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J Biol Chem, Vol. 274, Issue 40, 28371-28378, October 1, 1999

Bombesin and Platelet-derived Growth Factor Induce Association of Endogenous Focal Adhesion Kinase with Src in Intact Swiss 3T3 Cells^*

Eduardo Perez Salazar and Enrique Rozengurt^§

From the Department of Medicine, School of Medicine and Molecular Biology Institute, UCLA, Los Angeles, California 90095

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Stimulation of quiescent Swiss 3T3 cells with bombesin induces a rapid increase in the formation of complexes between focal adhesion kinase (FAK) and Src family members, which can be extracted with a buffer containing Triton, deoxycholate, and SDS but not with a buffer containing Triton alone. An increase in complex formation between FAK and Src in response to bombesin could be detected within 1 min, reached a maximum after 10 min, and declined toward base-line levels after 60 min of bombesin treatment. Bradykinin, endothelin, and lysophosphatidic acid also stimulated FAK-Src complex formation. Bombesin stimulated FAK/Src association through a Ca²⁺ and phosphatidylinositol 3'-kinase-independent pathway that requires the integrity of the actin filament network and is partly dependent on functional protein kinase C. Treatment with the selective Src kinase inhibitor PP-2 inhibited both FAK activation and phosphorylation of FAK at Tyr⁵⁷⁷ induced by bombesin in intact cells. Platelet-derived growth factor at low concentrations (1-10 ng/ml) also induced FAK-Src complex formation via a pathway that depended on the integrity of the actin cytoskeleton and phosphatidylinositol 3'-kinase. Thus, G protein-coupled receptor agonists and platelet-derived growth factor promote complex formation between endogenous FAK and Src in attached cells through different signal transduction pathways.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Neuropeptides stimulate DNA synthesis and cell proliferation in cultured cells and are implicated as growth factors in a variety of fundamental processes including development, inflammation, tissue regeneration, and tumorigenesis (1-3). In particular, bombesin and its mammalian counterpart gastrin-releasing peptide bind to a G-protein coupled receptor (GPCR)¹ (4, 5) that promotes G_q-mediated activation of  isoforms of phospholipase C (6, 7) to produce two second messengers: inositol 1,4,5-trisphosphate that mobilizes Ca²⁺ from internal stores and diacylglycerol that activates PKC (8-10). Subsequently, bombesin induces activation of phosphorylation cascades including p42^MAPK/p44^MAPK and p70^S6K (11-14), leading to increased expression of immediate early response genes, stimulation of cell cycle events, and subsequent cell proliferation (10, 15-18).

The binding of bombesin to its heptahelical GPCR also induces rapid tyrosine phosphorylation of multiple substrates in Swiss 3T3 cells (10, 19, 20), including activation and tyrosine phosphorylation of FAK (10, 21-23) and a transient increase in the kinase activity of Src (23, 24) through different pathways (25). Tyrosine phosphorylation of FAK is also increased by diverse signaling molecules that mediate cell growth and differentiation, including bioactive lipids such as LPA (26-28), polypeptide growth factors such as PDGF and insulin growth factor (29-31), bacterial toxins (32, 33), activated variants of Src (34, 35), and extracellular matrix proteins (36-39). These results indicate that FAK is a point of convergence in a variety of signal transduction pathways (40, 41). The importance of FAK-mediated signal transduction is underscored by recent experiments showing that this tyrosine kinase is implicated in embryonic development (42) and in the control of cell migration (43-45), proliferation (43, 46), and apoptosis (47-49).

Tyrosine phosphorylation plays a critical role in promoting the recruitment of active signaling molecules into multiprotein signaling networks (50). Because the major site of FAK autophosphorylation, Tyr³⁹⁷, is potentially a high affinity binding site for the SH2 domain of Src, phosphorylation of this site could facilitate the formation of a FAK-Src signaling complex in which both kinases are active (35, 51). However, FAK tyrosine phosphorylation is not sufficient for FAK/Src association, indicating the requirement for additional signals (52). Transformation by oncogenic variants of Src or plating suspended cells onto fibronectin-coated dishes, an artificial paradigm of integrin receptor activation (53), induces complex formation between FAK and Src (54-59). In contrast, little is known about the assembly of FAK signaling complexes in response to physiological concentrations of GPCR agonists in attached cells.

In the present study, we demonstrate that the mitogenic GPCR agonists bombesin, endothelin, bradykinin, and LPA induce a rapid increase in the formation of FAK-Src complexes in quiescent Swiss 3T3 cells. Bombesin stimulates FAK/Src association through a Ca²⁺ and PI 3-kinase-independent pathway that requires the integrity of the actin filament network and is partly dependent on functional PKC. In contrast, PDGF stimulates biphasic FAK/Src association via a PI 3-kinase-dependent pathway. Thus, GPCRs and tyrosine kinase receptors promote complex formation between endogenous FAK and Src in attached cells through different signal transduction pathways.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- Stock cultures of Swiss 3T3 cells were maintained in DMEM, supplemented with 10% fetal bovine serum in a humidified atmosphere containing 10% CO₂ and 90% air at 37 °C. For experimental purposes, Swiss 3T3 cells were plated in 100-mm dishes at 6 × 10⁵ cells/dish in DMEM containing 10% fetal bovine serum and used after 6-8 days when the cells were confluent and quiescent.

Immunoprecipitation-- Quiescent cultures of Swiss 3T3 cells were washed twice with DMEM, equilibrated in the same medium at 37 °C for at least 15 min, and then treated with bombesin or other factors in DMEM for the times indicated. We used 2 × 10⁶ cells grown in 100-mm dishes containing 10 ml of DMEM for each experimental condition. The stimulation was terminated by aspirating the medium and solubilizing the cells in 1 ml of ice-cold 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 MgCl₂, 1 mM EGTA, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 100 mM NaF, and 1 mM phenylmethylsulfonyl fluoride. In some experiments, the cells were lysed in a buffer without added SDS and/or sodium deoxycholate, as indicated in Fig. 1A.

Lysates were clarified by centrifugation at 14,000 rpm for 10 min, and the pellets were discarded. After centrifugation, supernatants were transferred to fresh tubes, and proteins were immunoprecipitated at 4 °C for 4 h with protein A-agarose linked to polyclonal anti-Src family (SRC-2) antibodies as described previously (19, 21, 24, 60). Immunoprecipitates were washed three times with lysis buffer containing 1% Triton, 1% deoxycholate, and 0.1% SDS and extracted in 2× SDS-PAGE sample buffer (200 mM Tris-HCl, pH 6.8, 1 mM EDTA, 6% SDS, 2 mM EDTA, 4% 2-mercaptoethanol, 10% glycerol), by boiling 10 min and resolved by SDS-PAGE.

Western Blotting-- After SDS-PAGE, proteins were transferred to Immobilon membranes. After transfer, membranes were blocked using 5% nonfat dried milk in phosphate-buffered saline, pH 7.2, and incubated for 2 h at 22 °C with the anti-FAK Ab (C-20) (0.1 µg/ml), anti-Tyr(P) Ab (PY20) (0.2 µg/ml), or anti-FAK-Tyr(P)⁵⁷⁷ (0.1 µg/ml), as indicated. The membranes were washed three times with phosphate-buffered saline, pH 7.2, 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 phosphate-buffered saline, pH 7.2, 0.1% Tween 20, the immunoreactive bands were visualized using ECL detection reagents. Autoradiograms were scanned using a ScanJet 6100C/T scanner (Hewlett Packard), and the labeled bands were quantified using the Multi-Analyst software program (Bio-Rad).

In Vitro Kinase Assay-- FAK immunoprecipitates were washed three times with lysis buffer, two times with lysis buffer without added SDS and sodium deoxycholate, and twice with FAK kinase buffer (20 mM HEPES, pH 7.35, 3 mM MgCl₂). Then the pellets were dissolved in 40 µl of kinase buffer supplemented with 1 µM PP-2 to eliminate transphosphorylation of FAK by co-immunoprecipitated Src during the in vitro kinase assays, as recently described (23). The reactions were started by adding 10 µCi of [-³²P]ATP, carried out at 30 °C for 10 min, and stopped on ice by adding EDTA to a final concentration of 10 mM. After centrifugation, the pellets were washed twice with lysis buffer containing 5 mM EDTA, extracted for 5 min at 95 °C in 2× SDS-PAGE sample buffer, and analyzed by SDS-PAGE. The gels were fixed and dried, and autoradiography was performed at 80 °C. Autoradiograms were scanned using a ScanJet 6100C/T scanner (Hewlett Packard), and the labeled band was quantified using the Multi-Analyst software program (Bio-Rad).

Materials-- Bombesin, endothelin, bradykinin, LPA, cytochalasin D, and GF I were obtained from Sigma. Recombinant PDGF (BB homodimer), horseradish peroxidase-conjugated donkey antibodies to rabbit (NA 934), and ECL reagent were from Amersham Pharmacia Biotech. Thapsigargin, wortmannin, and PP-2 were obtained from Calbiochem-Novabiochem Ltd. FAK polyclonal Ab C-20, Src family polyclonal Ab SRC-2, and anti-Tyr(P) monoclonal Ab PY20 were from Santa Cruz Biotechnology, Inc. FAK-Tyr(P)⁵⁷⁷ was from BIOSOURCE. All other reagents used were of the purest grade available.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bombesin Induces the Formation of a Complex between FAK and Src in Swiss 3T3 Cells-- To determine whether bombesin induces the formation of a complex between endogenous FAK and Src family members (collectively referred to as Src, unless otherwise indicated) in Swiss 3T3 cells, quiescent cultures of these cells were treated with or without bombesin for 10 min and lysed in buffer solutions containing 1% Triton in the absence or in the presence of deoxycholate with or without SDS. The extracts were immunoprecipitated with anti-Src antibody SRC-2, which recognizes the C-terminal sequence (residues 509-533) of the family members Src, Yes, and Fyn (the Src family members expressed in fibroblasts), and the immune complexes were analyzed by SDS-PAGE followed by Western blotting with anti-FAK antibody. As illustrated in Fig. 1A (upper panel), anti-FAK Western blotting of Src immunoprecipitates revealed an association of endogenous FAK with Src when bombesin-stimulated cells were lysed in a buffer containing Triton, deoxycholate, and SDS. In contrast, we did not detect significant FAK immunoreactivity in Src immunocomplexes when the cells were lysed in a buffer containing 1% Triton (Fig. 1A), although a substantial amount of Src was recovered in these lysates (Fig. 1A, lower panel), in agreement with our previous results (23, 24) and with results obtained using cells replated onto fibronectin (52). All subsequent experiments were performed in Swiss 3T3 cells lysed with a buffer containing Triton, deoxycholate, and SDS.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Bombesin induces FAK/Src association in the Triton-insoluble fraction of Swiss 3T3 cells. A, confluent and quiescent cells were treated at 37 °C with 2 nM bombesin for 10 min and subsequently lysed using a buffer containing 1% Triton without or with 0.1% SDS and 1% sodium deoxycholate (DOC), as indicated. FAK/Src association was analyzed by immunoprecipitation using anti-Src polyclonal antibody SRC-2 followed by Western blotting with anti-FAK Ab. B, confluent and quiescent cells were treated for 10 min in the absence () or in the presence (+) of 2 nM bombesin (BOM). The cells were then lysed in a buffer containing 1% Triton, 1% deoxycholate, and 0.1% SDS, and the lysates were analyzed by immunoprecipitation with anti-FAK Ab followed by Western blotting with anti-SRC2 Ab. The membranes were further analyzed by Western blotting using anti-FAK Ab. The position of Src family proteins is indicated by an arrow. The broad band under the Src band is immunoglobulin heavy chain. The position of FAK is indicated by an arrow. C, confluent and quiescent cells were treated for 10 min in the absence () or in the presence (+) of 2 nM bombesin (BOM), 20 nM bradykinin (BK), 10 nM endothelin (END), and 2 µM LPA. The cells were then lysed, and the lysates were analyzed by immunoprecipitation with anti-Src Ab followed by Western blotting with anti-FAK Ab. The membranes were further analyzed by Western blotting using anti-SRC-2 Ab. The positions of FAK and Src are indicated by the arrows. The results are representative of at least three independent experiments.

To substantiate further the existence of a FAK/Src interaction induced by bombesin, cell lysates were immunoprecipitated using an antibody directed against FAK followed by Western blot analysis to detect interacting Src. As illustrated in Fig. 1B, Src immunoreactivity was present in FAK immunoprecipitates produced from bombesin-treated cells. Anti-FAK Western blotting of anti-FAK immunoprecipitates confirmed that similar amounts of FAK protein were recovered after stimulation with or without bombesin (Fig. 1B). We did not detect any FAK/Src association when cell lysates obtained with buffer containing Triton, deoxycholate, and SDS were immunoprecipitated with nonimmune antisera (data not shown). These results indicate that bombesin induces the formation of a complex between endogenous FAK and Src in intact Swiss 3T3 cells.

To examine whether activation of other GPCRs also increases FAK/Src association, quiescent Swiss 3T3 cells were stimulated with 20 nM bradykinin, 10 nM endothelin, or 2 µM LPA for 10 min and lysed. The extracts were immunoprecipitated with anti-Src antibody SRC-2, and the immune complexes were analyzed by SDS-PAGE followed by Western blotting with anti-FAK antibody. As shown in Fig. 1C (upper panel), treatment with these GPCR agonists induced a marked increase in the association of endogenous FAK with Src that was comparable to that promoted by bombesin. Western blotting with anti-Src antibody of anti-Src immunoprecipitates confirmed that similar amounts of Src protein were recovered after treatment in the absence or in the presence of these agonists (Fig. 1C, lower panel).

Kinetics of Complex Formation between FAK and Src in Response to Bombesin-- The kinetics of FAK/Src association stimulated by bombesin in Swiss 3T3 cells is shown in Fig. 2A. An increase in complex formation between FAK and Src in response to bombesin could be detected within 1 min, reached a maximum after 10 min, and declined toward base-line levels after 60 min of bombesin treatment. In contrast, tyrosine phosphorylation of FAK in response to bombesin was detectable within seconds and persisted longer than FAK/Src association (Fig. 2B). These results imply that FAK tyrosine phosphorylation is not sufficient for FAK-Src complex formation in bombesin-stimulated Swiss 3T3 cells.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Time course and dose response of bombesin-induced FAK/Src association in Swiss 3T3 cells. A, confluent and quiescent cells were treated at 37 °C with 2 nM of bombesin for various times as indicated and were subsequently lysed in a buffer containing Triton, deoxycholate, and SDS. FAK-Src association was analyzed by immunoprecipitation using anti-Src polyclonal antibody SRC-2 followed by Western blotting with anti-FAK Ab. The membrane was analyzed further by Western blotting using anti-SRC-2 Ab. B, confluent and quiescent cells were treated at 37 °C with 2 nM bombesin for various times as indicated and subsequently lysed. Tyrosine phosphorylation of FAK was analyzed by immunoprecipitation using anti-FAK Ab followed by Western blotting with anti-Tyr(P) antibody PY20. The membrane was analyzed further by Western blotting using anti-FAK Ab. C, confluent and quiescent cells were treated at 37 °C for 10 min either in the absence or presence of various concentrations of bombesin as indicated, and cell lysates were analyzed for FAK-Src association as described above. The membrane was analyzed further by Western blotting using anti-SRC-2 Ab. The positions of FAK and Src are indicated by the arrows. The autoradiograms shown are representative of at least three independent experiments. Quantification of FAK associated with Src in A and C and tyrosine phosphorylation of FAK in B was performed by scanning densitometry. Values shown are the mean ± S.E. of at least three independent experiments and are expressed as the percentage of the maximal increase in FAK-Src association (A and B) or in tyrosine phosphorylation of FAK (B) above control (unstimulated) values.

Bombesin induced FAK-Src complex formation in a concentration-dependent manner; half-maximal effect was elicited at a concentration of 0.3 nM (Fig. 2C). Immunoblotting with anti-Src antibody of anti-Src immunoprecipitates verified that similar amounts of Src were recovered after different conditions (times and concentrations) of bombesin treatment (Fig. 2, A-C, lower panels).

Role of PKC and Ca²⁺ in the Association between FAK and Src Induced by Bombesin-- Bombesin promotes rapid G_q-mediated activation of phospholipase C to produce the second messengers inositol 1,4,5-trisphosphate that mobilizes Ca²⁺ from internal stores and diacylglycerol that activates PKC. Consequently, we examined the role of PKC and Ca²⁺ in bombesin-stimulated FAK-Src complex formation in Swiss 3T3 cells. As shown in Fig. 3A, direct stimulation of PKC with PDB for 10 min increased the association of FAK with Src, indicating that PKC is a potential signaling pathway leading to the formation of a complex between FAK and Src.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   Role of PKC and Ca2+ in FAK/Src association induced by bombesin in Swiss 3T3 cells. A, confluent and quiescent cells were treated for 1 h either in the absence () or in the presence (+) of 3.4 µM GF 109203X. Cells were then incubated for a further 10 min either in the absence () or in the presence (+) of 2 nM bombesin (BOM) or 200 nM PDB, as indicated. The cells were then lysed, and the extracts were analyzed by immunoprecipitation with anti-Src Ab followed by Western blotting with anti-FAK Ab. The membranes were further analyzed by Western blotting using anti-SRC-2 Ab. B, confluent and quiescent cells were treated for 1 h either in the absence () or in the presence (+) of 3.4 µM GF 109203X. Cells were then incubated for a further 10 min either in the absence () or in the presence (+) of 2 nM bombesin (BOM) or 200 nM PDB, as indicated. The cells were then lysed, and the extracts were analyzed by immunoprecipitation using anti-Tyr(P) antibody PY20 followed by Western blotting with anti-FAK Ab. C, Swiss 3T3 cells were pretreated for 30 min in the absence () or in the presence (+) of 30 nM thapsigargin (TG) and/or EGTA as indicated. Cells were then incubated for a further 10 min either in the absence () or in the presence (+) of 2 nM bombesin (BOM), and cell lysates were analyzed for FAK/Src association as described above. The membranes were further analyzed by Western blotting using anti-SRC-2 Ab. The positions of FAK and Src are indicated by the arrows. The autoradiograms shown are representative of at least three independent experiments. Quantification of FAK associated with Src in A and C was performed by scanning densitometry. Values shown are the mean ± S.E. of at least three independent experiments and are expressed as the percentage of the maximal increase in FAK/Src association above control (unstimulated) values.

To examine whether PKC was required for stimulation of FAK-Src complex formation in response to bombesin, quiescent Swiss 3T3 cells were incubated with or without 3.4 µM GF I (also known as bisindolylmaleimide I or GF 109203X), a selective inhibitor of PKC (22, 61, 62), prior to stimulation with either PDB or bombesin. As illustrated in Fig. 3A, treatment with GF I attenuated FAK-Src complex formation induced by bombesin and completely prevented the association between these kinases induced by PDB (Fig. 3A, upper panel). We verified that similar amounts of Src were recovered after treatment with bombesin or PDB in the absence or in the presence of GF I (Fig. 3A, lower panel). In parallel cultures, treatment with GF I did not prevent FAK tyrosine phosphorylation in response to bombesin but abrogated PDB-induced FAK tyrosine phosphorylation (Fig. 3B). These results demonstrate that activation of PKC is required for maximal FAK/Src association in response to bombesin in Swiss 3T3 cells.

To investigate whether an increase in intracellular Ca²⁺ mediates FAK-Src complex formation induced by bombesin, quiescent Swiss 3T3 cells were treated with the tumor promoter thapsigargin in the absence or in the presence of EGTA. Thapsigargin specifically inhibits the endoplasmic reticulum Ca²⁺-ATPase and thereby depletes Ca²⁺ from intracellular stores (63). Treatment with 30 nM thapsigargin for 30 min abolished the increase in cytosolic Ca²⁺ induced by subsequently added bombesin (results not shown) but did not block the increase in FAK-Src complex formation induced by bombesin (Fig. 3C). Similarly, chelation of extracellular Ca²⁺ with EGTA to prevent Ca²⁺ influx did not affect FAK-Src complex formation in response to bombesin. Furthermore, a combination of thapsigargin and EGTA that completely prevents Ca²⁺ movements did not inhibit bombesin-induced FAK-Src complex formation. We verified that similar amounts of Src were recovered after treatment with thapsigargin with or without EGTA in the absence or in the presence of bombesin (Fig. 3C, lower panel). These results indicate that bombesin stimulates FAK/Src association through a signal transduction pathway that is independent of Ca²⁺ influx and mobilization in Swiss 3T3 cells.

Src Contributes to Maximal FAK Activation in Response to Bombesin-- Recently, we demonstrated that GPCR agonists including bombesin induce FAK activation (23), and the results presented above indicate that bombesin also stimulates the formation of a complex between FAK and Src in intact cells. Src associated with FAK is thought to phosphorylate FAK at additional sites including Tyr⁵⁷⁶ and Tyr⁵⁷⁷, which are located in the kinase catalytic domain of FAK and are required for maximal FAK activity (52, 64). Since the precise mechanism by which GPCR agonists up-regulate FAK activity is not understood, we tested the hypothesis that Src transphosphorylation of FAK contributes to maximal FAK activation induced by bombesin in intact cells.

The pyrazolopyrimidine PP-2 is a novel and selective inhibitor of the Src kinase family members (65). At concentrations that inhibit Src kinase activity, PP-2 has only a slight effect on FAK kinase activity (23) and thus provides a useful tool that discriminates between FAK and Src. To examine the role of Src in the regulation of FAK activity, cultures of Swiss 3T3 cells were treated in the absence or in the presence of 10 µM PP-2, challenged with or without bombesin, and then lysed. The extracts were immunoprecipitated with a FAK Ab, and the resulting immunocomplexes were incubated with [-³²P]ATP and analyzed by SDS-PAGE and autoradiography to determine FAK autophosphorylation. PP-2 (at 1 µM) was also added to the incubation mixture to eliminate transphosphorylation of FAK by co-immunoprecipitated Src during the in vitro kinase assays (23). As illustrated in Fig. 4A, treatment of intact cells with PP-2 markedly inhibited (by 67%) the increase in FAK kinase activity induced by bombesin stimulation. These results suggest that Src mediates the increase in FAK activity induced by bombesin in intact cells, probably by phosphorylating Tyr⁵⁷⁶/Tyr⁵⁷⁷ in the activation loop of FAK. To examine directly this hypothesis, we determined whether bombesin induces phosphorylation of endogenous FAK at Tyr⁵⁷⁷ in Swiss 3T3 cells, using a site-specific antibody that recognizes the phosphorylated state of this site. As shown in Fig. 4B, bombesin induced a marked increase in the phosphorylation of FAK Tyr⁵⁷⁷. Treatment of intact cells with 10 µM PP-2 inhibited the phosphorylation of Tyr⁵⁷⁷ of FAK in response to bombesin. We verified that similar amounts of FAK were recovered after treatment with bombesin with or without PP-2 (Fig. 4B, lower panel).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Role of Src activity in FAK/Src association induced by bombesin in Swiss 3T3 cells. A, confluent and quiescent cells were treated for 15 min either in the absence () or in the presence (+) of 10 µM of PP2. Cells were then incubated for a further 10 min either in the absence () or in the presence (+) of 2 nM bombesin (BOM), as indicated. The cells were then lysed, the extracts were incubated with anti-FAK Ab, and in vitro kinase reactions were performed as described under "Experimental Procedures." The position of FAK is indicated by an arrow. The autoradiogram shown is representative of at least three independent experiments. Quantification of the kinase activity of FAK was performed by scanning densitometry. Values shown are the mean ± S.E. of at least three independent experiments and are expressed as the percentage of the maximal increase in the kinase activity of FAK above control (unstimulated) values. B, confluent and quiescent cells were treated for 15 min either in the absence () or in the presence (+) of 10 µM of PP2. Cells were then incubated for a further 10 min either in the absence () or in the presence (+) of 2 nM bombesin (BOM), as indicated. The cells were then lysed, and the extracts were analyzed by immunoprecipitation using anti-FAK antibody followed by Western blotting with anti-FAK-Tyr(P)577 Ab. The membranes were further analyzed by Western blotting using anti-FAK Ab. The positions of FAK(P)577 and FAK are indicated by the arrows. The autoradiogram shown is representative of at least three independent experiments. Quantification of FAK(P)577 was performed by scanning densitometry. Values shown are the mean ± S.E. of at least three independent experiments and are expressed as the percentage of the maximal increase in FAK(P)577 above control (unstimulated) values. C, confluent and quiescent cells were treated for 15 min either in the absence () or in the presence (+) of 10 µM of PP2. Cells were then incubated for a further 10 min either in the absence () or in the presence (+) of 2 nM bombesin (BOM), as indicated. The cells were then lysed, and the extracts were analyzed by immunoprecipitation using anti-FAK antibody followed by Western blotting with anti-Tyr(P) antibody PY20. The membranes were further analyzed by Western blotting using anti-FAK Ab. The positions of FAK(P) and FAK are indicated by the arrows. The autoradiogram shown is representative of at least three independent experiments. Quantification of tyrosine phosphorylation of FAK was performed by scanning densitometry. Values shown are the mean ± S.E. of at least three independent experiments and are expressed as the percentage of the maximal increase in tyrosine phosphorylation of FAK above control (unstimulated) values. D, confluent and quiescent cells were treated for 15 min either in the absence () or in the presence (+) of 10 µM of PP2. Cells were then incubated for a further 10 min either in the absence () or in the presence (+) of 2 nM bombesin (BOM), as indicated. The cells were then lysed, and the extracts were analyzed by immunoprecipitation using anti-Src antibody followed by Western blotting with anti-FAK Ab. The membranes were further analyzed by Western blotting using anti-SRC2-Ab. The positions of FAK and Src are indicated by the arrows. The autoradiogram shown is representative of at least three independent experiments. Quantification of FAK associated with Src was performed by scanning densitometry. Values shown are the mean ± S.E. of at least three independent experiments and are expressed as the percentage of the maximal increase in FAK-Src association above control (unstimulated) values.

In parallel cultures, a similar treatment with PP-2 reduced (but did not abolish) the increase in the overall tyrosine phosphorylation of FAK (Fig. 4C). This is consistent with the hypothesis that PP-2 inhibits the phosphorylation of specific tyrosine residues of FAK by Src (e.g. Tyr⁵⁷⁷, as shown in Fig. 4B) but does not interfere with FAK autophosphorylation (at Tyr³⁹⁷). In agreement with this interpretation, treatment of Swiss 3T3 cells with 10 µM PP-2 did not inhibit the association of FAK with Src induced by bombesin (in fact, a small but consistent enhancement was noticed), indicating that the catalytic activity of Src is not necessary for the formation of this complex and that Tyr³⁹⁷ is phosphorylated and available to associate with Src in cells treated with PP-2 (Fig. 4D). We verified that similar amounts of Src were recovered after treatment with bombesin with or without PP-2 (Fig. 4D, lower panel).

The results shown in Fig. 4 support the hypothesis that FAK-Src complex formation induced by bombesin leads to the subsequent tyrosine phosphorylation of endogenous FAK (by Src) at additional sites (e.g. Tyr⁵⁷⁷) that are responsible for up-regulating FAK activity in intact cells.

The Integrity of the Actin Cytoskeleton Is Essential for Agonist-induced FAK/Src Association-- Treatment of the cells with cytochalasin D, which caps the barbed end of actin filaments and promotes their depolymerization, selectively inhibits the increase in FAK tyrosine phosphorylation in response to bombesin and other GPCR agonists (22, 24, 27-29, 32, 33) but does not prevent Src activation in response to these agents (24). Here, we examined whether cytochalasin D-mediated disruption of the actin cytoskeleton interferes with the association of FAK with Src induced by bombesin.

Quiescent Swiss 3T3 cells were exposed for 2 h to increasing concentrations of cytochalasin D and then stimulated with 2 nM bombesin for another 10 min. As shown in Fig. 5A, treatment with cytochalasin D completely blocked the association of FAK with Src induced by bombesin in intact cells in a concentration-dependent manner. Maximum inhibitory effect was achieved at 2.4 µM, a concentration that completely disrupts the actin cytoskeleton and the assembly of focal adhesions and abolishes the increase in the overall tyrosine phosphorylation of FAK (22). We verified that similar amounts of Src were recovered after treatment with increasing concentrations of cytochalasin D (Fig. 5A, lower panel).

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Cytochalasin D prevents agonist-induced FAK/Src association in Swiss 3T3 cells. A, confluent and quiescent cells were treated for 2 h in the absence (0) or in the presence of increasing concentrations of cytochalasin D (CYT D), as indicated and then stimulated without () or with (+) 2 nM bombesin (BOM) for a further 10 min. The cells were then lysed, and the lysates were analyzed by immunoprecipitation with anti-Src SRC-2Ab followed by Western blotting with anti-FAK Ab. The positions of FAK and Src are indicated by the arrows. The autoradiogram shown is representative of at least three independent experiments. Quantification of FAK associated with Src was performed by scanning densitometry. Values shown are the mean ± S.E. of at least three independent experiments and are expressed as the percentage of the maximal increase in FAK-Src association above control (unstimulated) values. B, confluent and quiescent cells were treated for 2 h in the absence () or in the presence (+) of 2.4 µM cytochalasin D (CYT D), as indicated, and then stimulated with 2 nM bombesin for a further 10 min. The cells were then lysed, and the extracts were analyzed by immunoprecipitation using anti-FAK antibody followed by Western blotting with anti-FAK-Tyr(P)577 Ab. The membranes were further analyzed by Western blotting using anti-FAK Ab. The positions of FAK(P)577 and FAK are indicated by the arrows. The autoradiogram shown is representative of at least three independent experiments. Quantification of FAK(P)577 was performed by scanning densitometry. Values shown are the mean ± S.E. of at least three independent experiments and are expressed as the percentage of the maximal increase in FAK(P)577 above control (unstimulated) values. C, confluent and quiescent cells were treated for 2 h in the absence () or in the presence (+) of 2.4 µM cytochalasin D (CYT D), as indicated, and then stimulated with 20 nM bradykinin (BK), 10 nM endothelin (END), and 2 µM LPA for 10 min. The cells were then lysed, and the lysates were analyzed for FAK/Src association as described above. The membranes were further analyzed by Western blotting using anti-SRC-2 Ab. The positions of FAK and Src are indicated by arrows. The results are representative of at least three independent experiments.

Pretreatment of quiescent Swiss 3T3 cells for 2 h with 2.4 µM cytochalasin D also prevented the increase in the phosphorylation of Tyr⁵⁷⁷ in Swiss 3T3 cells (Fig. 5B). Treatment with cytochalasin D also prevented FAK-Src complex formation in response to other GPCR agonists including endothelin, bradykinin, and LPA (Fig. 5C). We verified that similar amounts of Src were recovered after treatment with cytochalasin D and the different agonists (Fig. 5C, lower panel).

PDGF Induces FAK/Src Association: Bell-shaped Dose Response and Dependence on Functional PI 3-Kinase and Actin Cytoskeleton Organization-- Previous studies from our laboratory demonstrated that PDGF induces biphasic tyrosine phosphorylation of FAK through a pathway dependent on the integrity of the actin cytoskeleton (29, 66). It is also known that PDGF stimulates association of Src with specific tyrosine-phosphorylated residues of the PDGF receptor and promotes a sustained increase in Src activity (67). Here, we examined the effect of PDGF on the formation of FAK-Src complexes.

Lysates of quiescent cultures of Swiss 3T3 cells exposed to increasing concentrations of PDGF (1-30 ng/ml) for 10 min were immunoprecipitated with SRC-2 antibody, and the level of FAK in the resulting Src immunoprecipitates was assessed by Western blot analysis. As illustrated in Fig. 6A, PDGF stimulated association of FAK with Src following a striking bell-shaped dose-response relationship. A detectable increase was seen at 1 ng/ml PDGF, and maximal effect was achieved at 5 ng/ml PDGF. At higher concentrations of PDGF (e.g. 20 and 30 ng/ml), FAK-Src complex formation was reduced sharply. We confirmed that similar amounts of Src were recovered after treatment with increasing concentrations of PDGF (Fig. 6A, lower panel).

View larger version (29K): [in this window] [in a new window]   Fig. 6.   PDGF induces biphasic FAK/Src association via a PI 3-kinase-dependent pathway in Swiss 3T3 cells. A, confluent and quiescent cells were incubated for 10 min at 37 °C either in the absence or in the presence of increasing concentrations of PDGF, as indicated. The cells were then lysed, and the lysates were analyzed by immunoprecipitation with anti-Src Ab followed by Western blotting with anti-FAK Ab. B, confluent and quiescent cells were treated for 30 min either in the absence () or in the presence (+) of 60 nM wortmannin. Cells were then incubated for a further 10 min either in the absence () or in the presence (+) of either 2 nM bombesin (BOM) or 5 ng/ml of PDGF. The cells were then lysed, and the lysates were analyzed for FAK/Src association, as described above. C, confluent and quiescent cells were treated for 2 h either in the absence () or in the presence (+) of 2.4 µM cytochalasin D (CYT D). Cells were then incubated for a further 10 min either in the absence () or in the presence (+) of 5 ng/ml of PDGF. The cells were then lysed, and lysates were analyzed as described above. The membranes were further analyzed by Western blotting using anti-SRC-2 Ab. The positions of FAK and Src are indicated by the arrows. The autoradiograms shown are representative of at least three independent experiments. Quantification of FAK associated with Src was performed by scanning densitometry. Values shown are the mean ± S.E. of at least three independent experiments and are expressed as the percentage of the maximal increase in FAK-Src association above control (unstimulated) values.

PDGF induces actin recruitment into membrane ruffles (68, 69) and tyrosine phosphorylation of FAK (30) via a PI 3-kinase-dependent pathway, but the contribution of this pathway to FAK-Src complex formation induced by PDGF was unknown. To determine the role of PI 3-kinase in the association between FAK and Src induced by PDGF in Swiss 3T3 cells, quiescent cultures of these cells were pretreated for 30 min with wortmannin (60 nM), which binds to and inhibits the catalytic (110-kDa) subunit of PI 3-kinase (70, 71) and then stimulated for 10 min with 5 ng/ml PDGF. To verify the selectivity of the effect of wortmannin under our experimental conditions, parallel cultures of Swiss 3T3 cells were also stimulated with bombesin, which does not increase the levels of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in these cells (72). As shown in Fig. 6B, treatment with wortmannin prevented PDGF-induced FAK-Src complex formation but had only a slight effect on the association of FAK with Src induced by bombesin. We verified that similar amounts of Src were recovered after treatment with bombesin or PDGF in the absence or presence of wortmannin (Fig. 6B, lower panel).

Given that PI 3-kinase induces Rac-dependent reorganization of actin cytoskeleton into membrane ruffles (73, 74), leading to formation of focal contacts (75), we examined whether disruption of the actin cytoskeleton interferes with FAK-Src complex formation in response to PDGF. Quiescent Swiss 3T3 cells were pretreated with 2.4 µM cytochalasin D for 2 h and then stimulated with 5 ng/ml PDGF for another 10 min. As shown in Fig. 6C, treatment with cytochalasin D completely blocked FAK-Src complex formation in response to PDGF. Similar amounts of Src were recovered after treatment with cytochalasin D in the absence or presence of PDGF (Fig. 6C, lower panel).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results presented here demonstrate that stimulation with bombesin, other GPCR agonists (endothelin, bradykinin, and LPA), and PDGF promotes the formation of FAK-Src complexes in Swiss 3T3 cells. Association of Src with phosphorylated FAK has been demonstrated previously in v-Src-transformed cells and in cells replated on fibronectin-coated dishes, an assay of integrin receptor activation (54, 56, 57, 59). Our results show that bombesin, at nanomolar concentrations, induced complex formation between endogenous FAK and Src in attached cells i.e. without overexpressing any of these components and without subjecting the cells to detachment and subsequent replating.

The kinase activity of Src kinase family members (such as Src, Yes, and Fyn) is repressed when a key tyrosine residue in the carboxyl-terminal region (corresponding to Tyr⁵²⁷ of the chicken protein) is phosphorylated by Csk (reviewed in Ref. 50). Phosphorylation at Tyr⁵²⁷ creates a binding site for Src SH2 domain and allows an intramolecular interaction that locks Src in an inactive conformation. Our previous results demonstrated that bombesin induces a very rapid (peaking at 30-40 s) and transient activation of Src in Swiss 3T3 cells (24) probably via dephosphorylation of Tyr⁵²⁷ by a tyrosine phosphatase. Src activity returned to base line levels after 2 min of incubation (24). In contrast, the results presented here demonstrate that complexes between Src and FAK persisted for at least 30 min. Given that competition for the SH2 and/or SH3 domains of Src by high affinity allosteric ligands is an alternative mechanism that promotes enzymatic activation of this kinase (50, 76), the association of Src to FAK would lead to the formation of a molecular complex in which Src kinases are active. Taken together, our results suggest that GPCR agonists induce Src activation via at least two different mechanisms in Swiss 3T3 cells.

Recently, we demonstrated that GPCR agonists including bombesin induce FAK activation (23), but the precise mechanism by which GPCR agonists up-regulate FAK activity is not understood. Src associated to FAK is thought to phosphorylate FAK at additional sites including Tyr⁵⁷⁶ and Tyr⁵⁷⁷, which are located in the catalytic domain of FAK. The phosphorylation of these sites is required for maximal FAK activity in vitro (52, 64). Since bombesin stimulates complex formation between FAK and Src, we tested the hypothesis that Src mediates FAK transphosphorylation and activation in response to bombesin in intact cells. Our results show, for the first time, that bombesin induces phosphorylation of Tyr⁵⁷⁷ of FAK and that the selective Src inhibitor PP-2, at a concentration that markedly reduced the phosphorylation of Tyr⁵⁷⁷, also attenuated FAK activation in response to bombesin. These findings suggest that Src mediates the increase in FAK activity induced by bombesin in intact Swiss 3T3 cells.

Src plays a critical role in FAK signaling including migration and apoptosis (44, 48), and FAK-Src complexes are implicated in the tyrosine phosphorylation of the adaptor proteins p130^CAS and paxillin (77-79). It is therefore likely that FAK-Src complex formation is under tight regulation (52). Our results indicate that FAK tyrosine phosphorylation is necessary but not sufficient for promoting the formation of FAK-Src complexes, suggesting the need for additional signals. For example, the kinetics of FAK tyrosine phosphorylation did not coincide with FAK-Src complex formation in bombesin-stimulated cells. Specifically, tyrosine phosphorylation of FAK was detectable within seconds and persisted longer than FAK/Src association. Furthermore, bombesin stimulated FAK tyrosine phosphorylation through a signal transduction pathway that is largely independent of PKC (e.g. Ref. 22 and Fig. 3B). However, we show here that maximal FAK/Src association induced by bombesin requires a functional PKC pathway. These findings support the hypothesis that FAK tyrosine phosphorylation is not sufficient for triggering FAK/Src association and suggests that a PKC-dependent signaling pathway contributes to the formation of this complex.

Agonist-mediated increase in FAK tyrosine phosphorylation is accompanied by profound alterations in the organization of the actin cytoskeleton and in the assembly of focal adhesions (26, 27, 29, 80-82), the distinct areas of the plasma membrane where FAK is localized (38, 83). Treatment of the cells with cytochalasin D, which disrupts actin filaments, prevents the increase in FAK tyrosine phosphorylation in response to multiple agents, suggesting a mechanism involving the actin cytoskeleton and the focal adhesion plaques (22, 24, 27-29, 32, 33). In contrast, bombesin induces Src activation in cytochalasin D-treated cells, indicating that FAK and Src activation are mediated by different pathways (24).

Here we show that cytochalasin D profoundly inhibits FAK-Src complex formation induced by bombesin and other agonists. Previous studies demonstrated that treatment with cytochalasin D does not inhibit production of inositol phosphates, Ca²⁺ mobilization, and stimulation of PKC, Src, and p42^MAPK/p44^MAPK activation in response to bombesin (22, 24, 27, 46, 60). Thus, disruption of the actin cytoskeleton prevents FAK tyrosine phosphorylation, activation, and association with Src in a selective manner. These findings are consistent with a model in which FAK/Src association requires the integrity of the actin cytoskeleton and intact focal adhesion plaques.

Our results demonstrate that PDGF induces a striking biphasic association between FAK and Src, with maximal effect at only 5 ng/ml. At higher concentrations of PDGF, complex formation between FAK and Src is dramatically reduced. Previous studies demonstrated that PDGF, at low concentrations, stimulates PI 3-kinase activity and causes accumulation of actin in membrane ruffles, while at high concentrations, PDGF induces actin disorganization (29). PI 3-kinase activity is required for PDGF-induced formation of membrane ruffles (69) and for the increase in tyrosine phosphorylation of FAK (30, 66). Here, we show that the increase in complex formation between FAK and Src induced by PDGF requires functional PI 3-kinase and an intact actin cytoskeleton. We hypothesize that the stimulatory limb of the bell-shaped dose-response curve of PDGF-stimulated FAK/Src association is mediated by a PI 3-kinase-dependent pathway, whereas the inhibitory limb is caused by PDGF-induced disorganization of the actin cytoskeleton. Interestingly, bombesin does not increase the levels of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate in Swiss 3T3 cells (72), and, accordingly, our results show that the PI 3-kinase inhibitor wortmannin did not prevent FAK/Src association promoted by this GPCR agonist. We conclude that there are PI 3-kinase-dependent and -independent pathways leading to FAK/Src association in the same cell.

In conclusion, our results demonstrate that stimulation of Swiss 3T3 cells with bombesin, endothelin, bradykinin, LPA, and PDGF induces a rapid increase in the formation of a complex between FAK and Src. Bombesin stimulates FAK/Src association through a Ca²⁺- and PI 3-kinase-independent pathway that requires the integrity of the actin filament network and is partly dependent on functional PKC. In contrast, PDGF simulates biphasic FAK/Src association via a PI 3-kinase pathway. Our results demonstrate, for the first time, that agonists of either GPCRs or tyrosine kinase receptors promote FAK/Src association in attached cells through different signal transduction pathways.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a postdoctoral fellowship from Consejo Nacional de Ciencia y Tecnologia, Mexico.

^§ To whom all correspondence should be addressed: 900 Veteran Ave., Warren Hall Rm. 11-124, Dept. of Medicine, UCLA School of Medicine, Los Angeles, CA 90095-178622. Tel.: 310-794-6610; Fax: 310-267-2399.

	ABBREVIATIONS

The abbreviations used are: GPCR, G-protein-coupled receptor; DMEM, Dulbecco's modified Eagle's medium; FAK, focal adhesion kinase; LPA, lysophosphatidic acid, Ab, antibody; PAGE, polyacrylamide gel electrophoresis; PDB, phorbol 12,13-dibutyrate; PDGF, platelet-derived growth factor; PI 3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; PP-2, pyrazolopyrimidine 2; SH2 and SH3, Src homology domain 2 and 3, respectively; MAPK, mitogen-activated protein kinase; S6K, S6 kinase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES