Role of Dynamin, Src, and Ras in the Protein Kinase C-mediated Activation of ERK by Gonadotropin-releasing Hormone*

Outhiriaradjou BenardDagger , Zvi Naor§, and Rony SegerDagger

From the Dagger  Department of Biological Regulation, The Weizmann Institute of Science, Rehovot 76100, Israel and the § Department of Biochemistry, Tel Aviv University, Ramat Aviv 69978, Israel

Received for publication, August 3, 2000, and in revised form, October 23, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

G-protein-coupled receptors are a large group of integral membranal receptors, which in response to ligand binding initiate diverse downstream signaling. Here we studied the gonadotropin-releasing hormone (GnRH) receptor, which uses Gq for its downstream signaling. We show that extracellular signal-regulated kinase (ERK) activation is fully dependent on protein kinase C (PKC), but only partially dependent on Src, dynamin, and Ras. Receptor tyrosine kinases, FAK, Gbeta gamma , and beta -arrestin, which were implicated in some G-protein-coupled receptor signaling to MAPK cascades, do not play a role in the GnRH to ERK pathway. Our results suggest that the activation of ERK by GnRH involves two distinct signaling pathways, which converge at the level of Raf-1. The main pathway involves a direct activation of Raf-1 by PKC, and this step is partially dependent on a second pathway consisting of Ras activation, which occurs in a dynamin-dependent manner, downstream of Src.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Signal transduction elicited by hormones, growth factors, and several other extracellular stimuli involves a sequential activation of cytosolic protein kinases, which are collectively known as the mitogen-activated protein kinase (MAPK)1 cascades (reviewed in Refs. 1-4). In growth factor signaling, the key elucidated MAPK cascade is the one using the extracellular signal-regulated kinase (ERK, also known as p42/44 MAPK), which is initiated by the small GTP-binding protein, Ras. Upon stimulation, Ras assumes its active, GTP-bound form, and recruits the protein kinase Raf-1, which transmits the signal further via MEK, ERK, and p90RSK to various cytoplasmic and nuclear target molecules. Other MAPK cascades (reviewed in Refs. 5-7) are: (i) Jun N-terminal kinase (JNK) cascade, which utilizes MEKK1, MKK4/7, and JNKs to activate transcription factors such as c-Jun, ATF2, and Elk1; (ii) p38 MAPK cascade, which uses MEKKs, MKK3/6, and P38 alpha -delta to activate Elk, ATF2, CHOP, and MEF; and (iv) big MAPK (BMK, ERK5) cascade, which uses MEKK3, MEK5, and BMK to transmit EGF and oxidative stress signals.

The family of receptors coupled to G-proteins (GPCR) is the largest group of integral membranal receptors involved in signal transduction (reviewed in Refs. 8-12). Members of this group of receptors transmit their signals primarily via GTP-binding proteins (G-proteins), which are heterotrimeric proteins composed of alpha , beta , and gamma  subunits. The G-proteins are classified according to the subtype of their alpha  subunit into four groups: Gs, Gi, Gq, and G12. Upon activation, the heterotrimer dissociates into a GTP-bound, activated, alpha  subunit (Galpha ), and a dimer of Gbeta gamma . Whereas a small number of common mechanisms is responsible for receptor Tyr kinase-mediated activation of ERK (13), GPCRs are thought to activate ERK by divers signaling pathways. All four Galpha subunits (12), the beta gamma subunits (14), receptor-interacting proteins such as Src (15), beta -arrestin (16), dynamin (17), and transactivation of receptor Tyr kinases by GPCRs (18) are capable of initiating several downstream signaling pathways culminating in MAPK activation. The various MAPK cascades appear to be important components in the downstream signaling events initiated by the GPCRs, although different receptors often use different mechanisms for this purpose (12).

Gonadotropin releasing hormone (GnRH) is a hypothalamic decapeptide, which serves as a key regulator of the reproductive system. In the pituitary, the signals of GnRH are transmitted via a specific GPCR (GnRH receptor; GnRHR), which upon activation interacts with the heterotrimeric Gq protein (reviewed in Refs. 19, 20). This interaction then initiates a variety of intracellular signaling events that culminate in the production and secretion of the leutinizing hormone and follicle-stimulating hormone. In recent studies it was shown that the JNK, ERK, and p38 MAPK cascades are activated in response to GnRH stimulation of alpha T3-1 cells (21-28). The mechanism of JNK activation by GnRH has been shown to involve sequential activation of protein kinase C (PKC), Src, CDC42, and MEKK1 (28); and the mechanism of p38 MAPK activation by GnRH seems also to be PKC-dependent (27). The mechanism of ERK activation was studied by several laboratories over the past few years and was shown to involve PKC, and an unknown protein-tyrosine kinase (PTK) that is only partially sensitive to the PTK inhibitor genistein (reviewed in Ref. 12).

Here we show that the activation of ERK by GnRH in the alpha T3-1 cells involves two distinct signaling pathways, which converge at the level of Raf-1. One of these pathways involves a direct activation of Raf-1 by PKC, whereas the other pathway involves activation of Raf-1 by Src and Ras. Transactivation of EGF receptor, or activation through either beta -arrestin or Gbeta gamma , which play a role in the signaling of many other GPCRs to the ERK cascade, are not involved in the GnRHR to ERK pathway. On the other hand, dynamin seems to be important for this pathway, because it is essential for the activation of Ras, in a PKC-independent manner. Thus, although the GnRHR to ERK signaling is mainly mediated by Gq-PKC, another pathway involving dynamin is required for the Src-mediated activation of Ras, which supports the step of Raf-1 activation by PKC.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Buffers-- Homogenization buffer (buffer H) consisted of 50 mM beta -glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.1 mM sodium orthovanadate, 1 mM benzamidine, aprotinin (10 µg/ml), leupeptin (10 µg/ml), and pepstatin (2 µg/ml). Buffer A consists of 50 mM beta -glycerophosphate (pH 7.3), 1.5 mM EGTA, 1 mM EDTA, 1 mM DTT, 0.1 mM sodium orthovanadate. Radioimmune precipitation buffer consisted of 137 mM NaCl, 20 mM Tris, pH 7.4, 10% (v/v) glycerol, 1% Triton X-100 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS, 2.0 mM EDTA, 1.0 mM phenylmethylsulfonyl fluoride, and 20 µM leupeptin. Ral buffer consisted of 40 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1% Nonidet P-40, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 250 mM phenylmethylsulfonyl fluoride, 10 mM NaF, and 1 mM sodium orthovanadate.

Stimulants, Inhibitors, Antibodies, and Miscellaneous Reagents-- [D-Trp6]GnRH, a stable GnRH analog (GnRH-a), genistein (PTK inhibitor), enolase, protein A-Sepharose, and protein G-Sepharose were obtained from Sigma Chemical Co. (St. Louis, MO). GF109203X, PD098059, SB203580, Wortmannin, and 12-O-tetradecanoylphorbol-13-acetate where purchased from Calbiochem. AG18 and AG1478 and PP1 were also obtained from Calbiochem. Polyclonal anti-hemagglutinin epitope antibody was from Santa Cruz Biotechnology. Monoclonal anti-GFP was from Roche Molecular Biochemicals. Mouse monoclonal anti-active MAPK antibody (DP-ERK antibody) was from Sigma, Israel (Rehovot, Israel). Antiphosphotyrosine antibody was from Santa Cruz Biotechnology. Antibody to delta -epitope of PKC was a gift from Dr. Chaya Brodie (Bar-Ilan University, Israel).

Plasmids-- N-terminally truncated FAK (DN-FAK; FRNK) was cloned in pCDNA1 using BamHI/XhoI sites. HA-ERK2, N-17 Ras, and L-61 Ras were prepared as previously described (29). Mammalian expression vectors containing wild-type dynamin, dominant negative dynamin (K44A-dynamin), beta -arrestin2 in pCMV5, and dominant negative beta -arrestin (V54D-beta -arrestin2) in pCDNA3 were a gift from Dr. M. Caron (Duke University, Durham, NC). GFP-Ras was a gift from Dr. Y. Klug (Tel Aviv University, Israel), and MEK1 was cloned in pEGFP-N1 in-frame with GFP using an ApaI and BamHI restriction site. CD8-tagged beta -ARK was from Dr. Zvi Vogel (Weizmann Institute of Science, Israel). PKCepsilon with a delta -epitope tag was a gift from Dr. Chaya Brodie (Bar-Ilan University, Israel). DN-EGF receptor (K721A) in pCDNA3 was provided by Dr Y. Yarden (Weizmann Institute of Science, Israel).

Transfection, Stimulation, and Harvesting of alpha T3-1 Cells-- Subconfluent alpha T3-1 cells were transfected with 5 µg of the examined plasmid. In case of cotransfection, 5 µg of each plasmid were used. The transfection was carried out using the calcium phosphate technique. The total amount of plasmid was adjusted to 10 µg with vector DNA in the control experiments. The transfection efficiency was 10-30%, as determined by transfection with a plasmid that contained beta -Gal and appropriate staining. Two days after transfection, the cells were serum-starved for 16 h and incubated for the desired time intervals with GnRH-a in the presence or absence of various inhibitors. After stimulation, cells were washed twice with ice-cold PBS, washed once with buffer A, and subsequently harvested in ice-cold buffer H. Cell lysates were centrifuged (20,000 × g, 20 min), and the supernatant was assayed for protein content.

Western Blot Analysis-- Cell supernatants, which contained the cytosolic proteins, were collected, and aliquots from each sample (20 µg) were separated on 10% SDS-PAGE followed by Western blotting with the appropriate antibodies. Alternatively, immunoprecipitated antibodies were boiled in sample buffer and subjected to SDS-PAGE and Western blotting. The blots were developed with alkaline phosphatase or horseradish peroxidase-conjugated anti-mouse or anti-rabbit Fab antibodies (Jackson Laboratories).

Immunoprecipitation with Anti-HA Antibodies-- For immunoprecipitation, protein A-Sepharose was mixed with HA antibodies at 37 °C for 1 h, after which the beads were washed twice with PBS and twice in buffer H. Cell lysates (300 µg) were added to the beads and swirled end to end at 4 °C for 2 h. The immunocomplexes were washed once with radioimmune precipitation buffer, washed twice with 0.5 M LiCl in 100 mM Tris, pH 8.0, and finally washed with buffer A.

Ras Activation Assay-- Cells were stimulated and washed as described above. Ras activation was assayed as described previously (30). In brief, cells were lysed in Ral buffer, and lysates (300 µg of protein) were incubated with 20 µg of GST-Raf (RBD) and were washed three times in a buffer containing 20 mM Hepes, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, and 10% glycerol. The amount of Ras pulled-down was then assessed by Western blotting using anti-Ras antibody. To study Ras activity in transfected cells, a construct of GFP-Ras was used. After GnRH stimulation, the cells were lysed in Ral buffer and 300 µg of protein was subjected for further treatment. The active, GTP-bound form of Ras was precipitated by the GST-Raf (RBD, 20 µg) and washed as above, and the activated GFP-Ras was then detected with anti-GFP antibody.

Src Activity-- Cell lysates (400-500 µg of protein in homogenization buffer containing 1% Triton X-100) were incubated with anti-c-Src-antibodies precoupled to protein A-Sepharose and swirled end to end at 4 °C. The immunocomplexes were washed once with radioimmune precipitation buffer, twice with 0.5 M LiCl in 0.1 M Tris-HCl, pH 8.0, and once with buffer A. The washed immunoprecipitate were resuspended in a kinase assay buffer (28), and the c-Src activity was determined using acid-denatured enolase (3 µM) as substrate in the presence of 20 µM [gamma -32P]ATP (8000 cpm/pmol). The enzymatic reactions were terminated by the addition of sample buffer. The samples were then subjected to SDS-PAGE and autoradiography.

FAK Activity-- Cell lysates (400-500 µg of protein in buffer H + 1% triton X-100) were incubated with anti-FAK antibody coupled to protein A-Sepharose and swirled end to end at 4 °C for 2 h. The immunoprecipitates were washed as above and mixed with sample buffer. Active FAK was determined by Western blot analysis using anti-phosphotyrosine antibody.

Raf-1 Activity-- The activity of Raf-1 was determined by immunoprecipitation using an anti-C-terminal Raf-1 antibody and a subsequent in vitro kinase assay with recombinant MEK. This was performed in the same reaction mixture described for MEK except that 2 µg of recombinant MEK1 were used instead of ERK in each reaction. The reactions were terminated by the addition of sample buffer, and the samples were subjected to SDS-PAGE analysis. The gels were blotted onto nitrocellulose membrane, and the phosphorylation of MEK was assessed by x-ray autoradiography.

MEK Activity-- The activity of transfected GFP-tagged MEK was assessed by immunoprecipitation with anti-GFP antibody followed by a subsequent in vitro kinase reaction. In brief, alpha T3-1 cells transfected with GFP-MEK were stimulated with GnRH-a and harvested in buffer H. The cell lysates were incubated with 5 µg/assay of GFP antibody precoupled to protein G-Agarose and were washed as described for immunoprecipitation. Immunocomplex kinase reaction was carried out in a reaction mixture containing 1 µg of recombinant ERK, 10 µM MgCl2, 1.5 µM DTT, 75 mM beta -glycerophosphate, pH 7.3, 0.075 µM sodium vanadate, 3 µM PKI peptide, 1.25 mM EGTA, 10 µM calmidazolium, and 20 µM [gamma -32P]ATP (300 cpm/pmol) for 20 min at 30 °C. The reactions were terminated by the addition of sample buffer, and the samples were subjected to SDS-PAGE analysis. The gels were blotted onto nitrocellulose membrane, and the phosphorylation of ERK was assessed by x-ray autoradiography.

PKC Activity-- The activity of transfected delta -epitope-tagged PKCepsilon was assessed using cellular fractionation. In brief, transfected alpha T3-1 cells were stimulated with GnRH-a, homogenized in buffer H, and centrifuged at 15,000 × g. Pellets containing plasma membranes were washed twice in buffer H and suspended in buffer H containing Triton X-100. Translocated PKC in the membranes was determined by Western blot analysis using antibody to the PKCdelta -epitope.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Role of PTKs in ERK Activation by GnRH-- Various MAPK cascades (ERK, JNK, and BMK) are activated in response to GnRH stimulation of alpha T3-1 cells. We have previously shown that the stimulation of JNK activity by GnRH is mediated by a unique pathway, which includes sequential activation of PKC, Src, CDC42, and probably also MEKK1 (28). PKC was implicated also in the activation of ERK by GnRH (25), but the other components involved in this pathway remained unclear. In this study we used anti-doubly phosphorylated ERK (DP-ERK) antibody to detect its phosphorylation and activation upon GnRH-a treatment of alpha T3-1 cells. ERK phosphorylation was detected 5 min after GnRH-a treatment (Fig. 1A), peaked at 15 min, and was slightly reduced 15 min later. No change was detected in the total amount of ERK as judged by the equal staining with anti-general ERK antibody (Fig. 1A). These results appear similar to the trend of ERK activation by GnRH (Ref. 25 and data not shown), indicating that the anti-DP-ERK antibody can serve as a tool to study ERK activation in alpha T3-1 cells.



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Fig. 1.   The effect of PKC and PTK inhibitors on ERK activation by GnRH. A, sensitivity of ERK activation by GnRH to various inhibitors: Subconfluent alpha T3-1 cells were pretreated (15 min) with 200 µM genistein (Gen), 3 µM GF109203X (GF), 25 µM PD98059 (PD), 25 nM wortmannin (Wor), or 20 µM SB203580 (SB) before stimulation or left untreated as control. GnRH-a (10-7 M) was added for 0, 5, 15, and 30 min and the activated form of ERK was determined by Western blot analysis with anti-diphospho ERK antibody (DP). The total amount of ERK was detected with the 7884 antibody (Ref. 60; total). These results are an average of four separate experiments. B, sensitivity of ERK activation by GnRH to inhibitors of PTKs: Subconfluent alpha T3-1 cells were pretreated with 100 µM AG18, 5 µM AG1478, or 5 µM PP1 for 15 min before stimulation or left untreated as control. Addition of GnRH-a and analysis of active ERK was carried out as above. These results are an average of three separate experiments.

To study the mechanism of ERK activation we first used inhibitors of various intracellular signaling cascades. As previously reported (25), the PTK inhibitor genistein partially (40%) inhibited, and the PKC inhibitor GF109203X abolished the GnRH-induced ERK activation. The MEK inhibitor PD98059 inhibited not only GnRH-stimulated ERK-activity but abolished also its basal activity, whereas the PI3K inhibitor wortmannin and the p38 MAPK inhibitor SB203580 had no significant effect on ERK activation. The results suggest that GnRH signaling toward ERK is mainly transmitted via PKC and to a lesser extent also by PTKs. Interestingly, the moderate effect of genistein on ERK activation was achieved under conditions where it completely abolished the GnRH activation of JNK (28). One possibility for this differential effect is that the PTK involved in the GnRH to ERK pathway is distinct from Src, which is the PTK operating in the GnRH-JNK pathway (28), and this distinct PTK is only mildly sensitive to genistein. To test this possibility, we examined the effects of additional PTK inhibitors on ERK activation by GnRH-a. We found (Fig. 1B) that the specific inhibitor of Src, PP1, which abolished endogenous src activation (data not shown), inhibited the GnRH-induced ERK activation to a similar extent as genistein. Similarly, the general PTK inhibitor AG18 had a small effect (~25%) on the GnRH-induced ERK activation. However, the EGF receptor inhibitor AG1478, which abolished the EGF-induced ERK activation in alpha T3-1 cells (data not shown), had no effect on ERK activation by GnRH-a. The data suggest that Src is partially involved in the activation of ERK by GnRH but probably not via transactivation of EGF or other growth factor receptors as suggested for other GPCRs (31, 32).

Src, but Not FAK or EGF Receptor, Plays a Role in GnRH to ERK Signaling-- Signaling by Src is often mediated via the focal adhesion kinase (FAK), which is usually instrumental in integrin signaling (33). We found that Src and FAK are activated in response to GnRH in alpha T3-1 cells, although the onset of Src activation appeared before that of FAK (Refs. 25 and 28 and Fig. 2). A useful tool in the study of GnRH signaling in alpha T3-1 cells is the co-overexpression of either constitutively active or dominant negative forms of upstream components together with a tagged form of the examined kinase (28). Therefore, we overexpressed hemagglutinin epitope-tagged ERK (HA-ERK2) in alpha T3-1 cells, stimulated the cells with GnRH, lysed the cells, immunoprecipitated the HA-containing proteins with anti-HA antibodies, and blotted with anti-DP-ERK antibodies. ERK activation measured by this method was essentially identical to that found for the endogenous ERK, indicating that overexpression can also serve as a useful tool in the study of ERK activation by GnRH. To study the role of Src/FAK in the GnRH to ERK signaling we co-overexpressed a dominant negative form of FAK (FRNK (34)) with the HA-ERK2 in alpha T3-1 cells. This FAK construct, which inhibits the activity of the endogenous FAK in alpha T3-1 cells (data not shown), had no effect on GnRH-induced ERK activation (Fig. 2B). This lack of effect, together with the late onset of FAK activation by GnRH-a stimulation, indicates that FAK is not involved in GnRH to ERK signaling. On the other hand, the C-terminal Src kinase (CSK), which acts as a dominant interfering mutant of Src (28, 35) had a partial inhibitory effect on the GnRH to ERK pathway. This is demonstrated by a ~35% inhibition in the activation of ERK by GnRH-a caused by overexpression of CSK in alpha T3-1 cells (Fig. 2B). This result, together with the ~40% inhibition cause by PP1, strongly suggests that Src is partially involved in the activation of ERK by GnRH in alpha T3-1 cells.



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Fig. 2.   Activation of Src and FAK by GnRH-a and the effect of Src and FAK on ERK activation by GnRH-a. A, activation of Src and FAK by GnRH: Subconfluent alpha T3-1 cells were treated with GnRH-a (10-7 M) for the indicated times. Stimulation was terminated by washing the cells with an ice-cold PBS followed by Src and FAK immunoprecipitation. Activity of Src was determined using acid-denatured enolase. FAK activity was detected by anti-phosphotyrosine antibody. B, the effect of CSK, which inhibits Src activity, and of dominant negative FAK on GnRH-a stimulation of ERK: Subconfluent alpha T3-1 cells were cotransfected with CSK and HA-ERK2 for Src experiment and with dominant negative-FAK and HA-ERK2 for the FAK experiment. Two days after transfection, the cells were serum-starved for 16 h and then either treated with GnRH-a (10-7 M) or left untreated. Activated HA-ERK2 was determined with anti-diphospho antibody (DP). The amount of immunoprecipitated HA-ERK2 was determined by Western blot analysis with anti-HA antibody. Activation (-fold) (GnRH-stimulated/Basal for each of the constructs) is shown in the bar graph in the bottom. The results in the bar graphs are the average of three experiments.

It was recently reported that EGF receptor might be involved in GnRH signaling (36). However, our initial results indicated that there is no inhibition of GnRH-induced ERK activation by the inhibitor of EGF receptor AG1478 (Fig. 1B). To further confirm the lack of participation of EGF receptor in GnRH-ERK signaling, we cotransfected the dominant negative mutant of EGF receptor (K721A) with HA-ERK2 into alpha T3-1 cells. The cells were then serum-starved and activated with either EGF or GnRH-a followed by the determination of ERK activation. The dominant negative EGF receptor had no influence on ERK activation by GnRH-a (Fig. 3A) under conditions where it prevented ERK activation by EGF (Fig. 3B). These results, together with the lack of effect of AG1478 strongly suggest that EGF receptor is not involved in the activation of ERK by GnRH.



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Fig. 3.   The effect of EGF receptor on ERK activation by GnRH-a. Subconfluent alpha T3-1 cell were cotransfected with dominant negative (DN)-EGF receptor and HA-ERK2. Two days after transfection, the cells were serum-starved for 16 h and then either treated with GnRH-a (10-7 M; A), EGF (50 ng/ml; B), or left untreated (where indicated). Activated HA-ERK2 was determined with anti-diphospho antibody. The amount of immunoprecipitated HA-ERK2 was determined by Western blot analysis with anti-HA antibody. The results in the bar graph represent the average of three experiments.

Activation of Raf-1 and Ras by GnRH-- To understand the nature of the partial involvement of Src in the GnRH to ERK pathway, we studied the effect of GnRH on the upstream components of the ERK cascade, including the protein serine/threonine kinase Raf-1. After treatment of alpha T3-1 cells with GnRH-a and inhibitors, the cells were lysed, Raf-1 was immunoprecipitated, and its activity toward recombinant MEK was measured as described (37). Raf-1 activity was stimulated within 5 min after GnRH activation (data not shown); its activity peaked 10 min after stimulation and declined thereafter (Fig. 4). Similar to the inhibition of ERK activation by GnRH, we found that GF109203X completely inhibited Raf-1 activation by GnRH-a, whereas PP1 and genistein had a partial inhibitory effect (~30% inhibition) and AG1478 had no significant influence on Raf-1 activation by GnRH-a. The small GTP-binding protein Ras, was also transiently activated by GnRH-a. Using a pulldown and a GTP loading assays, we found that activation of Ras was detected within 2 min from activation, peaked at 5-10 min, and declined thereafter (Fig. 5 and data not shown). However, the mechanism involved in this activation seems to be distinct from that of Raf-1 and ERK as judged from the differential sensitivity to the various inhibitors used. Thus, the Src inhibitors genistein and PP1 abolished the activation of Ras by GnRH-a, GF109203X had only a partial effect, and the EGF receptor inhibitor AG1478 had no effect upon Ras activation. These data indicate that, although both Ras and Raf-1 are activated in response to GnRH in alpha T3-1 cells, the upstream mechanism that leads to this activation is different. Indeed, when a dominant negative form of Ras (N-17 Ras) was transfected into the alpha T3-1 cells, it only partially (30%) inhibited ERK activation by GnRH-a (Fig. 6), whereas a constitutively active form of Ras (L61-Ras) caused a large elevation of ERK (18- to 22-fold above basal level). Therefore, although Ras is capable of activating the Raf-1/ERK pathway, GnRH activation of Raf-1/ERK is only partially Ras-dependent. The most plausible explanation for these data is that the main pathway operates via direct activation of Raf-1 by PKC (38), and that this activation requires only a minor contribution of activated Ras as previously suggested for the activation of Raf-1 by 12-O-tetradecanoylphorbol-13-acetate (39).



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Fig. 4.   Activation of Raf-1 by GnRH-a. Subconfluent alpha T3-1 cells were pretreated (15 min) with 200 µM genistein, 3 µM GF109203X (GF), 5 µM PP1, or 5 µM AG1478 before stimulation or left untreated as control. GnRH-a (10-7 M) was added for the indicated times. Cell extracts were immunoprecipitated with anti-Raf-1 C-terminal antibody, and Raf-1 activity was determined by phosphorylation of recombinant MEK. The bar graph in the bottom panel represents an average of three separate experiments.



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Fig. 5.   Activation of Ras by GnRH-a. Subconfluent alpha T3-1 cells were pretreated (15 min) with 200 µM genistein (Gen), 3 µM GF109203X (GF), 5 µM PP1, or 5 µM AG 1478 or left untreated as control. GnRH-a (10-7 M) was added for the indicated times. Activated Ras in the cell lysate was determined by Ras pulldown assay using GST-RBD as described under "Material and Methods." The results in the bar graph are average of three separate experiments.



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Fig. 6.   Effect of Ras on ERK activation by GnRH-a. alpha T3-1 cells were cotransfected with HA-ERK2 together with either N-17 Ras or L61-Ras. Two days after transfection, the cells were serum-starved for 16 h and then either treated with GnRH-a (10-7 M) for the indicated times or left untreated. Activated HA-ERK2 was visualized with anti-diphospho antibody (DP). The amount of immunoprecipitated HA-ERK2 was determined by Western blot analysis with anti-HA antibody (HA). The results in the bar graph are average of three experiments. Activation (-fold) (GnRH-stimulated/Basal for each construct) is indicated in the bar graph.

The Involvement of Dynamin, but Not beta -Arrestin or Gbeta gamma , in ERK Activation by GnRH-- Recently, the beta gamma subunits of G-proteins as well as beta -arrestin and dynamin have been implicated in the Galpha -independent GPCR-ERK signaling (reviewed in Ref. 12). For example, it was shown that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by Gbeta gamma -induced activation of Ras (40). To study the possible role of the beta gamma subunit in GnRH to ERK signaling, we used a chimera of CD8 (which allows anchoring to the membrane) fused to the C terminus of beta -adrenergic receptor kinase (ARK-C), which contains a Gbeta gamma binding domain (CD8-ARK-C). It has been previously shown that this chimera acts as a scavenger of the beta gamma dimer (41, 42). Although this construct was able to inhibit GPCR signaling toward ERK in COS7 cells, its overexpression had no significant effect on either the basal or the GnRH-induced activation of ERK in alpha T3-1 cells (Fig. 7), indicating that the signaling from GnRHR to ERK utilizes a Gbeta gamma -independent pathway. Another protein that was implicated in the signal transmission of GPCRs is beta -arrestin, which acts as a mediator of receptor internalization (43). Recently, it was also shown that beta -arrestin can act as a scaffold protein and transmit the signals of Gq-coupled receptors toward ERK by forming a complex that contains internalized receptor, Raf-1, and activated ERK (44). We examined the possible involvement of beta -arrestin using either an inactive form of this protein (V54D-beta -arrestin2), which inhibits the activity of all endogenous beta -arrestins (43), or by overexpressing wild-type beta -arrestin2, which should increase ERK activation by GPCRs (16, 45). Thus, we coexpressed these two constructs in alpha T3-1 cells together with HA-ERK2 and followed HA-ERK activation using anti-phospho ERK antibodies. As seen in Fig. 7, neither form of beta -arrestin had any effect on ERK activation by GnRH. Both the Gbeta gamma subunits and beta -arrestin do not seem to participate in the process of ERK activation by GnRH, although they can influence GPCR signaling in other systems.



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Fig. 7.   Effect of beta gamma subunits and beta -arrestin on ERK activation by GnRH-a. alpha T3-1 cells were cotransfected with HA-ERK2 together with either the scavenger of beta gamma -subunits (CD8 fused to beta -ARK; CD8), beta -arrestin2 (Arre), or dominant negative beta -arrestin2 (DnArre). Two days after transfection, the cells were serum-starved for 16 h and then either treated with GnRH-a (10-7 M) for the indicated times or left untreated. Activated HA-ERK2 was determined with anti-diphospho ERK antibody (DP) as described under "Materials and Methods." The amount of immunoprecipitated HA-ERK2 was determined by Western blot analysis with anti-HA antibody (HA). The results in the bottom bar graph are an average of three experiments. Activation (-fold) (GnRH-stimulated/basal for each construct) is indicated in the bar graph.

As well as beta -arrestin, dynamin seems to be a key regulator of the internalization processes of GPCR (46). As such, dynamin has also been implicated in GPCR signaling, including the activation of MAPKs by several GPCRs (16). The role of this protein in the alpha T3-1 cells was examined, as described for beta -arrestin, by co-overexpression of either wild-type or a dominant negative form of dynamin (K44A-dynamin (16, 47)) together with HA-ERK2. Although the wild-type form of dynamin had no significant influence on the activation of ERK by GnRH, the dominant negative form of dynamin partially inhibited (45%) both the basal and the GnRH-induced ERK activation (Fig. 8). This inhibition of both basal and GnRH-stimulated activities is similar to the results obtained with dominant negative Ras (Fig. 6) and to some extent also to the results with CSK, suggesting that dynamin, Src, and Ras operate on the same signaling pathway.



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Fig. 8.   Effect of dynamin on ERK activation by GnRH-a. alpha T3-1 cells were cotransfected with HA-ERK2 together with either dynamin (Dyn) or dominant negative dynamin (DN-Dyn). Two days after transfection, the cells were serum-starved for 16 h and then either treated with GnRH-a (10-7 M) for the indicated times, with peroxovanadate (Na3VO4 (100 µM) and H2O2 (200 µM); VOOH), or left untreated. Activated HA-ERK2 was determined with anti-diphospho ERK antibody (DP). The amount of immunoprecipitated HA-ERK2 was determined by Western blot analysis with anti-HA antibody (HA). The results in the bottom bar graph are an average of three experiments. Activation (-fold) (GnRH-stimulated/basal for each of the constructs) is indicated in the bar graph.

Dynamin, but Not beta -Arrestin, Is Involved in Ras Activation by GnRH-- To confirm that beta -arrestin is not involved in GnRH-induced stimulation of ERK and to further study the role of dynamin in this process, we undertook to explore the influence on other components of the GnRH to ERK signaling pathway. First we examined whether beta -arrestin has any effect on the GnRH activation of Ras. Thus, either wild-type or dominant negative forms of beta -arrestin2 were cotransfected into alpha T3-1 cells together with GFP-Ras. After GnRH-a stimulation, the active, GTP-bound form of Ras was precipitated by the Ras binding domain of Raf-1 (Raf-RBD), and activated GFP-Ras was then detected with anti-GFP antibody. Similar to the effect on ERK2, no effect of the beta -arrestin constructs on Ras activation could be detected under the conditions used (Fig. 9). Therefore, as was predicted by the lack of effect on ERK, beta -arrestin does not seem to play a role also in the GnRH-induced signaling toward Ras. This observation is in agreement with the lack of beta -arrestin involvement in GnRH-induced internalization of the GnRHR (48), suggesting that beta -arrestin does not play a significant role in GnRH signaling.

Unlike beta -arrestin, the other internalization mediator, dynamin appears to be involved in GnRH-induced GnRHR internalization (48) and, as demonstrated above (Fig. 8), in the activation of ERK by GnRH. We then undertook to elucidate the possible mechanism by which dynamin transmits the GnRH signals toward the downstream components of the ERK cascade. First, the possible involvement of dynamin in Ras activation by GnRH was examined by cotransfecting either wild-type or dominant negative (K44A) forms of dynamin together with GFP-Ras. Similar to the effect on ERK, the wild-type dynamin had no influence on Ras activity under the conditions examined. However, the dominant negative form of dynamin completely abrogated the GnRH activation of Ras under the conditions examined, indicating that dynamin lies upstream of Ras in the pathway that leads from the GnRHR. Similar to ERK, MEK activity was only partially inhibited by the dominant negative and to some extent also by wild-type dynamin (Fig. 9B). The fact that the inhibition of MEK activation by dominant negative dynamin was very similar to the inhibition of ERK activity makes it unlikely that dynamin influences the MEK-ERK level of the cascade as previously suggested in other systems (49). Moreover, unlike the inhibition of Ras, ERK, and MEK activities by dominant negative dynamin, there was no influence of this construct on the GnRH-induced membranal translocation of PKCepsilon (Fig. 9B), indicating that dynamin acts independently of this PKC. Because PKCepsilon is one of the main PKC isoforms that participate in GnRH signaling in alpha T3-1 cells (Ref. 50 and data not shown), the role of dynamin in the activation of Ras seems to be confined to its influence on the Src/Ras step without any significant influence on Raf-1 stimulation by PKC (Fig. 10).



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Fig. 9.   Effect of beta -arrestin on GnRH activation of Ras and effect of dynamin on GnRH activation of Ras, PKC, and MEK. A, effect of beta -arrestin on GnRH activation of Ras: alpha T3-1 cells were cotransfected with either wild-type (WT) or dominant negative (DN) forms of beta -arrestin together with GFP-Ras. Two days after transfection, the cells were serum-starved for 16 h and either treated with GnRH-a (10-7 M for 10 min,) or left untreated. Activated GFP-Ras was determined using Ras precipitation with GST-RBD as described under "Materials and Methods." The results in the bottom bar graph are an average of two experiments. Activation (-fold) (GnRH-stimulated/basal for each of the constructs) is indicated in the bar graph. B, effect of dynamin on GnRH activation of Ras, PKC, and MEK: alpha T3-1 cells were cotransfected with either wild type (WT-Dyn) or dominant negative dynamin (DN-Dyn) together with GFP-Ras, GFP-MEK, or epitope tagged-PKCepsilon . Two days after transfection, the cells were serum-starved for 16 h and then either treated with GnRH-a (10-7 M for 10 min for Ras and PKC and 4 min for MEK) or left untreated. Activated GFP-Ras was determined using a Western blot with anti-GFP antibody following Ras precipitation using GST-RBD as described under "Materials and Methods." Activity of transfected GFP-MEK was measured by immunoprecipitation with anti-GFP antibody and subsequent in vitro kinase reaction using recombinant ERK as a substrate (see "Materials and Methods"). To determine PKC translocation to the membrane, membranal fractions were collected and membranal (active) PKC was determined by Western blot analysis using antibody to the PKC epitope tag. The results in the bar graph represents the average of three experiments and represent the percentage of -fold activation of GnRH-stimulated enzyme in cells transfected with dominant negative or wild type dynamin as compared with activation (-fold) in cells transfected without dynamin constructs.



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Fig. 10.   Schematic representation of GnRH signaling toward the MAPK cascades. Broken lines indicate an indirect activation, and the solid line indicates a direct activation. Dyn, dynamin.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

GPCRs comprise the largest group of integral membranal proteins, which transmit their signals primarily via four groups of G-proteins, Gs, Gi, Gq, and G12 and also via receptor-interacting molecules and transactivation of growth factor receptors. The G-proteins and the other components are capable of transmitting signals from the receptor to MAPK cascades via distinct pathways, which often form an elaborate network of signaling cascades (12). However, the exact mechanisms, which are involved in each of these pathways, are still mostly obscure. Here we studied the receptor for GnRH, which was shown to operate via Gq or G11 in pituitary cells (51). This system is of particular interest because, in pituitary cells, GnRH does not promote growth and differentiation and does not constitute a stress signal and therefore might utilize a unique signaling pathway to activate the MAPK cascades (12, 20).

In previous studies we and others have shown that the ERK (21, 25), JNK (28), and p38MAPK (27) cascade are activated by GnRH in a PKC-dependent manner. However, although JNK activation seems to be fully dependent on Src and only partially (~70%) on PKC, ERK activation was shown to be fully dependent on PKC but only partially (~30%) on PTK. Because PTKs have been shown to play an important role in the activation of ERK by GPCR (12), the small effect of PTK inhibitors observed in our studies was surprising. We have ruled out the possibility that the PTK involved in the GnRH to ERK activation is only partially sensitive to genistein. This was demonstrated by showing that none of the general PTK inhibitors used here inhibit ERK activation by more than 40%. Moreover, the Src inhibitor PP1 and the inhibitory kinase CSK, which fully inhibit Src activity, also cause similar inhibitions. Because Src is activated by GnRH and has a major role in the transmission of signals to the JNK cascade, the most likely explanation for our results is that the activation of ERK is supported to some extent by Src, although the main pathway leading to ERK activation is PTK-independent.

PTK-induced activation of ERK is known to involve the small GTP-binding protein Ras. Interestingly, although GnRH causes a significant activation of Ras, a dominant negative form of Ras had only a minor inhibitory effect on ERK activation. Because the effect was similar to that exerted by Src, it is likely that Ras acts downstream of Src in a pathway that partially supports the activation of ERK (Fig. 10). Indeed, Ras activation by GnRH was abolished by the Src inhibitor, PP1. Interestingly, it has been previously demonstrated (39) that stimulation of PKC in COS-7 cells led to activation of Ras and formation of Ras·Raf-1 complexes, but the activation of Raf-1 by PKC was not completely blocked by dominant negative Ras. These data indicate that PKC activates Raf-1 by a mechanism distinct from that initiated by PTKs and that only a small amount of activated Ras is needed to allow the activation of Raf-1 by PKC. The fact that ERK activation is only partially dependent on Src but is fully dependent on PKC agrees well with this suggested model. Thus, we believe that, upon GnRH stimulation, Raf-1 is mainly activated by PKC but requires a small amount of active Ras. This possibility was demonstrated before in other experimental systems (38, 39) and explains the partial inhibition of ERK by interfering mutants of Src and Ras (Fig. 10). It has previously been shown that calcium may be involved in the activation of ERK by GnRH in alpha T3-1 cells (25, 52). According to our model this effect is probably on the Src/dynamin/Ras branch of the pathway and not on the PKC-Raf-1 step, which was shown to be independent of calcium influx (52) and not on the Raf-1-MEK-ERK steps, which were shown to be calcium-independent in several cellular systems (1).

It should be noted that a recent study claimed that the activation of ERK by GnRH in alpha T3-1 cells is inhibited equally by PKC and PTK inhibitors and that Ras activation by GnRH is mediated by EGF receptor (36). The reason for these slight discrepancies between our results and the results reported by Grosse et al. (36) is not clear. However, one possible explanation could be the different lengths of serum starvation, which modify the content of many signaling components (53). We found that the minimal time needed for a complete removal of MAP kinase phosphatases and complete quiescence in alpha T3-1 cells is 14 h (data not shown). Alternatively, it is also possible that the alpha T3-1 cells are modified under different growing conditions to form distinct subpopulations that exhibit different repertoire of signaling molecules as was shown also in other cell lines such as PC12. These explanations can be used also for the different results obtained in regard to the activation of JNK by GnRH in different laboratories (54).

Another point of interest is the mechanism of Src activation by GnRH. We have previously shown that Src is activated by GnRH via a mechanism that is partially (~70%) dependent on PKC (28). Similarly, we found that the activation of Ras is only partially dependent on PKC, but fully dependent on Src, supporting the notion that Ras is activated by Src. Such pathway of sequential activation of Src-Ras-ERK was reported also for some other GPCRs (12, 55-57). However, the fact that Src and Ras are only partially dependent on PKC raises the question as to what might be the other pathway involved in the activation of Src/Ras. An answer to this question may come from the recent observations that, upon GPCR stimulation, Src can be activated in a Galpha -independent manner and therefore we undertook to study the role of these additional signaling molecules as outlined below. Many GPCRs were shown to transmit their signal through transactivation of either EGF receptor- or cytoskeleton-associated PTKs (FAK and PYK). Our results indicate that those components are not involved in GnRH signaling to ERK. Thus, EGF receptor does not seem to be activated in response to GnRH (25), and the specific inhibitor of EGF receptor (AG1478) or dominant negative form of the EGF receptor had no effect on GnRH-induced ERK stimulation. Moreover, FAK does not seem to participate in GnRH to ERK signaling, because a dominant negative FAK had no effect on ERK activation by GnRH. Finally, PYK does not seem to be expressed to any detectable level in alpha T3-1 cells as judged by immunoblotting and immunoprecipitation experiments (data not shown), and therefore is unlikely to participate in the GnRH to ERK pathway.

Although the Galpha subunits are important transducers of GPCR signaling, dissociated beta gamma subunits have been implicated in the transmission of GPCRs signaling as well. Thus, beta gamma dimers can act via PTKs (such as Src), via a direct activation of Ras or via a direct activation of either the protein serine/threonine kinase KSR-1 or activation of phosphatidylinositol 3-kinase (58, 59). In addition, GPCRs can operate via beta -arrestin- and dynamin-mediated internalization (16), and beta -arrestin may serve as a scaffold for additional signaling molecules and initiate a second wave of G-protein-independent, heptahelical receptor-mediated signals that activate the MAPK cascades (12). Interestingly, in the alpha T3-1 system, we found that neither a scavenger of beta gamma dimer nor the dominant negative form of beta -arrestin affect the GnRH-induced ERK activation. However, overexpression of dominant negative dynamin reduced the activation of both basal and GnRH-induced ERK activation indicating that dynamin, unlike the other upstream components examined, may participate in the PKC-independent activation of Src/Ras.

Recently it was shown that, in addition to its role in the internalization of GPCRs, dynamin is also necessary for the direct activation of ERK by MEK (49). This may indicate that the inhibition by the dominant negative dynamin is downstream of Raf-1 and not upstream of the Src/Ras pathway. To determine the site of dynamin action, we examined its role on several components of the cascade. Interestingly, we found that GnRH-induced Ras activity was significantly inhibited (~80%) by the dominant negative dynamin, whereas PKC activity was not affected under the same conditions. Because Src activation is mostly dependent on PKC (~70%), inhibition by the dominant negative dynamin upstream of Src at the PKC-independent pathway should have resulted in only ~30% inhibition of Ras activation, which is much smaller than the complete inhibition obtained. Moreover, the inhibition of the GnRH-induced MEK activation was similar to that of ERK activation. Therefore, we suggest that the dynamin is required for the process of Ras activation by Src, and the step of Raf-1 activation by PKC is probably not affected by the dominant negative dynamin under the conditions used.

In summary, we studied here the mechanism of ERK activation by GnRH in the pituitary derived alpha T3-1 cell line. We show that ERK activation is fully dependent on PKC but only partially dependent on Src, Ras, and dynamin. Because it has previously been shown that Raf-1 activation by PKC is only partially dependent on active Ras, our results are best explained by the involvement of two distinct pathways in the GnRH-mediated stimulation of Raf-1/ERK. One of these pathways involves a direct activation of Raf-1 by PKC; the other involves Ras activation by Src and dynamin.


    ACKNOWLEDGEMENTS

We thank Dr. Chaya Brodie (Bar Ilan University, Israel) for the kind gift of epitope-tagged PKC and antibody to the tag, and Dr. Yoel Klug (Tel Aviv University, Israel) for the GFP-Ras.


    FOOTNOTES

* This work was supported by grants from MINERVA, from the Israel Cancer Research Fund, from the Estate of Siegmund Landau, and from La Fondation Raphael et Regina Levy.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Tel.: 972-8-934-3602; Fax: 972-8-934-4116; E-mail: rony.seger@weizmann.ac.il.

Published, JBC Papers in Press, November 16, 2000, DOI 10.1074/jbc.M006995200


    ABBREVIATIONS

The abbreviations used are: MAPK, mitogen-activated protein kinase; beta -ARK, beta -adrenergic receptor kinase; CSK, C-terminal Src kinase; DP, doubly phosphorylated; ERK, extracellular signal-regulated kinase; FAK, focal adhesion kinase; FRNK, truncated FAK; GFP, green fluorescence protein; GnRH, gonadotropin-releasing hormone; GnRHR, GnRH receptor; GnRH-a, GnRH analog ([D-Trp6]-GnRH); GPCR, G-protein coupled receptor; HA, hemagglutinin; MEK, MAPK/ERK kinase; PKC, protein kinase C; PTK, protein-tyrosine kinase; RBD, Ras binding domain; JNK, c-Jun NH2-terminal kinase; EGF, epidermal growth factor; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; GST, glutathione S-transferase; BMK, big MAPK.


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DISCUSSION
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