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Molecular Endocrinology, doi:10.1210/me.2005-0277
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Molecular Endocrinology 20 (3): 619-630
Copyright © 2006 by The Endocrine Society

Activation of the Lutropin/Choriogonadotropin Receptor in MA-10 Cells Leads to the Tyrosine Phosphorylation of the Focal Adhesion Kinase by a Pathway that Involves Src Family Kinases

Tetsuya Mizutani, Koji Shiraishi, Toni Welsh and Mario Ascoli

Department of Pharmacology, Carver College of Medicine, The University of Iowa, Iowa City, Iowa 52242-1109

Address all correspondence and requests for reprints to: Dr. Mario Ascoli, Department of Pharmacology, 2-319B BSB, 51 Newton Road, The University of Iowa, Iowa City, Iowa 52242-1109. E-mail: mario-ascoli{at}uiowa.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We show that activation of the endogenous or recombinant lutropin/choriogonadotropin receptor (LHR) in mouse Leydig tumor cells (MA-10 cells) leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) and one of its substrates (paxillin). Using specific antibodies to the five tyrosine residues of FAK that become phosphorylated, we show that activation of the LHR increases the phosphorylation of Tyr576 and Tyr577, but it does not affect the phosphorylation of Tyr397, Tyr861, or Tyr925. Because FAK is a prominent substrate for the Src family of tyrosine kinases (SFKs) we tested for their involvement in the LHR-mediated phosphorylation of FAK-Tyr576. Src is not detectable in MA-10 cells, but two other prominent members of this family (Fyn and Yes) are present. The LHR-mediated phosphorylation of FAK-Tyr576 is readily inhibited by PP2 (a pharmacological inhibitor of SFKs) and by dominant-negative mutants of SKFs. Moreover, activation of the LHR in MA-10 cells results in the stimulation of the activity of Fyn and Yes, and overexpression of either of these two tyrosine kinases enhances the LHR-mediated phosphorylation of FAK-Tyr576. Studies involving activation of other G protein-coupled receptors, overexpression of the different G{alpha}-subunits, and the use of second messenger analogs suggest that the LHR-induced phosphorylation of FAK-Tyr576 in MA-10 cells is mediated by SFKs, and that this family of kinases is, in turn, independently or cooperatively activated by the LHR-induced stimulation of Gs and Gq/11-mediated pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE PHENOTYPE of 46XY individuals harboring germ line loss-of-function or gain-of-function mutations of the human lutropin/choriogonadotropin (CG) receptor (hLHR) implicates this receptor as an important player in the proliferation of Leydig cells (reviewed in Refs.1, 2, 3). In addition, the finding that a somatic gain-of-function mutation of the hLHR is associated with Leydig cell adenomas (4, 5) suggests that the LHR may even be oncogenic.

With this background in mind, we have initiated a series of studies designed to determine which mitogenic pathways are stimulated upon activation of the LHR in Leydig cells. To address this issue, we have again taken advantage of a mouse Leydig tumor cell line (MA-10) that retains many of the properties of their normal counterparts, including a low density of endogenous LHR (6, 7). MA-10 cells are also readily transfectable, thus allowing for robust and selective experimental manipulations that can be used to study signal transduction pathways such as the expression of dominant-negative or constitutively active mutants of signaling molecules (8). In addition, the gonadotropin-induced responses can be amplified by expression of the hLHR-wt (9) or mimicked in a gonadotropin-independent fashion by expression of constitutively active mutants of the hLHR (10).

Using MA-10 cells, we have recently shown that hCG activates a classic mitogenic pathway, the ERK1/2 cascade, largely through an increase in cAMP accumulation, which leads to the activation of Ras through a protein kinase A-dependent pathway (8). More limited studies suggest that a similar pathway is operative in primary cultures of rat Leydig cells (8, 11). In additional studies designed to understand how hCG may activate Ras, we found that hCG stimulates the phosphorylation of tyrosine residues of a prominent protein with a molecular mass of approximately 120 kDa. The studies presented here identify this protein as focal adhesion kinase (FAK) (12, 13, 14, 15) and suggest mechanisms by which hCG can stimulate tyrosine kinase cascades leading to the phosphorylation of this tyrosine kinase.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Activation of the Recombinant hLHR Expressed in MA-10 Cells Leads to the Phosphorylation of FAK in Tyrosine 576
Western blots of whole-cell lysates of MA-10 cells expressing the recombinant hLHR clearly show that addition of hCG leads to the rapid phosphorylation of tyrosine residues in a protein of approximately 120 kDa (Fig. 1Go). A protein of the same size, as well as the endogenous epidermal growth factor (EGF) receptor, are also phosphorylated on tyrosine residues by addition of EGF (Fig. 1Go). To determine the identity of this phosphoprotein, we prepared detergent extracts of MA-10 cells incubated with or without hCG and purified them using antiphosphotyrosine antibodies. The immunopurified proteins were resolved on a sodium dodecyl sulfate (SDS) gel that was subsequently stained with silver nitrate. The 120-kDa region (see arrow on the left side of Fig. 2AGo)1 was cut, dried, reduced, alkylated, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectroscopy. The fragmentation pattern obtained (Fig. 2BGo) clearly identified the protein in question as FAK or the closely related protein tyrosine kinase 2 (Fig. 2CGo).


Figure 1
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  		Fig. 1. hCG Stimulates the Tyrosine Phosphorylation of a Prominent 120-kDa Protein in MA-10 Cells Expressing the Recombinant hLHR %MA-10 cells were transfected with the hLHR and stimulated with hCG (1000 ng/ml) or EGF (100 ng/ml) for the times indicated. Western blots (WB) of whole-cell lysates were developed using a phosphotyrosine antibody as described in Materials and Methods. The additional prominent band phosphorylated on tyrosine residues upon addition of EGF (see arrow on the right side of the gel) comigrates with the EGF receptor as determined with EGF receptor antibodies.

 

Figure 2
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  		Fig. 2. Identification of the 120-kDa Tyrosine Phosphorylated Protein as FAK %MA-10 cells were transfected with the hLHR and incubated with or without hCG (1000 ng/ml) for 30 min as indicated. Tyrosine-phosphorylated proteins were immunoprecipitated using antiphosphotyrosine antibodies and resolved on a SDS gel that was silver-stained (A). The 120-kDa region of the gel was excised and dried. The dried gel piece was reduced, alkylated, digested with trypsin, and analyzed by MALDI-TOF (B). C, Matches of the MALDI-TOF pattern of the approximately 120-kDa phosphoprotein to the MALDI-TOF pattern of proteins with a molecular mass of approximately 120 kDa. With scores of 82 and 83, the 120-kDa band is likely to be FAK or PTK2 with a statistical significance of P ≤ 0.0005.

 
The identity of the 120-kDa phosphoprotein was subsequently confirmed by probing Western blots of control and hCG-stimulated cells with commercially available antibodies to protein tyrosine kinase 2, FAK, and the different phosphorylation sites of FAK (12, 13, 14, 15). Protein tyrosine kinase 2 was not detectable on Western blots of whole-cell lysates of MA-10 cells (data not shown), but FAK was readily detected (Fig. 3AGo). The results presented in Fig. 3AGo also show that 1) in control cells FAK is highly phosphorylated on Tyr397 and minimally phosphorylated on tyrosine residues 576, 577, 861, and 925; 2) hCG stimulation increases the phosphorylation of Tyr576 and Tyr577; and 3) EGF stimulation increases the phosphorylation of Tyr576, Tyr577, and Tyr925. In three independent experiments hCG induced a 2.4 ± 0.2 (mean ± SEM) and 2.5 ± 0.8-fold increase in the phosphorylation of Tyr576 and Tyr577, respectively. In EGF-treated cells the phosphorylation of Tyr576, Tyr577, and Tyr925 increased 2.4 ± 0.7, 2.6 ± 0.3, and 6.2 ± 2-fold, respectively.


Figure 3
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  		Fig. 3. EGF and hCG Stimulate the Phosphorylation of FAK at Different Tyrosine Residues %A, MA-10 cells were transfected with the hLHR and incubated with buffer only, hCG (1000 ng/ml x 30 min), or EGF (100 ng/ml x 5 min) as indicated. Western blots (WB) of whole-cell lysates were developed using specific FAK phosphopeptide antibodies to Tyr397, Tyr576, Tyr577, Tyr861, Tyr925, or an antibody to FAK as indicated. B, Lysates of MA-10 cells were prepared using cells attached to culture dishes (lane 1), cells that had been detached by trypsinization (lane 2), or cells that had been detached by trypsinization and then allowed to reattach to fibronectin-coated dishes (lane 3) as described in Materials and Methods. Western blots (WB) of whole-cell lysates were developed using a phosphopeptide antibody to Tyr397 or an antibody to FAK as indicated. Only the appropriate areas of the gel are shown, and the results are representative of at least three independent experiments.

 
The high degree of phosphorylation of FAK-Y397 is expected because the cells are attached to a culture dish, and this residue is autophosphorylated in response to cell adhesion (12, 13, 14, 15). To document this phenomenon in MA-10 cells, we examined the phosphorylation of FAK-Y397 in attached cells, in cells that had been detached from the dish, and in cells that had been detached from the dish and allowed to reattach. These experiments are presented in Fig. 3BGo and show that, as expected (12, 13, 14, 15), the phosphorylation of FAK-Y397 decreases when MA-10 cells are detached from the dish and rebounds as they reattach. The total levels of FAK do not change.

Because the phosphorylation of FAK at Tyr576 and Tyr577 results in the activation of its kinase activity, we also determined whether FAK targets become tyrosine phosphorylated in response to hCG stimulation. The two main targets of FAK are p130Cas and paxillin (12, 13, 14, 15), but we chose to examine only paxillin. Activated FAK promotes the phosphorylation of paxillin on tyrosine residues 31 and 118, and phosphopeptide antibodies to these phosphorylation sites are readily available (12, 13, 14, 15). An obvious increase in the tyrosine phosphorylation of paxillin can be detected when lysates of control and hCG-stimulated MA-10 cells are probed with an antibody to paxillin-phosphoY118 (Fig. 4Go). In three independent experiments hCG induced a 1.8 ± 0.3-fold increase (mean ± SEM) in the phosphorylation of paxillin-Tyr118.


Figure 4
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  		Fig. 4. hCG Stimulates the Phosphorylation of Paxillin at Tyr118 in MA-10 Cells Transfected with a Vector Coding for the hLHR %MA-10 cells were transfected with the hLHR and incubated with buffer only or hCG (1000 ng/ml) for 30 min as indicated. Western blots (WB) of whole-cell lysates were developed using a specific phosphopeptide antibody to paxillin phosphorylated on Tyr118 or to total paxillin as indicated. Only the appropriate areas of the gel are shown, and the results are representative of three independent experiments. C, Control.

 
Because these experiments were done using MA-10 cells transfected with the recombinant hLHR, we also compared the effects of hCG on the phosphorylation of FAK in MA-10 cells transfected with an empty vector or with the hLHR (6, 9). In this and all subsequent experiments, we chose to examine only the phosphorylation of FAK-Y576 because this antibody provides the best signal for hCG-induced FAK phosphorylation (Fig. 3Go). Figure 5Goshows that hCG stimulates the phosphorylation of FAK-Y576 in MA-10 cells expressing the endogenous mouse (m) LHR or the transfected hLHR and that maximal levels of phosphorylation are attained with 100 ng/ml hCG. As is the case with many hCG-mediated responses (9), however, the sensitivity and magnitude of the hCG-induced phosphorylation of FAK-Y576 are greatly enhanced in MA-10 cells transfected with the hLHR (compare left and right panels in Fig. 5Go). In three independent experiments the magnitude of the hCG-induced phosphorylation of FAK-Y576 was 1.4 ± 0.1-fold (mean ± SEM) higher in hLHR-transfected cells than in empty vector-transfected cells. All subsequent experiments were done using hLHR-transfected cells, and the cells were stimulated with 1000 ng/ml of hCG (to ensure that maximal responses were obtained). Selected experiments done with a lower concentration of hCG (100 ng/ml) produced similar results, however.


Figure 5
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  		Fig. 5. hCG Stimulates the Phosphorylation of FAK at Tyr576 in MA-10 Cells Transfected with an Empty Vector or a Vector Coding for the hLHR %MA-10 cells were transfected with an empty vector (EV) or the hLHR as indicated. Cells were incubated with increasing concentrations of hCG for 30 min, and Western blots (WB) of whole-cell lysates were developed using an phosphor-FAK antibody specific for Tyr576 or with an antibody to FAK as indicated. Only the appropriate areas of the gel are shown, and the results are representative of at least three independent experiments.

 
The hCG-Induced Phosphorylation of FAK-Y576 Is Mediated by the Src Family Kinases
Because FAK is a prominent substrate for the Src family kinases (12, 13, 14), we hypothesized that the hCG-induced phosphorylation of FAK-Y576 may be a reflection of the ability of hCG to activate one of these kinases.

To test this hypothesis, we first determined the expression of different Src family of tyrosine kinases (SFKs) in MA-10 cells using Western blots of whole-cell lysates. These experiments (Fig. 6Go) revealed that MA-10 cells have undetectable levels of Src, but they do express Fyn and Yes, two other prominent members of this family. We next examined the involvement of Fyn and Yes on the hCG-induced increase in FAK-Y576 phosphorylation using several different approaches. First, we tested the effects of PP2, a selective pharmacological inhibitor of SFKs (16), on the hCG-induced increase in FAK-Y576 phosphorylation. As shown in Fig. 7Go, this compound is an effective inhibitor of FAK-Y576 phosphorylation. In three independent experiments the hCG-induced phosphorylation of FAK-Y576 expressed as fold-over basal (mean ± SEM), was 1.9 ± 0.1 and 0.7 ± 0.1 in cells treated without or with PP2, respectively.


Figure 6
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  		Fig. 6. MA-10 Cells Express Fyn and Yes But Do Not Express Src %Western blots (WB) of whole-cell lysates of MA-10 cells or Jurkat cells (supplied by Upstate Biotechnology as positive control for the antibodies used) were developed using antibodies to Src, Fyn, or Yes as indicated. The entire blots are shown, and the results are representative of at least three independent experiments.

 

Figure 7
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  		Fig. 7. PP2, a Selective Src Family Kinase Inhibitor, Blocks the Increase in FAK-Y576 Phosphorylation Induced by hCG %MA-10 cells were transfected with the hLHR and preincubated in assay medium containing dimethylsulfoxide (control) or 10 µM PP2 (dissolved in dimethylsulfoxide) for 30 min. The cells were then incubated with or without hCG (1000 ng/ml) for an additional 30 min as indicated. Western blots (WB) of whole-cell lysates were developed using a phosphor-FAK antibody specific for Tyr576 or with an antibody to FAK as indicated. Only the appropriate areas of the gel are shown, and the results are representative of at least three independent experiments.

 
Cotransfection of MA-10 cells with a dominant-negative (i.e. kinase-deficient) Fyn also effectively inhibited the hCG-induced phosphorylation of FAK-Y576 (from 3.1 ± 0.2 to 1.2 ± 0.1, mean ± SEM of three independent experiments; Fig. 8Go). In contrast, cotransfection with a dominant-negative Yes had little or no effect. The extent of hCG-induced phosphorylation of FAK-Y576 in the cells transfected without or with the dominant-negative Yes was 3.1 ± 0.2 and 2.6 ± 0.2 (mean ± SEM of three independent experiments; Fig. 8Go). In a complementary set of experiments, we also measured the effects of overexpression of the wild-type Fyn or Yes. These results are also presented in Fig. 8Go and show that overexpression of either of these two tyrosine kinases enhances the basal and hCG-induced phosphorylation of FAK-Y576 about 2-fold. Finally, we measured the effects of hCG on the phosphotransferase activity of Fyn and Yes in immunoprecipates of MA-10 cells obtained with the appropriate kinase antibodies. These results are presented in Table 1aGond show a stimulatory effect of hCG on the activities of the immunoprecipitated Fyn or Yes. EGF stimulated Fyn activity to about the same extent as hCG, but it had no effect on Yes activity (Table 1Go).


Figure 8
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  		Fig. 8. Effects of Dominant-Negative (Kinase-Deficient) Mutants of Fyn and Yes and Their Wild-Type Counterparts on the Increase in FAK-Y576 Phosphorylation Induced by hCG %MA-10 cells were cotransfected with the hLHR together with an empty vector, dominant-negative (DN) mutants of Fyn or Yes (1 µg/35 mm well), or with the wild-type forms of Fyn or Yes (all used at 1 µg/35 mm well) as indicated. The cells were then incubated with or without hCG (1000 ng/ml) for 30 min before lysis. Western blots (WB) of whole-cell lysates were developed using a phosphor-FAK antibody specific for Tyr576 or an antibody to FAK or with antibodies to Fyn or Yes as indicated. Only the appropriate areas of the gel are shown, and the results are representative of at least three independent experiments.

 

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  		Table 1. hCG Stimulates Fyn and Yes Activity in MA-10 Cells

 
We conclude from these experiments that the hCG-induced phosphorylation of FAK-Y576 is mediated by the hCG-induced activation of Fyn and/or Yes.

The hCG-Induced Phosphorylation of FAK-Y576 Involves the Activation of G Protein-Dependent Pathways
Because ß-arrestin-mediated pathways have gained such prominence in mediating the ability of G protein-coupled receptors (GPCRs) to activate tyrosine kinase cascades (17), we initially tested for their involvement in the hCG-induced FAK-Y576 phosphorylation. Experiments involving the overexpression of the wild-type arrestin-2 or -3 or and their dominant-negative counterparts (17) showed that neither of these two manipulations, however, affected the hCG-induced FAK-Y576 phosphorylation (data not shown). Another way by which GPCRs may activate tyrosine kinase cascades involves the transactivation of the EGF receptor (18, 19). The possibility that the EGF receptor is phosphorylated in response to hCG stimulation is rendered unlikely by the experiment presented in Fig. 1Go, which shows that the endogenous EGF receptor is phosphorylated when MA-10 cells are exposed to EGF, but a protein of the same size is not tyrosine phosphorylated when they are exposed to hCG.

In addition to the classical ways of activation of the Src family kinases by phosphorylation/dephosphorylation of tyrosine residues (20, 21, 22), their activity can also be modulated by direct binding to G{alpha}s or G{alpha}i (23, 24) and possibly by phosphorylation of serine/threonine residues catalyzed by protein kinase A or C (22, 25, 26, 27). These pathways were also considered because the binding of hCG to the recombinant hLHR expressed in MA-10 cells results in the activation of Gs, Gi/o, and Gq/11 (9, 10). The LHR-induced activation of these G proteins leads to a G{alpha}s-mediated increase in cAMP accumulation and a G{alpha}q/11-mediated increase in inositol phosphate/diacylglycerol accumulation (9, 10).

The involvement of G protein-mediated pathways was initially tested by examining FAK-Y576 phosphorylation in MA-10 cells transfected with constitutively active mutants of several G{alpha}-subunits. Overexpression of constitutively active mutants of G{alpha}q or G{alpha}11 resulted in obvious increases in FAK-Y576 phosphorylation, but overexpression of G{alpha}s, G{alpha}i, and G{alpha}o had little or no effect (Fig. 9AGo). The expression of the transfected products was monitored by Western blotting (Fig. 9BGo) and/or by second messenger assays (Table 2Go). One or both of these methods clearly documents the expression of each of the transfected G{alpha}-subunits (Fig. 9BGo and Table 2Go).


Figure 9
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  		Fig. 9. Constitutively Active Mutants of G{alpha}s, G{alpha}q, or G{alpha}11 Increase the Phosphorylation of FAK at Tyr576 %MA-10 cells were transfected with an empty vector (EV) or with expression vectors for constitutively active mutants of the different G{alpha} subunits (all at 1 µg/35-mm well). The cells were incubated for 30 min in assay medium without any stimuli. Western blots (WB) of whole cell lysates were developed using a phosphor-FAK antibody specific for Tyr576 or with an antibody to FAK (A) or with antibodies to the different G{alpha}-subunits as indicated (B). Only the appropriate areas of the gel are shown. The numbers at the top show the increase in the phosphorylation of FAK-Tyr576 expressed as fold over basal (mean ± SEM of three independent experiments).

 

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  		Table 2. Effect of Constitutively Active Mutants of G{alpha}-Subunits on Second Messenger Levels in MA-10 Cells

 
These results suggest that the LHR-induced activation of Gq or G11 and possibly Gs and Gi or Go could mediate the effect of hCG on FAK-Y576 phosphorylation. The potential involvement of Gi or Go on the hCG-induced FAK-Y576 phosphorylation was excluded, however, with the use of pertussis toxin. We found (data not shown) that preincubation of MA-10 cells with pertussis toxin did not inhibit the effects of hCG on FAK-Y576 phosphorylation. Finally, the involvement of Gß{gamma} was probed by overexpression of a C-terminal domain of GRK2 (a scavenger of Gß{gamma}; see Ref.28). This manipulation also did not affect the hCG-induced FAK-Y576 phosphorylation (data not shown).

We conclude from these experiments that Gq/11 and possibly Gs (or the second messengers generated by the activation of their {alpha}-subunits) could mediate the effect of hCG on FAK-Y576 phosphorylation. To test for the involvement of second messengers on FAK-Y576 phosphorylation, we incubated MA-10 cells with cAMP analogs or with PMA (a surrogate for diacylglycerol) because these second messengers are generated in response to the LHR-induced activation of Gs or Gq/11. The results presented in Fig. 10Goshow that PMA and two cAMP analogs [8-bromo-cAMP (8-Br-cAMP) or 8-CPT-cAMP)] that activate protein kinase A and cAMP-dependent guanine nucleotide exchange factors can effectively stimulate FAK-Y576 phosphorylation. A cAMP analog that is selective for the cAMP-dependent guanine nucleotide exchange factors (8-CPT-2-Me-cAMP; see Refs.29 and 30), however, does not stimulate FAK-Y576 phosphorylation. The magnitude of the stimulation of FAK-Y576 phosphorylation obtained with PMA was comparable to that obtained with hCG. The magnitude of the stimulation of FAK-Y576 phosphorylation obtained with 8-Br-cAMP or 8-CPT-cAMP was slightly lower, however, than that obtained with hCG (Fig. 10Go).


Figure 10
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  		Fig. 10. Effects of Several Second Messenger Analogs and Hormones on the Phosphorylation of FAK at Tyr576 %A, MA-10 cells were transfected with the hLHR and incubated with buffer only, hCG (1000 ng/ml), 8-Br-cAMP (1 mM), 8-CPT-cAMP (1 mM), 8-CPT-2-Me-cAMP (1 mM), or PMA (100 nM) for 30 min as indicated. Western blots (WB) of whole-cell lysates were developed using a phosphor-FAK antibody specific for Tyr576 or with an antibody to FAK as indicated. Only the appropriate areas of the gel are shown. The numbers at the top show the increase in the phosphorylation of FAK-Tyr576 expressed as fold over basal (mean ± SEM of three independent experiments). B, MA-10 cells were cotransfected with the hLHR and the ETAR (each at 1 µg/35-mm well) and incubated with buffer only (C), hCG (1000 ng/ml), ET-1 (0.1 µM), 8-Br-cAMP (0.1 mM), and ET-1 (0.1 µM) + 8-Br-cAMP (0.1 mM) for 30 min as indicated. Western blots (WB) of whole-cell lysates were developed using a phosphor-FAK antibody specific for Tyr576 or with an antibody to FAK as indicated. Only the appropriate areas of the gel are shown, and the results are representative of at least three independent experiments. C, Control.

 
To further test for the involvement of these two pathways, we stimulated Gs and Gq/11 by activation of the transfected hLHR or selectively stimulated Gq/11 by activation of the transfected endothelin type A receptor (ETAR) (31).2 This selective or dual activation is documented by the results presented in Table 3Go, which show that activation of the transfected ETAR results in an increase in inositol phosphate accumulation without a change in cAMP accumulation, whereas activation of the transfected hLHR results in an increase in inositol phosphates and cAMP accumulation (9). The results presented in Fig. 10Go show that the GPCR-mediated stimulation of Gs and Gq/11 (with hCG) or the selective stimulation of Gq/11 [with endothelin 1 (ET-1)] can increase FAK-Y576 phosphorylation to about the same extent. Finally, to determine whether both of these two G protein-mediated pathways can cooperate to increase FAK-Y576 phosphorylation, we compared the magnitude of FAK-Y576 phosphorylation in MA-10 cells transfected with the ETAR and stimulated with ET-1 only, with 8-Br-cAMP only, or with a combination of both. The results presented in Fig. 10Go show that FAK-Y576 phosphorylation can be clearly enhanced by addition of 8-Br-cAMP to ET-1-stimulated MA-10 cells.


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  		Table 3. Effect of hCG and ET-1 on cAMP and Inositol Phosphate Accumulation in MA-10 Cells

 
When considered together, these experiments show that the effect of hCG on FAK-Y576 phosphorylation could be mediated independently or cooperatively by the hCG-induced activation of Gs and Gq/11-mediated pathways.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
FAK is a ubiquitous and abundant protein tyrosine kinase that was initially and simultaneously identified as a substrate for the viral Src oncogene and as a phosphotyrosine-containing protein that is enriched in focal contacts (reviewed in Refs.12, 13, 14, 15). During the process of cell adhesion, activation of integrin receptors results in the autophosphorylation of FAK-Y397, and this phosphotyrosine creates a binding site for a number of SH2-containing proteins including Src (12, 13, 14, 15). The binding of Src to FAK increases the activity of Src, and the bound Src catalyzes the phosphorylation of FAK-Y576 and -Y577. These two residues are located in the kinase activation loop of FAK, and their phosphorylation is required for maximal FAK activity. The bound Src also catalyzes the phosphorylation of FAK-Y861 and FAK-Y925. These two phosphotyrosines become docking sites for other SH2 domain-containing proteins that serve as adaptors in signal transduction pathways. The Src/FAK complex (a dual tyrosine kinase complex) then promotes the tyrosine phosphorylation of the proteins bound to the phosphotyrosine residues or other FAK domains. Two of the best-characterized FAK/Src complex substrates are paxillin and p130Cas (12, 13, 14, 15).

In addition, and independently of its kinase activity, FAK serves the role of adaptor or scaffold for a number of signaling molecules that participate in cell migration, adhesion, shape, survival, and multiplication (12, 13, 14, 15). In fact, some of the actions of FAK, such as changes in motility, may be due entirely to the adaptor properties of FAK rather than to its enzymatic activity (12, 13, 14, 15).

The results presented here show, for the first time, that addition of hCG to MA-10 cells expressing the endogenous mLHR or recombinant hLHR results in the phosphorylation of FAK-Y576 and FAK-Y577 and the phosphorylation of paxillin-Y118, which is a downstream target of the FAK-SFK complexes (Figs. 1–5GoGoGoGoGo). The phosphorylation of FAK-Y576 seems to be mediated by Fyn and possibly Yes, two members of the SKFs that are endogenously expressed in MA-10 cells. Our data show that hCG activates Fyn and Yes (Table 1Go), and that the hCG-induced phosphorylation of FAK-Y576 can be inhibited with a selective pharmacological inhibitor of the SFKs (PP2) and with a dominant-negative mutant of Fyn (Figs. 7Go and 8Go). A dominant-negative mutant of Yes was not effective, however, in inhibiting the hCG-induced phosphorylation of FAK-Y576 (Fig. 8Go). We do not have a clear explanation for the lack of effect of the dominant-negative Yes. We note, however, that the expression of this construct is relatively low compared with the expression of the dominant-negative Fyn (compare lower panels in Fig. 8Go). It is also possible that the lack of effect of the dominant-negative Yes is due to the mislocalization of the transfected protein or to an incomplete loss of kinase activity induced by the mutation. Thus, the involvement of Yes in the hCG-mediated phosphorylation of FAK-Y576 is presently unclear.

The finding that hCG stimulates the phosphorylation of FAK-Y576 and FAK-Y577 but does not stimulate the phosphorylation of FAK-Y397, FAK-Y861, or FAK-Y925 is of interest but not entirely unexpected. In vascular endothelial cells, the activation of two different GPCRs (the sphingosine-1 phosphate or thrombin receptors) results in a different pattern of FAK phosphorylation. Thrombin increases the phosphorylation of FAK-Y397, FAK-Y576, and FAK-Y925 whereas sphingosine-1 phosphate enhances only the phosphorylation of FAK-Y576 (33). As already mentioned above, FAK-Y397 is the main FAK residue phosphorylated upon activation of integrin receptors (see above and Fig. 3Go), and the lack of effect of hCG on the phosphorylation of this residue implies that hCG does not transactivate integrin receptors (see below). The lack of effect of hCG on the phosphorylation of FAK-Y861 and FAK-Y925 is also of interest because these two residues are also prominent substrates for Src and have important implications for some of the activities of FAK (12, 13, 14, 15). This is particularly true for the phosphorylation of FAK-Y925, which creates a docking site for Grb2. Grb2 is an adaptor protein that can also bind SOS, a guanine nucleotide exchange factor for Ras. Thus, the phosphorylation of FAK-Y925 links FAK to the activation of the ERK1/2 cascade (12, 13, 14, 15). Because hCG does not increase the phosphorylation of FAK-Y925, we can exclude the involvement of FAK as a mediator of the hCG-induced activation of Ras and ERK1/2 that was previously reported by us (8). As shown herein, an increase in the phosphorylation of FAK-Y925 can be readily detected upon addition of EGF (Fig. 3Go). As already mentioned above, stimulation of other cells with different agonists can result in the phosphorylation of different FAK residues (33), and the same phenomenon is reported here with EGF and hCG (Fig. 3Go). Such findings may be explained by the preferential activation of different tyrosine kinases by EGF and hCG.

A number of GPCRs (but not the LHR) have been previously shown to stimulate the phosphorylation and/or activities of nonreceptor tyrosine kinases such as the SKFs and FAK (reviewed in Refs.17, 18, 20, 22 , and 34). Pathways that may mediate the GPCR-induced activation of nonreceptor tyrosine kinases include 1) the transactivation of integrin receptors; 2) the transactivation of EGF receptors; 3) a direct physical association of GPCRs and the tyrosine kinases; 4) the formation of a tyrosine kinases/ß-arrestin/GPCR complexes; 5) direct activation of the tyrosine kinases by G{alpha}-subunits; and 6) less direct activation of the tyrosine kinases through serine/threonine phosphorylation catalyzed by second messenger-dependent kinases.

The possibility that the hCG-induced FAK phosphorylation is mediated by transactivation of integrin receptors appears unlikely because hCG does not stimulate the phosphorylation of FAK-Y397 (Fig. 3Go). FAK-Y397 is the principal FAK residue phosphorylated in response to the activation of integrin receptors (Fig. 3Go and Refs.12, 13, 14, 15). Because we cannot detect an increase in the phosphorylation of the endogenous EGF receptor upon addition of hCG to MA-10 cells (Fig. 1Go), we can also rule out an hCG-induced transactivation of the EGF receptor as a mechanism involved in the phosphorylation of FAK-Y576. The involvement of ß-arrestins as platforms for the LHR-mediated activation of SFKs and the phosphorylation of FAK-Y576 also appears unlikely as discussed in the main text of the paper. Finally, the ability of the ß2-adrenergic receptor to directly associate with and activate Src through a phosphotyrosine-SH2 domain interaction (35) implies that a similar mechanism could mediate the LHR-induced activation of Fyn and Yes. This possibility has not yet been investigated.

A number of results presented here suggest that the LHR-induced phosphorylation of FAK-Y576 is mediated by G protein-dependent pathways, specifically those involving Gs and Gq/11 (Figs. 9Go and 10Go). In fact, it appears that the LHR-induced activation of Gs and/or Gq/11 could be required for optimal phosphorylation of FAK-Y576 (Figs. 9Go and 10Go). Because Src and Lck (another member of the SFKs) have been shown to be direct effectors of some G{alpha}-subunits (23, 24), future experiments need to consider the possibility that Fyn and/or Yes are directly activated by the G{alpha}s and/or G{alpha}q/11 that are liberated in response to the hCG-induced activation of the recombinant LHR in MA-10 cells. Clearly, however, the second messengers generated by the LHR-induced activation of these two G protein families can stimulate the phosphorylation of FAK-Y576 in MA-10 cells (Fig. 10Go). Src has been shown to be phosphorylated on Ser12 by protein kinase C and on Ser17 by protein kinase A (22, 25). The phosphorylation of Ser12 has little or no effect on the activity of Src (25, 36), but the protein kinase A-mediated phosphorylation of Ser17 of Src appears to participate in the Src-dependent activation of Rap1 in several cell types (26, 27). Ser12 of Src (the protein kinase C phosphorylation site; see above) is replaced by a Thr in Fyn and Yes, whereas Ser17 of Src (the protein kinase A phosphorylation site) is replaced by a Gly in Fyn and a Thr in Yes. We are not aware of any information on the phosphorylation of these or other Ser or Thr residues in Fyn and/or Yes, but the possibility that a protein kinase A- and/or protein kinase C-mediated phosphorylation of Fyn and Yes is responsible for the effects of hCG reported here needs to be considered as well.

In summary, we have shown that the activation of the LHR in MA-10 cells results in the stimulation of the prominent nonreceptor tyrosine kinases Fyn and Yes, and the tyrosine phosphorylation of FAK and paxilllin. Although the exact mechanisms by which the LHR activates these kinases and the functional consequences of this activation are not yet fully understood, our results provide a solid foundation for future studies on these areas. Because tyrosine kinase cascades play such a prominent role in cell proliferation and the LHR is clearly a mitogen for Leydig cells (see Introduction), it is tempting to speculate that the pathway described here, as well as the ERK1/2 cascade, is involved in the LHR-mediated proliferation of Leydig cells. The hCG-induced activation of SFKs could also affect other LHR-mediated signaling events such as second messenger accumulation (18, 35) and/or steroidogenesis (37, 38).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmids and Cells
The origin and handling of MA-10 cells have been described previously (6). These cells are now maintained in gelatin-covered plasticware and RPMI-1640 medium supplemented with 15% horse serum, 20 mM HEPES, and 50 µg/ml gentamicin, pH 7.4 (growth medium) as described elsewhere (9).

The expression vector for the hLHR modified with the myc-epitope at the N terminus has been described previously (9). Expression vectors for the wild-type and kinase-deficient mutant (K229M) of human Fyn were generously provided by Dr. Marylin Resh of the Memorial Sloan Kettering Cancer Center (39). An expression vector for the wild-type mouse Yes was provided by Dr. Marius Sudol (Weis Center for Research, Geisinger Health System; see Ref.40). A kinase-deficient mutant of Yes (K303M) was prepared in our laboratory using this plasmid as a template by standard PCR methods. The expression vectors for arrestin-1, Flag-arrestin-2, and Flag-arrestin-3 have been described elsewhere (41, 42) These were generously provided by J. L. Benovic (Thomas Jefferson University, Philadelphia, PA) and modified by us with the FLAG epitope (42). Expression vectors for constitutively active (i.e. GTPase-deficient) mutants of G{alpha}s, G{alpha}q, G{alpha}11, G{alpha}i, and G{alpha}o and for the ETAR were purchased from the University of Missouri-Rolla (UMR) cDNA Resource Center (www.cDNA.org). An expression vector for the C-terminal end of GRK2 was described previously (43), and it was provided to us by Dr. Robert Lefkowitz (Duke University, Durham, NC).

Purification and Identification of the p120 Phosphoprotein
MA-10 cells plated in 100-mm dishes were stimulated as described below, washed and lysed in RIPA buffer (150 mM NaCl, 50 mM Tris, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.4) supplemented with an EDTA-free protease inhibitor cocktail from Roche Applied Science (Indianapolis, IN), 1 mM NaF, and 1 mM sodium orthovanadate. The lysates from two dishes (~2 mg of protein) were clarified by centrifugation and immunoprecipitated with 5 µl of an antiphosphotyrosine antibody (4G10 from Upstate Biotechnology, Inc., Lake Placid, NY) that had been prebound to 50 µl of protein G Sepharose (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). After an overnight incubation at 4 C, the beads were collected by centrifugation, washed three times with 1-ml aliquots of RIPA buffer, three times with 1-ml aliquots of the RIPA buffer with 1 M NaCl, and then again three times with 1-ml aliquots of RIPA buffer. Finally, the bound proteins were eluted with 50 µl of SDS sample buffer, boiled, and resolved on a 7.5% SDS gel. The gel was stained with the SilverQuest kit from Invitrogen (San Diego, CA), and the appropriate region was cut out and dried. The dried gel piece was reduced, alkylated, digested with trypsin, and analyzed by MALDI-TOF mass spectroscopy at the Molecular Analysis Facility of the Carver College of Medicine of the University of Iowa (http://www.medicine.uiowa.edu/maf/).

Analysis of Protein Expression and Phosphorylation by Western Blotting
MA-10 cells were plated on 35-mm wells and transfected 1 d after plating, as indicated in the figures and tables. Transfections were done in 1 ml of OPTIMEM supplemented with 700 µg/ml CaCl2·2 H2O. Each well was transfected with a maximum of 2 µg of plasmid and Lipofectamine at a ratio of 4–6 µl/µg of DNA (9). After a 3-h incubation each well received 150 µl of horse serum, and the incubation was continued for another 16–24 h. The medium was then replaced with assay medium (RPMI-1640 medium supplemented with 1 mg/ml BSA, 20 mM HEPES, and 50 µg/ml gentamicin, pH 7.4), and the cells were incubated in this medium for another 16–18 h. On the day of the assay the medium was replaced with 1 ml of fresh assay medium, and hormones and other compounds were added as indicated in the figure legends. The transfection efficiency under these conditions is about 25% (9).

At the end of the stimulation period the medium was aspirated and the cells were lysed with 100 µl of RIPA buffer supplemented with protease and phosphatase inhibitors, as described above. The resulting lysates were clarified by centrifugation and assayed for protein content using the BCA protein assay kit from Bio-Rad Laboratories, Inc. (Hercules, CA). Equal amounts of protein from each lysate (30 µg) were then resolved on 7.5% or 10% SDS-polyacrylamide gels and transferred electrophoretically to polyvinylidene difluoride membranes (9). The membranes were incubated with primary antibodies using variable conditions (see below) followed by a second, constant 1-h incubation with a 1:3000 dilution of a secondary antibody covalently coupled to horseradish peroxidase (Bio-Rad Laboratories, Inc.). Finally, immune complexes were visualized and quantified using the Super Signal West Femto Maximum Sensitivity detection system (Pierce Chemical Co., Rockford, IL) and a Kodak digital imaging system (Eastman Kodak Co., Rochester, NY). FAK phosphorylated at tyrosine residues 397, 576, 577, 861, or 925 was detected using phospho-specific antibodies purchased from Biosource Technologies, Inc. (Camarillo, CA) or Cell Signaling Technology (Beverly, MA) using a 2-h incubation of the membranes with a 1:1000 or a 1:2000 dilution of antibody. Total FAK was detected with an antibody from Upstate Biotechnology (Lake Placid, NY) also using a 2-h incubation of the membranes with a 1:2000 dilution of antibody. Antibodies to Src, Fyn, and Yes were also from Upstate Biotechnology and were used to develop blots during an overnight incubation at 4 C at dilutions of 1:250, 1:1000, and 1:1000, respectively. The blot shown in Fig. 1Go was developed during an overnight incubation at room temperature with an antiphosphotyrosine antibody (4G10, also from Upstate Biotechnology) used a 1:1000 dilution. The different G{alpha} subunits were visualized by incubating the membranes with the appropriate primary antibodies (described in Ref.10) at a dilution of 1:300 for G{alpha}s, 1:200 for G{alpha}q/11 and 1:1000, for G{alpha}i-3/G{alpha}o, antibodies, respectively. Paxillin and paxillin phosphorylated on Tyr118 were visualized with antibobies from Cell Signaling Technology used at a dilution of 1:1000.

For the experiment presented in Fig. 3BGo, three groups of cells were used. The first group was kept attached to the culture dish as usual and incubated in assay medium for 30 min before lysis. The second group of cells was detached from the dish with trypsin as we routinely do for subculturing (6), recovered by centrifugation, resuspended in assay medium containing 50 µg/ml of soybean trypsin inhibitor, and incubated for 30 min with occasional shaking before lysis. The third groups of cells was detached and resuspended like the second group, but then they were allowed to attach to a culture dish coated with fibronectin for 30 min before lysis. Fibronectin coating was accomplished by adding a 50 µg/ml solution of fibronectin (in assay medium) to the dishes for 45 min. This solution was removed before the cells were allowed to attach.

When needed, quantitation of the phosphorylated FAK, phosphorylated paxillin, and their total counterparts was accomplished using the software of the Kodak digital imaging system described above. The signal obtained with the antibodies that recognize the phosphorylated proteins was then divided by the signal obtained with the antibodies to the total protein (i.e. FAK or paxillin). This ratio was defined as 1 (basal) in the cells incubated without any stimuli, and all data from stimulated cells were expressed as fold over this basal phosphorylation ratio.

Kinase Assays
Cells were plated in 100-mm dishes and transfected with 10 µg of the hLHR-wt expression vector. The medium was replaced 1 d after transfection with assay medium (see above), and the cells were incubated in this medium for 16–18 h. The medium was then replaced again, and the cells were incubated in 1 ml of assay medium containing the appropriate stimuli for 30 min as indicated in Table 1Go. At the end of this incubation the medium was aspirated and the cells were lysed in 700 µl of lysis buffer (1% Nonidet P-40 in 150 mM NaCl, 25 mM Tris-Cl, pH 7.5) supplemented with protease and phosphatase inhibitors as described above. The lysates were clarified by centrifugation and assayed for protein content using the BCA protein assay kit from Bio-Rad Laboratories Inc. Aliquots (500 µl) of the lysates containing identical amounts of protein (700–1000 µg) were immunoprecipitated for 4 h at 4 C with 3 µl of Fyn or Yes antibodies (see above) that had been prebound to 30 µl of a 50% suspension of protein G Sepharose. The immune complexes bound to the Sepharose beads were recovered by centrifugation and washed twice with 500 µl aliquots of lysis buffer, twice with lysis buffer supplemented with 1 M NaCl, and finally three times with a buffer containing 50 mM Tris-Cl, pH 7.2; 62.5 mM MgCl2; 12.5 mM MnCl2; 1 mM EGTA; 125 µM orthovanadate; and 1 mM dithiothreitol. The washed, packed protein G beads (15 µl) were mixed with 10 µl of kinase reaction buffer (100 mM Tris-Cl, pH 7.2; 125 mM MgCl2; 25 mM MnCl2; 2 mM EGTA; 250 µM orthovanadate; and 2 mM dithiothreitol) or with 10 µl of a 600 µM solution of a Src substrate peptide (Upstate Biotechnology) in kinase reaction buffer. Each tube then received 10 µl of water and 10 µl of a 1 mCi/ml solution of [32P-{gamma}]ATP (from PerkinElmer, Norwalk, CT) in 75 mM MnCl2, 500 µM ATP, 25 mM ß-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 20 mM 3-[N-morpholino]propanesulfonic acid (pH 7.2). After 30 min at 30 C, each tube received 20 µl of 40% trichloroacetic acid, and the incubation was continued for 5 min at room temperature. Aliquots (25 µl) of each reaction were then spotted on small squares of P81 phosphocellulose paper. These were washed five times (5 min each) with 5 ml of 0.75% phosphoric acid and once with 3 ml of acetone (3 min) before counting on a liquid scintillation counter. The counts per min found in the samples incubated without the Src substrate peptide (blanks) were subtracted from those containing the substrate.

Hormones and Supplies
Purified hCG (CR-127, ~13,000 IU/mg) was purchased from Dr. A. Parlow and the National Hormone and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases, and purified recombinant hCG3 was provided by Ares Serono (Randolph, MA). Cell culture media were obtained from Invitrogen. Other cell culture supplies and reagents were obtained from Corning and Invitrogen, respectively. 8-Br-cAMP, PMA, arginine vasopressin, pertussis toxin, ET-1, and recombinant EGF were from Sigma. 8-CPT-cAMP and 8-CPT-2-Me-cAMP were from Calbiochem. All other chemicals were obtained from commonly used suppliers.

Other Methods
Inositol phosphates and cAMP determinations were done as described elsewhere (9).


   FOOTNOTES
 
First Published Online November 17, 2005

Abbreviations: 8-Br-cAMP, 8-Bromo-cAMP; CG, choriogonadotropin; 8-CPT-cAMP, 8-(4-chlorophenylthio)cAMP; 8-CPT-2-Me-cAMP, 8-(4-chlorophenylthio)-2'-O-methyl cAMP; EGF, epidermal growth factor; ET-1, endothelin 1; ETAR, endothelin type A receptor; FAK, focal adhesion kinase; GPCR, G protein-coupled receptor; LHR, lutropin/choriogonadotropin receptor; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; PMA, phorbol-12-myristate-13-acetate; SDS, sodium dodecyl sulfate; SFKs, Src family of tyrosine kinases.

This work was supported by the National Cancer Institute (CA-40629).

1 The apparent lack of effect of hCG on the phosphorylation of the 120-kDa protein shown in Fig. 2AGo is probably due to the relative insensitivity of the silver staining procedure used to stain the gel. Back

2 Similar results (data not shown) were obtained by activation of the endogenous arginine vasopressin receptor present in MA-10 cells (8 32 ). Back

3 Both preparations were used in this study and were found to be indistinguishable. Back

Received for publication July 7, 2005. Accepted for publication November 9, 2005.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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