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. Author manuscript; available in PMC: 2006 Mar 1.
Published in final edited form as: Mol Endocrinol. 2005 Nov 17;20(3):619–630. doi: 10.1210/me.2005-0277

Activation of the lutropin/choriogonadotropin receptor (LHR) in MA-10 cells leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) by a pathway that involves Src family kinases*

Tetsuya Mizutani 1, Koji Shiraishi 1, Toni Welsh 1, Mario Ascoli 1,✉
PMCID: PMC1382007  NIHMSID: NIHMS7445  PMID: 16293639

Abstract

We show that activation of the endogenous or recombinant 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-mediate phosphorylation of FAK-Tyr576. Studies involving activation of other G protein-coupled receptors, overexpression of the different Gα 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

The phenotype of 46XY individuals harboring germ line loss-of-function or gain-of-function mutations of the hLHR implicates this receptor as an important player in the proliferation of Leydig cells (reviewed in refs. 1–3). 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 retain 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 ~120 kDa. The studies presented here identify this protein as FAK (12–15) and suggest mechanisms by which hCG can stimulate tyrosine kinase cascades leading to the phosphorylation of this tyrosine kinase.

Results

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 ~120 kDa protein (Figure 1). A protein of the same size as well as the endogenous EGF receptor are also phosphorylated on tyrosine residues by addition of EGF (Figure 1). To determine the identity of this phosphoprotein we prepared detergent extracts of MA-10 cells incubated with or without hCG and purified them using anti-phosphotyrosine antibodies. The immunopurified proteins were resolved on an SDS gel that was subsequently stained with silver nitrate. The 120 kDa region (see arrow on the left side of Figure 2A)1 was cut, dried, reduced, alkylated, digested with trypsin and analyzed by MALDI-TOF mass spectroscopy. The fragmentation pattern obtained (Figure 2B) clearly identified the protein in question as FAK or the closely related protein tyrosine kinase 2 (Figure 2C).

Figure 1. Human CG stimulates the tyrosine phosphorylation of a prominent 120 kDa protein in MA-10 cells expressing the recombinant hLHR.

Figure 1

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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 EGFR as determined with EGFR antibodies.

Figure 2. Identification of the 120 kDa tyrosine phosphorylated protein as FAK.

Figure 2

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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 anti-phosphotyrosine antibodies and resolved on a SDS gel that was silver-stained (panel 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 (panel B). Panel C shows the matches of the MALDI-TOF pattern of the ~120 kDa phosphoprotein to the MALDI-TOF pattern of proteins with a molecular mass of ~120 Kda. With scores of 82 and 83, the 120 kDa band is likely to be FAK or PTK2 with a statistical significance of ≤ 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 (12–15). Protein tyrosine kinase 2 was not detectable on Western blots of whole cell lysates of MA-10 cells (not shown) but FAK was readily detected (Figure 3A). The results presented in Figure 3A also show that (a) in control cells FAK is highly phosphorylated on Tyr397 and minimally phosphorylated on tyrosine residues 576, 577 861 and 925; (b) hCG stimulation increases the phosphorylation of Tyr576 and Tyr577; and (d) 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. EGF and hCG stimulate the phosphorylation of FAK at different tyrosine residues.

Figure 3

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A. MA-10 cells were transfected with the hLHR and incubated with buffer only, hCG (1000 ng/ml × 30 min) or EGF (100 ng/ml × 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 (12–15). 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 Figure 3B and show that, as expected (12–15), 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.

Since the phosphorylation of FAK at Tyr576 and Tyr577 results in the activation of its kinase activity we also determined if FAK targets become tyrosine phosphorylated in response to hCG stimulation. The two main targets of FAK are p130Cas and paxillin (12–15) 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 (12–15). 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 (Figure 4). In three independent experiments hCG induced a 1.8 ± 0.3-fold increase (mean ± SEM) in the phosphorylation of paxillin-Tyr118.

Figure 4. Human CG stimulates the phosphorylation of paxillin at Tyr118 in MA-10 cells transfected with a vector coding for the hLHR.

Figure 4

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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.

Since 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 (Figure 3). Figure 5 shows that hCG stimulates the phosphorylation of FAK-Y576 in MA-10 cells expressing the endogenous mLHR or the transfected hLHR and that maximal levels of phosphorylation are attained with 100 ng/ml hCG. As it 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 Figure 5). 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. Human CG stimulates the phosphorylation of FAK at Tyr576 in MA-10 cells transfected with an empty vector or a vector coding for the hLHR.

Figure 5

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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 phosphoFAK 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

Since FAK is a prominent substrate for the Src family kinases (12–14) 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 SFKs in MA-10 cells using Western blots of whole cell lysates. These experiments (Figure 6) 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 Figure 7 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. MA-10 cells express Fyn and Yes, but do not express Src.

Figure 6

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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. PP2, a selective Src family kinase inhibitor, blocks the increase in FAK-Y576 phosphorylation induced by hCG.

Figure 7

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MA-10 cells were transfected with the hLHR and preincubated in assay medium containing DMSO (control) or 10 μM PP2 (dissolved in DMSO) 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 phosphoFAK 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.

Co-transfection 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, Figure 8). In contrast co-transfection 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, Figure 8). 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 Figure 8 and show that overexpression of either of these two tyrosine kinases enhances the basal and hCG-induced phosphorylation of FAK-Y576 about 2-fold. Lastly, 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 1 and 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 1).

Figure 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.

Figure 8

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MA-10 cells were co-transfected 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 prior to lysis. Western blots (WB) of whole cell lysates were developed using a phosphoFAK 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.

Table 1.

Human CG stimulates Fyn and Yes activity in MA-10 cells

MA-10 cells expressing the hLHR were incubated with buffer only, hCG (1000 ng/ml) or EGF (100 ng/ml) for 30 min as indicated. Fyn and Yes were immunoprecipitated from whole cell lysates and the phosphotransferase activity present in the immunoprecipitates was measured as described in Materials and Methods.

Kinase assayed Cells stimulated with
hCG (fold-over-basal) EGF (fold-over-basal)
Fyn 1.54 ± 0.12* (n = 7) 1.36 ± 0.05* (n = 4)
Yes 1.26 ± 0.05* (n = 6) 1.04 ± 0.08 (n = 3)
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Phosphotransferase activity is expressed as fold-over-basal (i.e., the ratio of phosphotransferase activity measured in immunoprecipitates of stimulated cells over that measured in immunoprecipitates from cells incubated with buffer only). Each number is the mean ± SEM of the indicated number of experiments. Asterisks denote a statistically significant difference (p < 0.05, paired t test) from basal activity.

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

Since βarrestin-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 affected the hCG-induced FAK-Y576 phosphorylation, however (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 Figure 1 which show 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 (20–22) their activity can also be modulated by direct binding to Gαs or Gαi (23, 24) and possibly by phosphorylation of serine/threonine residues catalyzed by protein kinase A or C (22, 25–27). 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 Gαs-mediated increase in cAMP accumulation and a Gαq/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 Gα subunits. Overexpression of constitutively active mutants of Gαq or Gα11 resulted in obvious increases in FAK-Y576 phosphorylation but overexpression of Gαs, Gαi and Gαo had little or no effect (Figure 9A). The expression of the transfected products was monitored by Western blotting (Figure 9B) and/or by second messenger assays (Table 2). One or both of these methods clearly documents the expression of each of the transfected Gα subunits (Figure 9B and Table 2).

Figure 9. Constitutively active mutants of Gαs, Gαq, or Gα11 increase the phosphorylation of FAK at Tyr576.

Figure 9

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MA-10 cells were transfected with an empty vector (EV) or with expression vectors for constitutively active mutants of the different Gα 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 phosphoFAK antibody specific for Tyr576 or with an antibody to FAK (Panel A) or with antibodies to the different Gα subunits as indicated (Panel 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).

Table 2.

Effect of constitutively active mutants of Gα subunits on second messenger levels in MA-10 cells

Transfected construct cAMP (pmol/106cells) Inositol phosphates (cpm/106cells)
Empty vector 37 ± 7 1531 ± 361
Constitutively active Gαs 3474 ± 485* 3552 ± 702*a
Constitutively active Gαq 40 ± 6 35936 ± 8764*
Constitutively active Gα11 39 ± 2 28929 ± 6453*
Constitutively active Gαi 38 ± 5 1677 ± 251
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MA-10 cells were transfected with an empty vector or constitutively active forms of the indicated Gα subunits (1 μg of plasmid/35 mm well) as described in Materials and Methods and the legend to Figure 9. The cells were incubated in assay medium without any stimuli for 30 min or 60 min prior to the measurement of cAMP and inositol phosphates, respectively, as described in Materials and Methods. Each number is the mean ± SEM of three independent transfections. Asterisks denote statistically significant difference (p < 0.05, paired t test) from cells transfected with an empty vector.

a

We did not investigate the reason why G αs-transfected cells have increased levels of inositol phosphates. The ability of cAMP analogs to increase inositol phosphate levels in other cell types has been described, however (44).

These results suggest that the LHR-induced activation Gq, or G11 and possibly Gs and Gi or Go could mediate the effect 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. Lastly, the involvement of Gβγ was probed by overexpression of a C-terminal domain of GRK2 (a scavenger of Gβγ, 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 Figure 10 show that PMA and two cAMP analogs (8Br-cAMP or 8CPT-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 ref. 29, 30) does not stimulate FAK-Y576 phosphorylation, however. 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 8Br-cAMP or 8CPT-cAMP or was slightly lower than that obtained with hCG, however (Figure 10).

Figure 10. Effects several second messenger analogs and hormones on the phosphorylation of FAK at Tyr576.

Figure 10

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A--MA-10 cells were transfected with the hLHR and incubated with buffer only, hCG (1000 ng/ml), 8Br-cAMP (1 mM), 8-CPT-cAMP (1 mM), 8-CPT-2Me-cAMP (1 mM) or PMA (100 nM) for 30 min as indicated. Western blots (WB) of whole cell lysates were developed using a phosphoFAK 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), 8Br-cAMP (0.1 mM) and ET-1 (0.1 μM) + 8Br-cAMP (0.1 mM) for 30 min as indicated. Western blots (WB) of whole cell lysates were developed using a phosphoFAK 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.

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 ETAR (31)2. This selective or dual activation is documented by the results presented in Table 3 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 Figure 10 show that the GPCR-mediated stimulation of Gs and Gq/11 (with hCG) or the selective stimulation of Gq/11 (with ET-1) can increase FAK-Y576 phosphorylation to about the same extent. Finally, to determine if 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 8Br-cAMP only, or with a combination of both. The results presented in Figure 10 show that FAK-Y576 phosphorylation can be clearly enhanced by addition of 8Br-cAMP to ET1-stimulated MA-10 cells.

Table 3.

Effect of hCG and ET-1 on cAMP and inositol phosphate accumulation in MA-10 cells

MA-10 cells were co-transfected with the hLHR and the ETAR expression vectors (1 μg each/35 mm well). The transfected cells were then incubated in 1 ml of assay medium supplemented with buffer only, hCG (1000 ng/ml), or ET-1 (0.1 μM). Cyclic AMP and inositol phosphates were measured at the end of a 30 min or 60 min incubation, respectively, as described in Materials and Methods.

Additions cAMP (pmol/106cells) Inositol phosphates (cpm/106cells)
Buffer 52 ± 13 1861 ± 242
hCG 2021 ± 50* 13399 ± 1861*
ET-1 70 ± 22 23263 ± 2233*
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Each number is the mean ± SEM of three independent transfections. Asterisks denote statistically significant differences (p < 0.05, paired t test) from cells incubated with buffer only.

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

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. 12–15). 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 (12–15). 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 (12–15).

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 (12–15). 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 (12–15).

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 (Figures 1–5). 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 1), 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 (Figures 7 and 8). A dominant-negative mutant of Yes was not effective in inhibiting the hCG-induced phosphorylation of FAK-Y576, however (Figure 8). 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 to the expression of the dominant-negative Fyn (compare lower panels in Figure 8). It is also possible that the lack of effect of the dominant-negative Yes is due to the misslocalization 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 Figure 3) 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 the activities of FAK (12–15). 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 (12–15). Since 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 (Figure 3).

A number of GPCRs (but not the LHR) have been previously shown to stimulate the phosphorylation and/or activities of non-receptor tyrosine kinases such as the SKFs and FAK (reviewed in refs. 17, 18, 20, 22, 34). Pathways that may mediate the GPCR-induced activation of non-receptor tyrosine kinases include (a) the transactivation of integrin receptors; (b) the transactivation of EGF receptors; (c) a direct physical association of GPCRs and the tyrosine kinases; (d) the formation of a tyrosine kinases/βarrestin/GPCR complexes; (e) direct activation of the tyrosine kinases by Gα subunits; and (f) 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 (Figure 3). FAK-Y397 is the principal FAK residue phosphorylated in response to the activation of integrin receptors (Figure 3 and refs. 12–15). Since we cannot detect an increase in the phosphorylation of the endogenous EGF receptor upon addition of hCG to MA-10 cells (Figure 1) 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 βarrestins 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. Lastly, the ability of the β2-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 (Figures 9 and 10). In fact, it appears that the LHR-induced activation of Gs and/or Gq/11 could be required for optimal phosphorylation of FAK-Y576 (Figures 9 and 10). Since Src and Lck (another member of the SFKs) have been shown to be direct effectors of some Gα subunits (23, 24) future experiments need to consider the possibility that Fyn and/or Yes are directly activated by the Gαs and/or Gαq/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 (Figure 10). 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 of that a protein kinase A and/or 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 non-receptor 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. Since 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, are 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

Plasmids and cells

The origin and handling of MA-10 cells have been described (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 mycepitope at the N-terminus has been described (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 (41) ((42). These were generously provided by J.L. Benovic (Thomas Jefferson University) and modified by us with the FLAG epitope (42). Expression vectors for constitutively active (i.e., GTPase-deficient) mutants of Gαs, Gαq, Gα11, Gαi and Gαo and for the endothelin type A receptor (ETAR) were purchased from the 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).

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% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 7.4) supplemented with an EDTA-free protease inhibitor cocktail from Roche Applied Science (www.roche-applied-science.com), 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 anti-phosphotyrosine antibody (4G10 from Upstate Biotechnology) that had been pre-bound to 50 μl of protein G Sepharose (Santa Cruz Biotechnology). After an overnight incubation at 4°C the beads were collected by centrifugation, washed 3 times with 1 ml aliquots of RIPA buffer, 3 times with 1 ml aliquots of the RIPA buffer with 1 M NaCl and then again 3 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 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 as indicated in the figures and tables one day after plating. Transfections were done in 1 ml of OPTIMEM supplemented with 700 μg/ml CaCl2.2H2O. 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 three-hour incubation each well received 150 μl of horse serum and the incubation was continued for another 16–24 hours. The medium was then replaced with assay medium (RPMI-1640 medium supplemented with 1 mg/ml bovine serum albumin, 20 mM Hepes and 50 μg/ml gentamicin, pH 7.4) and the cells were incubated in this medium for another 16–18 hours. 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., Hercules, CA). Finally, immune complexes were visualized and quantified using the Super Signal West Femto Maximum Sensitivity detection system (Pierce Chemical Inc, 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 (www.biosource.com) or Cell Signaling Technology (www.cellsignal.com) using a 2 hour incubation of the membranes with a 1:1,000 or a 1:2,000 dilution of antibody. Total FAK was detected with an antibody from Upstate Biotechnology (www.upstatebiotech.com) also using a 2 hour incubation of the membranes with a 1:2,000 dilution of antibody. Antibodies to Src, Fyn and Yes were also from Upstate Biotechnology (www.upstatebiotech.com) and were used to develop blots during an overnight incubation at 4°C at dilutions of 1:250, 1:1,000, and 1:1,000, respectively. The blot shown in Figure 1 was developed during an overnight incubation at room temperature with an anti-phosphotyrosine antibody (4G10, also from Upstate Biotechnology) used a 1:1,000 dilution. The different Gα subunits were visualized by incubating the membranes with the appropriate primary antibodies (described in ref. 10) at a dilution of 1:300 for Gαs, 1:200 for Gαq/11 and 1:1000 for Gαi-3/Gαo, antibodies, respectively. Paxillin and paxillin phosphorylated on Tyr118 were visualized with antibobies from Cell Signaling Technology (www.cellsignal.com) used at a dilution of 1:1,000.

For the experiment presented in Figure 3B 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 prior to 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 prior to 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 prior to lysis. Fibronectin coating was accomplished by adding a 50 μg/ml solution of fibronectin (in assay medium) to the dishes for 45 minutes. 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. One day after transfection the medium was replaced with assay medium (see above) and the cells were incubated in this medium for 16–18 hours. 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 1. At the end of this incubation the medium was aspirated and the cells were lysed in 700 μl of lysis buffer (1% NP40 in 150 mM NaCl, 25 mM TrisCl, 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. (Hercules, CA). Five hundred μl aliquots 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 pre-bound 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 TrisCl 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 TrisCl 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-γ]ATP (from Perkin Elmer) in 75 mM MnCl2, 500 μM ATP, 25 mM β-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol, 20 mM MOPS, pH 7.2. After 30 min at 30°C each tube received 20 μl of 40% TCA and the incubation was continued for 5 min at room temperature. Twenty five μl aliquots of each reaction were then spotted on small squares of P81 phosphocellulose paper. These were washed 5 times (5 minutes each) with 5 ml of 0.75% phosphoric acid and once with 3 ml of acetone (3 min) prior to counting on a liquid scintillation counter. The cpm 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 was obtained from Invitrogen. Other cell culture supplies and reagents were obtained from Corning and Invitrogen, respectively. 8Br-cAMP, PMA, arginine vasopressin (AVP), pertussis toxin, endothelin 1 (ET-1) and recombinant EGF were from Sigma. 8CPT-cAMP and 8CPT-2Me-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

*

Supported by a grant from the National Cancer Institute (CA-40629).

1

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

2

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

3

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

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