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Endocrinology Vol. 139, No. 9 3876-3885
Copyright © 1998 by The Endocrine Society

ARTICLES

Prostaglandin F2{alpha} Stimulates the Raf/MEK1/Mitogen-Activated Protein Kinase Signaling Cascade in Bovine Luteal Cells1

Dong-bao Chen2, Suzanne D. Westfall, Hon W. Fong, Mark S. Roberson and John S. Davis

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Upon binding to its G protein-coupled transmembrane receptors, the actions of PGF2{alpha} 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{alpha}. 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{alpha} (1 µM) and the pharmacological PKC activator phorbol myristate acetate (PMA, 20 nM). Kinetic analysis revealed that PGF2{alpha} 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{alpha} (1 µM) and PMA (20 nM). Additionally, both PGF2{alpha} (1 µM) and PMA (20 nM) stimulated phosphorylation of Raf-1, MEK1, and p42mapk in 32P-labeled cells. Our data demonstrate that PGF2{alpha} activates the Raf/MEK1/p42/44mapk signaling cascade in bovine luteal cells and that the actions of PGF2{alpha} are mimicked by the PKC activator PMA. Activation of the Raf/MEK1/MAPK signaling cascade by PGF2{alpha} in luteal cells provides a mechanism to transduce signals initiated by PGF2{alpha} 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
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PGF2{alpha} EXERTS diverse physiological functions throughout the body. In a number of mammals (reviewed in Refs. 1, 2), PGF2{alpha} 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{alpha} receptors (FP receptors) are present on luteal cell membranes (3). An obligatory role of the FP receptor in mediating the actions of PGF2{alpha} 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{alpha}-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{alpha}-induced luteolysis in vivo (11) and the actions of PGF2{alpha} in vitro (12). However, a gap exists in our knowledge between the initial signaling events and the physiological sequelae observed in response to PGF2{alpha}. The mechanism(s) by which PGF2{alpha} 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{alpha}. Watanebe et al. (22) demonstrated that the MAPK signaling cascade was coupled to PGF2{alpha}-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{alpha} was apparently independent of PMA-sensitive isoforms of PKC (22). Hakeda et al. (23) recently found that PGF2{alpha} activated the MAPK pathway in osteoblastic MC3T3-E1 cells via PKC-dependent activation of Raf-1. Our preliminary data indicate that PGF2{alpha} 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{alpha} in the corpus luteum. We report herein that PGF2{alpha} and PMA activate the Raf/MEK1/p42mapk and p44mapk signaling cascade in bovine luteal cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Protein A-conjugated agarose beads were obtained from Upstate Biotechnology (Lake Placid, NY). Rabbit polyclonal anti-Raf-1-conjugated agarose beads, rabbit polyclonal anti-ERK2-conjugated agarose beads, rabbit polyclonal anti-ERK1-conjugated agarose beads, rabbit polyclonal anti-A-Raf, rabbit polyclonal anti-B-Raf, and polyclonal anti-Raf-1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti-{alpha}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). [{gamma}-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{alpha}, 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 [{gamma}-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 [{gamma}-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 [{gamma}-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 {alpha}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 [{gamma}-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 {alpha}PanERK were then sequentially immunoprecipitated from the same cell extracts. The cell extracts were incubated with anti-MEK1 or {alpha}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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunoblotting of A-Raf, B-Raf, and Raf-1 in bovine luteal cells
Mammalian cells contain three different Raf genes encoding the serine/threonine protein kinases A-Raf, B-Raf, and Raf-1 that function as MAPK kinase kinases to initiate the p42/42mapk signaling cascade (31). To characterize the effects of PGF2{alpha} 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. 1Go). A-Raf and B-Raf antibodies cross-reacted with a lower molecular mass protein of approximately 52 kDa.



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  		Figure 1. Immunoblot analysis of A-Raf, B-Raf, and Raf-1 in bovine luteal cells. Bovine luteal cells were lysed with a nondenaturing buffer. Total cell lysates (30 µg/lane) were size-fractionated on 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with polyclonal rabbit antibodies against A-Raf (1:250), B-Raf (1:250), and Raf-1 (1:250), followed by antirabbit peroxidase-conjugated IgG (1:4000). Bound antibodies were visualized with the ECL reagents according to manufacturer’s instructions. Results from one of four independent experiments are shown.

 
PGF2{alpha} activates Raf-1 and B-Raf, but not A-Raf
Activated Raf directly phosphorylates MEK1 at two serine residues within a conserved regulatory region between the DFG and A(/S)PE motifs (14). In turn, activated MEK1 phosphorylates p42mapk and p44mapk at threonine and tyrosine residues within a conserved regulatory region between the TEY motif (14). Raf activation can be measured in a two-step immunocomplex assay. This assay allows measurement of Raf activity (phosphorylation of MEK1) in the first step and measurement of MEK1 activity (phosphorylation of K52R) in the second step. The first step of the assay employs endogenous Raf immunocomplexes and catalytically active recombinant MEK1. The second step involves the MEK1 derived from the first step and a full-length, but catalytically inactive, p42mapk (K52R) to assess MEK1 activation. Figure 2AGo 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. 2AGo and previously (25, 26).



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  		Figure 2. Activation of A-Raf, B-Raf, and Raf-1 by PGF2{alpha} and PMA in bovine luteal cells. Serum-starved bovine luteal cells were washed and equilibrated with fresh M-199–0.1% BSA for 1 h, then treated with PGF2{alpha} (1 µM) or PMA (20 nM) for 5 min. Total cell lysates were made with a cold nondenaturing buffer. The enzymatic activities of immunoprecipitated A-Raf, B-Raf, and Raf-1 from cell lysates (200 µg protein) were then measured by [32P]ATP incorporation into [His]6-tagged MEK1 (1 µg) and its subsequent phosphorylation of [His]6-tagged K52R (0.1 µg) by the two-step immunocomplex kinase assay as described in Materials and Methods. A, An autoradiogram from one of three independent experiments with Raf-1 is shown. Lane 1, Raf-1 immunoprecipitates cannot phosphorylate K52R in the absence of MEK1. Lane 2, MEK1 phosphorylates K52R in the absence of cellular protein. Lanes 3–5, Raf-1 initiated phosphorylation of MEK1 and K52R in Raf-1 immunocomplexes isolated from cells treated with control medium (lane 3), PMA (lane 4), and PGF2{alpha} (lane 5). B, Data from three independent experiments are expressed as the mean ± SEM. In each immunocomplex assay, the levels of [32P]ATP incorporation into MEK1 and K52R were obtained after subtraction of their corresponding levels of phosphorylation in the absence of Raf immunocomplexes. Autophosphorylation of MEK1 was 128 ± 19 cpm, and phosphorylation of K52R in the presence of MEK1 was 215 ± 35 cpm. *, P < 0.05.

 
In Raf-1 and B-Raf immunoprecipitates, PGF2{alpha} (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. 2BGo). 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{alpha} and PMA had no effect on A-Raf/MEK1 protein kinase activity (Fig. 2BGo).

PGF2{alpha} stimulates Raf-1/MEK1 kinase activity in a time- and concentration-dependent manner
We further characterized the response to PGF2{alpha} by examining the temporal and concentration responses of PGF2{alpha} on Raf-1 activation. Treatment of luteal cells with PGF2{alpha} (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{alpha} (2.2- and 5-fold, respectively) were observed after 5–10 min of treatment. Thereafter, the PGF2{alpha}-induced Raf kinase activity gradually decreased and returned to the basal level at 30 min (Fig. 3AGo). When the luteal cells were treated with increasing concentrations of PGF2{alpha} for 5 min, PGF2{alpha} 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{alpha} (Fig. 3BGo). Similar concentration-dependent responses were observed for PGF2{alpha}-induced inositol phosphate accumulation, an indication of phospholipase C activation in bovine luteal cells (8, 9, 10).



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  		Figure 3. Temporal and concentration-dependent Raf-1 activation in response to PGF2{alpha} in bovine luteal cells. Cell lysates were prepared from bovine luteal cells at the indicated times after treatment with 1 µM PGF2{alpha} (A) or were prepared after treatment with increasing concentrations (0.001–10 µM) of PGF2{alpha} for 5 min (B). Raf-1 was immunoprecipitated from the cell lysates (200 µg). Raf-1/MEK1 kinase activity in the immunoprecipitates was measured as described in Fig. 2Go. Results are the mean ± SEM from three independent experiments.

 
PGF2{alpha} stimulates phosphorylation of p42mapk and p44mapk in a time- and concentration-dependent manner
Phosphorylation of p42mapk and p44mapk can be measured by Western blot analysis with {alpha}PanERK 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{alpha} can induce a mobility shift of p42mapk and p44mapk. Indeed, we observed that PGF2{alpha} rapidly stimulated the phosphorylation of p42mapk and p44mapk in luteal cells (Fig. 4Go). 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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  		Figure 4. Temporal and concentration-dependent responses of PGF2{alpha} on phosphorylation of p42mapk and p44mapk. Cell lysates prepared from serum-starved bovine luteal cells at the indicated times after treatment with PGF2{alpha} (1 µM; A) or prepared 5 min after treatment with increasing concentrations (0.001–10 µM) of PGF2{alpha} as described in Fig. 2Go. Total cell lysates (20 µg/lane) were size-fractionated with 10% SDS-PAGE and electrically transferred to a PVDF membrane. Phosphorylation of p42mapk and p44mapk was analyzed by immunoblotting with a {alpha}PanERK antibody (1:500) based on their mobility shift on SDS-PAGE upon activation. Upper panel autoradiograms represent longer exposures (45 sec), showing the mobility shift of p44mapk. Lower panel autoradiograms represent shorter exposures (15 sec), showing the mobility shift of p42mapk. Results from one of five similar independent experiments are shown.

 
Western blot analysis revealed that p42mapk and p44mapk were the major PGF2{alpha}-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 {alpha}PanERK antibody (Fig. 4Go) and the antiphosphotyrosine specific MAPK antibody (Fig. 5Go). Additionally, two unknown proteins with molecular masses of approximately 85 and 57 kDa were also observed in {alpha}PanERK immunoblots. These two proteins were not altered in response to PGF2{alpha} treatment.



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  		Figure 5. Tyrosine phosphorylation of p42mapk and p44mapk in response to PGF2{alpha} and PMA. Cell lysates prepared from serum-starved bovine luteal cells after treatment with PGF2{alpha} (1 µM) and PMA (20 nM) for 5 min. Total cell lysates (20 µg/lane) were size-fractionated with 10% SDS-PAGE and electrically transferred to a PVDF membrane. Tyrosine phosphorylation of p42mapk and p44mapk was analyzed with a Phospho-plus MAPK antibody (1:1000), which only recognizes the tyrosine 204-phosphorylated forms of p42mapk and p44mapk. The results represent one of four similar independent experiments.

 
Time-course studies revealed that PGF2{alpha} (1 µM) stimulated phosphorylation of p42mapk and p44mapk. The stimulatory effects of PGF2{alpha} on p42mapk and p44mapk phosphorylation were maximal after 5–10 min of treatment and returned to basal levels after 30 min (Fig. 4AGo). The effects of PGF2{alpha} on p42mapk and p44mapk phosphorylation were also concentration dependent. When luteal cells were treated with increasing concentrations (0.001–10 µM) of PGF2{alpha} for 5 min, maximal effects of PGF2{alpha} on the phosphorylation of p42mapk and p44mapk were observed with 0.1–1 µM PGF2{alpha} (Fig. 4BGo). The profiles of time courses and concentration responses of PGF2{alpha}-induced phosphorylation of p42mapk and p44mapk were consistent with those of PGF2{alpha}-induced Raf-1/MEK1 kinase activity, as shown in Fig. 3Go.

PGF2{alpha} 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{alpha} (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. 5Go).

PGF2{alpha} 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{alpha} (1 µM) for 5 min stimulated Elk-1 phosphorylation by 5- and 4.5-fold in p42mapk and p44mapk immunoprecipitates, respectively (Fig. 6Go). 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. 6Go).



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  		Figure 6. Activation of p42mapk and p44mapk in response to PGF2{alpha} and PMA. Cell lysates prepared from serum-starved bovine luteal cells after treatment with PGF2{alpha} (1 µM) and PMA (20 nM) for 5 min. Total cell lysates were made and subjected (200 µg protein) to p42mapk and p44mapk immunoprecipitation. The p42mapk and p44mapk immunocomplexes were incubated with GST-Elk-1 (0.5 µg) and [{gamma}-32P]ATP for 20 min at 30 C. The reaction mixtures were then analyzed with 10% SDS-PAGE followed by autoradiography. 32P incorporation into GST-Elk-1 was quantified by liquid scintillation counting of the corresponding bands. The left and right panels summarize activation of p42mapk and p44mapk, respectively, in response to PGF2{alpha} and PMA. A representative autoradiogram showing phosphorylation of GST-Elk-1 by activated p42mapk and p44mapk is included in their corresponding panels. The results are expressed as the mean ± SEM from three independent experiments. *, P < 0.05.

 
PGF2{alpha} and PMA phosphorylate Raf-1, MEK1, and MAPK in situ
Having demonstrated the activation of the Raf-1/MEK1/MAPK signaling cascade components in PGF2{alpha}-treated cells in vitro, we investigated the in situ phosphorylation of Raf-1, MEK1, and ERKs by PGF2{alpha} and PMA as described in Materials and Methods. Compared with untreated control cells, treatment with PGF2{alpha} (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{alpha} and PMA for 5 min, respectively. 32P incorporation into p42mapk immunoprecipitates increased 47% and 46% in cells treated with PGF2{alpha} and PMA for 5 min, respectively (Fig. 7Go).



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  		Figure 7. In situ phosphorylation of Raf-1, MEK1, and p42mapk in response to PGF2{alpha} and PMA. Serum-starved bovine luteal cells were prelabeled with 32P (1 mCi/ml) in phosphate-free DMEM for 1 h. The cells were then treated with PGF2{alpha} (1 µM) and PMA (20 nM) for 5 min and lysed with a nondenaturing buffer. Raf-1, MEK1, and p42mapk were sequentially immunoprecipitated from the total cell lysates as described in Materials and Methods. The immunoprecipitates were analyzed with 10% SDS-PAGE followed by autoradiography. The results represent averages (bars) from two (open circles) independent experiments. A representative autoradiogram showing the phosphorylation of Raf-1, MEK1, and p42mapk, respectively, is included in their corresponding panels.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Evidence has accumulated demonstrating that the actions of PGF2{alpha} 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{alpha} in the ovary. Our present data clearly demonstrate that PGF2{alpha} activates the Raf/MEK1/p42mapk/p44mapk signaling cascade in the corpus luteum. Activation of this cytoplasmic protein kinase pathway by PGF2{alpha} may provide a mechanism to link signaling events initiated by the PGF2{alpha} 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{alpha} and PKC{epsilon}. 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{alpha} and PMA, a pharmacological activator of PKC. Furthermore, in intact cells prelabeled with [32P]orthophosphate, PGF2{alpha} and PMA both increased phosphorylation of Raf-1. A role for PKC in PGF2{alpha}-activated Raf-1 in the corpus luteum is supported by the observations of Hakeda et al. (23), who demonstrated that PGF2{alpha}-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{alpha} 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{alpha} 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{alpha} 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{alpha} 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{alpha}, have been reported to be linked to MAPK activation via formation of a Ras-GTP complex (22). More recently, G protein ß{gamma}-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{alpha} in myometrial cells (45). The G protein {alpha}-subunits Gs{alpha}, Gq{alpha}, G11{alpha}, and Gi{alpha} are expressed in the corpus luteum (46). Although the specific G protein subunits involved in the action of PGF2{alpha} in luteal cells are not known, the fact that PGF2{alpha} activates phophatidylinositol-specific phospholipase C suggests the involvement of Gq{alpha} or Gi{alpha}. In other cells (22) PGF2{alpha}-induced activation of phospholipase C is believed to be mediated by Gq{alpha}. Further studies to characterize the role of G proteins and p21Ras in PGF2{alpha}-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{alpha} stimulated the phosphorylation and activation of Raf, MEK1, p42mapk, and p44mapk in luteal cells. We observed similar concentration and temporal response relationships for PGF2{alpha}-induced Raf, MEK1, and MAPK activation. The concentrations of PGF2{alpha} required to activate MAPK signaling were similar to the concentrations of PGF2{alpha} required for activation of phospholipase C (8). Activation of phospholipase C occurs within seconds of treatment with PGF2{alpha} 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{alpha} on each component of the MAPK signaling cascade were transient, returning to basal levels within 30 min of treatment with PGF2{alpha}. This suggests that this signaling pathway rapidly desensitizes in the presence of PGF2{alpha}. 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{alpha}-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{alpha}-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{alpha}-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{alpha} 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{alpha} stimulates PG synthesis [increased in cyclooxgenase-2 messenger RNA (mRNA)] in ovine large luteal cells (49). It seems likely, therefore, that PGF2{alpha}-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{alpha}-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{alpha} rapidly increases c-fos and c-jun mRNA levels in bovine luteal cells (43, 51). Additionally, Bertrand and Stormshak have recently shown that PGF2{alpha} 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{alpha}-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{alpha} 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{alpha} activation of the Raf/ERK pathway is correlated with cell proliferation. In NIH-3T3 cells, PGF2{alpha} activates p42mapk and p44mapk, via a Ras-dependent pathway, to exert its mitogenic effects (22). Similarly, in osteoblastic MC3T3–1 cells PGF2{alpha}-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{alpha} 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{alpha} injection (59) is associated with increased apoptosis in vivo. Thus, it is reasonable to infer that PGF2{alpha} should activate the JNK and p38mapk MAPK subfamilies in the corpus luteum. Indeed, we have recently observed that PGF2{alpha} also activates JNK-1 in cultured bovine luteal cells (60). However, the significance of JNK-1 activation in vitro is unclear, as PGF2{alpha} does not affect the viability of bovine luteal cell cultures (57). As PGF2{alpha} 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 PGF2{alpha}in 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{alpha} 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{alpha} action in the corpus luteum, as illustrated in Fig. 8Go. PGF2{alpha} 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{alpha}-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{alpha}-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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  		Figure 8. Proposed intracellular signaling actions of PGF2{alpha} in luteal cells. PGF2{alpha} initiates its actions on luteal cells at the plasma membrane by binding to its specific transmembrane G protein-coupled receptors. Ligand receptor activation of phospholipase C (PLC) results in the cleavage of membrane phosphoinositides into two second messengers, i.e. diacylglycerol (DAG) and inositol 1,4,5-trisphosphate. These early signaling events activate Raf-1 and initiate the Raf-1/MEK1/p42mapk and p44mapk signaling cascade. PGF2{alpha}-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 Raf-1/MEK1/p42mapk and p44mapk signaling cascade might mediate the luteolytic effects of PGF2{alpha}in vivo by stimulating the transcriptional activation of tissue-remodeling genes with promoter AP-1-binding sites. The roles of intracellular calcium, PKC, and Ras in activation of MAPK signaling are unclear (?). MAPK activation may serve to active other signaling events, such as phospholipase A2 (PLA2). {alpha}ß{gamma}, G protein subunits; Ca2+, intracellular calcium; SRE, serum response element; SRF, serum response factor.

 


   Acknowledgments
 
The authors are grateful for the helpful discussions with Drs. B. A. Keel and J. V. May. We thank Paul Dent and Thomas W. Sturgill, University of Virginia Health Sciences Center, for providing [His]6-tagged MEK1 and K52R.


   Footnotes
 
1 This work was supported by NIH Grant 22248, the Research Service of the Department of Veterans Affairs, the Lalor Foundation, the Women’s Research Institute, and the Wesley Medical Research Institutes. The data were presented in part at the 29th Annual Meeting of the Society for the Study of Reproduction, July 26–30, 1996, London, Ontario, Canada (Abstract 345). Back

2 Lalor Foundation postdoctoral fellow. Back

Received February 10, 1998.


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 Introduction
 Materials and Methods
 Results
 Discussion
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