Src Family Kinases Are Required for Integrin-mediated but Not for G Protein-coupled Receptor Stimulation of Focal Adhesion Kinase Autophosphorylation at Tyr-397*

Plating suspended Swiss 3T3 cells onto fibronectin-coated dishes promoted phosphorylation of endogenous focal adhesion kinase (FAK) at Tyr-397, the major autophosphorylation site, and at Tyr-577, located in the activation loop, as revealed by site-specific antibodies that recognize the phosphorylated form of these residues. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 (PP-2) markedly reduced the phosphorylation of both Tyr-397 and Tyr-577 induced by fibronectin. Furthermore, fibronectin-mediated FAK phosphorylation at Tyr-397 was dramatically reduced in SYF cells (deficient in Src, Yes, and Fyn expression). Stimulation of Swiss 3T3 cells with bombesin also induced a rapid increase in the phosphorylation of endogenous FAK at Tyr-397. In contrast to the results obtained with fibronectin, PP-2 did not prevent FAK Tyr-397 phosphorylation stimulated by bombesin at a concentration (10 μm) that suppressed bombesin-induced FAK Tyr-577 phosphorylation. Similarly, PP-2 did not prevent Tyr-397 phosphorylation in Swiss 3T3 cells stimulated with other G protein-coupled receptor agonists including vasopressin, bradykinin, endothelin, and lysophosphatidic acid. Lysophosphatidic acid also induced FAK phosphorylation at Tyr-397 in SYF cells. Our results identify, for first time, the existence of Src-dependent and Src-independent pathways leading to FAK autophosphorylation at Tyr-397 stimulated by adhesion-dependent signals and G protein-coupled receptor agonists in the same cell.

A rapid increase in the tyrosine phosphorylation of the nonreceptor tyrosine kinase FAK 1 (1,2), which localizes to focal adhesion plaques, has been identified as a prominent early event in cells stimulated by diverse signaling molecules that regulate cell proliferation, migration, and apoptosis (3)(4)(5). In particular, FAK is activated and tyrosine-phosphorylated in response to integrin clustering induced by cell adhesion or antibody cross-linking (1, 2, 6 -9). In addition, FAK is rapidly tyrosine-phosphorylated in cells stimulated by mitogenic neuropeptide agonists including bombesin (10 -16) and bioactive lipids including LPA (17)(18)(19) that act via heptahelical GPCRs, polypeptide growth factors (20 -24), bacterial toxins (25,26), and activated variants of pp60 Src (27,28). The importance of FAK-mediated signal transduction is underscored by experiments implicating this tyrosine kinase in embryonic development (29) and in the control of cell migration (30 -33), proliferation (30,34), and apoptosis (35,36). In addition, there is increasing evidence linking overexpression of FAK to the invasive properties of cancer cells (37,38). It is increasingly recognized that the tyrosine phosphorylation and activation of FAK is an important point of convergence in the action of integrins, GPCR agonists, growth factors, and oncogenes (5,39,40). However, the molecular pathways leading to FAK tyrosine phosphorylation in response to multiple extracellular factors remain incompletely understood.
Plating suspended cells onto fibronectin-coated dishes, a paradigm of integrin receptor activation (41), induces adhesion-dependent phosphorylation of FAK at multiple sites including tyrosines 397, 576, 577, 861, and 925 (32,(42)(43)(44)(45). Tyr-397, the only apparent autophosphorylation site (46 -51), has emerged as a critical residue in FAK-mediated signaling (5). The autophosphorylation of FAK at Tyr-397 creates a high affinity binding site for the SH2 domain of Src family kinases including Src, Yes, and Fyn and leads to the formation of a signaling complex between FAK and Src family kinases (46, 47, 49 -52). A model has recently been proposed that envisages reciprocal catalytic activation of FAK and Src family kinases in response to adhesiondependent signals. Src family kinases associated with FAK are thought to phosphorylate FAK at additional sites including Tyr-576 and Tyr-577 that are located in the activation loop of the kinase catalytic domain of FAK (32,42,53), thereby promoting maximal FAK catalytic activation. Because phenylalanine mutation of Tyr-576 and Tyr-577 reduced adhesion-mediated FAK autophosphorylation (at Tyr-397), it has been proposed that activation loop phosphorylation of FAK by Src stimulates intermolecular phosphorylation at Tyr-397, thereby leading to signal amplification at sites of integrin-mediated cell adhesion (32). Therefore, in this model Src family kinases are thought to play a major role leading to autophosphorylation of FAK at Tyr-397 as part of an adhesion-dependent signaling response. Recently, we demonstrated that GPCR agonists including bombesin and LPA also induce rapid activation of Src family kinases (54) and promote the formation of a FAK/Src signaling complex (55). However, it is not known whether Src family kinases are also required for promoting FAK phospho-rylation at Tyr-397 in cells stimulated with GPCR agonists, as would be predicted by the signal amplification model.
In the present study, we report that stimulation with the mitogenic GPCR agonists bombesin, bradykinin, endothelin, vasopressin, and LPA induced rapid phosphorylation of endogenous FAK at Tyr-397 in intact Swiss 3T3 cells, an effect comparable to that stimulated by integrin-mediated cell adhesion. Treatment with the selective Src family kinase inhibitor PP-2 inhibited the phosphorylation of Tyr-397 induced by fibronectin. Furthermore, integrin-mediated FAK phosphorylation at Tyr-397 was dramatically reduced in SYF cells (deficient in Src, Yes, and Fyn expression). In striking contrast, PP-2, at a concentration that abolished activation loop phosphorylation, did not prevent FAK Tyr-397 phosphorylation in Swiss 3T3 cells stimulated by bombesin, LPA, or other GPCR agonists. In addition, LPA also induced FAK phosphorylation at Tyr-397 in SYF cells. Our results demonstrate, for the first time, that the signaling events leading to the phosphorylation of FAK at Tyr-397 induced by GPCR agonists are Src-independent and thus can be distinguished from those stimulated by integrin receptors that require Src family kinase activity.

EXPERIMENTAL PROCEDURES
Cell Culture-Stock cultures of Swiss 3T3 cells were maintained in DMEM, supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO 2 and 90% air at 37°C. For experimental purposes, Swiss 3T3 cells were plated in 100-mm dishes at 6 ϫ 10 5 cells/dish in DMEM containing 10% fetal bovine serum and used after 6 -8 days when the cells were confluent and quiescent.
SYF cells (deficient in Src, Yes, and Fyn expression) and YF cells (deficient in Yes and Fyn expression) were obtained from ATCC (CRL-2459 and CRL-2497). Stock cultures of these cells were maintained in DMEM, supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO 2 and 90% air at 37°C. For experimental purposes, SYF and YF cells were plated in 100-mm dishes at 7 ϫ 10 5 cells/dish in DMEM containing 10% fetal bovine serum and were used after 5 days when the cells were confluent.
Cell Stimulation with Bombesin and Other Agonists-Confluent and quiescent Swiss 3T3 cells were washed twice with DMEM, equilibrated in the same medium at 37°C for at least 30 min, and then treated with bombesin or other agonists for the times indicated. The stimulation was terminated by aspirating the medium and solubilizing the cells in 1 ml of ice-cold RIPA buffer containing 50 mM HEPES, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10% glycerol, 1.5 mM MgCl 2 , 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM NaF, and 1 mM phenylmethylsulfonyl fluoride.
Confluent cultures of SYF and YF cells were incubated for 40 h in DMEM containing 0.1% fetal bovine serum and then treated as described above for Swiss 3T3 cells.
Cell Stimulation with Fibronectin or Adherence to Poly-L-lysine-Confluent and quiescent Swiss 3T3 cells were harvested by limited trypsin/EDTA treatment (0.05% trypsin, 2 mM EDTA in PBS). The trypsin was inactivated by soybean trypsin inhibitor (0.5 mg/ml) with 0.25% bovine serum albumin in DMEM. Cells were collected by centrifugation, resuspended in DMEM containing 0.1% bovine serum albumin, and held in suspension for 1 h at 37°C. Cell culture dishes (10 cm) were pre-coated with fibronectin purified from bovine plasma (10 g/ml) or poly-L-lysine (100 g/ml) in PBS overnight at 4°C, rinsed with PBS, and warmed to 37°C for 1 h prior to plating. Suspended cells were distributed onto ligand-coated dishes and incubated at 37°C, and at various times following plating, protein extracts were made in RIPA buffer as described previously. Confluent cultures of SYF and YF cells were incubated for 40 h in DMEM containing 0.1% fetal bovine serum and then treated as described for Swiss 3T3 cells.
Western Blotting-After SDS-PAGE, proteins were transferred to Immobilon membranes. After transfer, membranes were blocked using 5% nonfat dried milk in PBS, pH 7.2, and incubated overnight at 4°C with the anti-FAK-Tyr(P)-397 Ab (0.1 g/ml) or anti-FAK-Tyr(P)-577 Ab (0.1 g/ml), as indicated. The membranes were washed three times with PBS, 0.1% Tween 20 and then incubated with secondary antibodies (horseradish peroxidase-conjugated donkey antibodies to rabbit, NA 934) (1:5000) for 1 h at 22°C. After washing three times with PBS, 0.1% Tween 20, the immunoreactive bands were visualized using ECL detection reagents. Autoradiograms were scanned using the GS-710 Calibrated Imaging Densitometer (Bio-Rad), and the labeled bands were quantified using the Quantity One software program (Bio-Rad).
Materials-Bombesin, endothelin, bradykinin, vasopressin, LPA, bovine fibronectin, and poly-L-lysine were obtained from Sigma. Horseradish peroxidase-conjugated donkey antibodies to rabbit, NA 934, and ECL reagents were from Amersham Pharmacia Biotech. PP-2 and PP-3 were obtained from Calbiochem-Novabiochem. FAK polyclonal Ab C-20 was from Santa Cruz Biotechnology (Santa Cruz, CA). The phosphospecific polyclonal Abs to Tyr-397 and Tyr-577 of FAK were obtained from BIOSOURCE International (Camarillo, CA). All other reagents used were of the purest grade available.

RESULTS AND DISCUSSION
The Role of Src Family Kinase Activity in FAK Phosphorylation at Tyr-397 in Response to Fibronectin-In order to examine the role of Src family kinases in integrin-mediated phosphorylation of FAK at Tyr-397, we determined the effect of the pyrazolopyrimidine PP-2, a selective inhibitor of Src family kinase members (56,57), on the phosphorylation FAK at Tyr-397 induced by fibronectin. Previously, we demonstrated that this compound, at concentrations that completely inhibited the catalytic activity of Src family kinases, did not interfere with FAK autophosphorylation activity (54,57). Cultures of Swiss 3T3 cells placed in suspension were treated in the absence or in the presence of increasing concentrations of PP-2 (0.1-10 M) and then plated onto dishes coated with either fibronectin or poly-L-lysine for 30 min. Cell lysates were immunoprecipitated with anti-FAK Ab, and the immune complexes were analyzed by SDS-PAGE followed by Western blotting with a phosphospecific antibody directed against the autophosphorylation site of FAK (anti-FAK-Tyr(P)-397 Ab). As shown in Fig. 1A, treatment of cells with PP-2 strikingly inhibited the phosphorylation of FAK at Tyr-397 in a concentration-dependent fashion. Maximal inhibition was obtained at 10 M. In contrast, treatment with 10 M PP-3, a structurally related but inactive analog of PP-2, did not interfere with integrin-mediated FAK phosphorylation at Tyr-397 (Fig. 1B).
FAK autophosphorylation at Tyr-397 creates a high affinity binding site for the SH2 domain of Src, and Src associated with FAK is thought to phosphorylate FAK at additional sites including Tyr-576 and Tyr-577, which are located in the kinase catalytic domain of FAK. Treatment with 10 M PP-2 completely blocked the phosphorylation of FAK Tyr-577 induced by fibronectin (inset, Fig. 1B, right). These results suggest that Src family kinases are required for fibronectin-induced FAK phosphorylation at both activation loop and autophosphorylation sites.
FAK Phosphorylation at Tyr-397 in Response to Fibronectin Is Greatly Diminished in Cells Deficient in Src, Yes, and Fyn (SYF Cells)-Recently, Klinghoffer et al. (58) reported that integrin-induced tyrosine phosphorylation of FAK was markedly reduced in SYF cells, but the phosphorylation of specific sites was not analyzed. In order to substantiate that Src family kinases are required for FAK Tyr-397 phosphorylation induced by fibronectin, as suggested by the pharmacological results shown in Fig. 1A, we examined integrin-induced FAK Tyr-397 phosphorylation in SYF cells and in cells deficient in Yes and Fyn but not in Src (YF cells). As illustrated in Fig. 2, plating YF cells (which express c-Src) onto fibronectin induced a marked increase in FAK Tyr-397 phosphorylation as compared with either parallel cultures plated on poly-L-lysine or to SYF cells plated on fibronectin. The increase in FAK Tyr-397 phosphorylation induced by fibronectin in YF cells was virtually abrogated by treatment with 10 M PP-2. In contrast, plating SYF cells on fibronectin induced only a small (but measurable) increase in FAK Tyr-397 phosphorylation, as compared with SYF cells plated on poly-lysine. Interestingly, the small increase in fibronectin-induced FAK Tyr-397 phosphorylation in the absence of Src, Yes, and Fyn was not affected by treatment with 10 M PP-2. Taken together, the results presented in Figs. 1 and 2 indicate that Src family kinases are required for maximal integrin-induced FAK autophosphorylation and substantiate the specificity of PP-2 as an Src family inhibitor since this agent did not exert any inhibitory effect on FAK Tyr-397 phosphorylation in cells lacking Src, Yes, and Fyn.
Bombesin Stimulates FAK Phosphorylation at Tyr-397 through an Src-independent Pathway-Although an increase in the phosphorylation of FAK at Tyr-397 in response to an integrin stimulus has been well documented (45,59), the effect of GPCR agonists on the phosphorylation of this residue has not been demonstrated. In order to determine whether bombesin induces FAK phosphorylation at Tyr-397 in Swiss 3T3 cells, quiescent cultures of these cells were treated with 10 nM bombesin for various times and lysed. The extracts were im- munoprecipitated with a polyclonal anti-FAK Ab, which recognizes the C-terminal sequence of FAK, and the immune complexes were analyzed by SDS-PAGE followed by Western blotting using a site-specific antibody (anti-FAK-Tyr(P)-397) that recognizes the phosphorylated state of FAK at Tyr-397.
As shown in Fig. 3A (upper panel), bombesin stimulation of Swiss 3T3 cells induced a rapid and marked increase in the phosphorylation of FAK at Tyr-397. Densitometric scanning showed that the phosphorylation of this residue reached a maximum 2 min after the addition of bombesin to intact cells. Immunoblotting with anti-FAK antibody of FAK immunoprecipitates verified that similar amounts of FAK were recovered after different times of bombesin treatment (Fig. 3A, lower  panel). These results demonstrate that bombesin induces FAK phosphorylation at Tyr-397, the major autophosphorylation site of FAK that plays a critical role in FAK signaling.
To determine whether Src family kinase activity is required for FAK phosphorylation at Tyr-397 induced by bombesin, cultures of Swiss 3T3 cells were treated in the absence or in the presence of increasing concentrations of PP-2 and then stimulated with this agonist. As shown in Fig. 3B, exposure to PP-2 did not reduce the level of phosphorylation at Tyr-397 induced by bombesin stimulation, even at a concentration (10 M) that prevented integrin-induced phosphorylation of FAK at Tyr-397 and Tyr-577 (Fig. 1). In fact, the level of bombesin-induced FAK Tyr-397 phosphorylation in cells treated with PP-2 was indistinguishable from that obtained in control (untreated) cultures or in cultures treated with the inactive analog PP-3 (Fig. 3B,   FIG. 3. Bombesin stimulates FAK phosphorylation at Tyr-397 through an Src-independent pathway in Swiss 3T3 cells. A, confluent and quiescent Swiss 3T3 cells were stimulated with 10 nM bombesin for various times at 37°C, as indicated and subsequently lysed. FAK phosphorylation at Tyr-397 was analyzed by immunoprecipitation using anti-FAK antibody C-20 followed by Western blotting with anti-FAK-Tyr(P)-397 Ab. B, confluent and quiescent cells were treated for 15 min in the absence (0) or in the presence of increasing concentrations of PP-2, as indicated, and then stimulated without or with 10 nM bombesin (BOM, closed symbols) for a further 10 min and subsequently lysed. FAK phosphorylation at Tyr-397 was analyzed by immunoprecipitation using anti-FAK antibody C-20 followed by Western blotting with anti-FAK-Tyr(P)-397. Inset, confluent and quiescent Swiss 3T3 cells were treated for 15 min either in the absence (Ϫ) or in the presence (ϩ) of 10 M of PP-2 or 10 M of PP3. Cells were then incubated for a further 10 min either in the absence (Ϫ) or in the presence (ϩ) of 10 nM of bombesin (BOM), as indicated. The cells were then lysed and the extracts were incubated with anti-FAK antibody C-20, followed by Western blotting with anti-FAK-Tyr(P)-397 Ab. C, confluent and quiescent Swiss 3T3 cells were treated for 15 min in the absence or in the presence of increasing concentrations of PP-2 as indicated, and then stimulated without or with 10 nM of bombesin for a further 10 min and subsequently lysed. FAK phosphorylation at Tyr-577 was analyzed by immunoprecipitation using anti-FAK antibody C-20 followed by Western blotting with anti-FAK-Tyr(P)-577 Ab. In all cases, the membranes were analyzed further by Western blotting using anti- inset). We verified that similar amounts of FAK were recovered after treatment with or without bombesin and with or without PP-2 and PP-3 (Fig. 3B, lower panels).
Western blotting of FAK immunoprecipitates with the sitespecific antibody anti-FAK-Tyr(P)-577 revealed that bombesin stimulation also induced phosphorylation of FAK at Tyr-577 in Swiss 3T3 cells (Fig. 3C). Treatment of Swiss 3T3 cells with increasing concentrations of PP-2 prevented phosphorylation of FAK at Tyr-577 induced by bombesin in a concentration-dependent manner. Half-maximal and maximal inhibitory effects were achieved at 2 and 10 M, respectively. We verified that similar amounts of FAK were recovered after treatment with increasing concentrations of PP-2 (Fig. 3C). Thus, PP-2, at a concentration that completely blocked FAK activation loop phosphorylation (Fig. 3C), did not prevent FAK autophosphorylation at Tyr-397 stimulated by bombesin (Fig. 3B). Figs. 1-3 indicate that Src family kinases are required for fibronectin but not for bombesin receptor stimulation of FAK phosphorylation at Tyr-397. In order to substantiate further the existence of Src-dependent and -independent pathways leading to phosphorylation of FAK at Tyr-397, we examined the effect of PP-2 on the phosphorylation of FAK at Tyr-397 in Swiss 3T3 cells stimulated with either bombesin or fibronectin for various lengths of time.

Differential Contribution of Src Family Kinase Activity to FAK Phosphorylation at Tyr-397 in Response to Bombesin and Fibronectin-The results presented in
As shown in Fig. 4A, exposure to PP-2 markedly attenuated the phosphorylation of FAK at Tyr-397 in cells plated onto fibronectin-coated dishes at all the times examined, supporting the participation of Src in FAK autophosphorylation induced by adhesion-dependent signals. In contrast, treatment with PP-2 did not affect the phosphorylation of FAK at Tyr-397 in response to bombesin stimulation for various times (Fig. 4B). We verified that similar amounts of FAK were recovered after different times of exposure to fibronectin, bombesin, or PP-2 ( Fig. 4, lower panel). These results substantiate further the notion that Src family kinases are required for fibronectin but not for bombesin receptor stimulation of FAK phosphorylation at Tyr-397.
Role of Src in FAK Phosphorylation at Tyr-397 and Tyr-577 in Response to Vasopressin, Bradykinin, LPA, and Endothelin-The preceding results with bombesin prompted us to determine the role of Src family kinase activity in the phosphorylation of FAK at Tyr-397 and Tyr-577 induced by activation of other endogenously expressed GPCRs in Swiss 3T3 cells. Cultures of these cells were treated in the absence or in the presence of 10 M PP-2 and then stimulated with 20 nM vasopressin, 20 nM bradykinin, 2 M LPA, or 20 nM endothelin for 10 min and lysed. The lysates were immunoprecipitated with anti-FAK Ab, and the immune complexes were analyzed by SDS-PAGE followed by Western blotting with either anti-FAK-Tyr(P)-397 or anti-FAK-Tyr(P)-577 Abs. As shown in Fig. 5, cell stimulation with these agonists induced a marked increased in the phosphorylation of FAK at both Tyr-397 and Tyr-577. Treatment with 10 M PP-2 completely prevented the increase in the phosphorylation of FAK at Tyr-577 (Fig. 5A) but did not affect the phosphorylation of FAK at Tyr-397 in response to vasopressin, bradykinin, LPA, or endothelin (Fig. 5B). We verified that similar amounts of FAK were recovered after treatment with these GPCR agonists with or without PP-2.
Since previous results indicated that SYF and YF cells express receptors for LPA (58), we also examined whether this agonist induces FAK Tyr-397 phosphorylation in these cells. As illustrated in Fig. 6, FAK Tyr-397 phosphorylation was enhanced by LPA stimulation in both SYF cells and YF cells to the same degree. In addition, treatment with PP-2 did not exert any detectable inhibitory effect on LPA-induced FAK Tyr-397 phosphorylation in either SYF or YF cells. These results indicate that activation loop phosphorylation by Src family kinases is not required for the tyrosine phosphorylation of FAK at Tyr-397 triggered by a variety of GPCR agonists.
Concluding Remarks-A rapid increase in the overall tyrosine phosphorylation of the non-receptor tyrosine kinase FAK has been extensively documented as an early event in the action of multiple extracellular stimuli that modulates cell growth, motility, differentiation, and apoptosis in a variety of cell types. It is increasingly recognized that the function of FAK in signal transduction depends on the phosphorylation of specific residues in this enzyme. In particular, autophosphorylation of FAK at Tyr-397 has emerged as a crucial event in FAK-mediated signal transduction. The phosphorylation of FAK at Tyr-397 triggers the formation of molecular complexes with other signaling proteins including Src family kinases (5), the p85 regulatory subunit phosphatidylinositol 3-kinase (60), phospholipase C␥-1 (61), the adapter proteins Grb-7 (62) and Shc (53), and the tumor suppressor PTEN (63). These findings suggest that FAK autophosphorylation promotes the activation of multiple effector pathways.
The best characterized function of FAK autophosphorylation at Tyr-397 is the creation of a high affinity binding site for the SH2 domain of Src family members. Given that competition for the SH2 and/or SH3 domains of Src by high affinity allosteric ligands promotes enzymatic activation of this kinase (64,65), the association of Src with FAK should lead to the formation of a molecular complex in which Src kinases are activated. A model has recently been proposed that envisages reciprocal catalytic activation of FAK and Src family kinases in response to adhesion-dependent signals (32). In the framework of this model, Src activated by binding to FAK Tyr-397 phosphorylates FAK at Tyr-576 and -577 which are located in the activation loop of the kinase catalytic domain of FAK (42,53) and thereby promotes maximal FAK catalytic activity. Because FAK mutants in which Tyr-576 and Tyr-577 were substituted by phenylalanines show substantially reduced FAK Tyr-397 phosphorylation, relative to that of FAK, it has been proposed that activation loop phosphorylation FAK by Src family kinases stimulates intermolecular phosphorylation of FAK at Tyr-397, thereby leading to signal amplification at sites of integrinmediated cell adhesion (32,45). The results presented here demonstrate that treatment with the selective Src family kinase inhibitor PP-2, at concentrations that suppress activation loop phosphorylation, markedly attenuates FAK Tyr-397 phosphorylation in Swiss 3T3 cells plated onto fibronectin-coated dishes. Moreover, FAK phosphorylation at Tyr-397 in response to integrin engagement is drastically reduced in triple mutant cells lacking Src, Yes, and Fyn. Taken together, these findings are consistent with a model in which integrin-induced FAK autophosphorylation at Tyr-397 requires Src family kinase function.
Despite its importance, little is known about the effect of GPCR agonists on the phosphorylation of FAK at specific tyrosines, especially Tyr-397, the major site of autophosphorylation. Our results demonstrate that stimulation of intact Swiss 3T3 cells with bombesin induces a rapid increase in FAK phosphorylation at Tyr-397. Stimulation with other GPCR agonists including endothelin, vasopressin, bradykinin, and LPA also stimulates the phosphorylation of FAK at specific residues, the critical Tyr-397 as well as Tyr-577, as revealed using antibodies that specifically recognized these residues in their phosphorylated form. Our findings show that activation of GPCRs induces rapid multisite tyrosine phosphorylation of endogenously expressed FAK in attached Swiss 3T3 cells, i.e. without overexpressing FAK or Src and without subjecting the cells to detachment and subsequent replating.
In view of the results obtained with integrin-stimulated cells, we also examined whether Src family kinases are required for FAK phosphorylation at Tyr-397 in response to bombesin or other GPCR agonists. Our results demonstrate that treatment with PP-2, a selective inhibitor of Src family kinases, completely prevents the increase in FAK Tyr-577 phosphorylation induced by bombesin, in line with the notion that Src family kinases mediate phosphorylation of the activation loop of FAK. However, in striking contrast to the results obtained with integrin-stimulated cells, treatment with PP-2, at a concentration that abolished bombesin-induced FAK phosphorylation at Tyr-577, did not affect the phosphorylation of Tyr-397 in response to this agonist. Similarly, we found that PP-2 abrogated Tyr-577 phosphorylation in response to bradykinin, endothelin, vasopressin, and LPA but did not prevent the increase in FAK phosphorylation at Tyr-397 induced by these GPCR agonists. In agreement with these results, FAK Tyr-397 phosphorylation was enhanced by LPA stimulation in both SYF cells and YF cells. Thus, GPCR-mediated FAK phosphorylation at Tyr-397, unlike that promoted by integrin binding to fibronectin, is independent of Src-mediated activation loop phosphorylation.
In conclusion, the results presented here indicate that phosphorylation of FAK at Tyr-397 induced by bombesin and other GPCRs is Src-independent, whereas the phosphorylation of this residue promoted via integrin-mediated cell adhesion requires Src family kinase function. Our results identify, for first time, the existence of distinct pathways leading to FAK phosphorylation at Tyr-397 stimulated by GPCR agonists and adhesion-dependent signals in the same cell.