Activation of the Protein Kinase Akt/PKB by the Formation of E-cadherin-mediated Cell-Cell Junctions

E-cadherins are surface adhesion molecules localized at the level of adherens junctions, which play a major role in cell adhesiveness by mediating calcium-dependent homophylic interactions at sites of cell-cell contacts. Recently, E-cadherins have been also implicated in a number of biological processes, including cell growth and differentiation, cell recognition, and sorting during developmental morphogenesis, as well as in aggregation-dependent cell survival. As phosphatidylinositol (PI) 3-kinase and Akt play a critical role in survival pathways in response to both growth factors and extracellular stimuli, these observations prompted us to explore whether E-cadherins could affect intracellular molecules regulating the activity of the PI 3-kinase/Akt signaling cascade. Using Madin-Darby canine kidney cells as a model system, we show here that engagement of E-cadherins in homophylic calcium-dependent cell-cell interactions results in a rapid PI 3-kinase-dependent activation of Akt and the subsequent translocation of Akt to the nucleus. Moreover, we demonstrate that the activation of PI 3-kinase in response to cell-cell contact formation involves the phosphorylation of PI 3-kinase in tyrosine residues, and the concomitant recruitment of PI 3-kinase to E-cadherin-containing protein complexes. These findings indicate that E-cadherins can initiate outside-in signal transducing pathways that regulate the activity of PI 3-kinase and Akt, thus providing a novel molecular mechanism whereby the interaction among neighboring cells and their adhesion status may ultimately control the fate of epithelial cells.

The maintenance of structural and functional integrity of epithelia requires highly dynamic cell-to-cell and cell-to-matrix interactions, which are mediated by adhesion mechanisms involving different types of cell-surface receptors. Among them, cadherins and integrins play a major role, as they are able to recognize and interact with other cell adhesion receptors on neighboring cells or with proteins of the extracellular matrix, respectively (1)(2)(3). E-cadherins belong to the family of integral membrane glycoproteins promoting homophylic calciumdependent cell-cell interactions and are well characterized adhesion receptors found within adherens-type junctions in epithelia. The extracellular domain of E-cadherins is able to mediate per se calcium-dependent homotypic interactions at sites of cell-cell contacts, while its highly conserved intracytoplasmic tail is involved in the strengthening of the homophylic adhesions by binding a set of related proteins called catenins which, in turn, link the complex to the actin cytoskeleton and elicit certain nuclear responses (4,5). Recently, the dynamic aspects of cell adhesion and its relationship to physiological and pathophysiological events have been intensively investigated. They include cell growth and differentiation, cell recognition and sorting during developmental morphogenesis (reviewed in Ref. 2), and a role in certain pathological processes, including the correlation between loss of E-cadherins at the level of cell surface and enhanced cell invasiveness in vitro (6 -9) and tumor progression in vivo (10,11).
Several lines of evidence indicate that the E-cadherin-mediated adhesion system is subject to regulation from the cytoplasmic side in response to intracellular events (9,(12)(13)(14)(15)). In contrast, the generation of signals at the level of adherens junctions as a consequence of E-cadherin engagement has been thus far poorly investigated, although newly available evidence suggest that E-cadherins may participate in transducing outside-in signals (16). Of interest, it has been reported recently that E-cadherins can mediate aggregation-dependent cell survival in a variety of experimental settings (17)(18)(19). As the Akt kinase is an integral component of survival pathways utilized by both growth factors and extracellular stimuli (20 -23), these observations prompted us to investigate whether E-cadherins could affect the activity of signaling molecules controlling Akt function. In this study, we used an in vitro model for the disruption and subsequent re-formation of E-cadherin-dependent interactions in epithelial MDCK 1 cells to explore the possibility that E-cadherin-mediated cellular aggregation could result in Akt activation. We provide evidence that engagement of E-cadherins in homophylic adhesion with neighboring cells promotes a remarkable PI 3-kinase-dependent increase in the state of activation of Akt and the rapid translocation of Akt to the nucleus. We also demonstrate that engagement of E-cadherins is necessary and sufficient for the induction of Akt activity upon adherens junction assembly, and co-immunoprecipitation experiments demonstrate a physical association between PI 3-kinase and E-cadherin-containing multiprotein complexes in response to cell-cell contact formation, thus providing a likely mechanism for Akt activation. Overall, these findings indicate that E-cadherins may initiate outside-in signal transducing pathways, thus supporting an active role for E-cadherins in the control of key early post-aggregation events. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18

EXPERIMENTAL PROCEDURES
Cell Culture and Expression Plasmids-An expression vector for hemagglutinin-(HA) tagged Akt (pCEFL-HA-Akt) has been reported elsewhere (22). Early passage MDCK cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For transfections, cells were grown at 70% confluence in 60-mm cell culture dishes and transfected with 4 g of HA-tagged PKB/Akt cDNA by the calcium phosphate precipitation technique and selected with 500 g/ml G418. Transfected clones were maintained in 500 g/ml G418 to provide selection pressure.
Antibodies and Immunologic Detection Methods-Antibodies specific for E-cadherins, purchased from Transduction Laboratories (Lexington, KY), were used for immunoprecipitation and Western blotting. Antibodies to the extracellular domain of E-cadherin molecule (DECMA-1 clone, Sigma) were used for immunostaining and for antibody inhibition experiments. An anti-HA-specific monoclonal antibody (HA11, Babco, Richmond, CA) and a goat polyclonal anti-Akt antibody (C20, Santa Cruz Biotechnologies, Inc.) were used to detect ectopic and endogenously expressed Akt, respectively. Antibodies against the p85 regulatory subunit of PI 3-kinase and anti-phosphotyrosine (anti-Tyr(P), G410 clone) antibodies were from Upstate Biotechnology Inc. (Lake Placid, NY). Anti-mouse and anti-rabbit secondary antibodies coupled to horseradish peroxidase were from Cappel Laboratories (Durham, NC). Co-immunoprecipitation and Western blotting experiments were performed as described previously (22).
Akt and PI 3-Kinase Assays-MDCK cells were serum-starved overnight in the presence of 10 mM Hepes, and E-cadherin-mediated cellto-cell contacts were disrupted by treatment with EGTA to a final concentration of 4 mM for 30 -40 min at 37°C. Thereafter, intercellular interactions were allowed to re-establish in the presence of fresh, calcium-containing medium (final concentration CaCl 2 ϳ1.8 mM) (24). At different time points after calcium restoration, cells were harvested, lysed on ice, and assayed for Akt activity in immunocomplex kinase reactions, using histone 2B as a substrate in the presence of [␥-32 P]ATP (22). Comparable immunoprecipitation and loading of wild-type and epitope-tagged Akt kinase were determined by immunoblotting membranes with anti-Akt and anti-HA antibodies, respectively.
For measurement of PI 3-kinase activity, whole cell extracts were incubated with anti-Tyr(P) antibodies for 2 h at 4°C under constant agitation. The PI 3-kinase assay was subsequently performed as described previously (25) by evaluating the ability of the immunoprecipitates to phosphorylate PI to yield phosphatidylinositol 3-phosphate (PI3P). After thin layer chromatography, 32 P-labeled phospholipids were detected by autoradiography. In some experiments, the PI 3-kinase inhibitor wortmannin (50 nM, Sigma) was used. Autoradiograms were quantified on a PhosphorImager using the ImageQuant software.
Immunofluorescence Microscopy-MDCK cells were grown on glass coverslips to confluence, washed with PBS, and fixed for 20 min at room were serum-starved and treated with 4 mM EGTA for 30 -40 min and then calcium was restored for 5-60 min, as indicated. As controls, starved cells were left untreated (control) or stimulated with 100 nM EGF for 5 min. After calcium restoration, lysates were immunoprecipitated with anti-Akt or anti-HA antibodies and used for Akt kinase reaction as described under "Experimental Procedures." 32 P-Labeled substrate (histone 2B (H2B)) is indicated. Autoradiograms showing the time course of Akt activity correspond to representative experiments. Similar results were obtained in three to five independent experiments. Data represent the average (mean Ϯ S.D.) of three to five independent experiments, expressed as fold increase with respect to untreated cells (control). C, MDCK cells were stably transfected with HA-Akt, as described above. Following starvation, cells were left untreated (panel a), treated with 4 mM EGTA (panel b), or restored in calcium-containing medium for 30 min after EGTA treatment (panel c). Cells exposed to 100 nM EGF for 5 min were used as positive control (panel d). After stimulation, cells were fixed, permeabilized, stained with anti-HA monoclonal antibodies followed by a fluorescein isothiocyanate-conjugated secondary antibodies and prepared for microscope analysis at a ϫ 63 magnification. temperature in 3.7% formaldehyde in PBS. Cells were then permeabilized with 0.5% Triton X-100 for 10 min. After washing with PBS, cells were blocked with 1% bovine serum albumin in PBS and incubated with the appropriate primary antibody for 1 h at room temperature. Rat anti-E-cadherin (1:1000) and mouse anti-HA (1:50) antibodies were used to detect E-cadherin and HA-tagged Akt, respectively. Following incubation with the corresponding secondary antibodies (1:200) conjugated with tetramethylrhodamine B isothiocyanate or fluorescein isothiocyanate, respectively, the cells were washed, mounted in anti-fade medium (Molecular Probes, Eugene, OR), and examined under an UV microscope (Zeiss Axiophot) at a ϫ 40 or ϫ 63 magnification using the appropriate filters. Images were processed using Adobe photoshop.

E-cadherin-mediated Adherens Junction Formation Elicits
Akt/PKB Activation-Using epithelial MDCK cells, we first evaluated whether adherens junction assembly could affect the state of activation of endogenous Akt. As epithelial cells require Ca 2ϩ to form homophylic cell-cell adhesions, a simple method to study the adhesive properties of surface molecules involves the disruption of Ca 2ϩ -dependent homotypic boundaries among cells by EGTA-treatment and the re-establishment of cell-cell contacts by the subsequent restoration of Ca 2ϩ ions (24). Whereas control cells present a typical pattern of E-cadherin immunostaining at the level of cell-cell contacts (Fig. 1A, panel  a), in cells treated with the calcium chelator, EGTA, E-cadherins appear to be diffusely distributed (Fig. 1A, panel b). In this case, the loss of E-cadherins at the level of cell-cell contacts is most likely due to their redistribution throughout the cell surface rather than to the internalization of E-cadherins, as reported for other types of cadherins (26). After restoration of calcium, de novo formation of adherens junctions could be observed as early as 5-10 min (Fig. 1A, panel c), and the process appeared to be almost complete after 30 -40 min (Fig. 1A,  panel d).
To explore the possibility that E-cadherin engagement leads to Akt activation, MDCK cells were grown as long confluent cultures by maintaining cells in complete medium for at least 24 -48 h after reaching confluence, to optimize cell-cell contacts as well as to minimize the influence of integrin-extracellular matrix interactions (27). Cells were then serum-starved for 18 -24 h and cell-cell contacts disrupted by the treatment with EGTA (4 mM) for 30 -40 min. Subsequently, adherens junctions were allowed to re-form in the presence of fresh calcium-containing medium, and, at the indicated time points, cells were lysed and assayed for Akt kinase activity. As shown in Fig. 1B, under these experimental conditions, the reassembly of adherens junctions induced a remarkable elevation of Akt kinase activity. Kinetic studies demonstrated a rapid increase in Akt   FIG. 2. E-cadherin-mediated adhesion is necessary for enhanced Akt activity. Serum-starved MDCK cells untransfected (A) or stably expressing HA-Akt (B) were treated with 4 mM EGTA to disrupt E-cadherin-mediated cell-cell contacts. Cells were pretreated for 30 min with the indicated dilutions of anti-E-cadherin antibodies (DECMA-1 clone) and lysed after 30 min of calcium restoration. As controls, serumstarved cells were left untreated, treated with 100 nM EGF for 5 min, or treated with EGF and anti-E-cadherin antibodies, as indicated. Cellular lysates were assayed for Akt activity, as described under "Experimental Procedures." 32 P-Labeled substrate (histone 2B (H2B)) is indicated. Values represent the average Ϯ S.D. of triplicate samples from a typical experiment expressed as fold induction relative to controls. Nearly identical results were obtained in three additional experiments.

FIG. 3. Akt activation by E-cadherin-dependent aggregation is wortmannin-sensitive. MDCK cells were serum-starved overnight.
After EGTA treatment for 30 -40 min and subsequent calcium restoration for 30 min, kinase activity of endogenous (A) and ectopically expressed (B) Akt was evaluated in the presence or absence of 50 nM wortmannin, which was added 20 min prior to calcium stimulation. As controls, cells were left untreated or treated with 100 nM EGF for 5 min, as indicated. After immunoprecipitation with anti-Akt or anti-HA antibodies, Akt kinase assays were performed. Autoradiograms correspond to representative experiments. Data represent the mean Ϯ S.D. from three to five separate experiments.

FIG. 4. Adherens junction assembly induces PI 3-kinase activation and association of the p85/PI 3-kinase subunit to the E-cadherin multiprotein complex.
A, MDCK cells were serumstarved overnight, treated with 4 mM EGTA for 30 -40 min, and then calcium was restored in serum-free medium, as indicated. Serumstarved cells were left untreated (control) or exposed to 100 nM EGF for 5 min. After lysis, PI 3-kinase activity was assayed in anti-Tyr(P) immunoprecipitates, as detailed under "Experimental Procedures." The chromatographic mobility of 32 P-labeled PI3P is indicated. Autoradiogram corresponds to a representative experiment that was repeated three times. B, MDCK cells were treated as described above. E-cadherins were immunoprecipitated from total cell lysates with anti-Ecadherin-specific monoclonal antibodies. The presence of the p85/PI 3-kinase regulatory subunit in anti-E-cadherin immunoprecipitates was detected with specific anti-p85 polyclonal antibodies. As controls, untreated (control) or EGF-treated cells were also included. Top, the immunoblot depicts the amount of p85 recovered in anti-E-cadherin immunoprecipitates. Bottom, equal amounts of E-cadherins were observed in each immunoprecipitated sample. Similar results were obtained in three independent experiments. activity as early as 15 min after calcium restoration, with a peak at 30 -40 min followed by a decrease over a 1-h time course. Stable expression of a HA-tagged Akt in transfected MDCK cells revealed to be a useful tool to enhance the detectability of Akt activation upon E-cadherin engagement. Kinetics of activation of ectopically expressed Akt (Fig. 1B, panel b) was similar to that observed for the endogenous enzymatic activity (Fig. 1B, panel a), thus suggesting that endogenous levels of E-cadherins and Akt are sufficient to sustain a potent Akt activity following adherens junction assembly under physiological conditions.
Activation of Akt following E-cadherin Engagement Leads to Akt Translocation to the Nucleus-Following activation by a PI 3-kinase-mediated pathway, Akt has been shown previously to translocate to the nucleus (28), where it participates in the regulation gene expression (29). On the basis of these observations, we examined whether E-cadherin engagement could lead to a change in the subcellular distribution of an ectopically expressed Akt. Serum-starved cells stimulated with EGF (100 nM) for 5-10 min were used as a positive control (Fig. 1C, panel  d). Whereas Akt appeared to be diffusely distributed in the cytoplasm of untreated and EGTA-treated cells (Fig. 1C, panels  a and b), respectively, calcium restoration caused a marked translocation of Akt to the nucleus (Fig. 1C, panel c).
Akt Activation in Response to Adherens Junction Assembly Requires E-cadherin Engagement-To confirm that Akt activation is not due to the manipulation of calcium levels but to the ability of calcium restoration to mimic the physiological engagement of E-cadherins in cell-cell contacts, we took advantage of the availability of function-perturbing anti-E-cadherin antibodies, DECMA-1 clone, which have been described previously to be effective in blocking E-cadherin mediated adherens junction formation (30). As illustrated in Fig. 2 (A and B, sixth  lanes), pretreatment with anti-E-cadherin antibodies, that hinders the formation of adherens junctions, led to a dramatic suppression of Akt activity upon calcium restoration. This suggests that E-cadherin-dependent homophylic interactions among cells are strictly required for induction of Akt activity. In contrast, the presence of anti-E-cadherin antibodies did not affect EGF-induced Akt activation (Fig. 2, A and B, third lanes), thus establishing the specificity of the experimental approach and suggesting that E-cadherin-mediated Akt activation occurs through a growth factor-independent mechanism. Unexpectedly, during the course of dose-response experiments with blocking antibodies, we observed that high antibody dilutions caused a dramatic increase in the activity of Akt (Fig. 2,  A and B, eighth lanes). Antibody immobilization experiments, using MDCK cells suspended in 4 mM EDTA and plated on cell culture dishes precoated with different antibody dilutions, confirmed these observations (data not shown). The most straightforward explanation for these seemingly conflicting results relies on the fact that in the presence of a vast excess of DECMA-1 antibodies the majority of the antibody-bound Ecadherins would be expected to remain in a monomeric, inactive form. However, at high antibody dilutions each molecule of antibody would be expected to bind two molecules of E-cadherin, thus causing lateral dimerization and clustering of Ecadherins, which can mimic E-cadherin activation by calciumdependent homophylic interactions (31,32). Together, these observations suggest that E-cadherin engagement is necessary and sufficient for Akt activation in response to adherens junction assembly.
Role of PI 3-Kinase in the Activation of Akt in Response to E-cadherin-mediated Cellular Aggregation-The Akt/PKB kinase represents a downstream target of PI 3-kinase in a pathway critical for signaling cell survival in response to several stresses or growth factor deprivation (20 -22). We therefore used a PI 3-kinase inhibitor, wortmannin, to address the possible involvement of PI 3-kinase in the induction of Akt kinase activity by adherens junction assembly. As shown in Fig. 3, preincubation of cells with wortmannin (50 nM) for 30 min before addition of calcium completely abolished Akt activation, with Akt kinase levels falling even below the unstimulated background in wild-type as well as in stably transfected MDCK cells. These observations suggested that the functional activity of PI 3-kinase is required for the activation of Akt in response to E-cadherin engagement.
Formation of Adherens Junctions Causes Association of PI 3-Kinase with E-cadherin-containing Protein Complexes-Agonist activation of PI 3-kinase frequently involves the translocation of this enzyme to the plasma membrane where it can gain access to its lipid substrates (reviewed in Ref. 33). Thus, it was tempting to speculate that a similar mechanism may underlie the activation of Akt upon organization of E-cadherinmediated cell-cell contacts. In preliminary experiments we found that the p85 regulatory subunit of PI 3-kinase was tyrosine-phosphorylated as a function of time after calcium restoration, overlapping with the kinetics of Akt activation (data not shown). Therefore, we decided to evaluate directly the pattern of activation of PI 3-kinase after immunoprecipitation of MDCK cell lysates with anti-Tyr(P) antibodies. Of interest, the profile of PI 3-kinase activity after calcium restoration mirrored that of Akt, as illustrated in Fig. 4A. We next tested the possibility that Akt activation in response to E-cadherin engagement could involve the docking of PI 3-kinase to the adherens junction complex. As shown in Fig. 4B, upon calcium restoration, PI 3-kinase was found to be associated to the E-cadherin immunoprecipitates in a time-dependent manner, as judged by Western blotting with specific anti-p85/PI 3-kinase antibodies.
Taken together, overall results of this study indicate that one of the molecular events resulting from the E-cadherin-mediated cellular aggregation is the rapid activation of a PI 3-kinase/Akt cascade. Although the mechanism whereby E-cadherin engagement stimulates this biochemical route is not fully understood, it most likely involves a yet to be identified tyrosine kinase, which, when activated in response to E-cadherin mediated-cellular aggregation, might facilitate the recruitment of PI 3-kinase to E-cadherin-containing complexes at the level of the plasma membrane. Indeed, the recovery of the p85 regulatory subunit of PI 3-kinase in co-immunoprecipitation experiments with anti-E-cadherin antibodies, along with the kinetic pattern of PI 3-kinase activity detected in anti-Tyr(P) immunoprecipitates, strongly argue in favor of this hypothesis.
As PI 3-kinases are known to play a central role in a number of cellular processes, including mitogenic signaling and cell survival, cytoskeletal remodeling, as well as metabolic control and vesicular trafficking (reviewed in Ref. 33), our present findings may have broad implications to the understanding of epithelial cell biology. In this scenario, E-cadherins might function as "relationship molecules" between the extracellular and the intracellular environment, initiating the transduction of intracellular signaling pathways stimulating the PI 3-kinase/ Akt cascade. This might provide an interesting mechanism whereby the adhesion status of the cells may control the cell fate, including cell survival or death by apoptosis, as well as other critical events occurring during the stepwise organization of the epithelium.