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Stimulates the Raf/MEK1/Mitogen-Activated Protein Kinase Signaling Cascade in Bovine Luteal Cells1
The Women’s Research Institute, Departments of Obstetrics and Gynecology and Internal Medicine, University of Kansas School of Medicine-Wichita (D.-b.C., H.W.F., J.S.D.), and the Research Service of the Department of Veterans Affairs (S.D.W., J.S.D.), Wichita, Kansas 67214; and the Department of Physiology, Cornell University (M.S.R.), Ithaca, New York 14853
Address all correspondence and requests for reprints to: John S. Davis, Ph.D., The Women’s Research Institute, Department of Obstetrics and Gynecology, University of Kansas School of Medicine-Wichita, 1010 North Kansas, Wichita, Kansas 67214. E-mail: jdavis3{at}kumc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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on the corpus luteum are initiated by
the phospholipase C/diacylglycerol-inositol 1,4,5-trisphosphate
(InsP3)/Ca2+-protein kinase C (PKC) pathway.
However, little is known about the downstream intracellular signaling
events that can lead to transcriptional activation in response to
PGF2. The present study was conducted to examine the
involvement of the mitogen-activated protein kinase (MAPK) signaling
cascade in the corpus luteum. Three isoforms of the Raf family of
oncoprotein kinases (A-Raf, B-Raf, and Raf-1 or c-Raf) were detected in
bovine luteal cells. Raf-1 and B-Raf, but not A-Raf, were activated by
PGF2 (1 µM) and the pharmacological PKC
activator phorbol myristate acetate (PMA, 20 nM). Kinetic
analysis revealed that PGF2 rapidly and transiently
activated Raf-1. In vitro protein kinase assays
demonstrated that activation of Raf-1 and B-Raf resulted in the
phosphorylation and activation of MAPK kinase (MEK1), which
subsequently phosphorylated p42mapk. As determined by
hyperphosphorylation, tyrosine phosphorylation, and enzymatic activity,
p42mapk and p44 mapk were rapidly and
transiently activated by both PGF2 (1 µM)
and PMA (20 nM). Additionally, both PGF2 (1
µM) and PMA (20 nM) stimulated
phosphorylation of Raf-1, MEK1, and p42mapk in
32P-labeled cells. Our data demonstrate that
PGF2 activates the Raf/MEK1/p42/44mapk
signaling cascade in bovine luteal cells and that the actions of
PGF2 are mimicked by the PKC activator PMA. Activation
of the Raf/MEK1/MAPK signaling cascade by PGF2 in luteal
cells provides a mechanism to transduce signals initiated by
PGF2 receptors on the cell surface into the nucleus.
Activation of the Raf/MEK1/MAPK signaling cascade may be associated
with transcriptional activation of luteal genes possessing activator
protein-1-binding sites. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EXERTS diverse
physiological functions throughout the body. In a number of mammals
(reviewed in Refs. 1, 2), PGF2 is believed to be the
trigger that initiates the demise of the corpus luteum (luteolysis) in
the absence of pregnancy, which allows the estrous cycle to resume.
Ligand binding studies have revealed that high affinity
PGF2 receptors (FP receptors) are present on luteal cell
membranes (3). An obligatory role of the FP receptor in mediating the
actions of PGF2 during luteolysis has been recently
confirmed in mice lacking the gene encoding FP receptors (4). Molecular
cloning studies demonstrate that the FP receptor in the corpus luteum,
like other prostanoid receptors (5), is a member of the large class of
heterotrimeric guanine nucleotide-binding protein (G protein)-coupled
receptors (6, 7). PGF2-induced luteolysis is believed to
be initiated through ligand receptor activation of the phospholipase
C/diacylglycerol and inositol 1,4,5-trisphosphate
(InsP3)/Ca2+-protein kinase C (PKC) signaling
system (8, 9, 10). This concept is supported by a number of reports
demonstrating that the PKC activator phorbol myristate acetate (PMA)
can in part mimic PGF2-induced luteolysis in
vivo (11) and the actions of PGF2 in
vitro (12). However, a gap exists in our knowledge between the
initial signaling events and the physiological sequelae observed in
response to PGF2. The mechanism(s) by which
PGF2 regulates gene expression in the corpus luteum are
unknown. Biochemical studies in vertebrate cells together with genetic analysis in Caenorhabiditis elegans and Drosophila melanogaster have defined an ubiquitous family of cytoplasmic serine/threonine protein kinases, the mitogen-activated protein kinases (MAPKs), that are involved in the transduction of diverse extracellular signals into nuclear signals that regulate gene expression. The classical MAPKs consist of the extracellular signal-regulated kinases ERK2 and ERK1 (p42mapk and p44mapk, respectively), which are central elements of the growth factor receptor tyrosine kinase- and p21Ras-directed mitogenic signal transduction pathway (reviewed in Ref. 13). Regulation of p42mapk and p44mapk occurs via an evolutionary conserved protein kinase cascade involving the oncoprotein serine/threonine kinase Raf (MAPK kinase kinase). The intermediaries between Raf and p42mapk and p44mapk are the dual specificity MAPK kinases (MEK1 or MKK1) that activate MAPK by phosphorylation on both tyrosine and threonine residues (14). Once activated, MAPK transmits extracellular signals through either translocation into the nucleus (15) and phosphorylation of various transcription factors, i.e. c-Myc (16), c-Jun (17), and p62TCF/Elk-1 (18), etc., to regulate gene expression or recruitment to the cell membrane and regulation of the activity of receptor tyrosine kinases (19) and cytoplasmic phospholipase A2 (20). MAPK also regulates the activity of other cytoplasmic protein kinases to further participate in signal transduction networks, i.e., p90 ribosomal kinases, MAPK-activated protein, MEK, and Raf-1 (13).
Recent studies have shown that the Raf/MEK/MAPK signaling cascade also
mediates the cellular response to various ligands that activate G
protein-coupled receptors (reviewed in Ref. 21), including
PGF2. Watanebe et al. (22) demonstrated that
the MAPK signaling cascade was coupled to PGF2-induced
mitogenesis in NIH-3T3 cells, presumptively initiated via the formation
of a p21Ras-GTP complex and subsequent activation of MAPK
kinase (MEK1) and p42mapk. Of interest were results
demonstrating that MAPK activation by PGF2 was
apparently independent of PMA-sensitive isoforms of PKC (22). Hakeda
et al. (23) recently found that PGF2
activated the MAPK pathway in osteoblastic MC3T3-E1 cells via
PKC-dependent activation of Raf-1. Our preliminary data indicate that
PGF2 can also activate p42mapk in primary
cultures of bovine luteal cells (24). The present study was designed to
determine the signaling components of the MAPK pathway activated by
PGF2 in the corpus luteum. We report herein that
PGF2 and PMA activate the Raf/MEK1/p42mapk
and p44mapk signaling cascade in bovine luteal cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PanERK and monoclonal anti-MEK1 antibodies were obtained from
Transduction Laboratories (Lexington, KY). Phospho-plus MAPK antibody
was purchased from New England BioLabs (Beverly, MA).
[
-32P]ATP (37 MBq; 10 mCi/ml) and
[32P]orthophosphate (370 MBq; 150 mCi/ml) were obtained
from DuPont (Boston, MA). Tissue culture plasticware was obtained from
Corning (Corning, NY). FCS, medium 199 (M-199), and phosphate-free DMEM
were purchased from Life Technologies (Grand Island, NY).
Electrophoresis reagents were purchased from Bio-Rad Laboratories
(Hercules, CA). Enhanced chemiluminescence (ECL) kits were obtained
from Amersham (Arlington Heights, IL). Immobilon-p [polyvinylidene
difluoride (PVDF)] membrane was obtained from Millipore (Bedford, MA).
PGF2, PMA, prestained protein mol wt makers, and all
other chemicals and reagents were purchased from Sigma Chemical Co.
(St. Louis, MO). Recombinant [His]6-tagged-MEK1 (MEK1) and recombinant [His]6-tagged K52R (K52R, a full-length but catalytically inactive p42mapk) were prepared as described previously (25, 26). The transcriptional activation domain of human Elk-1 (amino acids 307–428) was obtained by PCR from HeLa cell complementary DNA and confirmed by nucleotide sequence analysis (27). The Elk-1 trans-activation domain was cloned into pGEX3 to produce a glutathione-S-transferase (GST)-Elk-1 fusion gene. GST-Elk-1 fusion was expressed in bacteria and partially purified as previously described (28).
Cell isolation and culture
Bovine ovaries from early pregnancy (fetal crown rump length,
<15 cm) were collected from a local slaughterhouse (Quality Meats,
Wellington, KS) and transported to the laboratory in cold M-199. The
luteal tissue was dissociated with collagenase as described previously
(29). Cell viability was determined by trypan blue exclusion, and only
those cell preparations with viability greater than 90% were used.
Luteal cells were plated (105/cm2) in M-199
with 5% FCS for 18 h at 37 C in a humidified atmosphere of 5%
CO2 in air. The cells were serum starved for 48 h
before experiments by replacing medium with serum-free M-199
supplemented with 0.1% BSA (fraction V) and 5 µg/ml bovine
insulin.
Experimental conditions and cell extracts
After equilibration with fresh M-199 plus 0.1% BSA for 1
h, the cells were treated as described in the figure legends.
Experiments were stopped by rapidly rinsing the monolayers twice with
cold PBS. The cells were lysed with cold nondenaturing lysis buffer A
[10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1
mM EDTA, 1 mM EGTA, 1% Triton X-100,
0.5% Nonidet P-40, 50 mM NaF, 1 mM
Na3VO4, 1 mM
phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, and 10 µg/ml pepstatin A] on ice with continuous shaking
for 15 min. The total cell lysates were collected with a cell scraper,
vortexed vigorously, and clarified by centrifugation (5 min,
15,000 x g). The protein content of the supernatant
was measured by a Bio-Rad procedure using BSA as the standard. Aliquots
of the total cell lysates were subjected to immunoprecipitation or were
added to Laemmli buffer and frozen at -80 C until Western blot
analysis could be performed.
Western blot analysis
After SDS-PAGE size-fractionation, proteins (20 µg/lane) were
electrically (100 V, 1.5 h) transferred to PVDF membranes.
Nonspecific binding was blocked with 5% fat-free milk in TBST [50
mM Tris-HCl (pH 7.5), 0.15 M NaCl, and 0.05%
Tween-20] overnight at 4 C, after which the membranes were incubated
with appropriate amounts of primary antibodies in TBST-3% BSA (for
monoclonal antibodies) or in TBST-5% fat-free milk (for polyclonal
antibodies) at room temperature for 1 h. After three washes (5 min
each) with 15 ml TBST, the membranes were incubated for 1 h with
antimouse or antirabbit peroxidase conjugated IgG, respectively. The
membranes were washed with TBST, and bound antibodies were detected
with the ECL reagents according to the manufacturer’s
instructions.
Immunoprecipitation
Clarified cell extracts (200 µg total protein) were precleared
by coincubation with 1 µl normal goat serum and 20 µl protein
A-conjugated agarose beads for 1 h at 4 C. After centrifugation
(15,000 x g, 4 C, 5 min), the precleared cell extracts
were transferred to new tubes. Immunoprecipitation was then performed
in precleared cell extracts at 4 C as described below. For Raf-1
immunoprecipitation, cell extracts were incubated with PBS-washed
rabbit polyclonal anti-Raf-1-conjugated agarose beads (5 µg) in lysis
buffer A with continuous rotation for 2 h. The beads
(immunocomplex) were collected by centrifugation. After three washes
with buffer A, three washes with LiCl solution (0.5 M LiCl
and 100 mM Tris-HCl, pH 8.0), and two washes with kinase
assay buffer B [50 mM ß-glycerophosphate (pH 7.3), 1.5
mM EGTA, 1 mM dithiothreitol, and 0.09%
Brig35], the beads were resuspended in 40 µl kinase buffer B. For
A-Raf and B-Raf immunoprecipitation, cell extracts were incubated with
rabbit polyclonal anti-A-Raf and rabbit polyclonal anti-B-Raf
antibodies (5 µg) in buffer A overnight with continuous rotation.
Protein A-conjugated agarose beads (20 µl) were then added, and the
samples were incubated for an additional 1 h with continuous
rotation. The immunocomplexes were collected and washed as described
for Raf-1 immunoprecipitation, and finally resuspended in 40 µl of
kinase buffer B. Aliquots of A-Raf, B-Raf, and Raf-1 immunoprecipitates
were prepared in Laemmli buffer for subsequent Western blot analysis to
verify that the same amounts of immunoprecipitated A-Raf, B-Raf, and
Raf-1 were used in the kinase assay (data not shown). For
p42mapk and p44mapk immunoprecipitation, 10
µl polyclonal anti-ERK2 antibody-conjugated agarose beads and
polyclonal anti-ERK1 antibody-conjugated agarose beads were used,
respectively, and the same procedure for Raf-1 immunoprecipitation was
applied.
Two-step immunocomplex Raf/MEK1 kinase assay
Raf kinase activity in A-Raf, B-Raf, and Raf-1
immunoprecipitates was measured by the ability to incorporate
[-32P]ATP into recombinant MEK1, and the ability of
phosphorylated (activated) MEK1 to subsequently phosphorylate the
synthetic kinase negative ERK2 (K52R). Briefly, 15 µl A-Raf, B-Raf,
or Raf-1 immunoprecipitates was coincubated with 10 µCi
[-32P]ATP and 30 µM ATP in buffer C [20
mM Tris-HCl (pH 7.4), 0.5 mM MnCl2,
and 5 mM MgCl2] in the presence of 1 µg MEK1
(final volume, 20 µl) at 30 C for 20 min. The reaction mixtures were
chilled on ice for 5 min and then centrifuged (15,000 x
g, 5 min). A portion of the supernatant (15 µl) containing
activated MEK1 was transferred to a new reaction tube containing 0.1
µg K52R, 5 µCi [-32P]ATP, and 30 µM
ATP in buffer C (final volume, 20 µl) and incubated at 30 C for 10
min. The reaction was stopped by addition of Laemmli buffer, and
proteins were size-fractionated on 10% SDS-PAGE. After Coomassie blue
staining, the gels were dried. Autoradiography was performed on dried
gels to visualize phosphorylated MEK1 and K52R bands. The MEK1 and K52R
bands were cut out and quantified by liquid scintillation counting.
Phosphorylation status of p42mapk and
p44mapk
The hyperphosphorylation of p42mapk and
p44mapk was assessed by a gel shift assay based upon the
slower migration rate of the phosphorylated active forms of
p42mapk and p44mapk than the nonphosphorylated
inactive forms on SDS-PAGE (30). The active and inactive forms of
p42mapk and p44mapk were detected by Western
blot analysis with PanERK antibodies (1:500). Tyrosine
phosphorylation of p42mapk and p44mapk was
measured by Western blot analysis with a Phospho-plus MAPK antibody
(1:1000; New England BioLabs, Beverly, MA) according to the
manufacturer’s instruction. This antibody was produced by immunizing
rabbits with a synthetic phosphotyrosine peptide (recombinant keyhole
limpet hemocyanin coupled) corresponding to residues 196–209
[DHTGFLTEY(p)VATRWC] of human p44mapk. It
detects p42mapk and p44mapk only when activated
by phosphorylation at tyrosine204.
p42mapk and p44mapkimmunocomplex protein kinase assay
p42mapk and p44mapk immunoprecipitates
were resuspended in 40 µl kinase buffer B. In the presence of 0.5
µg GST-Elk-1, 20 µl of the immunoprecipitates were coincubated with
10 µCi [-32P]ATP and 30 µM ATP in
buffer C for 30 min at 30 C. The reaction was stopped by addition of
Laemmli buffer. The reaction mixture was size-fractionated with 10%
SDS-PAGE, after which the gels were stained with Coomassie blue.
Autoradiography was performed on dried gels to visualize phosphorylated
GST-Elk-1. Elk-1 bands were cut out, and 32P incorporation
was quantified by liquid scintillation counting.
Phosphorylation of Raf-1/MEK1/MAPK in situ
Luteal cells (1 x 106/well, six-well plate)
were washed and labeled with [32P]orthophosphate (1
mCi/ml) in fresh phosphate-free DMEM for 1 h. The cells were then
treated for 5 min as described in the figure legends. After rapidly
rinsing twice with cold PBS, the cells were lysed with buffer A as
described above, and aliquots were taken for scintillation counting to
normalize the 32P counts subjected to immunoprecipitation.
The cell extracts were precleared, and Raf-1 was immunoprecipitated as
described above. MEK1 and PanERK were then sequentially
immunoprecipitated from the same cell extracts. The cell extracts were
incubated with anti-MEK1 or PanERK antibodies (5 µg) for 2 h
at 4 C with continuous rotation, after which 20 µl protein
A-conjugated agarose beads were added. After an additional 1-h
incubation, the immunocomplex beads were collected by centrifugation
and washed three times with buffer A and twice with LiCl solution.
Raf-1/MEK1/MAPK were eluted from the beads with Laemmli buffer. Elutes
were size-fractionated with 10% SDS-PAGE. Autoradiography was
performed on dried gels, and the Raf-1, MEK1, and MAPK bands were cut
out and quantified by liquid scintillation counting.
Statistical methods
The results are expressed as the mean ± SEM of
the average responses in multiple experiments, each performed with
different cell preparations. Data were analyzed by ANOVA followed by
multiple range testing or by t tests for paired
comparisons.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on Raf
activation in the corpus luteum, we determined the presence of Raf
proteins in bovine luteal cells by using specific rabbit polyclonal
antibodies raised against different Raf epitopes corresponding to amino
acids 629–648 for A-Raf, 632–650 for B-Raf, and 587–606 for Raf-1.
Immunoblotting revealed that 69- to 71-kDa A-Raf, 69- to 71-kDa B-Raf,
and 74-kDa Raf-1 were present in bovine luteal cells (Fig. 1
). A-Raf and B-Raf antibodies
cross-reacted with a lower molecular mass protein of approximately 52
kDa.
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activates Raf-1 and B-Raf, but
not A-Raf demonstrates the
utility of this assay. As expected, autophosphorylation was observed
with MEK1, but not the catalytically inactive K52R, in the absence of
cellular protein (data not shown). Phosphorylation of K52R by Raf was
mediated by MEK1, because Raf immunoprecipitates were incapable of
phosphorylating K52R in the absence of exogenous MEK1. These data
suggest an orderly sequence in the activation of Raf, MEK1, and
p42mapk, as shown in Fig. 2A and previously (25, 26).
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(1
µM) treatment for 5 min provoked significant increases in
the phosphorylation of MEK1 (Raf kinase activity) and K52R (MEK1 kinase
activity) compared with untreated control cells (Fig. 2B
). Treatment of
luteal cells with PMA (20 nM) for 5 min had similar
stimulatory effects on the activation of Raf-1, B-Raf, and MEK1.
Conversely, treatment with PGF2 and PMA had no effect on
A-Raf/MEK1 protein kinase activity (Fig. 2B).
PGF2 stimulates Raf-1/MEK1 kinase
activity in a time- and concentration-dependent manner
We further characterized the response to PGF2 by
examining the temporal and concentration responses of
PGF2 on Raf-1 activation. Treatment of luteal cells with
PGF2 (1 µM) rapidly increased the activity
of Raf-1. Within 1 min of treatment, phosphorylation of MEK1 and K52R
increased 0.8- and 2-fold, respectively. Maximal effects of
PGF2 (2.2- and 5-fold, respectively) were observed after
5–10 min of treatment. Thereafter, the PGF2-induced Raf
kinase activity gradually decreased and returned to the basal level at
30 min (Fig. 3A). When the luteal cells
were treated with increasing concentrations of PGF2 for
5 min, PGF2 stimulated Raf-1/MEK1 kinase activities in a
concentration-dependent manner. Maximal increases in Raf-1/MEK1 protein
kinase activities were observed in cells treated with 1
µM PGF2 (Fig. 3B). Similar concentration-dependent
responses were observed for PGF2-induced inositol
phosphate accumulation, an indication of phospholipase C activation in
bovine luteal cells (8, 9, 10).
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stimulates phosphorylation of
p42mapk and p44mapk in
a time- and concentration-dependent mannerPanERK antibody based on
the reduced migration rate (mobility shift) of the phosphorylated
active forms compared with that of the nonphosphorylated inactive forms
(3) in SDS-PAGE. We used a similar approach to investigate whether
PGF2 can induce a mobility shift of p42mapk
and p44mapk. Indeed, we observed that PGF2
rapidly stimulated the phosphorylation of p42mapk and
p44mapk in luteal cells (Fig. 4). We obtained optimal separation of the
phosphorylated and nonphosphorylated forms by using 10% SDS-PAGE with
an acrylamide/bisacrylamide ratio of 29.8:0.2.
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-responsive
isoforms of the ERK subfamily of MAPK in bovine luteal cells.
Additionally, the levels of p42mapk were greater than the
levels of p44mapk, as shown by Western blot analysis with
both the PanERK antibody (Fig. 4) and the antiphosphotyrosine
specific MAPK antibody (Fig. 5).
Additionally, two unknown proteins with molecular masses of
approximately 85 and 57 kDa were also observed in PanERK
immunoblots. These two proteins were not altered in response to
PGF2 treatment.
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(1 µM)
stimulated phosphorylation of p42mapk and
p44mapk. The stimulatory effects of PGF2 on
p42mapk and p44mapk phosphorylation were
maximal after 5–10 min of treatment and returned to basal levels after
30 min (Fig. 4A). The effects of PGF2 on
p42mapk and p44mapk phosphorylation were also
concentration dependent. When luteal cells were treated with increasing
concentrations (0.001–10 µM) of PGF2 for
5 min, maximal effects of PGF2 on the phosphorylation of
p42mapk and p44mapk were observed with 0.1–1
µM PGF2 (Fig. 4B). The profiles of time
courses and concentration responses of PGF2-induced
phosphorylation of p42mapk and p44mapk were
consistent with those of PGF2-induced Raf-1/MEK1 kinase
activity, as shown in Fig. 3.
PGF2 and PMA stimulate tyrosine
phosphorylation of p42mapk and
p44mapk
MAPK activation results from the phosphorylation of
threonine/tyrosine residues in the threonine-glutamine-tyrosine motif
(13, 14). When analyzed with an antiphosphotyrosine-specific MAPK
antibody, we also observed that treatment with PGF2 (1
µM) for 5 min markedly stimulated the tyrosine
phosphorylation of p42mapk and p44mapk in
luteal cells. PMA (20 nM) had similar stimulatory effects
on the tyrosine phosphorylation of p42mapk and
p44mapk (Fig. 5).
PGF2 and PMA increase the enzymatic
activity of p42mapk and
p44mapk
Activation of the MAPK signaling cascade regulates gene expression
in part by the ability of MAPK to phosphorylate and activate direct
downstream transcription factors of the Ets family, including Elk-1
(18). We employed the transcription factor Elk-1 as a specific
substrate to determine the activities of p42mapk and
p44mapk. Elk-1 phosphorylation was determined in
p42mapk and p44mapk immunocomplex kinase assays
obtained from control and stimulated luteal cells. Treatment with
PGF2 (1 µM) for 5 min stimulated Elk-1
phosphorylation by 5- and 4.5-fold in p42mapk and
p44mapk immunoprecipitates, respectively (Fig. 6). Activation of PKC by PMA also
increased Elk-1 phosphorylation. When treated for 5 min, PMA (20
nM) stimulated Elk-1 phosphorylation by 4.3- and 5.5-fold
in p42mapk and p44mapk immunoprecipitates,
respectively (Fig. 6).
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and PMA phosphorylate Raf-1, MEK1,
and MAPK in situ -treated cells
in vitro, we investigated the in situ
phosphorylation of Raf-1, MEK1, and ERKs by PGF2 and PMA
as described in Materials and Methods. Compared with
untreated control cells, treatment with PGF2 (1
µM) and PMA (20 nM) for 5 min increased
32P incorporation into Raf-1 immunoprecipitates by 50% and
33%, respectively. 32P incorporation into MEK1
immunoprecipitates increased 69% and 67% in cells treated with
PGF2 and PMA for 5 min, respectively. 32P
incorporation into p42mapk immunoprecipitates increased
47% and 46% in cells treated with PGF2 and PMA for 5
min, respectively (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on the corpus luteum are initiated at the plasma
membrane through the stimulation of phospholipase C, resulting in
inositol trisphosphate-induced intracellular Ca2+
mobilization and diacylglycerol activation of PKC (8, 9, 10, 31). To our
knowledge, little is known about the postmembrane signaling events
stimulated by PGF2 in the ovary. Our present data
clearly demonstrate that PGF2 activates the
Raf/MEK1/p42mapk/p44mapk signaling cascade in
the corpus luteum. Activation of this cytoplasmic protein kinase
pathway by PGF2 may provide a mechanism to link
signaling events initiated by the PGF2 receptor to the
regulation of nuclear transcriptional activity. Mammalian cells contain three Raf genes encoding the serine/threonine protein kinases A-Raf, B-Raf, and C-Raf (Raf-1) that function as MAPK kinase kinases to initiate the p42mapk and p44mapk signaling cascade (31). Raf-1 is expressed ubiquitously, whereas A-Raf is thought to be restricted to the urogenital system, and B-Raf is known to be expressed largely in the brain and testes (32). Our data demonstrate the presence of each of the Raf proteins in the corpus luteum. Sequence comparisons reveal that Raf proteins share three conserved regions, termed CR1, CR2, and CR3 (33, 34, 35). CR-1, the regulatory domain near the NH2-terminus, contains the major region required for Ras-GTP-Raf interaction (36, 37). This region in Raf-1 also contains a cAMP-dependent serine phosphorylation site that is associated with reduced affinity of Raf for Ras (38). CR2 consists of a stretch of 20 amino acids rich in serine and threonine residues, which are potential targets for Raf autophosphorylation and phosphorylation by other serine/threonine kinases, including PKC. CR3 comprises the catalytic domain on the COOH-terminus half of the protein. The catalytic domain of Raf-1 contains serine (Ser499) and tyrosine phosphorylation residues. These phosphorylation sites are known to be regulated by PKC (39, 40) and Src (37), respectively. Thus, multiple mechanisms exist for the regulation of Raf activation.
Ras-dependent activation of Raf-1 occurs at the plasma membrane in the
presence of membrane lipids and/or other protein kinases (41). Most
studies regarding the regulation of Raf protein kinases in vertebrate
cells have been conducted on Raf-1, and the regulation of A-Raf and
B-Raf isoforms has not been well characterized (38). A-Raf exhibits
substantial homology to Raf-1 within the kinase domain (42), whereas
B-Raf lacks two tyrosine phosphorylation sites (Tyr340 and
Tyr341) that are involved in Ras-dependent activation of
Raf-1 by tyrosine kinases (34, 37). A recent report demonstrated that
synergistic signals from Ras-GTP and Src induced maximal
activation of Raf-1 and A-Raf, whereas formation of Ras-GTP
was sufficient for maximal activation of B-Raf (37). Additionally, Cai
et al. (40) demonstrated that Raf-1 was effectively
activated by phosphorylation on serine residues by PKC and PKC
.
However, they also noted that Raf-1 activated by PKC could be further
enhanced by tyrosine phosphorylation. Our present data suggest that PKC
is involved in Raf activation in the corpus luteum. We observed similar
patterns of Raf-1 and B-Raf activation in response to
PGF2 and PMA, a pharmacological activator of PKC.
Furthermore, in intact cells prelabeled with
[32P]orthophosphate, PGF2 and PMA both
increased phosphorylation of Raf-1. A role for PKC in
PGF2-activated Raf-1 in the corpus luteum is supported
by the observations of Hakeda et al. (23), who demonstrated
that PGF2-stimulated Raf/MAPK signaling via a
PKC-dependent mechanism in osteoblastic MC3T3-E1 cells. However, our
results and those of Hakeda et al. (23) differ from a report
indicating that PGF2 action in NIH-3T3 cells was
mediated by PKC-independent activation of Raf-1 (22). As indicated
above, multiple mechanisms of Raf activation might be operational in
the corpus luteum. In this regard, our preliminary results indicate
that depletion of conventional isoforms of PKC by chronic treatment
with PMA only partially inhibits Raf-1 activation in bovine luteal
cells (43). A recent report indicates that cAMP and protein kinase A
may play a role in B-Raf activation in PC-12 cells (44). As neither
PGF2 nor PMA increases cAMP in bovine luteal cells (9),
activation of B-Raf by cAMP is unlikely to be involved in the mechanism
by which PGF2 activates Raf. Unlike our results with
Raf-1 and B-Raf, neither PGF nor PMA activated A-Raf in our study. The
reason for this differential activation of Raf isoforms by
PGF2 and PMA in luteal cells is unclear and requires
further investigation.
Mechanisms other than PKC-mediated phosphorylation may play a role in
Raf activation. Raf-1 activation by G protein-coupled receptors appears
to be mediated through G protein subunits; however, the exact pathways
appear to be dependent upon the specific cell types involved (reviewed
in Ref. 21). In NIH-3T3 cells, FP receptors, perhaps via
Gq, have been reported to be linked to MAPK activation
via formation of a Ras-GTP complex (22). More recently, G protein
ß-subunit-mediated tyrosine phosphorylation of the adaptor protein
Shc and the guanine nucleotide exchange factor SOS has been implicated
in p21ras-mediated Raf-1 activation by
PGF2 in myometrial cells (45). The G protein
-subunits Gs, Gq, G11,
and Gi are expressed in the corpus luteum (46). Although
the specific G protein subunits involved in the action of
PGF2 in luteal cells are not known, the fact that
PGF2 activates phophatidylinositol-specific
phospholipase C suggests the involvement of Gq or
Gi. In other cells (22) PGF2-induced
activation of phospholipase C is believed to be mediated by
Gq. Further studies to characterize the role
of G proteins and p21Ras in PGF2-induced Raf activation
in luteal cells are necessary to identify their involvement in the
signaling event(s) that lead to Raf activation.
The present series of experiments demonstrated that PGF2
stimulated the phosphorylation and activation of Raf, MEK1,
p42mapk, and p44mapk in luteal cells. We
observed similar concentration and temporal response relationships for
PGF2-induced Raf, MEK1, and MAPK activation. The
concentrations of PGF2 required to activate MAPK
signaling were similar to the concentrations of PGF2
required for activation of phospholipase C (8). Activation of
phospholipase C occurs within seconds of treatment with
PGF2 and precedes activation of the Raf/MEK1/MAPK
signaling cascade, suggesting that the MAP kinase signaling cascade
lies downstream of phospholipase C activation. Of interest, however,
were the observations that the stimulatory effects of
PGF2 on each component of the MAPK signaling cascade
were transient, returning to basal levels within 30 min of treatment
with PGF2. This suggests that this signaling pathway
rapidly desensitizes in the presence of PGF2. The
observation that the activities of Raf, MEK1, and MAPK are coordinately
regulated suggests that desensitization occurs at or precedes the
activation of Raf. As PGF2-induced accumulation of
inositol phosphates in these cells is continuous for more than 30 min
(8), the desensitization of MAP kinase signaling cannot be explained by
a reduction in PGF2-induced phospholipase C activity.
Although phosphorylation of Raf by PKC (40) and tyrosine kinases (37)
appears to represent early receptor-initiated events for Raf
activation, a recent report suggests that hyperphosphorylation of Raf
reduces the ability of Raf to associate with the plasma membrane and
contributes to desensitization of the MAP kinase signaling cascade
(47). Interestingly, in these studies Raf desensitization appeared to
require activation of p42mapk. Thus, p42mapk
may directly or indirectly modulate the activity of Raf. The molecular
mechanisms responsible for PGF2-induced desensitization
of MAP kinase signaling in luteal cells remain to be established.
The exact role of MAPK in the corpus luteum awaits discovery. A few
possibilities are suggested by the present data. An important cytosolic
substrate for MAPK is phospholipase A2 (13), a key enzyme
that releases arachidonic acid from membrane phospholipids for
prostanoid synthesis. MAPK phosphorylates and activates phospholipase
A2 (20). In fact, PGF2 is capable of
activating phospholipase A2 in the rat corpus luteum (48).
Of considerable interest is the recent report by Tsai et al.
demonstrating that PGF2 stimulates PG synthesis
[increased in cyclooxgenase-2 messenger RNA (mRNA)] in ovine large
luteal cells (49). It seems likely, therefore, that
PGF2-activated p42mapk and
p44mapk could participate in intraluteal PG synthesis by
phosphorylating and activating phospholipase A2.
An important component of the MAPK signaling cascade is its role in
regulation of transcriptional activity. Once activated,
p42mapk and p44mapk translocate from the
cytoplasm into the nucleus (15) and phosphorylate various transcription
factors (13). Our studies clearly demonstrate that
PGF2-activated luteal MAPKs can phosphorylate Elk-1
in vitro. The Ets family transcription factor, Elk-1, is
well documented as a physiological substrate for p42mapk
and p44mapk. Elk-1 phosphorylation stimulates the
transcription of the immediate early response gene c-fos by
facilitating the formation of a ternary complex with serum response
element and the serum response factor (18). The c-Fos protein
heterodimerizes with c-Jun to form the activator protein-1 (AP-1)
transcription factor and plays an important role in regulation of the
expression of numerous genes possessing AP-1-binding sites (reviewed in
Ref. 50). In this regard, our preliminary data demonstrate that
PGF2 rapidly increases c-fos and
c-jun mRNA levels in bovine luteal cells (43, 51).
Additionally, Bertrand and Stormshak have recently shown that
PGF2 treatment in vivo increases the
expression of c-jun mRNA (52) in the corpus luteum. In
keeping with this evidence, we postulate that phosphorylation of Elk-1
or other Ets family members mediates PGF2-regulated
expression of specific genes in the corpus luteum through AP-1
transcription factors. Conversely, c-Fos and c-Jun have been implicated
in the repression of gene transcription by interactions at DNA-binding
sites other than AP-1 (53). Thus, the ability of PGF2 to
increase expression of c-fos and c-jun may
stimulate the expression of some genes and inhibit the expression of
others.
Similar to the Raf/MEK/MAPK pathway, multiple isoforms of MAPK along
with their unique MAPK kinase kinase, MAPK kinase, and MAPK
phosphorylation cascades coexist in mammalian cells. These parallel
pathways regulate diverse and even opposite cellular events (reviewed
in Ref. 54). MAPK family members identified to date can be assembled
into three subfamilies: ERKs, the Jun NH2-terminus kinase
(JNKs) or stress-activated protein kinases, and p38mapk.
The ERK subfamily of MAPK (p42mapk and p44mapk)
appears to play a central role in mitogenic signaling correlated to
cell survival (55) and proliferation (21), whereas activation of the
JNK and p38mapk subfamilies has been recently correlated to
cell death or apoptosis (56). Recent reports in other cell lines
suggest that PGF2 activation of the Raf/ERK pathway is
correlated with cell proliferation. In NIH-3T3 cells,
PGF2 activates p42mapk and
p44mapk, via a Ras-dependent pathway, to exert its
mitogenic effects (22). Similarly, in osteoblastic MC3T3–1 cells
PGF2-induced mitogenesis is correlated with activation
of the Raf-1/p42mapk and p44mapk pathway (23).
However, in the corpus luteum activation of this signaling cascade
appears not to be correlated to cell proliferation, as
PGF2 alone does not stimulate DNA synthesis (Davis,
J. S., unpublished data) or cell proliferation (57) in serum-free
cultures of bovine luteal cells in vitro. In contrast, the
process of structural luteolysis in the bovine corpus luteum either in
the late estrous cycle (58) or as a result of PGF2
injection (59) is associated with increased apoptosis in
vivo. Thus, it is reasonable to infer that PGF2
should activate the JNK and p38mapk MAPK subfamilies in the
corpus luteum. Indeed, we have recently observed that
PGF2 also activates JNK-1 in cultured bovine luteal
cells (60). However, the significance of JNK-1 activation in
vitro is unclear, as PGF2 does not affect the
viability of bovine luteal cell cultures (57). As PGF2
actions on bovine luteal cells in vitro are not directly
associated with proliferation or cell death, it seems likely that the
Raf/MEK1/p42mapk/p44mapk signaling pathway may
be important for the regulation of other luteal processes,
i.e. regulation of gene expression during luteal development
and regression (61). Moreover, the luteolytic actions of PGF2in vivo may require the presence or absence of other
factors (i.e. growth factors, cytokines, and steroids) and
cell-cell communications to be fully expressed.
In summary, the present study describes a cytoplasmic protein kinase
signaling cascade that is activated by PGF2 and PMA in
luteal cells. Combined with previous reports (reviewed in Refs. 10, 62), the present data allow us to extend the intracellular signaling
mechanism of PGF2 action in the corpus luteum, as
illustrated in Fig. 8.
PGF2 binds to its specific transmembrane G
protein-coupled receptors. Ligand receptor activation of phospholipase
C results in the cleavage of membrane phosphoinositides and activation
of PKC. These initial signaling events activate Raf-1 to initiate the
Raf-1/MEK1/p42mapk and p44mapk signaling
cascade. PGF2-activated p42mapk and
p44mapk translocate into the nucleus and phosphorylate
transcription factors, such as Elk-1, to regulate the expression of the
early response gene c-fos. We hypothesize that the
PGF2-induced Raf-1/MEK1/p42mapk and
p44mapk signaling cascade might mediate its effects
in vivo through regulation of the transcriptional activation
of genes possessing AP-1-binding sites.
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Acknowledgments |
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Footnotes |
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2 Lalor Foundation postdoctoral fellow.
Received February 10, 1998.
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