Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1.

Gab-1 is a multiple docking protein that is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met, hepatocyte growth factor/scatter factor receptor, and epidermal growth factor receptor. We have now demonstrated that cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1 and that disruption of cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreased cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas the expression of E-cadherin specifically induced tyrosine phosphorylation of Gab-1. A relatively selective inhibitor of Src family kinases reduced cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increased it. Disruption of cell-cell adhesion, which reduced tyrosine phosphorylation of Gab-1, also reduced the activation of mitogen-activated protein kinase and Akt in response to cell-cell adhesion. These results indicate that E-cadherin-mediated cell-cell adhesion induces tyrosine phosphorylation by a Src family kinase of Gab-1, thereby regulating the activation of Ras/MAP kinase and phosphatidylinositol 3-kinase/Akt cascades.

at cell-cell adhesion sites of MDCK cells (36,37). In addition, HGF/SF or a phorbol ester, TPA, induces disruption of cell-cell adhesion, which is accompanied by endocytosis of both E-cadherin and c-Met. Thus, Gab-1 and the cadherin-catenin system could interact with each other.
In this study, we show that cell-cell adhesion stimulates tyrosine phosphorylation of Gab-1 and that this effect may be mediated through E-cadherin and a Src family kinase. Thus, cell-cell adhesion as well as growth factors regulates the activation of the downstream signaling pathway of Gab-1.

EXPERIMENTAL PROCEDURES
Antibodies-The FLAG-tagged human cDNA of Gab-1 was provided by Dr. T. Hirano (Osaka University, Suita, Japan) (38). MDCK cells were supplied by Dr. W. Birchmeier (Max-Delbruck Center for Molecular Medicine, Berlin, Germany). Human recombinant HGF/SF was provided by Dr. T. Nakamura (Osaka University, Suita, Japan). The anti-Gab-1 rabbit polyclonal Ab was obtained from Upstate Biotechnology (Lake Placid, NY) and also provided by Dr. T. Hirano (Osaka University). An anti-FLAG mouse mAb (M2) was from Eastman Kodak. The horseradish peroxidase-conjugated anti-phosphotyrosine mAb (PY20) was obtained from Santa Cruz Biotechnology. (Santa Cruz, CA). The anti-tyrosine-phosphorylated form of MAP kinase rabbit polyclonal Ab, the anti-MAP kinase rabbit polyclonal Ab, the anti-serine-phosphorylated form of Akt rabbit polyclonal Ab, and the anti-Akt rabbit polyclonal Ab were obtained from New England BioLabs (Beverly, MA). Other materials and chemicals were obtained from commercial sources.
Cell Culture-MDCK cells, mouse mammary tumor MTD-1A cells, and mouse keratinocyte 308R cells were maintained at 37°C in a humidified atmosphere of 10% CO 2 and 90% air in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Life Technologies, Inc.), 100 units/ml penicillin, and 100 g/ml streptomycin. L, CL, and EL cells were kindly supplied by Drs. S. Tsukita and A. Nagafuchi (Kyoto University, Kyoto, Japan). These cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. For transient transfection experiments, L, CL, and EL cells were seeded at a density of 1 ϫ 10 5 cells/dish onto 10-cm dishes and transfected with 1 g of pCMV vector containing the FLAG-tagged Gab-1 cDNA by the use of LipofectAMINE and PLUS Reagent (Life Technologies, Inc.) at 24 h after seeding. The recombinant adenovirus, Ax1CATcsk-⌬K, was obtained from Dr. M. Okada (Osaka University). MDCK cells were infected with the adenovirus vector at the indicated multiplicity of infection as described previously (39).
Immunoprecipitation and Immunoblotting-All cultured cells (in a 10-cm plate) were frozen in liquid nitrogen and then lysed on ice in 1 ml of an ice-cold lysis buffer (20 mM Tris-HCl at pH 7.6, 140 mM NaCl, 2.6 mM CaCl 2 , 1 mM MgCl 2 , 1% (v/v) Nonidet P-40, and 10% (v/v) glycerol) containing 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, and 1 mM sodium vanadate. Whole cell lysates were centrifuged at 10,000 ϫ g at 4°C for 15 min, and the resulting supernatants were subjected to immunoprecipitation and immunoblotting. Briefly, the supernatants were incubated at 4°C with the anti-Gab-1 poyclonal Ab bound to protein G-Sepharose beads (2 g of Ab/20 l of beads) (Amersham Pharmacia Biotech) for 4 h, after which the beads were washed twice with 1 ml of WG buffer (50 mM Hepes-NaOH at pH 7.6, 150 mM NaCl, and 0.1% (v/v) Triton X-100) and resuspended in an SDS sample buffer. SDS-polyacrylamide gel electrophoresis and immunoblotting with various Abs were performed using an ECL detection kit (Amersham Pharmacia Biotech).
Phosphorylation of GST-Gab-1 in Vitro-A GST fusion protein containing the COOH-terminal half of Gab-1 (amino acids 421-694) was generated and purified using glutathione-Sepharose beads (Amersham Pharmacia Biotech). GST fusion proteins (0.1 g), which were immobilized on glutathione-Sepharose beads, were washed twice with kinase assay buffer (50 mM Hepes-NaOH at pH 7.6, 3 mM MnCl 2 , 10 mM MgCl 2 , 1 mM dithiothreitol) and incubated with partially purified Src kinase (Upstate Biotechnology) at 24°C for 30 min in 50 l of a kinase assay buffer in the absence or presence of 10 mM ATP. The reaction mixtures were then centrifuged at 10,000 ϫ g at 4°C for 5 min, and the resulting supernatants were mixed with the SDS sample buffer, boiled, and subjected to SDS-PAGE. The extent of tyrosine phosphorylation of GST proteins was analyzed by immunoblotting with the horseradish peroxidase-conjugated anti-phosphotyrosine mAb PY20.

Cell-Cell Adhesion-dependent Tyrosine Phosphorylation of
Gab-1-MDCK cells were serum starved in the culture medium containing 2 mM Ca 2ϩ for up to 24 h, after which the whole cell lysates were prepared and subjected to immunoprecipitation with the anti-Gab-1 polyclonal Ab. Immunoblotting of the resulting immunoprecipitates with the anti-phosphotyrosine mAb PY20 revealed that Gab-1 was tyrosine phosphorylated in the serum-starved MDCK cells (Fig. 1A). When the Ca 2ϩ concentration in the culture medium was switched from 2 mM to 2 M, the MDCK cells gradually detached from each other, and dissociation of cells was complete at 4 h after the Ca 2ϩ switch as reported previously (36, 37 and data not shown). The tyrosine phosphorylation of Gab-1 was decreased in a time of incubation-dependent manner and almost undetectable at 4 h after the Ca 2ϩ switch from 2 mM to 2 M (Fig. 1A). After MDCK cells The whole cell lysates were then subjected to immunoprecipitation (IP) with the anti-Gab-1 polyclonal Ab (␣Gab-1), and the resulting immunoprecipitates were subjected to immunoblotting with the horseradish peroxidase-conjugated anti-phosphotyrosine mAb PY20 (␣PY) (upper panel). The same blot was reprobed with the anti-Gab-1 polyclonal Ab (lower panel) to ensure that similar amounts of endogenous Gab-1 were present in each lane. High, MDCK cells were deprived of serum for 12 h in medium containing 2 mM Ca 2ϩ . Low, MDCK cells were deprived of serum in the medium containing 2 mM Ca 2ϩ for 12 h and subsequently cultured in medium containing 2 M Ca 2ϩ for the indicated time. Low 3 High, cells pretreated with 2 M Ca 2ϩ for 4 h were cultured further in the medium containing 2 mM Ca 2ϩ for the indicated time. B, serum-deprived MDCK cells were incubated in the presence or absence of 10 ng/ml HGF/SF for 0 -18 h. The whole cell lysates were then subjected to immunoprecipitation with the anti-Gab-1 polyclonal Ab, and the resulting immunoprecipitates were subjected to immunoblotting with either the horseradish peroxidase-conjugated anti-phosphotyrosine mAb PY20 (upper panel) or the anti-Gab-1 polyclonal Ab (lower panel). The results shown are representative of three independent experiments.
were incubated with 2 M Ca 2ϩ for 4 h, the concentration of Ca 2ϩ in the culture medium was increased to 2 mM. The dissociated cells then gradually formed cell-cell adhesion (data not shown), which was accompanied by tyrosine phosphorylation of Gab-1 (Fig. 1A). The same filter was reprobed with the anti-Gab-1 polyclonal Ab to confirm that a similar amount of Gab-1 was present in each lane (Fig. 1A).
Stimulation of MDCK cells with HGF/SF caused spreading and subsequent scattering of the cells as described previously (40,41). The HGF/SF-induced dissociation of cells also decreased the tyrosine phosphorylation of Gab-1 in the culture medium containing 2 mM Ca 2ϩ (Fig. 1B). In addition to MDCK cells, tyrosine phosphorylation of Gab-1 was also observed in mouse mammary tumor MTD-1A cells and mouse keratinocyte 308R cells, both of which were serum starved in the culture medium containing 2 mM Ca 2ϩ (Fig. 2). Furthermore, the marked decrease in the extent of tyrosine phosphorylation of Gab-1 was observed in response to the disruption of cell-cell adhesion of these two cell lines by the low Ca 2ϩ treatment (Fig.  2). Stimulation of 308R cells with HGF/SF also caused scattering of the cells and reduced the tyrosine phosphorylation of Gab-1 (data not shown). These results suggest that cell-cell adhesion reversibly induces the tyrosine phosphorylation of Gab-1.
Involvement of E-Cadherin in Cell-Cell Adhesion-dependent Tyrosine Phosphorylation of Gab-1-When the concentration of Ca 2ϩ in the culture medium was increased from 2 M to 2 mM in the presence of anti-E-cadherin mAb, which blocks the adhesion activity of E-cadherin, no recovery of cell-cell adhesion resulted in a loss of the tyrosine phosphorylation of Gab-1 in MDCK cells (Fig. 3A). In addition, minimal tyrosine phosphorylation of Gab-1 was observed in both L cells that did not express E-cadherin and CL cells that expressed claudin, an integral membrane protein responsible for tight junction strand formation (42), whereas it was evident in EL cells that expressed human E-cadherin (43) in the culture medium containing 2 mM Ca 2ϩ (Fig. 3B). It has been shown that when MDCK cells preincubated at 2 M Ca 2ϩ for 2 h are incubated further with TPA, a tight junction-like structure is observed, and ZO-1, but not E-cadherin or ␤-catenin, accumulates there (44). The addition of TPA after the incubation of MDCK cells with a low concentration of Ca 2ϩ failed to re-increase the tyrosine phosphorylation of Gab-1 (Fig. 3C). Thus, these data suggest that E-cadherin mediates the cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1.
Involvement of a Src Family Kinase in the Cell-Cell Adhesiondependent Tyrosine Phosphorylation of Gab-1-To investigate further the mechanism by which cell-cell adhesion induces the tyrosine phosphorylation of Gab-1, we next examined the effects of 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo [3,4-D]pyrimidine, a compound reported to act as a relatively selective inhibitor of Src family kinases (45). 4-Amino-5-(4methylphenyl)-7-(t-butyl)pyrazolo [3,4-D]pyrimidine markedly decreased the tyrosine phosphorylation of Gab-1 in the culture medium containing 2 mM Ca 2ϩ (Fig. 4A). We also examined the effect of overexpression of a kinase-inactive mutant of Csk (Csk-⌬K) on the tyrosine phosphorylation of Gab-1 in MDCK cells. Csk is a Src-like tyrosine kinase that inhibits the activity of Src family kinases by catalyzing the phosphorylation of a COOH-terminal tyrosine residue (46,47). Recombinant adenovirus vectors were used to express Csk-⌬K in MDCK cells as described previously (39). This mutant Csk has been shown to act in a dominant-negative manner (39). MDCK cells were infected with either Ax1CAT-lacZ or Ax1CAT-csk-⌬K (Fig. 4B). The extent of the tyrosine phosphorylation of Gab-1 was increased markedly by the expression of Csk-⌬K in the culture medium containing 2 mM Ca 2ϩ compared with that in the control cells (Fig. 4B). Partially purified Src kinase tyrosine phosphorylated the GST-COOH-terminal region of Gab-1 (amino acid 421-694), which contained five tyrosine residues (Tyr-447, Tyr-472, Tyr-589, Tyr-627, and Tyr-659) (data not shown). Therefore, these results suggest that a Src family kinase mediates the cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1.
Effects of Cell-Cell Adhesion on the Downstream Signaling of Gab-1-We next examined the effects of cell-cell adhesion on the downstream signaling of Gab-1. Decreasing the Ca 2ϩ concentration of the medium induced a marked reduction of the tyrosine phosphorylation of Gab-1 (Fig. 5A). We then determined the activation of MAP kinase by immunoblotting the whole cell lysates with the anti-tyrosine-phosphorylated MAP kinase polyclonal Ab. The significant activation of MAP kinase in MDCK cells without any stimulation was observed when cells were cultured in the culture medium containing 2 mM Ca 2ϩ (Fig. 5B). In contrast, reduction of the Ca 2ϩ concentration in the medium inhibited MAP kinase activation markedly compared with that observed in the 2 mM Ca 2ϩ medium (Fig. 5B). Tyrosine-phosphorylated Gab-1 binds PI3-kinase (48,49), which subsequently activates Akt/PKB kinase, a downstream target of PI3-kinase (50). Thus, we next examined the effect of reduction of medium Ca 2ϩ on Akt activation, as determined by immunoblotting the whole cell lysates with the anti-serinephosphorylated Akt polyclonal Ab. The significant activation of Akt was observed in MDCK cells without any stimulation when cells were cultured in the culture medium containing 2 mM Ca 2ϩ , whereas the reduction of the medium Ca 2ϩ concentration inhibited Akt activation markedly (Fig. 5C). These data suggest that cell-cell adhesion is involved in the activation of MAP kinase and Akt presumably through the tyrosine phosphorylation of Gab-1.

DISCUSSION
In the present study, we have demonstrated that cell-cell adhesion reversibly induces the tyrosine phosphorylation of Gab-1 in cultured epithelial cells. The extent of the tyrosine phosphorylation of Gab-1 is increased markedly in EL cells compared with that observed in L cells, whereas the anti-E- cadherin mAb markedly reduces the tyrosine phosphorylation of Gab-1 in MDCK cells. Thus, cell-cell adhesion induces the tyrosine phosphorylation of Gab-1 in an E-cadherin-dependent manner. Several proteins such as ␤-catenin, ␥-catenin, p120 ctn , which are all located at AJs, have also been shown to be tyrosine phosphorylated (20 -22), although the mechanism underlying their tyrosine phosphorylation is not fully understood. Thus, Gab-1 might be a new member of the group of proteins that are localized at AJs and undergo tyrosine phosphorylation in response to cell-cell adhesion.
We have also explored the mechanism by which cell-cell adhesion stimulates the tyrosine phosphorylation of Gab-1. 4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo [3,4-D]pyrimidine, a relatively selective inhibitor of Src family kinases, reduces the tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increases its response. These data suggest that a Src family kinase, at least in part, contributes to the tyrosine phosphorylation of Gab-1 in response to cell-cell adhesion. In fact, Src family kinases have been found at AJs (51). In addition, tyrosine phosphorylation of AJ proteins, such as ␤-catenin, ␥-catenin, and p120 ctn , has been shown to be increased markedly in response to cell-cell adhesion in mouse keratinocytes (30). In contrast, in fyn-deficient keratinocytes, the tyrosine phosphorylation of these AJ proteins has been shown to be decreased markedly, and structural and functional abnormalities at the cell-cell adhesions similar to those caused by tyrosine kinase inhibitors have been observed (30). Thus, Src family kinases may generally contribute to regulate the tyrosine phosphorylation of AJ proteins in response to cell-cell adhesion.
We have also demonstrated that the tyrosine phosphorylation of Gab-1 by cell-cell adhesion correlates well with the activation of MAP kinase and Akt activation in response to cell-cell adhesion. Activation of Akt by the formation of Ecadherin-mediated cell-cell junctions has also been demonstrated in MDCK cells (52). It has been reported that cell-cell adhesion, which is mediated by E-cadherin, can promote cell survival in a variety of cell types (53,54). Compelling evidence suggests that both Ras/MAP kinase and PI3-kinase/Akt cascades are involved in cell survival (55). Thus, our present results provide an interesting mechanism whereby E-cadherinmediated cell-cell adhesion stimulates the tyrosine phosphorylation of Gab-1, which then induces the activation of both Ras/ MAP kinase and PI3-kinase/Akt cascades. This mechanism could be involved in cell-cell adhesion-dependent cell survival.
It remains unknown how the E-cadherin-mediated cell-cell adhesion induces the Src family kinase-mediated tyrosine phosphorylation of Gab-1. It is possible that the E-cadherinmediated cell-cell adhesion induces the activation of a Src family kinase. In contrast, the E-cadherin-mediated cell-cell adhesion promotes the recruitment of this kinase to the place where Gab-1 is localized. In fact, it has been shown that the binding of extracellular matrix to integrin also induces tyrosine phosphorylation of several proteins by Src, which is recruited to integrin-based focal adhesions through its binding to FAK, another focal adhesion-associated tyrosine kinase (56). Further efforts are clearly necessary to clarify the molecular mechanism by which the E-cadherin-mediated cell-cell adhesion leads to the tyrosine phosphorylation of Gab-1.