p120ctn Binds to the Membrane-proximal Region of the E-cadherin Cytoplasmic Domain and Is Involved in Modulation of Adhesion Activity*

Cadherins are transmembrane glycoproteins involved in Ca2+-dependent cell-cell adhesion. Previously, we showed that the conserved membrane-proximal region of the E-cadherin cytoplasmic domain negatively regulates adhesion activity. In this report, we provide several lines of evidence that p120ctn is involved in this negative regulation. p120ctn binds to the membrane-proximal region of the nonfunctional carboxyl-terminally deleted E-cadherin protein. An additional internal deletion in this region prevented the association with p120ctn and activated the protein, as seen in an aggregation assay. Furthermore, the nonfunctional E-cadherin can be activated through coexpression of p120ctn proteins with amino-terminal deletions, which eliminate several potential serine/threonine phosphorylation sites but do not affect the ability to bind to cadherins. Finally, we show that staurosporine, a kinase inhibitor, induces an increased electrophoretic mobility of p120ctn bound to E-cadherin polypeptides, activates the nonfunctional E-cadherin protein, and converts the wild-type E-cadherin and an E-cadherin-α-catenin chimeric protein from a cytochalasin D-sensitive to a cytochalasin D-insensitive state. Together, these results indicate that p120ctn is a modulator of E-cadherin-mediated cell adhesion.

The cadherins are a family of transmembrane glycoproteins that play essential roles in the initiation and stabilization of cell-cell contacts (1)(2)(3)(4). The extracellular domain of cadherins is responsible for specific homophilic binding (5), whereas the carboxyl-terminal region of the cytoplasmic domain interacts with intracellular proteins termed catenins (6,7). Each cadherin molecule can bind to either ␤-catenin or ␥-catenin (plakoglobin), which in turn binds to ␣-catenin (8 -11). ␣-Catenin is an actin-binding protein (12) that interacts with other actinbinding proteins, i.e. ␣-actinin (13), ZO-1 (14), and vinculin (15,16). Such interactions link cadherins to the actin cytoskeleton. Binding of the cadherin-catenin complexes to the actin cytoskeleton has been proposed to be essential for the binding activity. Deletion or truncation of the cytoplasmic domain of cadherin results in a loss of function, despite its continued expression on the cell surface (7,17). Additionally, cells expressing normal E-cadherin but lacking ␣-catenin do not ag-gregate (18), and cell-cell adhesion can be restored by transfection of these cells with the ␣-catenin cDNA (19,20). Although these studies have defined protein-protein interactions that are important for cell adhesion, the mechanisms involved in regulation of cell adhesion remain poorly understood. p120 ctn is a recently described component of the cadherin adhesion complex (21)(22)(23) that seems to be able to associate directly with cadherins (24), but its function in the complex remains unknown. p120 ctn , like ␤-catenin, is a member of the armadillo family of proteins, having 10 copies of the 42-amino acid armadillo repeat (25), and a number of different but closely related isoforms have been identified (26). p120 ctn was first discovered as a protein the phosphorylation of which on tyrosine residues was correlated with transformation in cells transfected with pp60 v-Src (27). p120 ctn is also tyrosine-phosphorylated following the stimulation of cells by growth factors, epidermal growth factor, colony-stimulating factor, and platelet-derived growth factors (28,29). In addition to phosphorylation on tyrosine residues in transformed cells and in response to growth factors, constitutive phosphorylation of p120 ctn on serine and to a lesser extent on threonine residues in both normal and src-transformed cells was noticed (29). It was also shown that in Madin-Darby canine kidney cells, p120 ctn is phosphorylated primarily on serine residues, with some phosphothreonine but no detectable phosphotyrosine (30). Dephosphorylation of these residues, caused by either activation of protein kinase C or the addition of kinase inhibitors such as staurosporine, has been correlated with faster migration of p120 ctn during SDS-PAGE 1 and precedes the permeability increase across epithelial cell monolayers (30), raising the possibility that the phosphorylation/dephosphorylation of p120 ctn on serine/threonine residues modulates intercellular junctions.
Recent experiments revealed that the membrane-proximal region of the cadherin cytoplasmic domain plays a role(s) in the regulation of its activity. In the case of C-cadherin, it supports lateral clustering and adhesive strengthening (31), whereas in the case of E-cadherin, it prevents dimerization of the extracellular domain of the protein and thereby negatively regulates adhesion activity (32). Thus, carboxyl-terminally truncated mutant E-cadherin proteins retaining the membrane-proximal region are inactive in cell adhesion, but deletion of the region results in activation of the nonfunctional E-cadherin polypeptides. Although its precise role in the regulation remains to be determined, the membrane-proximal region seems to interact with p120 ctn (31)(32)(33)(34). Therefore, we investigated the potential role of p120 ctn in this negative regulation and obtained several lines of evidence that p120 ctn is involved in modulation of E-cadherin-mediated cell adhesion.

EXPERIMENTAL PROCEDURES
cDNA Construction-Mammalian expression vectors containing mouse E-cadherin cDNAs encoding the wild-type, mutant proteins E⌬C71 or EC0 (Fig. 1a) and an E-cadherin-␣-catenin chimeric protein (E␣C) were described previously (32,35). The cDNA encoding another E-cadherin mutant protein, E⌬C71⌬604 -615 (Fig. 1a), was constructed as described below and cloned into the same expression vector, pCAGGSneo (36) (a gift from Dr. K. Yamamura, Kumamoto University, Kumamoto, Japan). To construct E⌬C71⌬604 -615, two additional EcoRI sites were generated in the E⌬C71 cDNA at the positions encoding amino acid residues 604 -605 and 614 -615, by means of the polymerase chain reaction described previously (32). For this, the following two combinations of sense and antisense primers were used: X1 (TAT-ACCGCTCGAGAGCCG) and EN (CGGAATTCATCATAGTAATACA-CATTGTCCC), and EC (CGGAATTCAGCCAGCTGCACAGGGGC) and ⌬C71 (ATCTTAATTACCGATGAAGTTTCCAATTTC). Sense primer X1 contains a XhoI restriction sequence, whereas sense primer EC and antisense primer EN each contain a EcoRI restriction sequence near the 5Ј-end. The cDNA fragments were assembled into the pBluescript II KS(ϩ) vector using the XhoI-EcoRV site. After confirming the sequence, the cDNA was cloned into the expression vector for E-cadherin, from which the XhoI-EcoRV fragment of the E-cadherin cDNA that encodes the carboxyl-terminal 373 amino acids of E-cadherin had been removed.
cDNA for mouse p120 ctn (the 1A isoform) was kindly provided by Drs. J. Stappert and R. Kemler (Max-Planck Institut fü r Immunbiologie, Freiburg, Germany). Carboxyl-terminal epitope-tagged p120 (p120HA) was generated by the addition of the sequence that encodes the nine amino acid hemagglutinin (HA) epitope for the anti-peptide mAb 12CA5. cDNAs encoding two amino-terminal deletion mutant p120 proteins, p120⌬N1HA and p120⌬N2HA (see Fig. 5a), were constructed by deleting in-frame segments between two restriction sites, NcoI and EcoRI or SmaI and SmaI, respectively. These cDNAs were cloned into the pCAGGSneo vector For the blot overlay assay, the entire E-cadherin cytoplasmic domain or parts of it were expressed as fusion proteins with glutathione Stransferase (GST) (see Fig. 4a). For this, cDNA fragments were generated by either digestion at appropriate restriction enzyme sites in the cDNA or polymerase chain reaction with the following oligonucleotides as primers: N5 (CGGGATCCGGAGGAGAACGGTG) and N3 (AACTG-CAGTCAAGTCACTTCCGGTCGGG), and C5 (TGACCCGGGAGGTG-GAGAAGAAGAC) and C3 (GCGTCGACTTAAGGGGGTGCCGTGGG). The cDNA fragments were cloned into pGEX4T vectors (Amersham Pharmacia Biotech), and the fusion proteins were purified as described previously (11).
Cells and Transfection-Mouse fibroblastic L-tk Ϫ cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. L cells (5 ϫ 10 5 ) were transfected with the expression vectors (10 g) by the calcium phosphate method as described previously (6). G418resistant clones were isolated and examined for E-cadherin expression by immunofluorescence staining as described previously (6). Positive cells were subcloned and used for further studies. To obtain transfectants expressing both the mutant E-cadherin (E⌬C71) and mutant p120 ctn proteins, either pC-p120HA, pC-p120⌬N1HA, or pC-p120⌬N2HA (15 g) was introduced into an L cell clone expressing the mutant E-cadherin (E⌬C71L cells) by the calcium phosphate method. Because E⌬C71L cells already contained the neomycin gene, another plasmid (pStk) containing the herpes simplex virus thymidine kinase gene (37) (1.5 g) was cotransfected. After selection in HAT (hypoxanthine, aminopterine, and thymidine; Life Technologies, Inc.) medium, single colonies were isolated and analyzed for the expression of the p120 ctn protein by immunoblotting with anti-HA mAb.
Cell Aggregation Assay-The cell aggregation assay was performed as described previously (7). In brief, cells were incubated for 10 min at 37°C in Hepes-buffered saline containing 0.01% trypsin (type XI, Sigma) and 2 mM CaCl 2 . After the addition of soybean trypsin inhibitor (Sigma), the cells were washed and resuspended. After incubation for 30 min at 37°C with constant rotation at 70 rpm, the cells were fixed by adding an equal volume of 6% formaldehyde in PBS.
Antibodies-A mouse mAb against p120 ctn (pp120) was purchased from Transduction Laboratories (Lexington, KY). DECMA-1, a rat mAb to E-cadherin (38), was used for immunoblotting and immunofluorescence staining, and rabbit anti-E-cadherin antibodies (6) were used for immunoprecipitation. A mAb against GST was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). A mAb (12CA5) directed against HA and a mAb against phosphotyrosine (4G10) were kindly provided by Dr. A. Yoshimura (Kurume University, Fukuoka, Japan).
Immunoblotting and Immunoprecipitation-Immunoblot analysis was carried out as described previously (32). Immunoprecipitation was carried out as described previously (32) with the following modifications. The cells (5 ϫ 10 6 ) were lysed with either R lysis buffer (10 mM Tris-HCl buffer, pH 7.4, containing 0.5% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 0.1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 25 g/ml aprotinin), or PI lysis buffer to preserve phosphorylated amino acid residues in p120 ctn (25 mM Tris-HCl buffer, pH 7.4, containing 0.5% Nonidet P-40, 2 mM EDTA, 10 mM sodium pyrophosphate, 10 mM NaF, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 25 g/ml aprotinin). The E-cadherincatenin complex was collected with rabbit anti-E-cadherin antibodies, which had been preabsorbed to protein A-Sepharose CL4B (Amersham Pharmacia Biotech). The immune complex was washed with the same buffer four times and then boiled for 5 min in the SDS-PAGE sample buffer. For treatment with alkaline phosphatase, the immunoprecipitate was washed three times with AP buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM MgCl 2 , and 1 mM phenylmethylsulfonyl fluoride), and then incubated with 20 units of calf intestine alkaline phosphatase (Takara Shuzo Co., Ltd., Ohtsu, Japan) in 200 l of AP buffer. To check the specificity of the phosphatase, a phosphatase inhibitor (100 mM ␤-glycerophosphate, 25 mM NaF, 4 mM EDTA, and 1 mM Na 3 VO 4 ) was used (39). After 1 h incubation at 30°C with occasional mixing, the immunocomplex was washed with PI buffer and boiled with SDS-PAGE sample buffer.
Blot Overlay Assay-The blot overlay assay was carried out as described previously (11) except that anti-p120 antibodies were used to detect the GST-p120 fusion proteins bound to the GST-E-cadherin cytoplasmic domain fusion proteins that had been separated by SDS-PAGE and transferred to nitrocellulose membranes and that the proteins were visualized with an ECL detection kit.
Immunofluorescence Staining-Cells were fixed with 3% formaldehyde in PBS for 20 min at room temperature. After three washes with PBS containing 50 mM NH 4 Cl, the cells were soaked in a blocking solution (PBS containing 5% fetal calf serum) for 15 min and then permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were incubated with antibodies as described previously (32).
Phosphate Labeling and Phosphoamino Acid Analysis-Cells were incubated overnight in phosphate-free Dulbecco's modified Eagle's medium containing 1% fetal calf serum (dialyzed against 0.9% NaCl and 10 mM Hepes buffer, pH 7.5) and 250 Ci/ml [ 32 P]orthophosphate (NEN Life Science Products). The cells were then treated with or without 100 nM staurosporine for 60 min, lysed, and subjected to immunoprecipitation as described above. Following transfer to polyvinylidene difluoride membranes (Immobilon, Millipore Corp., Bedford MA) and immunoblotting to assess the recoveries of proteins, the area of the filter containing p120 ctn was excised. The proteins were hydrolyzed at 110°C for 1 h in 5.7 M HCl to release phosphoamino acids. Following lyophilization, phosphoamino acids were separated and detected as described (40).

Expression of Mutant E-cadherin Polypeptides on L
Cells-To examine the potential role of p120 ctn in the modulation of E-cadherin-mediated cell adhesion, we expressed the wild-type E-cadherin as well as mutant E-cadherin polypeptides E⌬C71 and EC0 on L cells. We chose L cells in the present study because L cells seem to exhibit less proteolytic activity toward p120 ctn compared with K562 cells, in which degradation of p120 ctn during immunoprecipitation experiments was observed (32). The wild-type E-cadherin has a cytoplasmic domain of 151 amino acids at its carboxyl terminus. E⌬C71 is a mutant E-cadherin polypeptide with a carboxyl-terminal deletion of 71 amino acid residues, whereas EC0 is a mutant polypeptide completely lacking the cytoplasmic domain (Fig.  1a). When expressed on L cells, these proteins migrated as polypeptides of the expected molecular weights upon immunoblot analysis (Fig. 1b). Consistent with our previous experiments on these proteins expressed on K562 cells (32), EC0 expressed on L cells exhibits activity in a cell aggregation assay but E⌬C71 was inactive (Fig. 2). Therefore, as in the case of K562 cells, the presence of the membrane-proximal region of p120 ctn and E-cadherin Modulation the E-cadherin cytoplasmic domain maintains the partially truncated E-cadherin polypeptide (E⌬C71) expressed on L cells in an inactive state. Immunoblot analysis of the E-cadherin immunoprecipitate with anti-p120 revealed that p120 ctn is associated with E-cadherin expressed on L cells (Fig. 1c). In the case of the E⌬C71 polypeptide, a reduced amount of p120 ctn (ϳ10% of the wild-type E-cadherin polypeptide) was coprecipitated, but no p120 ctn was detected in the EC0 precipitate. The largest isoform of p120 ctn isolated from L cells migrated as a protein of 110 kDa, which was slightly smaller than the reported size of p120 ctn in other cells (25). Different posttranslational modification may be responsible for the difference. p120 ctn Binds to the Membrane-proximal Region of the Ecadherin Cytoplasmic Domain-To demonstrate the direct binding of p120 ctn to the cytoplasmic domain of E-cadherin and to localize p120 ctn -binding site(s) in the latter, we expressed different regions of the domain as fusion proteins with GST (Fig. 3a). Their interaction with p120 ctn , which was also expressed as a GST fusion protein, was analyzed using the blot overlay assay. As shown in Fig. 3b, p120 ctn bound to the Ecadherin cytoplasmic domain fusion proteins containing residues 578 -728 (the entire cytoplasmic domain) and residues 578 -657 (the membrane-proximal region of the domain), but not to a fusion protein including residues 658 -728 (the membrane-distal region of the domain). Furthermore, p120 ctn bound to a cytoplasmic domain fusion protein containing residues 596 -628, but not to a fusion protein containing residues 605-671, eliminating the possibility that the region that was split to produce the membrane-proximal and membrane-distal regions contains an additional binding site for p120 ctn . The deletion of residues 604 -615 from the membrane-proximal region abolished its ability to bind to p120 ctn . These results suggest that the p120 ctn -binding site in the E-cadherin cytoplasmic domain is localized in the membrane-proximal region, i.e. not in the membrane-distal region, and that residues 604 -615 play a E⌬C71⌬604 -615 is a derivative of E⌬C71 and has an additional deletion of residues 604 -615. b, immunoblot detection of E-cadherin polypeptides. L cells expressing the wild-type E-cadherin (EL) or the mutant polypeptides with deletions of the cytoplasmic domain as in a were lysed in SDS sample buffer and then subjected to immunoblot analysis with DECMA-1, an E-cadherin mAb. c, immunoblot detection of p120 ctn coprecipitated with the E-cadherin polypeptides. Cells were lysed as described under "Experimental Procedures," and then E-cadherin was collected using E-cadherin antibodies. After SDS-PAGE and transfer to nitrocellulose membranes, the proteins were stained with either DECMA-1 or anti-p120 antibodies. The faint 115-kDa band recognized by DECMA-1 for the E⌬C71 immunoprecipitate seems to be an intracellular form of the E⌬C71 polypeptide containing a prosequence of ϳ15 kDa (37). The results shown are representative of at least three independent experiments.  ) were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and then incubated with anti-GST mAb or p120 ctn , which was also expressed as a GST fusion protein. The bound p120 ctn was detected with an anti-p120 mAb as described under "Experimental Procedures." c, lack of coprecipitation of p120 ctn with the E⌬C71 polypeptide containing a further deletion of residues 604 -615. p120 ctn was coprecipitated with the carboxyl-terminally truncated E-cadherin polypeptides retaining the membrane-proximal region (E⌬C71), but not with the E⌬C71 polypeptide with a deletion of residues 604 -615 (E⌬C71⌬604 -615). The results shown are representative of at least three independent experiments. p120 ctn and E-cadherin Modulation critical role in the binding.
The Membrane-proximal Region of the E-cadherin Cytoplasmic Domain Is Enough to Localize p120 ctn at Cell-cell Contacts-p120 ctn has been shown to be colocalized with E-cadherin and catenins at cell-cell contacts (21)(22)(23). Immunofluorescence staining with anti-p120 mAb revealed that p120 ctn is localized at cell-cell contacts in L cells expressing wild-type E-cadherin (EL) or a mutant E-cadherin polypeptide with the membrane-proximal region (E⌬C71L) (Fig. 4). In contrast, p120 ctn was undetectable at cell-cell junctions in L cells expressing the tail-less E-cadherin polypeptide (EC0L); instead, it was found throughout the cytoplasm (Fig. 4). Thus, the tail-less E-cadherin cannot recruit p120 ctn to the contacts. These results show that the membrane-proximal region of the E-cadherin cytoplasmic domain is enough to localize p120 ctn at cell-cell contacts.
Deletion of the p120 ctn -binding Site Activates Partially Truncated Nonfunctional E-cadherin Polypeptides-If the binding of p120 ctn to the membrane-proximal region of the E-cadherin cytoplasmic domain is involved in the failure of the mutant E-cadherin polypeptide (E⌬C71) to be functional in the cell aggregation assay, the deletion of the p120 ctn -binding site from the E⌬C71 polypeptide should activate the protein. Therefore we constructed a cDNA that encodes a mutant E-cadherin, designated E⌬C71⌬604 -615 (Fig. 1a). The E⌬C71⌬604 -615 polypeptide has an additional deletion of residues 604 -615, the residues shown to be required for p120 ctn -binding in the above experiments, within the E⌬C71 protein. This construct was introduced into L cells, and G418-resistant clones were isolated. Although the E⌬C71⌬604 -615 protein has the molecular weight expected from its construct and migrated faster than the E⌬C71 protein on SDS-PAGE (Fig. 3c), immunofluorescence staining with DECMA-1, an anti-E-cadherin mAb, revealed that each clone positive for DECMA-1 staining contained a mixture of two types of cells, one (10ϳ%) stained normally, like EL cells, and the other (ϳ90%) stained weakly. This unstable nature of expression of the E⌬C71⌬604 -615 protein persisted even after recloning. Therefore, we analyzed aggregation activity using L cell clones consisting of the two types of cells. Despite the presence of the weakly stained cells, L cells expressing the E⌬C71⌬604 -615 protein showed a certain degree of cell aggregation (Fig. 2). Immunofluorescence staining revealed that cells in the aggregates were stained strongly with DECMA-1, whereas the majority of the cells remaining as single ones were stained only weakly (data not shown). Therefore, the E⌬C71⌬604 -615 protein expressed on L cells seemed to be active in the aggregation assay. The lack of association of p120 ctn with the E⌬C71⌬604 -615 protein was confirmed by immunoblot analysis of the E-cadherin immuno-precipitate with anti-p120 ctn antibodies (Fig. 3c).
Coexpression of p120 ctn Proteins with the Amino-terminal Deletion Activates the Nonfunctional E-cadherin Polypeptides-Whereas the above results demonstrating p120 ctn binds to a site (including residues 604 -615) of the membrane-proximal region of the E-cadherin cytoplasmic domain and the deletion of this site from the nonfunctional E-cadherin (E⌬C71) activates the protein strongly suggested that p120 ctn was a crucial modulator of the protein, the possibility existed that another protein that can bind to the protein at this site could be modulating the aggregation activity. To confirm the role of p120 ctn in the modulation of the adhesion activity of the E⌬C71 protein, we constructed two amino-terminally truncated p120 ctn proteins (p120⌬N1 and p120⌬N2) (Fig. 5a) and expressed them in E⌬C71L cells. p120 ctn can be divided into three parts; an amino-terminal part, a central portion composed of the so-called armadillo repeats, and a small carboxyl-terminal tail. The armadillo repeats are responsible for its binding to cadherins (24,41).
E⌬C71L cells were transfected with these constructs together with the pStk vector containing the herpes simplex virus thymidine kinase gene or the pStk vector alone. After selection in HAT medium, single colonies were isolated and analyzed for expression of the p120 ctn protein by immunoblot- FIG. 4. Immunofluorescence staining of p120 ctn in L cells expressing the wild-type E-cadherin and mutant polypeptides. L cells expressing either the wild-type E-cadherin (a), the E⌬C71 protein (b), or the tail-less EC0 protein (c) were stained with anti-p120 mAb as described under "Experimental Procedures." Bar, 30 m.

FIG. 5.
Expression of the full-length and amino-terminally truncated p120 ctn polypeptides in E⌬C71L cells. a, schematic representation of the full-length and mutant p120 ctn constructs expressed in E⌬C71L cells. The armadillo repeats are indicated by the hatched boxes, and the reported region required for the association with cadherins (24, 41) is indicated by an arrow. b, immunoblot detection of p120 ctn polypeptides. E⌬C71L cells transfected with the pStk vector alone (E⌬C71/tkL), or E⌬C71L cells expressing the full-length p120 ctn (E⌬C71/p120L) or the mutant p120 ctn polypeptides (E⌬C71/p120⌬N1L or E⌬C71/p120⌬N2L) were lysed in the SDS sample buffer and then subjected to analysis with anti-HA or anti-p120 mAb. The results for two independent clones of each transfectant (indicated by numbers at the top of the gels) are shown. c, coprecipitation of the amino-terminally truncated p120 ctn polypeptides with the E⌬C71 polypeptides. E⌬C71/ p120L, E⌬C71/p120⌬N1L, or E⌬C71/p120⌬N2L cells were lysed as described under "Experimental Procedures," and then E-cadherin was collected using E-cadherin antibodies. After SDS-PAGE and transfer to nitrocellulose membranes, the proteins were stained with either DECMA-1 or anti-p120 mAb. Although the exogenously expressed fulllength p120 ctn polypeptide comigrated with the endogenous p120 ctn polypeptide exhibiting the lowest electrophoretic mobility, the mutant p120 ctn polypeptides migrated faster than the endogenous polypeptides. The amounts of these mutant polypeptides seem to be similar to those of the endogenous polypeptides. The results shown are representative of at least three independent experiments. p120 ctn and E-cadherin Modulation ting with anti-HA and anti-p120 antibodies (Fig. 5b). Immunoblot analysis of the E-cadherin immunoprecipitates with anti-p120 antibodies revealed that these truncated p120 ctn polypeptides are associated with the E⌬C71 polypeptides to similar degrees to the endogenous p120 ctn polypeptides (Fig.  5c). Aggregation assay of E⌬C71 cells expressing these truncated p120 ctn polypeptides (clone 3 of E⌬C71/p120⌬N1L cells and clones 8 and 3 of E⌬C71/p120⌬N2L cells) revealed that these cells form aggregates, whereas E⌬C71L cells transfected with the pStk vector alone (E⌬C71/tkL cells) and E⌬C71L cells expressing the full-length p120 ctn polypeptides (E⌬C71/p120L cells) showed no aggregation (Fig. 6). E⌬C71L cells expressing the p120⌬N1 polypeptides at lower levels (clone 5 of E⌬C71/ p120⌬N1L cells) showed only weak aggregation, suggesting that certain amounts of the mutant p120 ctn polypeptides are required for the activation of the EDC71 protein.
Staurosporine Induces an Increased Electrophoretic Mobility of p120 ctn and Activates the Nonfunctional E-cadherin Polypeptides-p120 ctn was originally described as a substrate for activated Src and is also tyrosine-phosphorylated following the stimulation of cells with growth factors, such as epidermal growth factor (27)(28)(29), but it has also been shown to be constitutively phosphorylated on serine/threonine residues in several cell lines (29,30). Therefore, we were interested in determining whether or not a change in the phosphorylation level of p120 ctn has some effect on the activity of the E⌬C71 protein expressed on L cells. For this, L cells expressing the wild-type E-cadherin and the mutant proteins were incubated with kinase inhibitors and then subjected to the aggregation assay. Of the kinase inhibitors tested, staurosporine (100 nM) enhanced the aggregation of E⌬C71L cells but not that of EL cells or EC0L cells (Fig. 7). The aggregation of the staurosporine-treated E⌬C71L cells is E-cadherin-dependent, because it was inhibited by DECMA-1 or 5 mM EGTA (data not shown). Genistein (100 ng/ml) or herbimycin A (0.5 g/ml) had no effect on the aggregation of these cells (data no shown). Staurosporine treatment seemed not to affect the association of p120 ctn with the E⌬C71 protein, but it altered the electrophoretic mobility of p120 ctn associated with the protein, as revealed on immunoblot analysis of E⌬C71 immunoprecipitates with anti-p120 ctn antibodies (Fig. 8a). To determine whether or not dephosphorylation in-creases the electrophoretic mobility of p120 ctn , the immunoprecipitates were treated with alkaline phosphatase (Fig. 8b). This treatment increased the electrophoretic mobility of p120 ctn in the absence but not in the presence of its inhibitor, indicating that dephosphorylation causes the faster migration. Immunoblotting of the immunoprecipitates with anti-phosphotyrosine antibodies revealed that p120 ctn associated with the E⌬C71 protein was not detectably phosphorylated on tyrosine residues before or after staurosporine treatment (data not shown). [ 32 P]Phosphoamino acid analysis revealed that phosphorylation of p120 ctn occurred exclusively on serine and to a lesser extent on threonine residues without any phosphorylation on tyrosine residues before staurosporine treatment (Fig. 8c). Following the addition of staurosporine, there was no apparent reduction in the levels of phosphorylation of serine residues (Fig. 8c), suggesting that dephosphorylation of a specific residue(s) is responsible for the mobility shift and activation of the nonfunctional E-cadherin.
The aggregation of E⌬C71L cells expressing either p120⌬N1 or p120⌬N2 was enhanced by staurosporine (data not shown). This may be explained by the presence of the endogenous p120 ctn . These constructs no longer showed the staurosporineinduced mobility shift (data not shown).
Disconnection of E-cadherin from the Actin Cytoskeleton Reduces the Aggregation Activity of E-cadherin, but Staurosporine Restores the Activity-Cadherin-mediated aggregation has been shown to require anchorage to an intact actin cytoskeleton. In the presence of cytochalasin D (CD), cells expressing the wild-type cadherin protein exhibited weak aggregation and little strengthening of cadherin-mediated adhesion (42,43). Consistent with these previous observations, aggregation of EL cells was inhibited by CD (Fig. 7). Likewise, aggregation of cells (E␣C) expressing a functional E-cadherin chimeric protein covalently linked with the carboxyl-terminal one-third (amino acid residues 612-906) of ␣-catenin, which has the ability to interact with an actin cytoskeleton via the carboxyl-terminal region of ␣-catenin (42), was affected by the presence of CD. These results showed that an intact actin cytoskeleton was required for the activity of both the wild-type E-cadherin and the E-cadherin-␣-catenin chimera and that the disconnection of these proteins from the actin cytoskeleton resulted in reduction of their aggregation activity. The aggregation of EC0L cells was not inhibited by CD (Fig. 7).
The cell aggregation activities of these CD-treated cells were restored, however, when staurosporin was given to the cells (Fig. 7). Staurosporine treatment induced a p120 ctn band shift for these cells similar to that observed for E⌬C71 cells but did not affect the association of p120 ctn with the wild-type E-cadherin or the E␣C protein, as revealed on immunoblot analysis of the immunoprecipitates with anti-p120 antibodies (Fig. 8a).

DISCUSSION
The membrane-distal region of the cadherin cytoplasmic domain contains the binding site for ␤-catenin and ␥-catenin (the catenin-binding site). The deletion of this region inactivates cadherins; i.e. they cannot mediate aggregation. Therefore, the complex formation with catenins has been proposed to be essential for cadherins to be functional in cell adhesion. Previously, we showed that the removal of the membrane-proximal region activates the nonfunctional E-cadherin containing the region and that the membrane-proximal region of E-cadherin is involved in the negative regulation of cell adhesion activity (32). In the present study, we showed that p120 ctn directly binds to this region and is involved in the regulation. It has been reported that in E-cadherin, p120 ctn binds to a different but juxtaposed region from that for ␤-catenin and plakoglobin within the last 37 carboxyl-terminal residues, because the deletion apparently abolished the ability of E-cadherin to coprecipitate p120 ctn (22). Our present and previous results (32), as well as those of others (33), revealed that a reduced amount of p120 ctn is coprecipitated with the carboxyl-terminally truncated E-cadherin and VE-cadherin proteins. Through blot overlay assays, we demonstrated the direct binding of p120 ctn to E-cadherin and localized the p120 ctn -binding site in the membrane-proximal region, including amino acids 604 -615, of the cytoplasmic domain. Although we cannot exclude the possibility that the carboxyl-terminal region of E-cadherin has another binding site for p120 ctn , which is dependent on the conformation for the binding and is destroyed by SDS-PAGE and subsequent transfer to a nitrocellulose membrane, we assume that this less likely. Immunofluorescence staining for p120 ctn of L cells expressing either the wild-type or carboxyl-terminally deleted mutant E-cadherin gave an essentially identical staining pattern; i.e. p120 ctn is localized mainly at cell-cell contacts, demonstrating that the mutant E-cadherin protein has the same ability to recruit p120 ctn to cell-cell contacts as the wildtype E-cadherin protein. If the amount of p120 ctn associated with the mutant E-cadherin is reduced to ϳ10% of that bound to the wild-type E-cadherin, as detected on immunoblot analysis of the E-cadherin immunoprecipitates, the staining at cell-cell contacts in L cells expressing the mutant E-cadherin should also be reduced, because in L cells expressing the tailless E-cadherin, the staining is found mainly in the cytoplasm. This is not, however, the case. Therefore, we assume that p120 ctn is associated with the mutant and wild-type E-cadherin proteins to similar extents but that p120 ctn is released from the mutant E-cadherin during cell lysis or during collection of the mutant E-cadherin. The reason for this is presently unknown. Therefore, the cytoplasmic domain of E-cadherin has at least two functional sites, one for ␤-/␥-catenin and the other for p120 ctn -binding. The ␤-/␥-catenin-binding site is present in the membrane-distal portion and the p120 ctn -binding site in the membrane-proximal part.
In this report, we provide several lines of evidence that p120 ctn is involved in modulation of E-cadherin. First, the removal of 12 amino acid residues from the p120 ctn -binding site in the inner membrane-proximal region of E-cadherin activated the partially truncated nonfunctional polypeptides. Second, coexpression of the amino-terminally deleted p120 ctn , but not that of the full-length p120 ctn , activated the nonfunctional E-cadherin polypeptides. These mutant p120 ctn proteins still retained the ability to bind to E-cadherin. These results suggested that the ability of these mutant proteins to activate the nonfunctional E-cadherin polypeptides was due to their competition with endogenous p120 ctn for association with the nonfunctional E-cadherin polypeptides, rather than their competition with some other proteins that may bind to the same site on E-cadherin. We did not detect, however, displacement of endogenous p120 ctn from the nonfunctional E-cadherin by the mutant p120 ctn proteins. The reason for this is presently unknown. Chemical cross-linking experiments revealed the presence of the E⌬C71 protein dimer in cells expressing the mutant p120 ctn proteins but not in cells expressing the fulllength p120 ctn protein. 2 It is therefore conceivable that these mutant p120 ctn proteins bound to the nonfunctional E-cadherin polypeptides could function as dominant-positive (activating) mutants through facilitating dimerization of the mutant E-2 M. Ozawa, unpublished results.
FIG. 8. Staurosporine treatment induces dephosphorylation of serine/threonine residues of p120 ctn associated with the E-cadherin polypeptides. a, electrophoretic mobility shift of p120 ctn coprecipitated with the E-cadherin polypeptides. Cells expressing either the wild-type E-cadherin, the E⌬C71 protein, or the E␣C chimeric protein were incubated in the presence or absence of staurosporine (100 nM) for 60 min. Cells were lysed as described under "Experimental Procedures," and then E-cadherin was collected using E-cadherin antibodies. After SDS-PAGE and transfer to nitrocellulose membranes, the proteins were stained with either DECMA-1 or anti-p120 antibodies. b, alkaline phosphatase treatment. The E⌬C71 immunoprecipitates were incubated in the presence (ϩ) or absence (Ϫ) of alkaline phosphatase (AP) and its specific inhibitor (PI), and then immunoblotted with anti-p120 mAb. c, phosphoamino acid analysis. Following phosphate labeling, immunoprecipitation, and visualization of labeled p120 ctn as above, the p120 ctn bands were excised, and the hydrolyzates were separated by two-dimensional electrophoresis as described under "Experimental Procedures." The results shown are representative of at least three independent experiments.
cadherin. Finally, dephosphorylation of serine/threonine residues in p120 ctn associated with E-cadherin polypeptides induced by staurosporine treatment and detected as a mobility shift is correlated with activation of the nonfunctional E-cadherin proteins and conversion of the wild-type E-cadherin protein and E-cadherin-␣-catenin chimeric protein from a CDsensitive to a CD-insensitive state. Although staurosporine did not cause apparent decrease in phosphoserine and phosphothreonine content in p120 ctn protein, it increased electrophoretic mobility of p120 ctn . Therefore, it seems likely that dephosphorylation of a specific serine/threonine residue(s) is responsible for the mobility shift and modulation of E-cadherin. Further studies are needed to identify the residues responsible for these observations. Together, these findings suggest that p120 ctn is a modulator of E-cadherin-mediated adhesion.
The functions of cadherins are regulated from the cytoplasmic side. Because the tail-less E-cadherin is fully active in cell aggregation assays, linkage to an actin cytoskeleton via catenins is not an absolute requirement for E-cadherin to be functional. Therefore, disconnection from the actin cytoskeleton, one of the possible sites for the regulation, is not enough to inactivate cadherins. Presumably, cells have developed a mechanism to maintain cadherins in a less active state in the absence of an actin linkage. Although association of p120 ctn with cadherins per se seems not to inactivate cadherins, serine/ threonine phosphorylation of the associated p120 ctn weakens the activity of the latter molecules. As shown in this study and by others (29,30), p120 ctn is constitutively phosphorylated on serine/threonine residues in unstimulated cells. Therefore, cadherins disconnected from the actin cytoskeleton seem to become immediately less active in these cells. The mechanism by which the serine/threonine-phosphorylated p120 ctn inactivates the disconnected cadherins is, however, unknown at present. Anchorage to the actin cytoskeleton via catenins may activate cadherins even on continued association of the phosphorylated p120 ctn with cadherins. Thus, the complex formation with catenins and the anchorage to the actin cytoskeleton overcome the negative modulation imposed by serine/threonine phosphorylation of p120 ctn in the regulation of the activity.
The amino acid residues of the p120 ctn -binding site of Ecadherin are relatively well conserved in other cadherins (2). Among these cadherins, E-cadherin (21-23, 32, this study), Nand P-cadherin (41), VE-cadherin (33), and C-cadherin (31) have been shown to coprecipitate with p120 ctn . Thus, the association of cadherins with p120 ctn appears to be important in the control of the activity states of multiple cadherin families. There have, however, been results that differ from those we present here. The carboxyl-terminally deleted mutant VE-cadherin or C-cadherin polypeptides retaining the membraneproximal region, which can thus associate with p120 ctn , expressed on Chinese hamster ovary cells have been reported to be able to promote cell aggregation (31,44). At present, we do not know the reasons for the discrepancies. As discussed previously, it is not, however, because of different methodologies that gave different results (32). To express and to assess the adhesion activity of the truncated VE-cadherin and C-cadherin polypeptides, Chinese hamster ovary cells were used. Therefore, if the serine/threonine phosphorylation of p120 ctn associated with these mutant cadherins in Chinese hamster ovary cells differs from that of p120 ctn associated with the mutant E-cadherin in L cells or K562 cells, different cell systems could give different results.