Insulin-like Growth Factor I Stimulates Tyrosine Phosphorylation of p130Cas, Focal Adhesion Kinase, and Paxillin

Addition of insulin growth factor-I (IGF-I) to quiescent Swiss 3T3 cells rapidly induced tyrosine phosphorylation of the p130Crk-associated substrate (p130Cas), a novel adaptor protein localized at focal adhesions. Half-maximal effect was obtained at 0.6 nm. IGF-I also promoted the formation of a complex between p130Cas and c-Crk and elicited a parallel increase in the tyrosine phosphorylation of p125Fak and paxillin. IGF-I-induced p130Cas, p125Fak, and paxillin tyrosine phosphorylation could be dissociated from mitogen-activated protein kinase kinase, p70S6K, and protein kinase C activation. In contrast, the structurally unrelated phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 markedly attenuated the increase in tyrosine phosphorylation of p130Cas, p125Fak, and paxillin induced by IGF-I. Cytochalasin D, which disrupts the network of actin microfilaments, completely prevented tyrosine phosphorylation of p130Cas, p125Fak, and paxillin and the formation of a p130Cas·Crk complex in response to IGF-I. Thus, our results identified a phosphatidylinositol 3-kinase-dependent pathway that requires the integrity of the actin cytoskeleton to induce tyrosine phosphorylation of p130Cas, p125Fak, and paxillin in response to IGF-I and suggest that tyrosine phosphorylation of these focal adhesion proteins, together with the recruitment of c-Crk into a complex with p130Cas, may play a novel role in IGF-I signal transduction.

The insulin-like growth factor-1 (IGF-I) 1 is implicated in many normal and abnormal processes including development, cell growth, and malignant transformation (1)(2)(3)(4). The initial event in IGF-I signaling involves its binding to the IGF-I receptor, an ␣2 ␤2 heterotetramer with ligand-dependent tyrosine kinase activity (5). The insulin receptor substrates 1 and 2 are rapidly phosphorylated on multiple tyrosine residues in response to IGF-I (6, 7) and act as docking proteins that bind, through their phosphorylated residues, SH2 domain-containing proteins which propagate the IGF-I signal (8). These include the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and the adaptor proteins Grb-2 and Shc which lead to activation of the Ras/Raf/ERK pathway (5,9). In addition, IGF-I also promotes cell migration of normal and tumor cells and induces the recruitment of actin into membrane ruffles (10 -13).
Changes in the organization of the actin cytoskeleton induced by many extracellular factors are accompanied by striking changes in the tyrosine phosphorylation of several signaling proteins localized at the focal adhesion plaques (14,15), the distinct sites of the plasma membrane that are in close contact with the extracellular matrix proteins (16). Specifically, the tyrosine phosphorylation of the non-receptor tyrosine kinase p125 Fak (17,18) and of the adaptor proteins p130 Cas (19) and paxillin (20,21) is increased by diverse signaling molecules that regulate cell growth, differentiation, and migration including neuropeptide agonists (22)(23)(24)(25)(26), bioactive lipids (26 -29), and growth factors (26, 30 -32). The increase in the tyrosine phosphorylation of focal adhesion proteins induced by these agents is critically dependent on the integrity of the actin cytoskeleton (24,27,28,30,33) and is mediated by PI 3-kinase-and PKCdependent and -independent pathways (24,27,31). Tyrosine phosphorylation of p125 Fak , p130 Cas , and paxillin is also induced by extracellular matrix proteins (17, 34 -36) and transforming variants of p60 src (37). Thus, the phosphorylation of these focal adhesion proteins represents a point of convergence in the action of growth factors, integrins, and oncogenes (14,38).
In contrast to other growth factors, insulin has been shown to decrease the level of tyrosine phosphorylation of p125 Fak and paxillin in a variety of cell types (39 -43) through pathways involving Csk (41), phosphotyrosine phosphatase 1D (43), and integrins (44). The influence of IGF-I on the tyrosine phosphorylation of p125 Fak and paxillin has been less studied and is the subject of conflicting reports (12,44,45). The effect of IGF-I on the tyrosine phosphorylation of p130 Cas , a novel adaptor protein implicated in transformation (19) and recently identified as a critical mediator of cell migration (46,47), is unknown.
In the present study we report, for the first time, that IGF-I induces a rapid and transient increase in the tyrosine phosphorylation of p130 Cas in Swiss 3T3 cells, a useful model system to elucidate signal transduction pathways in the action of growth factors (48). Our results also show that IGF-I promotes the formation of a complex between p130 Cas and c-Crk and elicits a coordinate increase in the tyrosine phosphorylation of p125 Fak and paxillin. IGF-I stimulates tyrosine phosphorylation of * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 1 The abbreviations used are: IGF-I, insulin-like growth factor I; anti-Tyr(P), anti-phosphotyrosine; DMEM, Dulbecco's modified Eagle's medium; mAb, monoclonal antibody; p125 Fak , p125 focal adhesion kinase; p130 Cas , p130 Crk-associated substrate; PAGE, polyacrylamide gel electrophoresis; PDBu, phorbol 12,13-dibutyrate; PI 3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; SH2 and SH3, Src homology domain 2 and 3, respectively; PDGF, platelet-derived growth factor. these focal adhesion proteins through a PI 3-kinase-dependent pathway that requires the integrity of the actin cytoskeleton.

EXPERIMENTAL PROCEDURES
Cell Culture-Stock cultures of Swiss 3T3 fibroblasts 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, 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.
Western Blotting-Treatment of quiescent cultures of cells with growth factors, cell lysis, and immunoprecipitation was performed as described above. After SDS-PAGE, proteins were transferred to Immobilon membranes. The membranes were blocked using 5% non-fat dried milk in phosphate-buffered saline, pH 7.2, and incubated for 2 h at 22°C with either the anti-Tyr(P) mAb (4G10, 1 g/ml) or the anti-p130 Cas mAb (1 g/ml), as indicated. Immunoreactive bands were visualized using 125 I-labeled sheep anti-mouse IgG (1:1000) followed by autoradiography. The autoradiograms were scanned using an LKB Ultrascan XL densitometer, and labeled bands were quantified using an Ultrascan XL internal integrator. The values were expressed as percentages of the maximum increase in tyrosine phosphorylation above control values.
p70 S6K Mobility Shift Assay-Activation of p70 S6K was determined by the appearance of slowly migrating forms in SDS-PAGE which results from phosphorylation of this enzyme on Thr-229 and Thr-389 and Ser-404 (49). Immunoblot analysis on cell lysates was performed using a rabbit polyclonal antibody which recognized both ␣I and ␣II isoforms of p70 S6K , with immunoreactive bands being visualized with iodinated protein A.
p42 mapk Mobility Shift Assays-Activation of p42 mapk can be determined by the appearance of slower migrating forms in SDS-PAGE gels, which results from the phosphorylation of specific threonine and tyrosine residues within its subdomain VIII. Quiescent cultures of Swiss 3T3 cells were treated with factors as indicated; the cells were lysed in 2ϫ SDS-PAGE sample buffer and analyzed by SDS-PAGE. Proteins were subsequently transferred to Immobilon membranes which were blocked as described above and incubated for 1 h at 22°C with a polyclonal p42 mapk antiserum (1:1000) in phosphate-buffered saline containing 3% non-fat dried milk. Immunoreactive bands were visualized using iodinated protein A followed by autoradiography.
Materials-IGF-I, bombesin, cytochalasin D, wortmannin, LY294002, and agarose-linked anti-mouse IgG were obtained from the Sigma. Recombinant PDGF (BB homodimer), 125 I-sheep anti-mouse IgG (15 mCi/mg), and 125 I-protein A were from Amersham Corp., United Kingdom. The 4G10 anti-Tyr(P) mAb was from Upstate Biotechnology, Inc. (Lake Placid, NY). Rapamycin and PD98059 were obtained from Calbiochem (Nottingham, UK). The mAbs directed against p130 Cas , c-Crk, or paxillin were from Transduction Laboratories, and the rabbit polyclonal antibodies against p125 Fak and p70 S6K were from Santa Cruz Biotechnology, Santa Cruz, CA. The polyclonal anti-p42 mapk antibody raised against a COOH-terminal peptide (EETARFQPGYRS) was a gift from Dr. D. Whitters.

IGF-I Stimulates Tyrosine
Phosphorylation of the Focal Adhesion Proteins p130 Cas , p125 Fak , and Paxillin-To determine whether IGF-I induces tyrosine phosphorylation of p130 Cas in Swiss 3T3 cells, quiescent cultures of these cells were stimulated with various concentrations of IGF-I for 5 min and lysed, and the extracts were immunoprecipitated with anti-p130 Cas antibody. The immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with anti-Tyr(P) mAb. As shown in Fig. 1A, IGF-I induced a marked increase in the tyrosine phosphorylation of p130 Cas in a dose-dependent manner. Half-maximum and maximum effects were obtained at 0.6 and 10 nM, respectively.
The kinetics of tyrosine phosphorylation of p130 Cas stimulated by IGF-I in Swiss 3T3 cells is shown in Fig. 1B. An increase in tyrosine phosphorylation of p130 Cas could be detected as early as 1 min after addition of 10 nM IGF-I, reaching a maximum after 2.5-5 min. Thereafter, tyrosine phosphorylation of p130 Cas declined gradually to almost base-line levels after 30 min of incubation. We verified that similar amounts of p130 Cas were recovered from lysates of cells treated without or with different concentrations of IGF-I for various times (results not shown).
p130 Cas has a cluster of 15 potential SH2-binding motifs, nine of which are sequences that are expected to have a preferential affinity for c-Crk-SH2 domain (19). Consequently, we examined whether IGF-I-induced tyrosine phosphorylation of p130 Cas could lead to the formation of a complex between endogenous c-Crk and p130 Cas in intact Swiss 3T3 cells. Anti-p130 Cas Western blotting of c-Crk immunoprecipitates revealed that IGF-I stimulated an association of p130 Cas with c-Crk that was time-dependent and parallel to the IGF-I-induced tyrosine phosphorylation of p130 Cas . We verified that similar amounts of c-Crk were recovered from lysates of cells treated without or with IGF-I (result not shown). The association of p130 Cas with c-Crk reached a maximum after 2.5-5 min of IGF-I stimulation and then declined ( Fig. 2A).
IGF-I also induced a rapid and transient increase in the tyrosine phosphorylation of the non-receptor tyrosine kinase p125 Fak and the adaptor protein paxillin which could be detected as early as 1 min after addition of 10 nM IGF-I, reaching a maximum after 5 min. Thereafter, tyrosine phosphorylation of p125 Fak and paxillin declined to almost base-line levels ( Fig.  2B). Similar amounts of p125 Fak and paxillin were recovered from lysates of cells treated without or with IGF-I (results not shown). Thus, IGF-I induces a rapid and parallel increase in the tyrosine phosphorylation of the focal adhesion proteins p130 Cas , p125 Fak , and paxillin and, concomitantly, promotes association of tyrosine-phosphorylated p130 Cas with c-Crk in Swiss 3T3 cells.
Role of ERK in IGF-I-induced Tyrosine Phosphorylation of p130 Cas , p125 Fak , and Paxillin-The Ras-ERK pathway has been involved in IGF-I-mediated cellular proliferation and tumorigenesis (50). Recent evidence has implicated p42 mapk (ERK-2) and p44 mapk (ERK-1) in the control of the actin cytoskeleton and cell migration via phosphorylation and activation of the myosin light chain kinase (51). To determine the contribution of the ERK pathway to IGF-I-mediated tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin, we used PD98059, a highly specific inhibitor of the ERK-activating enzyme mitogen-activated protein kinase kinase-1 (52). Quiescent cultures of Swiss 3T3 cells were pretreated with 15 M PD98059 for 2 h and then stimulated for 5 min with either 10 nM IGF-I or 10 nM bombesin (53). Lysates of the treated cells were immunoprecipitated with either p130 Cas or p125 Fak mAbs, and the immunoprecipitates were further analyzed by Western blotting with anti-Tyr(P) mAb. Lysates derived from parallel cultures were analyzed by Western blotting using a specific polyclonal antibody against the ERKs. In this assay, activation of p42 mapk and p44 mapk is determined by the appearance of slowly migrating forms that result from the phosphorylation of specific threonine and tyrosine residues within their catalytic domain. As shown in Fig. 3A, IGF-I induced a small but detectable mobility shift of the ERKs in Swiss 3T3 cells, whereas bombesin stimulated a robust ERK activation, in agreement with previous results (53). Treatment with 15 M PD98059 completely prevented the mobility shift of the ERKs induced by IGF-I and partially inhibited ERK activation in response to bombesin. In contrast, the increase in the tyrosine phosphorylation of p130 Cas and p125 Fak in response to IGF-I or bombesin was unaffected by pretreatment of the cells with the mitogen-activated protein kinase kinase-1 inhibitor. In other experiments, we also found that exposure to PD98059 did not prevent the increase in paxillin tyrosine phosphorylation in- duced by IGF-I (results not shown). Thus, IGF-I-induced tyrosine phosphorylation of focal adhesion proteins can be dissociated from the activation of the ERK pathway in Swiss 3T3 cells.
Effect of Wortmannin and LY294002 on IGF-I-stimulated Tyrosine Phosphorylation of p130 Cas , p125 Fak , and Paxillin-IGF-I induces actin recruitment into membrane ruffles via a PI 3-kinase-dependent pathway (11), but the contribution of this pathway to the tyrosine phosphorylation of focal adhesion proteins induced by IGF-I was unknown. To examine the role of PI 3-kinase in the tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin induced by IGF-I in Swiss 3T3 cells, quiescent cultures of these cells were pretreated for 20 min with wortmannin, which binds to and inhibits the catalytic (110 kDa) subunit of PI 3-kinase (54,55), and then stimulated for 5 min with 10 nM IGF-I. To verify the selectivity of the effect of wortmannin under our experimental conditions, parallel cultures of Swiss 3T3 cells were also stimulated with either PDGF (5 ng/ml) or the G protein-coupled receptor agonist bombesin which induces p130 Cas tyrosine phosphorylation via PI 3-kinasedependent and -independent pathways, respectively (26). As shown in Fig. 4, A and B, tyrosine phosphorylation of p130 Cas stimulated by either IGF-I or PDGF was markedly inhibited by preincubation of the cells with wortmannin up to 60 nM. In contrast, the tyrosine phosphorylation of p130 Cas stimulated by bombesin was not inhibited by wortmannin, at identical concentrations.
In order to substantiate the results obtained with wortmannin, we examined if the structurally unrelated compound LY294002, which selectively inhibits PI 3-kinase by a distinct mechanism (56), also prevents p130 Cas tyrosine phosphorylation in response to IGF-I. As shown in Fig. 4, A and B, LY294002 also attenuated the increase in the tyrosine phosphorylation of p130 Cas in response to either IGF-I or PDGF but not in response to bombesin. Pretreatment with either wortmannin or LY294002 also inhibited tyrosine phosphorylation of p125 Fak (Fig. 5A) and paxillin (Fig. 5B) induced by either IGF-I or PDGF. Thus, IGF-I stimulates p130 Cas , p125 Fak and paxillin tyrosine phosphorylation through a PI 3-kinase-dependent pathway in Swiss 3T3 cells.
Rapamycin Dissociates IGF-I-induced Tyrosine Phosphorylation of p130 Cas , p125 Fak , and Paxillin from p70 S6K -p70 ribosomal S6 kinase (p70 S6K ) is activated by many mitogenic stimuli, including growth factors and oncogene products, as a downstream target of PI 3-kinase (57). To verify that IGF-I activates PI 3Ј-kinase-dependent activation of p70 S6K in Swiss 3T3 cells, quiescent cultures of these cells were pretreated with 40 nM wortmannin for 20 min and then stimulated with 10 nM IGF-I for different times. As shown in Fig. 6A, IGF-I induced the appearance of slowly migrating forms of p70 S6K that are characteristic of the phosphorylated and activated form of the enzyme (49). Pretreatment of the cells with wortmannin, at a concentration (40 nM) that prevented tyrosine phosphorylation of focal adhesion proteins, markedly inhibited IGF-I-stimulated phosphorylation of p70 S6K . Consequently, it was important to clarify whether p70 S6K lies in the signaling pathway that mediates PI 3-kinase-dependent tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin in response to IGF-I.
The potent immunosuppressant rapamycin is a selective inhibitor of p70 S6K activation in many cell types (57), including Swiss 3T3 cells (58). The phosphorylation of Thr-229 and Thr-389 and Ser 404, which are responsible for the activation and mobility shift of p70 S6K , is prevented or reversed by treatment with rapamycin (49). We therefore examined the effect of rapamycin on the IGF-I-stimulated tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin. Quiescent cultures of Swiss 3T3 cells were pretreated with 10 nM rapamycin for 1 h and then stimulated with 10 nM IGF-I for different times. Cell lysates were analyzed for p130 Cas tyrosine phosphorylation. Lysates derived from parallel cultures were Western-blotted with an anti-p70 S6K polyclonal antibody. As shown in Fig. 6B, pretreatment of cells with rapamycin completely inhibited the p70 S6K mobility shift induced by IGF-I but had no effect on IGF-Istimulated tyrosine phosphorylation of p130 Cas , p125 Fak , or paxillin in parallel cultures. Furthermore, IGF-I induced p130 Cas , p125 Fak , and paxillin tyrosine phosphorylation more rapidly than p70 S6K activation, with maximal effects at 5 and 30 min, respectively. These results clearly dissociate p70 S6K activation from IGF-I-mediated tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin.

IGF-I-induced Tyrosine Phosphorylation of p130 Cas , p125 Fak , and Paxillin
Is PKC-independent-The second messengers phosphatidylinositol (3,4)-biphosphate and phosphatidylinositol (3,4,5)-triphosphate generated by PI 3-kinase activity have been proposed to activate the novel and atypical isoforms of PKC (59). In addition, phorbol esters have been shown to induce tyrosine phosphorylation of p125 Fak , p130 Cas , and paxillin in Swiss 3T3 cells through activation of PKC (23,24,26). To determine whether PKC activation is involved in IGF-I-induced tyrosine phosphorylation of focal adhesion proteins, PKC was selectively inhibited by pretreatment for 1 h with 3.5 M GF 109203X (also known as bisindolylmaleimide or GF1) (60, 61) and then the cultures were treated with or without 10 nM IGF-I for 5 min. As illustrated in Fig. 3B, IGF-I-induced tyrosine phosphorylation of either p130 Cas or p125 Fak was not prevented by pretreatment with GF 109203X. In other experiments, we also found that GF 109203X failed to prevent the increase in paxillin tyrosine phosphorylation induced by IGF-I (results not shown). We verified, using parallel cultures, that pretreatment with 3.5 M GF 109203X blocked PDBu stimulation of p130 Cas , p125 Fak , and paxillin tyrosine phosphorylation, in agreement with our previous results (23,24,26). Thus, PKC activation is not responsible for the rapid IGF-I-induced tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin in Swiss 3T3 cells.
The Integrity of the Actin Cytoskeleton Is Essential for IGF-I-induced Tyrosine Phosphorylation of p130 Cas , p125 Fak , and Paxillin-Another pathway that lies downstream of PI 3-kinase is the Rac-dependent reorganization of the actin cytoskeleton into membrane ruffles (11,(62)(63)(64). Given that tyrosine-phosphorylated p130 Cas , p125 Fak , and paxillin are found in focal contacts associated with stress fibers and membrane ruffles, we examined whether disruption of the actin cytoskeleton could interfere with the tyrosine phosphorylation of these focal adhesion proteins in response to IGF-I. Quiescent Swiss 3T3 cells were pretreated for 2 h with 1.2 M cytochalasin D, a concentration that is known to completely disrupt the actin cytoskeleton and the assembly of focal adhesion in Swiss 3T3 cells (24), and then stimulated with 10 nM IGF-I for another 5 min. As shown in Fig. 7A, treatment with cytochalasin D completely blocked IGF-I stimulation of p130 Cas , p125 Fak , and paxillin tyrosine phosphorylation.
Addition of PDGF, at high concentrations (e.g. 30 ng/ml), also disrupts the organization of the actin cytoskeleton in 3T3 cells (30). As illustrated in Fig. 7A, pretreatment of Swiss 3T3 cells with 30 ng/ml PDGF completely inhibited tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin by IGF-I. Thus, the integrity of the actin cytoskeleton is necessary for IGF-I-induced tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin.
To assess whether the complex formation between p130 Cas and c-Crk induced by IGF-I also depends on the integrity of the actin cytoskeleton, quiescent Swiss 3T3 cells were pretreated for 2 h with or without 1.2 M cytochalasin D and then stimulated with 10 nM IGF-I. Fig. 7B shows that treatment of Swiss 3T3 cells with cytochalasin D, at a concentration shown in Fig.  7A to inhibit p130 Cas tyrosine phosphorylation, prevented the association of p130 Cas with c-Crk induced by IGF-I in intact Swiss 3T3 cells.

DISCUSSION
There is increasing evidence indicating that IGF-I signaling is involved in multiple biological processes including cell migration, proliferation, and transformation, but the downstream targets that mediate these effects have not been fully identified. The findings presented here demonstrate that IGF-I induces tyrosine phosphorylation of the adaptor molecule p130 Cas in Swiss 3T3 cells. The rapidity and the low, physiological concentrations of IGF-I inducing this effect suggest that this event may be functionally important in the biological action of IGF-I. Our results also demonstrate that IGF-I induces tyrosine phosphorylation of p125 Fak and paxillin with kinetics that parallel those of p130 Cas tyrosine phosphorylation.
The signal transduction events initiated by the IGF-IR have been extensively investigated, but the pathway(s) leading to tyrosine phosphorylation of the focal adhesion proteins p130 Cas , p125 Fak and paxillin by IGF-I was unknown. Given that the mitogen-activated protein kinase kinase-1/ERK and PI 3-kinase pathways have emerged as major mediators of the biological effects promoted by IGF-I, we examined their role in IGF-mediated tyrosine phosphorylation of focal adhesion proteins. We found that IGF-I stimulated tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin independently of ERK activation. In contrast, the structurally unrelated PI 3-kinase inhibitors wortmannin and LY294002 markedly attenuated the increase in the tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin induced by IGF-I. Thus, our results identify a PI 3-kinase-dependent pathway in the tyrosine phosphorylation of these focal adhesion proteins in response to IGF-I stimulation.
PI 3-kinase activity is required for the formation of membrane ruffles by several motility-inducing growth factors (11,65), and the lipid products of this enzyme have been shown to induce actin reorganization and to increase cell migration (64). The small GTPbinding protein Rac is known to mediate the recruitment of actin into membrane ruffles induced by extracellular stimuli (66,67). Previous studies have demonstrated the existence of a signal transduction pathway in the action of PDGF and epidermal growth factor involving PI 3-kinase, Rac, and actin cytoskeletal reorganization that leads to the tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin (26,31,32). The results presented here show that treatment of Swiss 3T3 cells with either cytochalasin D or high concentrations of PDGF, at concentrations known to disrupt the organization of the actin cytoskeleton (24,30), abrogated the increase in the tyrosine phosphorylation of these focal adhesion proteins induced by IGF-I. Our results reveal a novel cross-talk between IGF-I and PDGF and indicate that an intact actin cytoskeleton is essential for IGF-I-induced tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin via the PI 3-kinase pathway. The integrity of the actin cytoskeleton is likely to be required for promoting the assembly of the focal contacts (16), the distinct sites of the plasma membrane where p130 Cas , p125 Fak , and paxillin are recruited and become tyrosine-phosphorylated.
The signaling pathways that lie downstream of PI 3-kinase also include the serine/threonine kinases p70 S6K (57) and the novel and atypical isoforms of PKC (59). Our results demonstrate that inhibition of either p70 S6K or PKC activation, with the immunosuppressant rapamycin or the specific PKC antagonist GF 109203X, respectively, did not prevent IGF-I-stimulated tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin. Thus, IGF-I-mediated tyrosine phosphorylation of focal adhesion proteins is PI 3-kinase-dependent but can be dissociated from activation of either p70 S6K or PKC.
The results presented here also demonstrate that the tyrosine phosphorylation of p130 Cas in response to bombesin is not prevented by either wortmannin or LY294002, at concentrations that virtually abolished the tyrosine phosphorylation of p130 Cas induced by IGF-I. These results imply that there is a PI 3Ј-kinase-dependent and PI 3Ј-kinase-independent signal transduction pathway stimulating the tyrosine phosphorylation of focal adhesion proteins in the same cells.
The molecular cloning of p130 Cas revealed an adaptor protein that contains an SH3 domain, proline-rich regions, and a cluster of 15 putative SH2-binding motifs (19). This suggests that tyrosine-phosphorylated p130 Cas may serve to promote the assembly of multiple SH2-containing molecules. In fact, p130 Cas forms stable complexes with other signaling proteins, including c-Crk and c-Src, in an SH2-dependent manner (19, 68 -70) and associates with p125 Fak , through its SH3 domain (71). Recent reports have shown that integrin-dependent cell adhesion, neuropeptide agonists, and low concentrations of PDGF and epidermal growth factor stimulate association of p130 Cas with c-Crk, an SH2 and SH3 domain-containing adaptor protein (26,32,72,73).
The results presented here demonstrate, for the first time, that IGF-I rapidly induces the formation of a p130 Cas ⅐c-Crk complex that is dependent on the integrity of the actin cytoskeleton. Interestingly, c-Crk has been implicated as a positive effector of IGF-I-mediated mitogenic signaling (74), but the mechanisms involved remain unclear. IGF-I has been shown to increase c-Crk dissociation from insulin receptor substrate-1 (75), resulting in a pool of c-Crk available for new molecular interactions. The assembly of a p130 Cas ⅐c-Crk complex, as shown by our results, is one of the new interactions of c-Crk triggered by IGF-I. The complex between p130 Cas and c-Crk may be important in regulating the subcellular distribution of c-Crk and/or the activity of new downstream effectors in IGF-I signal transduction pathways.
The findings presented here assume an added interest in view of the increasing evidence implicating p130 Cas and p125 Fak in cell migration, proliferation, and transformation. The adaptor protein p130 Cas also been implicated in agoniststimulated mitogenesis (26) and in cell transformation (19,76), and it has recently been identified as a mediator of p125 Fakmediated cell migration (46). In this context, it is interesting that the formation of a p130 Cas ⅐c-Crk complex is emerging as a critical switch in the promotion of cell migration (47). In addition, c-Crk binds to a number of signaling proteins through its SH3 terminal domain including C3G (77), a guanine nucleotide exchange factor for Rap-1, which is a small GTP-binding protein that induces mitogenesis in Swiss 3T3 cells (78). Thus, the increase in the tyrosine phosphorylation of p130 Cas and the induction of complex formation between p130 Cas and c-Crk may be important novel early events in IGF-I signal transduction leading to cell migration and mitogenesis.
Gene disruption experiments have demonstrated a critical role of p125 Fak in embryonic development, cell migration, and turnover of focal adhesions (79 -81). Furthermore, microinjection of dominant-negative fragments of p125 Fak which displaces endogenous p125 Fak from focal adhesions and prevents its activation inhibited cell motility and serum stimulation of DNA synthesis (82). In addition, there is increasing evidence linking overexpression of p125 Fak to cell migration (80) and to the invasive properties of cancer cells (83). IGF-I signaling is also of critical importance in cell proliferation, migration, and transformation (84). For example, IGF-I has been shown to stimulate tumor cell metastasis, a process that requires cell migration (13). Consequently, our results showing that IGF induces a coordinate increase in the level of tyrosine phosphorylation of p130 Cas , p125 Fak , and paxillin and the formation of a complex between p130 Cas and c-Crk through a PI 3-kinasedependent signaling pathway suggest novel mechanisms of action for this important growth promoting factor.