IMPACT ON GROWTH HORMONE SIGNALING J. Biol. Chem., May 14, 2004; 279(20): 20898 - 20905. [Abstract] [Full Text] [PDF]
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 H.-C. Chen and E. P. Feener MEK1,2 response element mediates angiotensin II--stimulated plasminogen activator inhibitor-1 promoter activation Blood, April 1, 2004; 103(7): 2636 - 2644. [Abstract] [Full Text] [PDF]
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M. D. Lewis, M. Horan, D. S. Millar, V. Newsway, T. E. Easter, L. Fryklund, J. W. Gregory, M. Norin, C.-J. Del Valle, J. P. Lopez-Siguero, et al. A Novel Dysfunctional Growth Hormone Variant (Ile179Met) Exhibits a Decreased Ability to Activate the Extracellular Signal-Regulated Kinase Pathway J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1068 - 1075. [Abstract] [Full Text] [PDF]
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 E. M. Dahly, M. E. Miller, P. K. Lund, and D. M. Ney Postreceptor Resistance to Exogenous Growth Hormone Exists in the Jejunal Mucosa of Parenterally Fed Rats J. Nutr., March 1, 2004; 134(3): 530 - 537. [Abstract] [Full Text] [PDF]
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G. P. Pilipuk, M. D. Galigniana, and J. Schwartz Subnuclear Localization of C/EBP{beta} Is Regulated by Growth Hormone and Dependent on MAPK J. Biol. Chem., September 12, 2003; 278(37): 35668 - 35677. [Abstract] [Full Text] [PDF]
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L. Ling, T. Zhu, and P. E. Lobie Src-CrkII-C3G-dependent Activation of Rap1 Switches Growth Hormone-stimulated p44/42 MAP Kinase and JNK/SAPK Activities J. Biol. Chem., July 11, 2003; 278(29): 27301 - 27311. [Abstract] [Full Text] [PDF]
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J. Woelfle, J. Billiard, and P. Rotwein Acute Control of Insulin-like Growth Factor-I Gene Transcription by Growth Hormone through Stat5b J. Biol. Chem., June 13, 2003; 278(25): 22696 - 22702. [Abstract] [Full Text] [PDF]
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R. C. Y. Choi, N. L. Siow, A. W. M. Cheng, K. K. Y. Ling, E. K. K. Tung, J. Simon, E. A. Barnard, and K. W. K. Tsim ATP Acts via P2Y1 Receptors to Stimulate Acetylcholinesterase and Acetylcholine Receptor Expression: Transduction and Transcription Control J. Neurosci., June 1, 2003; 23(11): 4445 - 4456. [Abstract] [Full Text] [PDF]
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Y. Huang, S.-O. Kim, J. Jiang, and S. J. Frank Growth Hormone-induced Phosphorylation of Epidermal Growth Factor (EGF) Receptor in 3T3-F442A Cells: MODULATION OF EGF-INDUCED TRAFFICKING AND SIGNALING J. Biol. Chem., May 23, 2003; 278(21): 18902 - 18913. [Abstract] [Full Text] [PDF]
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E. Yang, L. Lerner, D. Besser, and J. E. Darnell Jr. Independent and Cooperative Activation of Chromosomal c-fos Promoter by STAT3 J. Biol. Chem., April 25, 2003; 278(18): 15794 - 15799. [Abstract] [Full Text] [PDF]
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H. Fujino, W. Xu, and J. W. Regan Prostaglandin E2 Induced Functional Expression of Early Growth Response Factor-1 by EP4, but Not EP2, Prostanoid Receptors via the Phosphatidylinositol 3-Kinase and Extracellular Signal-regulated Kinases J. Biol. Chem., March 28, 2003; 278(14): 12151 - 12156. [Abstract] [Full Text] [PDF]
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S.-Q. Wu, T. Minami, D. J. Donovan, and W. C. Aird The proximal serum response element in the Egr-1 promoter mediates response to thrombin in primary human endothelial cells Blood, December 15, 2002; 100(13): 4454 - 4461. [Abstract] [Full Text] [PDF]
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S.-O. Kim, K. Loesch, X. Wang, J. Jiang, L. Mei, J. M. Cunnick, J. Wu, and S. J. Frank A Role for Grb2-Associated Binder-1 in Growth Hormone Signaling Endocrinology, December 1, 2002; 143(12): 4856 - 4867. [Abstract] [Full Text] [PDF]
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T. Zhu, L. Ling, and P. E. Lobie Identification of a JAK2-independent Pathway Regulating Growth Hormone (GH)-stimulated p44/42 Mitogen-activated Protein Kinase Activity. GH ACTIVATION OF Ral AND PHOSPHOLIPASE D IS Src-DEPENDENT J. Biol. Chem., November 15, 2002; 277(47): 45592 - 45603. [Abstract] [Full Text] [PDF]
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G. Piwien-Pilipuk, O. MacDougald, and J. Schwartz Dual Regulation of Phosphorylation and Dephosphorylation of C/EBPbeta Modulate Its Transcriptional Activation and DNA Binding in Response to Growth Hormone J. Biol. Chem., November 8, 2002; 277(46): 44557 - 44565. [Abstract] [Full Text] [PDF]
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E. L. K. Goh, T. Zhu, W.-Y. Leong, and P. E. Lobie c-Cbl Is a Negative Regulator of GH-Stimulated STAT5-Mediated Transcription Endocrinology, September 1, 2002; 143(9): 3590 - 3603. [Abstract] [Full Text] [PDF]
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 K. Fukuda, T. Shoda, H. Mima, and H. Uga Midazolam Induces Expression of c-Fos and EGR-1 by a Non-GABAergic Mechanism Anesth. Analg., August 1, 2002; 95(2): 373 - 378. [Abstract] [Full Text] [PDF]
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K. Rosenblum, M. Futter, K. Voss, M. Erent, P. A. Skehel, P. French, L. Obosi, M. W. Jones, and T. V. P. Bliss The Role of Extracellular Regulated Kinases I/II in Late-Phase Long-Term Potentiation J. Neurosci., July 1, 2002; 22(13): 5432 - 5441. [Abstract] [Full Text] [PDF]
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 G. P. Amorino, V. M. Hamilton, K. Valerie, P. Dent, G. Lammering, and R. K. Schmidt-Ullrich Epidermal Growth Factor Receptor Dependence of Radiation-induced Transcription Factor Activation in Human Breast Carcinoma Cells Mol. Biol. Cell, July 1, 2002; 13(7): 2233 - 2244. [Abstract] [Full Text] [PDF]
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W. R. Duan, M. Ito, Y. Park, E. T. Maizels, M. Hunzicker-Dunn, and J. L. Jameson GnRH Regulates Early Growth Response Protein 1 Transcription Through Multiple Promoter Elements Mol. Endocrinol., February 1, 2002; 16(2): 221 - 233. [Abstract] [Full Text] [PDF]
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R. A. Frost, G. J. Nystrom, and C. H. Lang Regulation of IGF-I mRNA and Signal Transducers and Activators of Transcription-3 and -5 (Stat-3 and -5) by GH in C2C12 Myoblasts Endocrinology, February 1, 2002; 143(2): 492 - 503. [Abstract] [Full Text] [PDF]
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J. C. Tsai, L. Liu, J. Zhang, K. C. Spokes, J. N. Topper, and W. C. Aird Epidermal growth factor induces Egr-1 promoter activity in hepatocytes in vitro and in vivo Am J Physiol Gastrointest Liver Physiol, November 1, 2001; 281(5): G1271 - G1278. [Abstract] [Full Text] [PDF]
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L. Guillemot, A. Levy, M. Raymondjean, and B. Rothhut Angiotensin II-induced Transcriptional Activation of the Cyclin D1 Gene Is Mediated by Egr-1 in CHO-AT1A Cells J. Biol. Chem., October 12, 2001; 276(42): 39394 - 39403. [Abstract] [Full Text] [PDF]
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L. N. N. Shoba, M. Newman, W. Liu, and W. L. Lowe Jr. LY 294002, an Inhibitor of Phosphatidylinositol 3-Kinase, Inhibits GH-Mediated Expression of the IGF-I Gene in Rat Hepatocytes Endocrinology, September 1, 2001; 142(9): 3980 - 3986. [Abstract] [Full Text] [PDF]
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 M. Shimada, T. Maeda, and T. Terada Dynamic Changes of Connexin-43, Gap Junctional Protein, in Outer Layers of Cumulus Cells Are Regulated by PKC and PI 3-Kinase During Meiotic Resumption in Porcine Oocytes Biol Reprod, April 1, 2001; 64(4): 1255 - 1263. [Abstract] [Full Text]
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K. K. Kaulsay, T. Zhu, W. F. Bennett, K.-O. Lee, and P. E. Lobie The Effects of Autocrine Human Growth Hormone (hGH) on Human Mammary Carcinoma Cell Behavior Are Mediated via the hGH Receptor Endocrinology, February 1, 2001; 142(2): 767 - 777. [Abstract] [Full Text] [PDF]
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K. Tsuruzoe, R. Emkey, K. M. Kriauciunas, K. Ueki, and C. R. Kahn Insulin Receptor Substrate 3 (IRS-3) and IRS-4 Impair IRS-1- and IRS-2-Mediated Signaling Mol. Cell. Biol., January 1, 2001; 21(1): 26 - 38. [Abstract] [Full Text]
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S. Jeay, G. E. Sonenshein, P. A. Kelly, M.-C. Postel-Vinay, and E. Baixeras Growth Hormone Exerts Antiapoptotic and Proliferative Effects through Two Different Pathways Involving Nuclear Factor-{{kappa}}B and Phosphatidylinositol 3-Kinase Endocrinology, January 1, 2001; 142(1): 147 - 156. [Abstract] [Full Text] [PDF]
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J. Westermarck, S. Li, P. Jaakkola, T. Kallunki, R. Grénman, and V.-M. Kähäri Activation of Fibroblast Collagenase-1 Expression by Tumor Cells of Squamous Cell Carcinomas Is Mediated by p38 Mitogen-activated Protein Kinase and c-Jun NH2-terminal Kinase-2 Cancer Res., December 1, 2000; 60(24): 7156 - 7162. [Abstract] [Full Text]
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K. W. Kang, J. H. Ryu, and S. G. Kim The Essential Role of Phosphatidylinositol 3-Kinase and of p38 Mitogen-Activated Protein Kinase Activation in the Antioxidant Response Element-Mediated rGSTA2 Induction by Decreased Glutathione in H4IIE Hepatoma Cells Mol. Pharmacol., November 1, 2000; 58(5): 1017 - 1025. [Abstract] [Full Text]
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M. R. Stofega, J. Herrington, N. Billestrup, and C. Carter-Su Mutation of the SHP-2 Binding Site in Growth Hormone (GH) Receptor Prolongs GH-Promoted Tyrosyl Phosphorylation of GH Receptor, JAK2, and STAT5B Mol. Endocrinol., September 1, 2000; 14(9): 1338 - 1350. [Abstract] [Full Text]
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L. Liang, J. Jiang, and S. J. Frank Insulin Receptor Substrate-1-Mediated Enhancement of Growth Hormone-Induced Mitogen-Activated Protein Kinase Activation Endocrinology, September 1, 2000; 141(9): 3328 - 3336. [Abstract] [Full Text] [PDF]
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K. K. Kaulsay, H. C. Mertani, K.-O. Lee, and P. E. Lobie Autocrine Human Growth Hormone Enhancement of Human Mammary Carcinoma Cell Spreading Is Jak2 Dependent Endocrinology, April 1, 2000; 141(4): 1571 - 1584. [Abstract] [Full Text] [PDF]
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G. Xiao, D. Jiang, P. Thomas, M. D. Benson, K. Guan, G. Karsenty, and R. T. Franceschi MAPK Pathways Activate and Phosphorylate the Osteoblast-specific Transcription Factor, Cbfa1 J. Biol. Chem., February 11, 2000; 275(6): 4453 - 4459. [Abstract] [Full Text] [PDF]
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T. Zhu and P. E. Lobie Janus Kinase 2-dependent Activation of p38 Mitogen-activated Protein Kinase by Growth Hormone. RESULTANT TRANSCRIPTIONAL ACTIVATION OF ATF-2 AND CHOP, CYTOSKELETAL RE-ORGANIZATION AND MITOGENESIS J. Biol. Chem., January 21, 2000; 275(3): 2103 - 2114. [Abstract] [Full Text] [PDF]
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S.-O. Kim, J. C. D. Houtman, J. Jiang, J. M. Ruppert, P. J. Bertics, and S. J. Frank Growth Hormone-induced Alteration in ErbB-2 Phosphorylation Status in 3T3-F442A Fibroblasts J. Biol. Chem., December 10, 1999; 274(50): 36015 - 36024. [Abstract] [Full Text] [PDF]
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J. A. Costoya, J. Finidori, S. Moutoussamy, R. Señaris, J. Devesa, and V. M. Arce Activation of Growth Hormone Receptor Delivers an Antiapoptotic Signal: Evidence for a Role of Akt in This Pathway Endocrinology, December 1, 1999; 140(12): 5937 - 5943. [Abstract] [Full Text]
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J. Liao, G. Piwien-Pilipuk, S. E. Ross, C. L. Hodge, L. Sealy, O. A. MacDougald, and J. Schwartz CCAAT/Enhancer-binding Protein beta (C/EBPbeta ) and C/EBPdelta Contribute to Growth Hormone-regulated Transcription of c-fos J. Biol. Chem., October 29, 1999; 274(44): 31597 - 31604. [Abstract] [Full Text] [PDF]
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J. A. Alberta, K. R. Auger, D. Batt, P. Iannarelli, G. Hwang, H. L. Elliott, R. Duke, T. M. Roberts, and C. D. Stiles Platelet-derived Growth Factor Stimulation of Monocyte Chemoattractant Protein-1 Gene Expression Is Mediated by Transient Activation of the Phosphoinositide 3-Kinase Signal Transduction Pathway J. Biol. Chem., October 22, 1999; 274(43): 31062 - 31067. [Abstract] [Full Text] [PDF]
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E. L. K. Goh, T. Zhu, S. Yakar, D. LeRoith, and P. E. Lobie CrkII Participation in the Cellular Effects of Growth Hormone and Insulin-like Growth Factor-1. PHOSPHATIDYLINOSITOL-3 KINASE DEPENDENT AND INDEPENDENT EFFECTS J. Biol. Chem., June 2, 2000; 275(23): 17683 - 17692. [Abstract] [Full Text] [PDF]
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N. Delesque-Touchard, S.-H. Park, and D. J. Waxman Synergistic Action of Hepatocyte Nuclear Factors 3 and 6 on CYP2C12 Gene Expression and Suppression by Growth Hormone-activated STAT5b. PROPOSED MODEL FOR FEMALE-SPECIFIC EXPRESSION OF CYP2C12 IN ADULT RAT LIVER J. Biol. Chem., October 27, 2000; 275(44): 34173 - 34182. [Abstract] [Full Text] [PDF]
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R. Duan, W. Xie, R. C. Burghardt, and S. Safe Estrogen Receptor-mediated Activation of the Serum Response Element in MCF-7 Cells through MAPK-dependent Phosphorylation of Elk-1 J. Biol. Chem., April 6, 2001; 276(15): 11590 - 11598. [Abstract] [Full Text] [PDF]
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C. Bjorbak, R. M. Buchholz, S. M. Davis, S. H. Bates, D. D. Pierroz, H. Gu, B. G. Neel, M. G. Myers Jr., and J. S. Flier Divergent Roles of SHP-2 in ERK Activation by Leptin Receptors J. Biol. Chem., February 9, 2001; 276(7): 4747 - 4755. [Abstract] [Full Text] [PDF]
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G. Piwien-Pilipuk, D. Van Mater, S. E. Ross, O. A. MacDougald, and J. Schwartz Growth Hormone Regulates Phosphorylation and Function of CCAAT/Enhancer-binding Protein beta by Modulating Akt and Glycogen Synthase Kinase-3 J. Biol. Chem., May 25, 2001; 276(22): 19664 - 19671. [Abstract] [Full Text] [PDF]
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T. de Jager, T. Pelzer, S. Muller-Botz, A. Imam, J. Muck, and L. Neyses Mechanisms of Estrogen Receptor Action in the Myocardium. RAPID GENE ACTIVATION VIA THE ERK1/2 PATHWAY AND SERUM RESPONSE ELEMENTS J. Biol. Chem., July 20, 2001; 276(30): 27873 - 27880. [Abstract] [Full Text] [PDF]
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A. Iwata, M. Maruyama, I. Kanazawa, and N. Nukina alpha -Synuclein Affects the MAPK Pathway and Accelerates Cell Death J. Biol. Chem., November 21, 2001; 276(48): 45320 - 45329. [Abstract] [Full Text] [PDF]
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