p130Cas, a substrate associated with v-Src and v-Crk, localizes to focal adhesions and binds to focal adhesion kinase.

p130Cas (rk ssociated ubstrate) has the structural characteristics of an adapter protein, containing multiple consensus SH2 binding sites, an SH3 domain, and a proline-rich domain. The structure of p130Cas suggests that it may act to provide a framework for protein-protein interactions; however, as yet, its functional role in cells is unknown. In this report we show that p130Cas is localized to focal adhesions. We demonstrate that p130Cas associates both in vitro and in vivo with pp125FAK (ocal dhesion inase), a kinase implicated in signaling by the integrin family of cell adhesion receptors. p130Cas also associates with pp41/43FRNK (pp125FAK-related, non-kinase), an autonomously expressed form of pp125FAK composed of only the C-terminal noncatalytic domain. We show that the association of p130Cas with pp125Fak and pp41/43FRNK is direct, and is mediated by the binding of the SH3 domain of p130Cas to a proline-rich sequence present in both the C terminus of pp125FAK and in pp41/43FRNK. In agreement with recent studies we show that p130Cas is tyrosine-phosphorylated upon integrin mediated cell adhesion. The association of p130Cas with pp125FAK, a kinase which is activated upon cell adhesion, is likely to be functionally important in integrin mediated signal transduction.

p130 Cas ( crk associated substrate) has the structural characteristics of an adapter protein, containing multiple consensus SH2 binding sites, an SH3 domain, and a proline-rich domain. The structure of p130 Cas suggests that it may act to provide a framework for protein-protein interactions; however, as yet, its functional role in cells is unknown. In this report we show that p130 Cas is localized to focal adhesions. We demonstrate that p130 Cas associates both in vitro and in vivo with pp125 FAK ( focal adhesion kinase), a kinase implicated in signaling by the integrin family of cell adhesion receptors. p130 Cas also associates with pp41/43 FRNK (pp125 FAK -related, non-kinase), an autonomously expressed form of pp125 FAK composed of only the C-terminal noncatalytic domain. We show that the association of p130 Cas with pp125 Fak and pp41/43 FRNK is direct, and is mediated by the binding of the SH3 domain of p130 Cas to a proline-rich sequence present in both the C terminus of pp125 FAK and in pp41/43 FRNK . In agreement with recent studies we show that p130 Cas is tyrosine-phosphorylated upon integrin mediated cell adhesion. The association of p130 Cas with pp125 FAK , a kinase which is activated upon cell adhesion, is likely to be functionally important in integrin mediated signal transduction.
Adhesion of cells to the extracellular matrix is mediated primarily by the integrin family of receptors (1). Binding of the integrin receptors to the extracellular matrix results in the clustering of integrins and the recruitment of a wide array of proteins to form polyprotein structures called focal adhesions. Focal adhesions serve to link the extracellular matrix with the actin cytoskeleton of the cell (2). In addition to carrying out a structural role, integrins can also activate a variety of signaling pathways, similar to that of growth factor receptors. These signals include tyrosine phosphorylation, elevation of intracellular pH and Ca 2ϩ , and lipid turnover (reviewed in Ref. 3). Consistent with the signaling capability of integrins, cell adhesion can influence a variety of cellular functions including cell growth, differentiation (4), and apoptosis (5).
Integrins are catalytically inactive and based on the paradigm of cytokine receptors it is thought that they signal via the recruitment of signaling molecules (3,6,7). A variety of signaling molecules have been identified in focal adhesions. These include the tyrosine kinases pp125 FAK 1 (8), pp60 Src (9), and C-terminal Src kinase (10), the serine/threonine kinases, protein kinase C (11) and mitogen-activated protein kinase, the small G proteins Ras, Rho, and Rac, protein tyrosine phosphatase 1D, phospholipase C␥, PI-3K, and adapter proteins such as paxillin and Grb2 (12)(13)(14). The recruitment to focal adhesions of all signaling molecules tested to date, except pp125 FAK , is inhibited by tyrosine kinase inhibitors or cytochalasin D (13). pp125 FAK is tyrosine-phosphorylated upon cell adhesion (15)(16)(17), which correlates with an increase in its catalytic activity (18,19). These observations are consistent with a model in which the early recruitment of pp125 FAK enables the tyrosine phosphorylation required by other signaling molecules for focal adhesion recruitment.
Cell adhesion leads to the tyrosine phosphorylation of a limited number of proteins, which in addition to pp125 FAK includes paxillin (20), tensin (21), and cortactin (22). It has been suggested that the tyrosine phosphorylation of these proteins may result in the creation of SH2 binding sites for other focal adhesion components, allowing their recruitment and leading to the generation of multiprotein complexes, important either structurally or for signaling from the focal adhesion (23,24). In this study we identify p130 Cas as another component of focal adhesions and demonstrate that p130 Cas is tyrosine-phosphorylated upon integrin mediated cell adhesion. p130 Cas is an adapter family protein consisting of an SH3 domain, a prolinerich domain, a central "substrate" domain composed of a cluster of potential SH2 interaction sites, and a C-terminal domain containing both consensus SH3 and SH2 binding sites for Src (25).
p130 Cas was originally identified as a protein highly tyrosine-phosphorylated during transformation mediated by both the v-src and v-crk oncogenes (26,27). This suggests that p130 Cas plays an important role in transformation mediated by both of these oncogenes. In order to better understand the role p130 Cas plays in cellular transformation, we analyzed its function in normal cells. In this study we report that p130 Cas is localized to focal adhesions, and that it associates with pp125 FAK in vivo. This interaction can be reconstituted in vitro and is mediated by the SH3 domain of p130 Cas binding to a proline rich containing sequence in the C-terminal domain of pp125 FAK . The interaction of p130 Cas with pp125 FAK , a kinase activated upon cell adhesion, is likely to be functionally important in integrin mediated signal transduction.

MATERIALS AND METHODS
Cell Culture, Cell Adhesion, and Protein Expression-Primary chicken embryo (CE) cells were prepared and cultured as described previously (28). pp125 FAK and pp41/43 FRNK were expressed in CE cells using the replication competent RCAS A retrovirus vector as described previously (15,29). To study the adhesion-dependent phosphorylation of p130 Cas , 100-mm bacterial plastic dishes were coated overnight at 4°C, or for 2 h at 37°C, with fibronectin (2.5 g/cm 2 ), or for 1 h at room temperature with poly-L-lysine (25 g/cm 2 ). Cells were removed from culture dishes by trypsinization and washed with phosphate-buffered saline containing soybean trypsin inhibitor. The cells were kept in suspension for approximately 30 min at which time 4 ϫ 10 6 cells/ 100-mm dish were plated onto coated dishes at 37°C for the indicated times in serum-free medium. Fibronectin, poly-L-lysine, and soybean trypsin inhibitor were all obtained from Sigma. Rat embryo fibroblast (REF) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, penicillin (100 units/ml), and streptomycin (100 g/ml) (Life Technologies, Inc.). Cells were lysed as described previously (30) using modified radioimmune precipitation buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.5% sodium deoxycholate), containing protease and phosphatase inhibitors (10 g/ml leupeptin, 100 g/ml phenylmethylsulfonyl fluoride, 0.045 unit/ml aprotinin, 1 mM sodium vanadate, 40 mM sodium fluoride, 40 mM p-nitrophenylphosphate). For the coimmunoprecipitation experiments, modified radioimmune precipitation buffer without sodium deoxycholate was used. Protein concentrations were determined using the BCA assay (Pierce).
Mutagenesis of pp125 FAK and Construction of GST Fusions of pp125 FAK and p130 Cas -The point mutations P715A, P718A, P715A/ P718A, P878A, P881A, and P878A/P881A were made in the context of a truncated form of pp125 FAK composed of amino acid residues 687-1054 ( Fig. 6, CtermII), using the Altered Sites in vitro mutagenesis system (Promega). The presence of the mutations was confirmed by dideoxy sequencing (U. S. Biochemical Corp.). The mutants were cloned into pGEX2TK (Pharmacia) for expression as bacterial fusion proteins. The quadruple point mutant P715A/P718A/P878A/P881A was generated in pGEX2TK-CtermII by splicing together the double point mutants P715A/P718A and P878A/P881A at an internal HindIII site.
For in vitro association experiments regions of p130 Cas were expressed as bacterial fusion proteins. GST fusion proteins of p130 Cas composed of the SH3 domain (amino acids 100 -158), the substrate domain (amino acids 211-512) and two different portions of the C terminus, amino acids 513-637 (mid-domain) and amino acids 638 -916 (C-TERM) ( Fig. 1) were generated by cloning the corresponding cDNA inframe into pGEX2TK. The generation of the GRAF ( GTPase regulator associated with FAK) SH3 is described elsewhere (32).
Immunofluorescence-To determine the subcellular localization of p130 Cas , REF cells, or CE cells were grown overnight on fibronectincoated coverslips (2.5 g/cm 2 ). The cells were fixed in 4% paraformaldehyde for 20 min, permeabilized for 5 min in 0.25% Triton X-100, and stained as described previously (33). Polyclonal antibodies CAS B and CAS F were used to detect p130 Cas . To ensure the staining was due to specific binding of the antibodies to p130 Cas , the antibody was depleted with glutathione beads complexed to either GST or the GST fusion protein against which the antibody was raised. Incubations were carried out at 4°C for 1 h, the beads were removed by centrifugation at 14,000 ϫ g for 3 min, and the supernatant was used for cell staining. Vinculin was detected using a monoclonal antibody (Sigma). pp125 FAK was detected using 2A7, a FAK-specific monoclonal antibody (8).
Protein Association-For in vitro association experiments, GST fusion proteins were expressed in bacteria and affinity-purified using glutathione-Sepharose (Pharmacia) (34). Equivalent amounts of the GST fusion proteins (approximately 5 g) or GST alone, immobilized on glutathione-Sepharose, were incubated with the indicated quantity of lysate for 1 h at 4°C with rotation. The beads were washed twice with modified radioimmune precipitation buffer and twice with Tris-buffered saline (50 mM Tris-HCl, pH 7.5, 150 mM NaCl). Associated proteins were eluted by boiling in Laemmli sample buffer, resolved by 8% SDS-PAGE and transferred to nitrocellulose for Western or Far-Western blotting. Immunoprecipitations were carried out by incubating the cell lysate with antibody for 2 h at 4°C. pp125 FAK was immunoprecipitated using either the FAK specific monoclonal antibody 2A7 or the polyclonal antisera BC3 (8). p130 Cas was immunoprecipitated with either a CASspecific monoclonal antibody (Transduction Laboratories) or a mix of the polyclonal antisera CAS B and F. The immune complexes were recovered by incubation for 1 h at 4°C with protein A-Sepharose beads, either alone or complexed to rabbit anti-mouse IgG, for the monoclonal antibody. The immune complexes were washed three times with modified radioimmune precipitation buffer and twice with Tris-buffered saline. Associated proteins were recovered by boiling for 5 min in Laemmli sample buffer, separated by 8% SDS-PAGE and subjected to either Western or Far Western blotting.
For Western blotting the indicated antibodies were used. Primary antibodies were detected using horseradish peroxidase-conjugated rabbit antimouse IgG or horseradish peroxidase-conjugated protein A, followed by enhanced chemiluminescence (ECL; Amersham Corp.). Far Western blotting and generation of the 32 P-labeled probe were carried out as described previously (35).

RESULTS
p130 Cas Localizes to Focal Adhesions-To determine the subcellular localization of p130 Cas , immunofluorescent labeling of REF cells was carried out using two p130 Cas specific polyclonal antisera, CAS B and CAS F. CAS B was raised against a GST fusion protein composed of residues 318 to 486 within the substrate domain of p130 Cas . CAS F was raised against a GST fusion protein composed of residues 670 to 896 within the C terminus of p130 Cas (Fig. 1). Both antibodies gave a staining pattern characteristic of focal adhesions (Fig. 2, panels A and C). To confirm the p130 Cas staining was localized to focal adhesions, costaining was carried out using a monoclonal antibody to vinculin, a well characterized component of focal adhesions. The staining of p130 Cas with both antibodies colocalized exactly with that of vinculin (compare panels A with B, and C with D). Coimmunostaining with 2A7, a monoclonal antibody specific for pp125 FAK , another component of focal adhesions, gave the same results (data not shown). To confirm the specificity of the antibodies for p130 Cas they were incubated prior to cell staining with glutathione-Sepharose beads, complexed with GST or the GST fusion protein to which they were raised. Preclearing with GST fusion protein resulted in a loss of staining, whereas GST alone had no effect on the ability of the antibodies to stain focal adhesions (data not shown). In addition, no staining of focal adhesions was observed when preimmune sera was used (data not shown). Immunostaining of CE cells gave a similar pattern of focal adhesion staining.
p130 Cas Associates in Vitro with pp125 FAK -To determine if p130 Cas was associated with any known components of focal adhesions, in vitro association experiments were carried out. On the basis of sequence analysis, putative functional domains have been delineated in p130 Cas (25); these include an SH3 domain, a "substrate domain" rich in potential SH2 interaction sites and a C-terminal domain containing putative Src SH2 and SH3 binding sites (Fig. 1). GST fusion proteins encompassing these putative functional domains of p130 Cas were made as described under "Materials and Methods." CE cell lysate was incubated with the GST fusion proteins or GST immobilized on glutathione beads, the beads were washed, and associated proteins were analyzed by Western blotting using a phosphotyrosine specific antibody. As shown in Fig. 3A the SH3 domain of p130 Cas formed a stable complex with the major tyrosinephosphorylated protein present in CE cell lysates (lanes 1 and  3). This protein was previously identified as pp125 FAK (8), a tyrosine kinase present in focal adhesions. To confirm the identity of this protein the association assay was repeated and Western blotting was carried out using BC3, a polyclonal antisera specific for pp125 FAK (8). As shown in Fig. 3B (lanes 4 -6) and no nonspecific association with GST was detected (lane 2).
In addition to being immunoreactive with pp125 FAK , BC3 also recognizes a pp125 FAK -related protein, pp41/43 FRNK . pp41/ 43 FRNK is an autonomously expressed form of pp125 FAK , composed of only the C-terminal noncatalytic region (15). Blotting of the GST fusion protein associated proteins with BC3 revealed that pp41/43 FRNK was also capable of specifically interacting with the SH3 domain of p130 Cas in vitro (Fig. 3B, bottom  panel, lane 3). This interaction was not detected on the phosphotyrosine blot because pp41/43 FRNK is phosphorylated only on serine residues (15). 2 Comparing lane 1 with lane 3 we estimate that approximately 20% of the cellular pp125 FAK and approximately 50% of pp41/43 FRNK was associated with p130 Cas .
To examine the specificity of the association of the p130 Cas SH3 with pp125 FAK and pp41/43 FRNK , equivalent amounts of GST fusion proteins of the SH3 domains of p130 Cas , Abl, Src, Fyn, Lyn, cortactin, and GRAF along with full-length Grb2 were incubated with lysate from CE cells and Western blotted for the presence of pp125 FAK and pp41/43 FRNK using BC3. Among these SH3 domains only p130 Cas and GRAF were capable of stably associating with both pp125 FAK and pp41/43 FRNK (Fig. 4, lanes 3 and 9), with p130 Cas exhibiting the most efficient binding. Lyn was capable of associating with pp125 FAK (lane 7); however, this binding appeared less efficient.
p130 Cas Binding to pp125 FAK Is Direct-To determine if the interaction of the SH3 domain of p130 Cas with pp125 FAK and pp41/43 FRNK is direct or is mediated by other proteins, a Far Western blot analysis was carried out. The GSTp130 Cas SH3 was purified and labeled with 32 P on a specific protein kinase A  site present on the GST portion of the fusion protein. The labeled fusion protein was used to probe lysates from either pp125 FAK or pp41/43 FRNK overexpressing CE cells, which were resolved by SDS-PAGE and transferred to nitrocellulose. The labeled p130 Cas SH3 bound predominantly to a single protein in lysates from each of these cells (Fig. 5, lane 1). This protein was the same molecular weight as that associated in vitro with the GSTp130 Cas SH3 in each these cell lysates (lane 5). That these proteins were pp125 FAK and pp41/43 FRNK was confirmed by probing BC3 immune complexes prepared from each of these lysates, with the labeled p130 Cas SH3 (lane 3). No proteins were detected in association with either GST (lane 4) or preimmune sera (lane 2). These results confirm that the SH3 domain of p130 Cas is capable of interacting directly with pp125 FAK and pp41/43 FRNK .
p130 Cas Interacts with a Proline-rich Sequence Present in the C Terminus of pp125 FAK -The association of the SH3 domain of p130 Cas with pp41/43 FRNK indicated that the site of p130 Cas interaction with pp125 FAK was within the C-terminal domain. The C terminus of pp125 FAK has two proline-rich sequences, P 712 PKPSRPGYPSP and P 874 PKKPPRPGAP which could act as binding sites for an SH3 domain (Fig. 6). To determine which of these regions was functionally important, proline to alanine  (Fig. 6). GST fusion proteins of these constructs were generated and assayed for their ability to associate with p130 Cas in CE cell lysate. Fig. 7 shows a representative Western blot of the association. Among the mutant proteins CtermIIP715A and CtermIIP715A/718A, which contain mutations within the proline-rich region proximal to the catalytic domain of pp125 FAK , reproducibly displayed reduced association to p130 Cas , localizing the binding site to the type II consensus motif P 712 PKPSR.
p130 Cas Associates with pp125 FAK in Cells-To demonstrate that p130 Cas and pp125 FAK are associated within the cell, coimmunoprecipitation experiments were carried out. pp125 FAK was expressed in CE cells infected with a retroviral RCAS A vector and immunoprecipitated from lysates using 2A7, a monoclonal antibody specific for pp125 FAK . As shown in Fig. 8A p130 Cas was present in these pp125 FAK immune complexes as revealed by blotting with a mixture of CAS specific antibodies (lane 3), whereas no p130 Cas was present in a control immune complex prepared with rabbit antimouse IgG (lane 2). Similarly immune complexes prepared using the p130 Cas -specific monoclonal antibody contained pp125 FAK as shown by Western blotting with BC3, a polyclonal antibody specific for pp125 FAK (Fig.  8B, lane 3). Rabbit anti-mouse IgG did not associate with pp125 FAK (lane 2). These results show that p130 Cas and pp125 FAK form stable complexes within cells.
p130 Cas Is Tyrosine-phosphorylated in Response to Integrin-mediated Cell Adhesion-The localization of p130 Cas to focal adhesions and its association with pp125 FAK prompted us to examine whether p130 Cas was tyrosine-phosphorylated upon integrin mediated cell adhesion. CE cells were placed in suspension, and plated onto fibronectin or poly-L-lysine-coated dishes for the indicated times ( Fig. 9). p130 Cas immune complexes were prepared using a mixture of two p130 Cas -specific polyclonal antisera, CAS B and CAS F. p130 Cas was tyrosinephosphorylated in continuously adherent cells (Fig. 9, lane 1). Upon placing the cells in suspension the level of tyrosine phosphorylation decreased significantly (lane 2). Replating the cells onto fibronectin, on which the cells attach and spread in an integrin dependent fashion, resulted in an increase in tyrosinephosphorylated p130 Cas (lanes 4, 6, 8, and 10). In contrast, replating onto poly-L-lysine, where the cells adhere in a nonspecific manner, caused no further increase in p130 Cas tyrosine phosphorylation over that observed in cells in suspension (lanes 3, 5, 7, and 9). These results indicate that p130 Cas is tyrosine-phosphorylated in an integrin-dependent manner.
The kinetics of p130 Cas phosphorylation and pp125 FAK phosphorylation upon adhesion to fibronectin were similar (data not shown).

DISCUSSION
The structural organization of focal adhesions is dictated by a complex network of protein-protein interactions, involving some well characterized proteins and other more poorly understood components. Here we show that p130 Cas , a substrate for The numbers below the schematic represent amino acid numbers. P represents proline-rich regions, the sequence of which is given underneath in single-letter code.
protein tyrosine kinases in v-src-and v-crk-transformed cells, is a component of focal adhesions and stably associates with the focal adhesion-associated tyrosine protein kinase, pp125 FAK . Previous studies examining the localization of p130 Cas , used an antibody against the tyrosine-phosphorylated form of p130 Cas and reported variable staining of focal adhesions, nuclei, and stress fibers (36). Using antibodies directed against purified bacterially produced Cas protein, we confirmed the focal adhesion localization of p130 Cas ; however, we observed no detectable staining of nuclei or stress fibers in REF or CE cells. An association of p130 Cas with pp125 FAK was initially suggested based on the results of a yeast two-hybrid screen designed to isolate pp125 FAK -interacting proteins (37). The results reported herein extend these observations and clearly show that p130 Cas and pp125 FAK can form stable complexes in vivo. Further, the association can be reconstituted in vitro and in this context is mediated predominantly by the binding of the SH3 domain of p130 Cas to the sequence P 712 PKPSR, a typical type II SH3 consensus binding site (38) present in the C terminus of pp125 FAK . The physiological relevance of the interaction of p130 Cas with pp125 FAK is thus supported by three observations; the colocalization of p130 Cas and pp125 FAK to the same cellular compartment, the isolation of stable complexes of p130 Cas and pp125 FAK from cells expressing pp125 FAK , and the ability to reconstitute the interaction of FAK and Cas in vitro.
The tyrosine phosphorylation of p130 Cas during cell adhesion to fibronectin indicates that the association of p130 Cas and pp125 FAK may be functionally significant and is in accord with recent reports (22,36,39). In addition to pp125 FAK , other tyrosine kinases, including the Src family kinases and C-terminal Src kinase, have been reported to localized to focal adhesions (9,10,13). pp125 FAK is an attractive candidate for mediating the tyrosine phosphorylation of p130 Cas , because it becomes phosphorylated upon adhesion with similar kinetics to that of p130 Cas , and the tyrosine phosphorylation of pp125 FAK has been shown to coincide with an increase in its kinase activity (18,19). Using pp125 FAK overexpressing cells we observed that p130 Cas is phosphorylated in a pp125 FAK -dependent fashion in vivo, upon inhibition of cellular tyrosine phosphatases by vanadate. 3 This observation is consistent with pp125 FAK playing a role, either directly or indirectly, via the activation of Src (23,40), in the tyrosine phosphorylation of p130 Cas by integrin activation. Further evidence for the functional importance of the binding of FAK to Cas is provided by the conservation of this interaction between Cas family members. The SH3 domain of p130 Cas is highly conserved in EFS ( embryonal Fyn-associated substrate), a recently isolated p130 Cas family member (41). A partial cDNA clone (designated FIPSH3-2) encoding the SH3 domain of EFS, was isolated along with p130 Cas during the yeast two hybrid screen designed to identify binding partners of pp125 FAK (37).
p130 Cas joins a small family of proteins which includes tensin, paxillin and cortactin, that become tyrosine-phosphorylated upon adhesion (20 -22). p130 Cas is rich in consensus SH2 binding sites, nine of which conform to the consensus for Crk binding. Consensus binding sites also exist for the SH2 domains of Src, tensin, Abl, Grb2, PI3K, and Nck. It has been shown that the substrate domain of Cas binds to v-Crk, whereas v-Src binds to the C-terminal domain (31). p130 Cas is also able to associate with Grb2 in an in vitro association assay. 3 Therefore tyrosine phosphorylation of p130 Cas upon adhesion could generate functional binding sites for any of these ligands. These molecules have been implicated in a variety of signaling pathways and their binding to p130 Cas could thus mediate the activation of these signaling pathways upon integrin dependent adhesion. Alternatively generation of a tensin binding site on p130 Cas could promote the assembly of focal adhesions by facilitating the binding of actin stress fibers (42).
p130 Cas was identified originally as a protein highly tyrosine-phosphorylated in v-src-and v-crk-transformed cells (26,27). A recent study has shown that overexpression of antisense mRNA for p130 Cas caused a morphological reversion of the transformed phenotype in cells transformed by ornithine decarboxylase, v-src and c-Ha-ras (43), suggesting p130 Cas plays a central role in transformation mediated by these oncogenes. The tyrosine phosphorylation of p130 Cas upon integrin mediated cell adhesion raises the possibility that one mechanism of the action of these oncogenes is in bypassing normal integrin stimulated signaling. Further elucidation of the role of p130 Cas in focal adhesion signaling and structure will be key to understanding its involvement in cell transformation.