©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Integrin-mediated Cell Adhesion Promotes Tyrosine Phosphorylation of p130, a Src Homology 3-containing Molecule Having Multiple Src Homology 2-binding Motifs (*)

Yoshihisa Nojima (1)(§), Noritsugu Morino (1), Toshihide Mimura (1), Ken Hamasaki (1), Hiroko Furuya (1), Ryuichi Sakai (2), Toshiya Sato (3), Kouichi Tachibana (3), Chikao Morimoto (3), Yoshio Yazaki (1), Hisamaru Hirai (1)

From the (1)Third Department of Internal Medicine, University of Tokyo, Tokyo 113 Japan, the (2)Molecular Biology Division, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-04, Japan, and the (3)Division of Tumor Immunology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

p130 (Cas) has been recently identified as a 130-kDa protein that is highly phosphorylated on tyrosine residues and is stably associated with p47 (v-Crk) and p60(v-Src) oncogene products in cells transformed by the respective genes. Cas is a novel signaling molecule having a single Src homology (SH) 3 domain and a cluster of multiple SH2-binding motifs. While the tight association of Cas with v-Crk and v-Src is strongly suggestive of a significant role in regulating cellular transformation, the function of Cas in normal untransformed cells is totally unknown. We report here that cell adhesion to fibronectin rapidly promotes tyrosine phosphorylation of Cas in human and rat fibroblast cell lines. The response was equally induced by cell adhesion to plates coated with vitronectin, laminin, and collagen but not by cell attachment to nonspecific substrate poly-L-lysine. The kinetic profile of Cas phosphorylation was almost identical with that of tyrosine phosphorylation of focal adhesion kinase pp125 (Fak), which is well known to be activated subsequent to integrin-mediated cell adhesion. Adhesion-dependent Cas phosphorylation was completely inhibited by treating cells with cytochalasin D, an agent that disrupts polymerization of actin stress fibers. These results suggest that tyrosine phosphorylation of Cas is stimulated by normal cell adhesion in close association with Fak phosphorylation and the formation of actin stress fibers. In v-Src- or v-Crk-transformed cells, however, the tyrosine phosphorylation of Cas is markedly increased in an adhesion-independent manner that is insensitive to treatment with cytochalasin D. Thus, Cas plays a role in signaling pathways mediated by cell adhesion as well as by transformation. We propose that Cas may amplify and propagate integrin-mediated signals by interacting with SH2-containing molecule(s).


INTRODUCTION

Integrins comprise the major class of receptors used by cells to interact with the extracellular matrix(1, 2) . Integrin/extracellular matrix protein interactions play a critical role in a variety of biological processes, including embryonic development, wound healing, tumor metastasis, and cell growth and differentiation(2, 3) . It is now evident that integrins can transduce biochemical signals across the plasma membrane to the cell interior(3) . Integrins regulate many intracellular signals including cytoplasmic alkalization, intracellular Ca levels, and induction of gene expression(3, 4, 5) . Recent evidence indicates that integrin-mediated signaling pathways also involve a cascade of tyrosine kinases and phosphorylation events (6-11). Engagement of cell surface integrins has been shown to rapidly stimulate tyrosine phosphorylation of several intracellular proteins, including paxillin, tensin, focal adhesion kinase pp125 (Fak), and mitogen-activated protein (MAP)()kinases(11, 12, 13, 14, 15, 16) . Among these, Fak has received the most attention, since it constitutes a novel family of protein tyrosine kinases and is activated by integrin clustering on the cell surface(9, 10, 11) . Once phosphorylated on its tyrosine residues, Fak has been shown to bind to SH2 domains of Src family proteins and of phosphatidylinositol 3-kinase (17-19). It is proposed that binding of these kinases to Fak may result in their enzymatic activation(18, 19) . Moreover, recent evidence indicates that integrin-mediated cell adhesion also promotes SH2-mediated association of the Grb2/Ash adaptor protein with Fak(15) . Since Grb2 is constitutively associated with the Ras GDP/GTP exchange protein Sos, the formation of FakGrb2Sos complex may lead to an accumulation of GTP-Ras followed by sequential activation of a serine/threonine kinase cascade including Raf, MAP kinase kinase, and MAP kinase(15) . Indeed, we and others have recently reported that MAP kinase activation occurs in response to integrin-mediated cell adhesion (14-16). All of these observations indicate that signal transduction events elicited by integrin ligation are similar to those stimulated by growth factor binding to receptor tyrosine kinases, in which protein-protein interactions through SH2 and SH3 domains are essential (20).

In the present study, we have identified pp130 (Cas) as another molecule that participates in integrin-mediated signaling cascades. Cas was originally identified as a 130-kDa protein that is highly phosphorylated on tyrosine residues in cells expressing transforming gene products p47 (v-Crk) and p60 (v-Src)(21, 22) . The amino acid sequence deduced from cDNA encoding this molecule revealed that Cas is a novel SH3-containing molecule with a cluster of multiple putative SH2-binding motifs(21) . Tyrosine phosphorylation of Cas occurs after cellular transformation by v-Crk and v-Src but not by other transforming genes such as v-K-ras. Cas forms a stable complex with v-Crk and v-Src in vivo in a phosphorylation-dependent manner. Moreover, immune complex kinase assays have shown that Cas is a major substrate component of both v-Crk and v-Src in vitro (21). These results have strongly suggested that Cas plays a role in signaling pathways of cellular transformation triggered by v-Crk and v-Src. Little is yet known, however, about physiological stimuli that promote Cas phosphorylation in normal untransformed cells. Here we demonstrate that cell adhesion to matrix proteins of normal fibroblast cells induces a significant increase in the tyrosine phosphorylation of Cas. Our findings suggest that Cas mediates integrin-mediated signals by assembling multiple SH2-containing molecules.


EXPERIMENTAL PROCEDURES

Reagents

Human fibronectin (FN), vitronectin, laminin and type I collagen, and rat FN were all purchased from Telios (San Diego, CA). Anti-phosphotyrosine antibody (anti-Tyr(P)) (4G10) was obtained from UBI Laboratories (Lake Placid, NY). Poly-L-lysine (PLL), and cytochalasin D were obtained from Sigma. Rabbit anti-p130 protein serum (anti-Cas2) was developed in our laboratory and described previously (21). Rabbit anti-serum against FAK was developed by immunizing rabbits with GST-human Fak C-terminal protein.()

Cell Culture

Human skin fibroblasts (HSF) were established as described previously(16) . Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 50 µg/ml streptomycin. 3Y1 is a rat fibroblast cell line and is maintained in 10% fetal calf serum/Dulbecco's modified Eagle's medium. SR-3Y1 is a 3Y1 cell line transformed by v-Src of Rous sarcoma virus(21) . 3Y1-Crk is an isolated clone of rat 3Y1 cells transfected with v-crk cDNA of an avian sarcoma virus, ASV-1, inserted in the pMV-7 expression vector(21) .

Cell Adhesion and Preparation of Cell Lysates

Preparation of culture dishes coated with adhesive ligands, PLL, and mAbs was described previously(8, 16) . Confluent cells were detached by treating with 0.05% trypsin/EDTA, followed by washing 3 times with serum-free Dulbecco's modified Eagle's medium. Cells were then plated onto dishes coated with different reagents as indicated and incubated at 37 °C for the indicated time periods in serum-free Dulbecco's modified Eagle's medium. Bound cells were lysed in situ with 1% Nonidet P-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM iodoacetamide, 10 mM NaF, 10 mM sodium pyrophosphate, and 0.4 mM sodium vanadate). After removing insoluble materials by centrifugation at 14,000 rpm for 10 min, protein concentrations in the supernatant were determined using micro BCA protein assay kit (Pierce, Rockford, IL). Cell lysates were stored at -70 °C until use.

Immunoblotting and Immunoprecipitation

Cell lysates were loaded on 7.5% SDS-polyacrylamide gels in reducing conditions. Proteins on the gel were electrotransfered to nitrocellulose membranes. Anti-Tyr(P) immunoblotting was performed according to methods described previously(16) . For immunoprecipitations, cell extracts were incubated with a 1/100 dilution of anti-Cas2 or anti-Fak sera for 1 h at 4 °C followed by additional incubation with protein A-Sepharose beads for 1 h at 4 °C. Beads were then washed 5 times with 1% Nonidet P-40 lysis buffer to remove unbound proteins. Immune complexes were treated with sample buffer and subjected to SDS-polyacrylamide gel electrophoresis for immunoblotting with anti-Tyr(P) or anti-Cas2.


RESULTS

Using HSF cells, we have previously reported that cell adhesion to FN, but not to the nonspecific substrate PLL, stimulates tyrosine phosphorylation of several intracellular proteins including 130- (pp130), 120- (pp120), and 42- (pp42) kDa proteins(16) . We identified pp120 and pp42 as Fak and MAP kinase, respectively. In the present study, we have attempted to examine whether pp130 is identical to Cas. Anti-Tyr(P) immunoblots of Nonidet P-40 lysates from HSF cells after adherence to FN-coated plates for 30 min demonstrate significantly higher tyrosine phosphorylation of 120-kDa (Fak) and broad 130-kDa (pp130) proteins than cells attached to PLL-coated plates (Fig. 1A, lanes1 and 2). The results are consistent with our previous report(16) , although pp42 is not obvious in this experiment. From these extracts, Cas was immunoprecipitated with rabbit anti-Cas serum (anti-Cas2) and immunoblotted with anti-Tyr(P) (Fig. 1A). Anti-Cas2 (lanes3 and 4) but not normal rabbit (lane5) sera precipitated a broad 130-kDa protein whose tyrosine phosphorylation was clearly enhanced by adhesion to FN. This 130-kDa phosphoprotein comigrated with pp130 in the total cell extracts. Moreover, anti-Cas2 completely immunodepleted pp130 from the total extracts of FN-adherent cells, while control serum did not (Fig. 1A, lanes6 and 7), suggesting that protein(s) immunoprecipitated by anti-Cas2 represent the vast majority of the pp130 in total cell lysates. To further confirm the identity of pp130 as Cas, phosphotyrosyl proteins were immunoprecipitated with anti-Tyr(P) from PLL- and FN-adherent cells and probed with anti-Cas2 immunoblotting (Fig. 1B). We found that pp130 in anti-Tyr(P) immunoprecipitates was clearly reactive with anti-Cas2. These results confirm that pp130 is identical to Cas.


Figure 1: Tyrosine phosphorylation of Cas is induced by cell adhesion to FN. A, HSF cells were allowed to adhere to PLL (P; lanes1 and 3) and FN (F; lanes2, 4, and 5) for 30 min. Total cell extracts (lanes1 and 2) and immunoprecipitates with anti-Cas2 (lanes3 and 4) or control sera (lane5) were all probed by immunoblotting with anti-Tyr(P). Supernatants of anti-Cas2 immunoprecipitates (lane6) and of control anti-serum immunoprecipitates (lane7) from FN-adherent cell extracts were also subjected to anti-Tyr(P) blotting. B, phosphotyrosyl proteins were immunoprecipitated with anti-Tyr(P) from PLL- (P; lane1) and FN-adherent (F; lane2) HSF cells and then probed with anti-Cas2. C, anti-Cas2 immunoprecipitates from HSF cells adhered to PLL (P; lanes1 and 3) and FN (F; lanes2 and 4) for 30 min were immunoblotted with either anti-Tyr(P) (lanes1 and 2) or anti-Cas2 (lanes3 and 4). D, HSF cells were allowed to adhere to plates coated with anti-MHC class I (lane1) and anti-integrin 1 (lane2) antibodies for 30 min. Cas was immunoprecipitated from these extracts and probed with anti-Tyr(P).



In normal fibroblast cells of rat (3Y1) and mouse (NIH3T3) origin, Cas has been detected as two bands at 115 and 125 kDa (Cas-A and Cas-B, respectively)(21) . Cellular transformation by v-Crk or v-Src, however, is associated with a decrease in Cas-A, and the simultaneous appearance of a broad 130 kDa band (Cas-C). Since tyrosine phosphorylation is found mostly in Cas-C, Cas-C appeared to be a modified form of Cas-A or Cas-B with a retarded gel motility secondary to extensive phosphorylation at multiple sites(21) . Most of the Cas in HSF is also present as 115- and 125-kDa proteins (Cas-A and Cas-B), as shown in Fig. 1C, lanes3 and 4. However, cells adherent to FN exhibit a faint broad band at 130 kDa that is hardly detectable in cells adherent to PLL. This band corresponds to tyrosine phosphorylated forms of Cas (Cas-C) (Fig. 1C, lanes1 and 2). In contrast to v-Crk- or v-Src-transformants(21) , HSF cell adhesion did not result in a significant change in the amounts of Cas-A/B. This suggests that only a small proportion of Cas is tyrosine phosphorylated upon FN adherence by normal untransformed cells. Tyrosine phosphorylation of Cas was similarly induced by HSF cell adhesion to culture plates coated with anti-1 integrin antibody (anti-4B4) but not with anti-MHC class I antibody (W6/32) (Fig. 1D, upperpanel). Duplicate filters were blotted with anti-Cas2 (Fig. 1D, lowerpanel). A faint band representing Cas-C was again detectable in cells adherent to anti-4B4-coated plates. Moreover, pretreatment of cells with soluble anti-1 integrin antibody inhibited tyrosine phosphorylation as well as cell adhesion to FN (data not shown). These results suggest that Cas phosphorylation induced by adherence to FN is at least partially dependent on 1 integrins. Taken together, our results indicate that cell adhesion to FN and ligation of 1 integrins by immobilized antibody stimulates tyrosine phosphorylation of Cas.

In addition to FN, other adhesive ligands including vitronectin, laminin, and collagen were also potent in inducing Cas phosphorylation (Fig. 2), indicating that the response is not specific to FN. Our findings rather suggest that tyrosine phosphorylation of Cas is a common event induced by cell adhesion through various integrins as is the case of adhesion-dependent Fak phosphorylation.


Figure 2: Tyrosine phosphorylation of Cas is induced by HSF adhesion to vitronectin, laminin, and collagen. Cas was immunoprecipitated with anti-Cas2 from lysates of HSF cells adhered to PLL (lane1), vitronectin (lane2), laminin (lane3), and collagen (lane4), followed by anti-Tyr(P) immunoblotting.



We next examined kinetics of Cas phosphorylation induced by HSF adhesion to FN. Tyrosine phosphorylation of Cas was detectable 10 min after starting cell culture on FN-coated plates and reached maximal levels in 40-80 min (Fig. 3A, upperpanel). Duplicate filters were probed with anti-Cas2 to confirm that the same amount of Cas was loaded in each lane (data not shown). We compared kinetics of tyrosine phosphorylation of Cas and Fak (Fig. 3A, lowerpanel) induced by FN adherence and present the results in Fig. 3B by densitometric analysis of the bands obtained by immunoblotting. The overall kinetics of phosphorylation of both proteins were quite similar, suggesting that phosphorylation of Fak and Cas are related in cell adhesion-mediated signaling pathways.


Figure 3: Adhesion-dependent tyrosine phosphorylation of Cas and Fak. A, Cas (upperpanel) and Fak (lowerpanel) were immunoprecipitated from lysates of nonadherent HSF cells and cells adhered to plates coated with FN for indicated periods. Immunoprecipitates were then immunoblotted with anti-Tyr(P). B, densitometric scanning of adhesion-dependent tyrosine phosphorylation of Cas (opencircles) and Fak (closedcircles). Data represent the percentage of control (nonadherent cells, 0 min).



Following attachment to substrata, cells alter their shape and spread by developing actin stress fibers(23) . Such morphological changes are generally preceded by tyrosine phosphorylation of proteins including Fak(11, 16) . Meanwhile, cytochalasin D that disrupts actin polymerization has been shown to inhibit adhesion-dependent tyrosine phosphorylation of Fak or MAP kinase(14, 16) . Thus, protein tyrosine phosphorylation induced by cell adhesion is closely linked with cytoskeletal organization. In the previous study(16) , we showed that adhesion-dependent tyrosine phosphorylation of pp130 in HSF cells was completely abrogated by treating cells with cytochalasin D. In accordance with this, the adhesion-dependent increase of Cas phosphorylation, determined by anti-Tyr(P) blotting of anti-Cas2 immunoprecipitates, was inhibited by cytochalasin D in a dose-dependent manner (Fig. 4). Duplicate filters were blotted with ant-Cas2, showing that cytochalasin D did not affect the amount of Cas (data not shown). Thus, cytoskeleton organization is required for adhesion-induced tyrosine phosphorylation of Cas as well as of Fak and MAP kinase.


Figure 4: Inhibition of adhesion-dependent Cas phosphorylation by treating cells with cytochalasin D. HSF cells in suspension were pretreated for 5 min with the indicated concentrations of cytochalasin D prior to being added to FN-coated dishes. The viability of HSF cells was not affected over this dose range of cytochalasin D. Cells were then allowed to adhere to FN for 30 min in the continuous presence of cytochalasin D (lanes3-6). Untreated cells were also cultured in PLL- and FN-coated plates in the absence of cytochalasin D (lanes1 and 2, respectively). Bound cells were lysed with Nonidet P-40 lysis buffer, immunoprecipitated with anti-Cas2, and immunoblotted with anti-Tyr(P).



Cas was originally identified as a protein highly phosphorylated on tyrosyl residues in v-Crk- and v-Src-transformed cells(21) . We examined, therefore, whether Cas phosphorylation was dependent on cell adhesion in these transformants. Following attachment to FN-coated plates, wild-type 3Y1 cells and 3Y1 cells transformed by v-Crk (3Y1-Crk) gradually developed membrane processes on the substrata (data not shown). After culturing for 30 min, more than 90% of cells changed their shape by spreading on the FN-coated plates, while cells plated on PLL maintained a rounded shape although they tightly bound to the substrata. These morphological changes suggest that cells are forming focal adhesions upon adhesion to FN, thereby developing actin stress fibers. In contrast, 3Y1 cells transformed by v-Src (SR-3Y1) poorly developed membrane processes even when cultured on FN-coated plates (data not shown). Most cells remained to have a rounded appearance, and the relatively weak cell spreading occurred in less than 20% of this transformant. Thus, morphological responses induced by cell adhesion were different between two types of transformants. Cas was immunoprecipitated from lysates (1 mg of total protein) of wild-type and transformed cells before (in suspension) and after adhesion to FN. Immunoprecipitates were then probed with anti-Tyr(P) immunoblotting. Like in HSF cells, tyrosine phosphorylation of Cas is significantly enhanced by adhesion to FN by wild-type 3Y1 cells (Fig. 5, lanes1 and 2). In both transformants, however, Cas was highly phosphorylated even when cells were in suspended conditions (lanes3 and 5). FN adherence did not affect levels of phosphorylation of Cas (lanes4 and 6). Anti-Cas2 immunoblots of the same sets of immunoprecipitates (data not shown) revealed that Cas in these transformants is mostly present as Cas-C and Cas-B with decreased amounts of Cas-A, as described previously(21) . The relative amounts of the different forms of Cas did not change after FN binding. Moreover, treating cells with 3 µM cytochalasin D, capable of suppressing adhesion-dependent Cas phosphorylation in normal 3Y1 cells to the basal levels, had no effect on the increased phosphorylation of Cas in the transformed cells (Fig. 5B). These results indicate that tyrosine phosphorylation of Cas is constitutively enhanced in these transformants, independent from cell adhesion, actin organization, and morphological alterations.


Figure 5: Cas is constitutively tyrosine phosphorylated in v-Src- and v-Crk-transformants in an adhesion-independent manner. A, wild-type 3Y1, SR-3Y1, and 3Y1-Crk were allowed to adhere to FN for 30 min. Cas was immunoprecipitated from extracts of these adherent cells (F; lanes2, 4, and 6) and from cells in suspension (S; lanes1, 3, and 5) and then probed with anti-Tyr(P) immunoblotting. B, cells adherent to FN in the absence (opencolumns) or in the presence (closedcolumns) of 3 µM cytochalasin D were immunoprecipitated with anti-Cas2 and probed with anti-Tyr(P) immunoblotting. These bands were analyzed by a densitometric scanner, and data were presented as the percentage of controls (in the absence of cytochalasin D; 100%) in each cell line.




DISCUSSION

In the present paper, we have shown that integrin-mediated adhesion of normal fibroblast cell lines stimulates tyrosine phosphorylation of Cas in close association with Fak phosphorylation and actin stress fiber formation. In contrast, extensive phosphorylation of Cas found in cells transformed by v-Crk and v-Src occurred independently from cell adhesion and subsequent changes of cell morphology. Stoichiometrically, the amount of Cas that undergoes tyrosine phosphorylation after FN-binding by normal fibroblasts was far less than that in transformed cells. Most of the Cas remained unphosphorylated in untransformed HSF and 3Y1 cells even after adhesion to FN. Therefore, one might argue that Cas does not play a major role in integrin-mediated signal transduction. It is possible, however, that the phosphorylated Cas, even in a small amount, may interact with signaling molecules having an affinity for Cas through their SH2 domains. Such multimolecular complexes may amplify and propagate integrin-mediated signals downstream. Demonstrating interactions between Cas and Cas-associated molecules and determining their downstream effects will be necessary to ascertain whether Cas is really an important player in the integrin-mediated signaling.

We have not yet identified the kinase(s) that phosphorylates Cas in the process of cell adhesion or transformation. Recently, in an attempt to search optimal peptide substrates for distinct tyrosine kinases using a degenerate peptide library, Songyang et al.(24) have identified the (I/V)YXXP sequence as a good candidate for the motif that preferentially binds to the kinase domain of the c-Abl cytoplasmic protein kinase(24) . Intriguingly, the (I/V)YXXP motif appears a dozen times in Cas(21) . This would predict that Cas should be an excellent substrate for c-Abl. c-Abl was also shown to constitutively bind to the SH3 domain of v-Crk (25), while v-Crk was stably associated with phosphorylated Cas(21) . Thus, the formation of trimolecular complex between these signaling molecules may result in the hyperphosphorylation of Cas in v-Crk-transformed cells. In addition, c-Abl possesses binding domains in its C-terminal portion for F-actin cytoskeletons(26) . In this regard, our finding that Cas phosphorylation was sensitive to disruption of actin stress fibers by cytochalasin D favors the view that c-Abl may be the responsible kinase. An alternative candidate for regulating Cas phosphorylation appears to be c-Src. Our data showed that tyrosine phosphorylation of Fak and Cas is closely related, suggesting that a common mechanism may operate in this process. Indeed, the SH2 domain of v-Src tightly binds to tyrosine-phosphorylated Cas and Fak both in vivo and in vitro(17, 18, 21) . A binding portion within Fak for Src-SH2 domain has been identified as Tyr-397, which corresponds to an autophosphorylation site of this kinase(18) . Moreover, Schlaepfer et al.(15) have recently demonstrated that integrin-mediated cell adhesion of NIH3T3 cells induced autophosphorylation of Fak and subsequent binding of Fak to c-Src. Fak and c-Src association has been postulated to result in the activation of Src kinase activity by dissociating a negatively regulatory C terminus of c-Src from its own SH2 domain(15, 18) . c-Src activated in this fashion may further phosphorylate Fak (15) and, on the other hand, may induce Cas phosphorylation. In addition to the (I/V)YXXP motif, a putative c-Abl substrate, Cas contains two candidate motifs (YDNV and YDYV) that are suitable for binding to the Src-SH2 domain(21) . In accordance with this, we have detected a trace amount of c-Src associating with phosphorylated Cas in 3Y1-Crk cells(21) . So far, however, we have failed to detect c-Src or any other kinase activities associating with Cas in normal fibroblast cells. This may be due to the relatively small amount of phosphorylated Cas contained in normal cells even after adhesion to FN (Fig. 5A). Thus, the kinases responsible for Cas phosphorylation during the process of integrin ligation remained to be determined.

In summary, herein we presented a novel aspect of integrin-mediated signal transduction. Our data further support the notion that cell adhesion and transformation share common signaling molecules(10) . This provides a fundamental basis for the regulation of cell growth and differentiation by the extracellular matrix protein. Identification of responsible kinases and downstream elements that bind phosphorylated Cas will be an important issue to fully understand signaling pathways triggered by integrin ligation.


FOOTNOTES

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

§
To whom correspondence should be addressed: The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. Tel.: 81-03-3815-5411; Fax: 81-03-5684-3987.

The abbreviations used are: MAP, mitogen-activated protein; HSF, human skin fibroblast; FN, fibronectin; PLL, poly-L-lysine; SH, Src homology; Tyr(P), phosphotyrosine.

K. Tachibana, T. Sato, N. D'Avirro, and C. Morimoto, unpublished results.


ACKNOWLEDGEMENTS

We thank Dr. David Rothstein (Yale University, New Haven, CT) for helpful discussions.


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