Tyrosine phosphorylation of p130Cas and cortactin accompanies integrin-mediated cell adhesion to extracellular matrix.

We show in this report that two v-src substrate proteins, p130 and cortactin, become tyrosine-phosphorylated during integrin-mediated cell adhesion to extracellular matrix substrata and upon cell attachment onto immobilized anti-integrin antibodies. This tyrosine phosphorylation does not occur when cells attach to polylysine or through antibodies against major histocompatibility complex. It also does not take place when adhesion-mediated reorganization of the actin cytoskeleton is inhibited with cytochalasin D. Tyrosine phosphorylation of p130 and cortactin coincides with tyrosine phosphorylation of focal adhesion kinase during integrin-mediated cell adhesion but is independent of cell adhesion in v-src-transformed cells. The tyrosine-phosphorylated sites in p130 and cortactin may serve as binding sites for proteins containing Src homology 2 domains, as is the case with two other integrin-regulated docking proteins, focal adhesion kinase and paxillin. Thus, these results suggest that ligand binding of integrins regulates the tyrosine phosphorylation state of multiple docking proteins. These proteins may mediate anchorage dependence of growth; their misregulation in v-src-transformed and other tumorigenic cells may be responsible for the anchorage independence of such cells.

We show in this report that two v-src substrate proteins, p130 Cas and cortactin, become tyrosine-phosphorylated during integrin-mediated cell adhesion to extracellular matrix substrata and upon cell attachment onto immobilized anti-integrin antibodies. This tyrosine phosphorylation does not occur when cells attach to polylysine or through antibodies against major histocompatibility complex. It also does not take place when adhesion-mediated reorganization of the actin cytoskeleton is inhibited with cytochalasin D. Tyrosine phosphorylation of p130 Cas and cortactin coincides with tyrosine phosphorylation of focal adhesion kinase during integrin-mediated cell adhesion but is independent of cell adhesion in v-src-transformed cells. The tyrosine-phosphorylated sites in p130 Cas and cortactin may serve as binding sites for proteins containing Src homology 2 domains, as is the case with two other integrin-regulated docking proteins, focal adhesion kinase and paxillin. Thus, these results suggest that ligand binding of integrins regulates the tyrosine phosphorylation state of multiple docking proteins. These proteins may mediate anchorage dependence of growth; their misregulation in v-src-transformed and other tumorigenic cells may be responsible for the anchorage independence of such cells.
Integrins are a family of heterodimeric transmembrane proteins that function as receptors for proteins of the extracellular matrix (ECM) 1 , such as fibronectin, vitronectin, collagens, and laminin (1,2). Upon ligand binding, integrins form clusters at sites of close cell-substrate contact termed focal adhesions, providing a linkage between the ECM and the cytoskeleton (3). This linkage is thought to arise from the interaction of the cytoplasmic domains of the integrins with cytoskeletal proteins such as ␣-actinin and talin (4,5). Focal adhesions are important not only as structural links between the ECM and the cytoskeleton, but also as sites of signal transduction from the ECM.
Elevated phosphotyrosine has been noted in focal adhesions in cells adhered on ECM, indicating that tyrosine kinase(s) are present and active at these sites (6). One of the kinases present in the focal adhesions is the focal adhesion protein-tyrosine kinase FAK (7)(8)(9). Upon coupling of integrins to the ECM, FAK becomes activated and tyrosine-phosphorylated (8, 10 -14), creating a binding site for the Src homology 2 (SH2)-domains of Src-family tyrosine kinases (15)(16)(17)(18). Src phosphorylates several other sites in FAK, which in turn can function as binding sites for other proteins containing SH2-domains (19). Association of FAK with C-terminal Src kinase (20) and Grb2 (18) has been reported, suggesting a role for FAK in connecting integrin ligand binding to downstream signaling pathways, such as activation of the mitogen-activated protein kinases (18,(21)(22)(23). Two other focal adhesion proteins besides FAK, namely paxillin and tensin (12,24), have been shown to contain elevated phosphotyrosine in response to cell adhesion. These proteins may also serve as a connection to the cytoskeleton and to recruit additional SH2-bearing signaling molecules to focal adhesions.
In this report, we have examined tyrosine phosphorylation of proteins in response to cell adhesion to ECM substrata. We show that two recently described signaling molecules, the v-src substrate proteins p130 Cas (25) and cortactin (26,27), become tyrosine-phosphorylated upon cell adhesion to ECM proteins and to immobilized anti-integrin antibodies in normal fibroblastic cells.

EXPERIMENTAL PROCEDURES
Reagents-Dulbecco's modified Eagle's medium (DMEM) was supplied by Life Technologies, Inc., fetal calf serum was from Tissue Culture Biologicals (Tulare, CA), and Glutamine Pen-Strep from Irvine Scientific (Santa Ana, CA). Cytochalasin D was from Sigma. Human plasma fibronectin was obtained from the Finnish Red Cross. Vitronectin was purified from human plasma as previously described (28). Anti-mouse MHC class I H-2 monoclonal antibody was from ATCC, and anti-rat MHC class I RT1A monoclonal antibody was from Pharmingen (San Diego, CA). Polyclonal rabbit anti-␣5␤1 and anti-␣v␤3 antibodies have been described earlier (29,30). Polyclonal rabbit anti-cortactin antibody was a gift from Dr. Tom Maciag (31) and polyclonal rabbit anti-p130 Cas antibody (Cas-2) from Dr. Hisamaru Hirai (25). Monoclonal anti-p130 Cas antibody, monoclonal anti-phosphotyrosine antibody py20, monoclonal anti-FAK antibody, and horseradish peroxidaseconjugated py20 were from Transduction Laboratories (Lexington, KY). Monoclonal anti-paxillin antibody was from Zymed (South San Francisco, CA). All other reagents were acquired from Sigma.
Cell Culture and Cell Adhesion-Rat embryo fibroblasts 52 (REF) cells, NIH 3T3 cells, c-src-transfected 3T3 cells, and v-src-transformed 3T3 cells were grown in DMEM supplemented with 10% fetal calf serum, 50 units/ml penicillin, and 50 g/ml streptomycin. Prior to experiments, cells were serum-starved for 14 h in DMEM containing 0.3% fetal calf serum in order to avoid inadvertent induction of tyrosine phosphorylation of paxillin, FAK, and p130 Cas by lysophosphatidic acid present in the serum (32). Experiments were also carried out without serum starvation; no differences were found in the results between these two conditions (not shown). Cells were detached by trypsinization followed by washing with soybean trypsin inhibitor. The cells were washed twice with DMEM containing 0.5% bovine serum albumin, and cell suspensions were incubated in DMEM, 0.5% bovine serum albumin at 37°C for 40 min on a rotator. Cells were then plated onto the dishes coated with various substrates and incubated at 37°C for the indicated times; cells referred as suspended cells under "Results" were held in suspension for an additional 20 min. In some experiments, cytochalasin D (1 M) was added 10 min prior to plating the cells. Dishes were coated with fibronectin (20 g/ml), vitronectin (20 g/ml), affinity-purified anti-␣5␤1 antibody (10 g/ml), affinity-purified anti-␣v␤3 antibody (10 g/ml), monoclonal anti-MHC antibodies (10 g/ml), or polylysine (20 g/ml) overnight, and blocked with 1% bovine serum albumin for 1 h prior to plating the cells.
Preparations of Cell Lysates, Immunoprecipitations, and Immunoblotting-Cells were washed with ice-cold phosphate-buffered saline and lysed in modified radioimmune precipitation buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% deoxycholate, 50 mM NaF, 0.5 mM Na 3 VO 4 , 0.1 unit/ml aprotinin, 10 g/ml leupeptin, and 4 g/ml pepstatin A). Lysates were clarified by centrifugation at 15,000 ϫ g for 15 min. Antibodies were added to lysates containing an equal amount of protein, and samples were rotated at 4°C for 1 h. To precipitate the antibody-antigen complexes, Gammabind-Sepharose (Pharmacia Biotech Inc.) was added to the lysates, and rotation was continued for 2 h. The immunoprecipitates, pelleted by microcentrifuging, were washed three times in wash buffer (ϭlysis buffer without SDS and deoxycholate). In immunodepletion experiments, the supernatant was subjected to three rounds of precipitation with the anti-FAK and anti-paxillin antibodies, and the supernatant cleared of FAK and paxillin (as determined by immunoblot analysis) was immunoprecipitated with anti-phosphotyrosine py20 antibody. The pellets were boiled in sample buffer and electrophoresed on 4 -12% precast SDS-polyacrylamide gel electrophoresis gels (Novex). After electrophoresis, proteins were transferred to nitrocellulose. Phosphotyrosine-containing proteins were visualized by incubating with horseradish peroxidase-conjugated anti-phosphotyrosine py20 antibody followed by enhanced chemiluminescence detection (ECL, Amersham Corp.). To detect FAK, p130 Cas , and cortactin, blots were probed with monoclonal FAK, monoclonal p130 Cas , and polyclonal anti-cortactin antibodies, followed by horseradish peroxidase-conjugated anti-mouse IgG and protein A, respectively. Immunoreactive bands were visualized by enhanced chemiluminescence.

Cell Adhesion Induces Tyrosine Phosphorylation of p130 Cas
and Cortactin-To investigate tyrosine phosphorylation of proteins in response to cell adhesion, REF cells were either kept in suspension or plated on dishes coated with fibronectin for 45 min. As shown in Fig. 1 (lanes 1 and 2), cell adhesion to fibronectin resulted in elevated tyrosine phosphorylation of proteins of 160, 120 -130, 115, and 70 -85 kDa in molecular mass. NIH 3T3 cells were also tested throughout the study, and similar results were obtained. Unless otherwise indicated, results shown in the figures are those obtained with REF cells. As expected based on the previous reports, one of the phosphorylated proteins in the 120 -130-kDa range was found to be FAK (8, 10 -13), while one of the 70 -85 kDa proteins was identified as paxillin (12) (data not shown). When lysates obtained from cells plated on fibronectin were depleted of FAK and paxillin by repeated immunoprecipitations with the appropriate antibodies, bands around 120 -130 and 70 -85 kDa could still be detected in anti-phosphotyrosine blot, suggesting that multiple proteins in these molecular mass ranges become tyrosine phosphorylated upon cell adhesion and integrin ligand binding (not shown).
To investigate the possibility that two v-src substrates, p130 Cas (25) and cortactin, an 80/85 kDa protein (26,27), would be among the unidentified phosphoproteins, samples from suspended and adherent REF cells were immunoprecipitated with antibodies against p130 Cas and cortactin, and they were analyzed by immunoblotting with an anti-phosphotyrosine antibody (Fig. 1, upper panel). When immunoprecipitated from suspended cells, p130 Cas and cortactin (lanes 3 and 7) exhibited very low levels of tyrosine phosphorylation. Cell adhesion to polylysine, to which cells can adhere in a nonspecific fashion, had no effect on the tyrosine phosphorylation of p130 Cas and cortactin (lanes 4 and 8). Similarly, no increase in tyrosine phosphorylation was observed when cells were allowed to adhere on dishes coated with anti-MHC antibodies (not shown). An increase in tyrosine phosphorylation was seen in cells plated on fibronectin or on anti-␣5␤1 antibodies (lanes 5, 6, 9, and 10). The amounts of p130 Cas and cortactin were essentially the same in all samples as shown when the same plot was stripped and reprobed with anti-p130 Cas and anti-cortactin antibodies (Fig. 1, lower panel). As it has been previously reported, p130 Cas was found to migrate as multiple bands, presumably due to differences in post-translational modifications (25). Increased tyrosine phosphorylation of p130 Cas and cortactin was also observed when cells were plated on dishes coated with vitronectin or anti-␣v␤3 antibodies (not shown).

Tyrosine Phosphorylation of p130 Cas and Cortactin Coincides with FAK Phosphorylation and Correlates with Actin Filament
Reorganization-To study the connection of cortactin and p130 Cas phosphorylation with actin filament reorganization, REF cells were allowed to adhere to fibronectin-coated dishes, and tyrosine phosphorylation of p130 Cas , cortactin, and FAK was assessed as a function of time (Fig. 2). At the time point zero, all of these proteins seem to contain little phosphotyrosine. After 15 min, most of the cells had adhered to fibronectin, but had not yet fully spread. As shown earlier, at this time FAK phosphorylation had reached maximal levels and seemed to plateau (11). The time course for p130 Cas and cortactin phosphorylation was indistinguishable from that of FAK. Thus, the phosphorylation of all these proteins seems to be coincident with cell adhesion, and maximal phosphorylation occurs around the time of cell spreading and actin filament reorganization. To determine whether reorganization of cytoskeleton is necessary for cell adherence to stimulate tyrosine phosphorylation of p130 Cas and cortactin, the spreading of cells plated onto a fibronectin substratum was inhibited with cytochalasin D; p130 Cas , cortactin, or FAK did not become tyrosine-phosphorylated in the presence of cytochalasin D (Fig. 2). Cytochalasin D treatment has earlier been demonstrated to prevent integrin- mediated tyrosine phosphorylation of FAK and tensin (24).

Cell Adhesion Is Not Required for Tyrosine Phosphorylation of p130 Cas and Cortactin in v-src-transformed Cells-Both
p130 Cas and cortactin, as well as FAK, paxillin, and tensin, were originally identified as tyrosine-phosphorylated proteins in v-src-transformed cells (33,34). Recently, it has been demonstrated that tyrosine phosphorylation and activation of FAK in v-src transformed cells is independent of cell adhesion (18). We tested whether the tyrosine phosphorylation of p130 Cas and cortactin in anchorage-independent v-src transformed cells requires integrin-mediated signaling and cell adherence to ECM substrata. As shown in Fig. 3, no differences were found in the levels of tyrosine phosphorylation of p130 Cas and cortactin between v-src cells held in suspension or plated on fibronectin, demonstrating that adhesion is not required for tyrosine phosphorylation of p130 Cas and cortactin in v-src cells. We also tested the adhesion-dependence of p130 Cas and cortactin tyrosine phosphorylation in NIH 3T3 cells which express enhanced levels of c-src (35). Overexpression of c-src in NIH 3T3 cells generates in vitro kinase activity levels comparable to those of v-src transformed cells. These cells display morphological characteristics intermediate between those of normal and v-srctransformed NIH 3T3 cells, but overexpression of c-src does not induce transformation of NIH 3T3 cells (35). As in the case of normal NIH 3T3 cells, but not of the v-src transformed cells, the tyrosine phosphorylation of p130 Cas and cortactin was dependent on cell adhesion in c-src overexpressing cells (Fig. 3). DISCUSSION Several proteins are tyrosine-phosphorylated in response to cell adhesion and spreading on extracellular substrata. Three of these proteins have previously been identified as FAK, paxillin, and tensin (8, 10 -13, 24, 37). In this study, we report on elevated phosphotyrosine on two additional proteins, p130 Cas and cortactin, as cells adhere to ECM substrates or to immobilized anti-integrin antibodies. We also show that this tyrosine phosphorylation coincides with tyrosine phosphorylation of FAK, requires organization of actin cytoskeleton, and is independent of cell adhesion in v-src-transformed cells.
p130 Cas was originally identified as a highly tyrosine-phosphorylated protein during cellular transformation by v-src (34,38), as well as by v-crk (39,40), that forms stable complexes with these oncoproteins. Recent molecular cloning of p130 Cas identified it as a novel SH3-containing signaling molecule with a cluster of multiple putative SH2-binding motifs (25). The unique structure of p130 Cas indicates the possible role of p130 Cas in assembling signals from multiple SH2-containing molecules, including Src and Crk. Therefore, p130 Cas may serve as a docking protein that tethers other proteins to a multicomponent complex, and integrin-mediated tyrosine phosphorylation of p130 Cas observed here may function to regulate such protein-protein interactions. Recently, FAK has been shown to interact with Src, Grb2, C-terminal Src kinase, and phosphatidylinositol 3-kinase in an adhesion and/or tyrosine phosphorylation-dependent manner (15-18, 20, 41), while paxillin forms complexes with FAK, Src, C-terminal Src kinase, and Crk (20,36,(42)(43)(44). Thus, ligand binding of integrins seems to regulate multiple putative docking proteins that connect to downstream signaling pathways.
Cortactin is an 80/85-kDa SH3-domain containing protein, that becomes phosphorylated on tyrosine in v-src-transformed cells and in cells stimulated with certain growth factors (26,27). Cortactin binds to F-actin and is enriched in cortical cell structures such as lamellipodia (27). It has therefore been suggested that cortactin may be important for microfilamentmembrane interactions and that it may also transduce signals from the cell surface to the cytoskeleton (27). Thus, one possibility is that the integrin-mediated tyrosine phosphorylation of cortactin observed here may play a role in actin remodeling during cell adhesion to ECM substrata. Previous results, however, indicate that tyrosine phosphorylation of cortactin does not influence F-actin binding (27). It is also possible that, similar to paxillin, FAK and p130 Cas , phosphorylation of cortactin may promote the binding of cortactin to cellular proteins containing SH2 domains, allowing cortactin to contribute to the assembly of signaling molecule complexes.
Our finding that p130 Cas and cortactin become tyrosinephosphorylated following cell adhesion to ECM substrates, but not to polylysine, is consistent with the tyrosine phosphorylation being mediated by integrins. This is further supported by the observation that cell adhesion to two different anti-integrin antibodies also results in elevated tyrosine phosphorylation of p130 Cas and cortactin. These findings also suggest that ligand binding to different integrins can activate the same tyrosine kinase(s) (the one(s) responsible for phosphorylating p130 Cas and cortactin).
In addition to REF and NIH 3T3 cells, we have also analyzed tyrosine phosphorylation of p130 Cas and cortactin in human 293 embryonic kidney carcinoma cell line, and in mouse embryonic and Rat-1 fibroblasts (not shown). Elevated tyrosine phosphorylation of p130 Cas in response to cell adhesion was seen in each of the cell lines we have tested so far. However, we have not been able to detect elevated tyrosine phosphorylation levels of cortactin in some of the cell lines tested, including chick embryo fibroblasts and human skin fibroblasts. Whether this truly reflects cell type specificity, or whether there are  1 and 2), v-src-transformed 3T3 cells (lanes 3 and 4), and c-src-transfected 3T3 cells (lanes 5 and 6) were either held in suspension (lanes 1, 3, and 5) or plated on fibronectin for 45 min (lanes 2, 4, and  6). Anti-p130 Cas and anti-cortactin immunoprecipitates were prepared from the cell lysates and subjected to phosphotyrosine analysis (upper panel). The blots were stripped and reprobed to confirm equal loading (lower panel).
changes that are below detection limits of our antibodies is not known.
We found that tyrosine phosphorylation of p130 Cas and cortactin coincides with tyrosine phosphorylation of FAK. It has been reported that paxillin and FAK become coordinately phosphorylated on tyrosine during cell spreading on fibronectin (12), and that paxillin is a substrate for FAK kinase activity in vitro and in vivo (36,45). It remains to be seen whether tyrosine phosphorylation of p130 Cas and cortactin requires integrin-mediated FAK activation, or whether it is a result of a separate, integrin-activated, but FAK-independent kinase pathway.
Tyrosine phosphorylation of p130 Cas and cortactin requires the presence of intact cytoskeleton, since the adhesion-induced tyrosine phosphorylation of these proteins can be prevented by cytochalasin D treatment. A similar situation has been observed with FAK and tensin (24). Because no direct association between integrins and kinases has been observed, it is possible that kinases associate with integrins through interactions with cytoskeletal complexes induced by the cross-linking of integrins (9). Thus, intact, functional cytoskeleton may be required to bring together the various components of this signaling complex, resulting in tyrosine phosphorylation of cellular proteins.
While this work was in progress, Petch et al. (46) reported on the adhesion-induced tyrosine phosphorylation of p130 Cas . They demonstrated that, as in the case of FAK, paxillin, and tensin, p130 Cas is also found in focal contacts. The focal contact colocalization of the proteins connected to integrin signaling emphasizes the role of these sites in the signal transduction from the ECM into the cell.
It has recently been demonstrated that tyrosine phosphorylation of FAK in v-src-transformed cells is independent of cell adhesion (18), and we show here that this is also the case with p130 Cas and cortactin. Based on the apparent correlation between cellular transformation and elevated levels of protein tyrosine phosphorylation in v-src transformed cells, it is believed that physiologically important Src substrates will be proteins normally involved in the control of cellular proliferation (47). Since it seems that a number of v-src substrates are in the integrin signal transduction pathway, it is equally possible that these proteins and integrin signals mediated by them are responsible for anchorage-dependence, which is lost in transformed cells. Identification of protein-protein interactions resulting from integrin-dependent tyrosine phosphorylation of cellular proteins and characterization of signaling pathways that become activated following these interactions may lead to better understanding of anchorage dependence of growth.