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Originally published In Press as doi:10.1074/jbc.M310576200 on October 14, 2003

J. Biol. Chem., Vol. 278, Issue 52, 52371-52378, December 26, 2003
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Two Regions of Cadherin Cytoplasmic Domains Are Involved in Suppressing Motility of a Mammary Carcinoma Cell Line*

Mary Fedor-Chaiken{ddagger}§, Thomas E. Meigs¶||, Daniel D. Kaplan¶**, and Robert Brackenbury{ddagger}

From the {ddagger}Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0521 and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, September 24, 2003 , and in revised form, October 9, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
E-cadherin has been termed an "invasion suppressor," yet the mechanism of this suppression is not known. In contrast, several reports indicate N-cadherin does not suppress but, rather, promotes cell motility and invasion. Here, by characterizing a series of chimeric cadherins we defined a previously uncharacterized region consisting of the transmembrane domain and an adjacent portion of the cytoplasmic segment that is responsible for the difference in ability of E- and N-cadherin to suppress movement of mammary carcinoma cells, as quantified from time-lapse video recordings. A mutation in this region enabled N-cadherin to suppress motility, indicating that both E- and N-cadherin can suppress, but the activity of N-cadherin is latent, presumably repressed by binding of a specific inhibitor. To define regions common to E- and N-cadherin that are required for suppression, we analyzed a series of deletion mutants. We found that suppression of movement requires E-cadherin amino acids 699–710. Strikingly, {beta}-catenin binding is not sufficient for and p120ctn is not involved in suppression of these mammary carcinoma cells. Furthermore, the comparable region of N-cadherin can substitute for this required region in E-cadherin and is required for suppression by the mutant form of N-cadherin that is capable of suppressing. Variations in expression of factors that bind to the two regions we have identified may explain previously observed differences in response of tumor cells to cadherins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The cadherin family of calcium-dependent adhesion molecules plays important roles in differentiation and morphogenesis (1). Alterations in cadherin expression also have been found to be an important determinant of tumor behavior (2). E-cadherin suppresses cell movement (3, 4), whereas loss of E-cadherin correlates with acquisition of invasive capacity in vitro (5, 6) and in carcinomas (7). Restoration of E-cadherin expression suppresses invasion in vitro (5, 6) and in a mouse tumor model (8). For these reasons, E-cadherin has been termed an invasion suppressor, but the mechanisms mediating these effects are poorly understood. In contrast, N-cadherin promotes rather than suppresses cell motility and invasion (9, 10), apparently by triggering or enhancing activation of the fibroblast growth factor receptor (9, 11). Because cadherins might suppress invasion through mechanical restraint or through alterations in cell behavior induced by signaling, the roles of adhesion and signaling in suppression of invasion have been the subject of substantial investigation.

These investigations have focused upon the cytoplasmic domain, which is highly conserved among classic cadherins and crucial for mediating cadherin function. Proteins that bind to the cadherin cytoplasmic domain in roughly stoichiometric amounts were termed catenins (12). {beta}-Catenin binds to a C-terminal region of cadherins referred to as the catenin binding domain (13) and also to {alpha}-catenin, which interacts with the actin cytoskeleton (14). A related molecule, p120ctn, binds to a second region close to the plasma membrane (15, 16). Both regions contribute to adhesion, although there is substantial cell type variation in precise roles (see (14, 17)). The catenin binding and p120ctn binding domains are highly conserved between E- and N-cadherin.

We investigated the role of these cytoplasmic domains in suppression of motility and found that the p120ctn binding domain was essential for E-cadherin to suppress motility of a rat astrocyte-like cell line (4). Three groups found that cytoplasmic p120ctn induced changes in cell morphology and small GTPase activity and suggested that E-cadherin may suppress motility by sequestering p120ctn (18–20). In contrast, Wong and Gumbiner (21) recently reported that p120ctn is not involved and instead suggest that modulation of {beta}-catenin association is responsible for control of cell invasion by E-cadherin. Activated {beta}-catenin stimulates motility of epithelial cells (22). There are apparent inconsistencies in these findings, and none of these studies offers an obvious explanation for the difference in suppressing capacity of E- and N-cadherin.

To explore the basis for this difference and to investigate the mechanisms by which E-cadherin suppresses cell movement, we have analyzed the effects of a series of E/N chimeric cadherins and deletion mutants on the motility of mammary carcinoma cells. This work revealed a region common to both E- and N-cadherin that is essential for suppression of the movement of these cells and a second region responsible for the difference in suppression capacity of E- and N-cadherin. Surprisingly, we found that N-cadherin is capable of suppressing motility, but this capacity is normally repressed or latent.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—MDA-MB-435 cells and MDA-MB-231 cells were obtained from Michael Kinch (MedImmune) and grown in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum and 60 µg/ml gentamycin. Cells were transfected using FuGENE 6 (Roche Applied Science). Cadherin constructs were co-transfected with pCMV-puro, and selection was performed using 1 µg/ml puromycin. Expression of cadherins at the cell surface was verified by immunofluorescent labeling and aggregation assays.

Antibodies—Rabbit polyclonal antibodies (795) specific for the E-cadherin extracellular domain were produced as described (23). Antibodies to p120ctn and {beta}-catenin were obtained from BD Biosciences Pharmingen. Monoclonal antibody 13A9 specific for the N-cadherin cytoplasmic domain (24) was a gift from Margaret Wheelock (University of Nebraska Medical Center). Horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies were from Jackson Laboratories.

Constructs—The mouse E-cadherin vector pBATEM2 (25), mouse N-cadherin vector pBATMNC (26), E-cadherin {Delta}594–618 (formerly {Delta}JM) vector pBATEM34 (27), and the E-cadherin 692T (formerly {Delta}CB) vector pBATEM21 (28) were obtained from Masatoshi Takeichi (Kyoto University). The enhanced GFP vector was from Clontech, and the p120ctn-GFP vector was obtained from Keith Burridge (18). The E-cadherin 584T (formerly {Delta}cyto) vector pBATEM66 (4) and the E-cadherin {Delta}699–710 (formerly {Delta}G12) vector pBATEM{Delta}G12 (23) were made in our laboratory as previously described.

A two-step PCR approach was used to generate a fragment containing the E-cadherin {Delta}581–593 deletion. This fragment was cloned into the unique BstEII and Bsu36I sites in pBATEM2, replacing the wild-type cadherin sequences, and the resulting construct was sequenced. The N-HA1 construct, in which amino acids 591–599 of wild type N-cadherin were replaced with the 9-amino acid HA tag YPYDVPDYA, was also made by a two-step PCR reaction that resulted in a fragment encoding amino acids 439–747 (COOH end) of N-cadherin plus 375 bases beyond the N-cadherin translation stop site, incorporating the HA tag in place of amino acids 591–599. This fragment was cloned into the EcoRV and NsiI sites of pBATMNC, replacing the wild type N-cadherin sequences, and the resulting clone was verified by sequencing. A similar PCR approach was used to make a fragment encoding the NHA {Delta}720–731 deletion. This fragment was cloned into the unique EcoRV and NsiI sites of pBATMNC.

Eight chimeric cadherin constructs, NEE, ENN, EEN, NNE, NEN, ENE, E657N, and E618N, were made using a two-step PCR approach and subsequent cloning into either the BstEII and Bsu36I sites of pBATEM2 or the EcoRV and NsiI sites of pBATMNC. The sequence of all PCR-generated fragments was determined. Table I shows the chimera junctions and the parent vectors.


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  		TABLE I
Sequence junctions of chimeric cadherin molecules

 
Aggregation Assays—Aggregation was assayed as described previously (23). Briefly, monolayers of cells at 60–90% confluence were trypsinized under conditions that preserve cadherins (29, 30). Approximately 2 x 106 cells were placed in vials and rotated at 90 rpm, 37 °C. Aggregates were photographed after 60 min.

Wound-filling Assays—Wound filling assays were done in two ways. For quantitative time-lapse analysis cells were grown to confluence on coverslips coated with laminin (Sigma), and a wound was made. The coverslip was placed in a Sykes Moore chamber, which was then filled with fresh medium containing 10 µg/ml mitomycin C (Sigma) to inhibit cell division. The chamber was placed on a heated stage on an Olympus IMT-2 microscope, and images were captured every 15 min for a total of 15 h using Scion Image. Movement of selected cells at the wound edge was tracked using Metamorph software, and distance moved in the x axis, the y axis, and in total was determined. For quick qualitative analysis cells were grown to confluence on tissue culture plates, and a wound was made by scraping the monolayer with a pipette tip. Images were captured using Scion Image software immediately after wounding and 18–24 h later. Morphology of the wound edge at 18–24 h was observed and compared with the wound edges of cells shown to be motile or suppressed by quantitative time-lapse analysis. For each cell line assayed, at least two independent clones were assessed by the qualitative assay, and at least one clone was assessed by the quantitative assay.

Statistical Analysis—For quantitative assessment of motility, two time-lapse video recordings were made of a clonal cell line, and 8 cells were tracked in each video. Means and S.D. were calculated for the x axis, y axis, and total movement data. Mean motilities were compared by one-way analysis of variance, and pairwise multiple comparisons were carried out using Tukey's adjustment for multiple comparison.

Immunoprecipitations and Immunoblotting—Immunoprecipitations and immunoblotting were carried out as previously described (4) except that cells were extracted with CSK buffer (10 mM PIPES, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.2% Triton X-100) plus 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 4 µm leupeptin, and 2 µg/ml pepstatin to maintain association of p120ctn with cadherins.

Subcellular Fractionation—Cytosolic and membrane fractions were prepared as described (18). Protein concentrations of the cytosolic fractions were determined, and for each cell line, 15 µg of cytosolic protein was loaded per well. For the membrane fractions, the volume loaded represented the same proportion of the total sample as the volume of cytosolic sample loaded.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
To identify regions of the cadherin cytoplasmic domain that are involved in suppression of motility, we examined the effect of mutant and chimeric cadherins on the motility of an MDAMB-435 mammary carcinoma cell line. Although some isolates of MDA-MB-435 cells express N-cadherin (9), the isolate used for all studies described here does not express E-cadherin or N-cadherin endogenously but does express low levels of protein(s) that react with a pan-cadherin antibody. The cells form only small loose aggregates in the presence of calcium (Fig. 1) and are motile. These MDA-MB-435 cells were transfected with each cadherin expression vector, and in each case, several permanent lines were established. The expression of cadherins was verified by immunofluorescent labeling and quantified by immunoblotting, which revealed that all of the lines transfected with E-cadherin vectors expressed amounts of E-cadherin that were roughly similar to each other and to MCF-10 cells, a relatively normal mammary epithelial line (31). Similarly, immunoblotting revealed that all of the N-cadherin lines expressed comparable amounts of N-cadherin. The ability of the cadherins to mediate aggregation was determined by comparison to control cells and E-cadherin- and N-cadherin-expressing cells as shown in Fig. 1, and then motility was assayed by observing the movement of cells into artificial "wounds" induced by scraping.



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  		FIG. 1.
Exogenous cadherin expression increases aggregation of MDA-MB-453 mammary carcinoma cells. MDA-MB-435 cells expressing a control vector (Control), an E-cadherin expression vector (E-cadherin), or an N-cadherin expression vector (N-cadherin) were allowed to aggregate in the presence of calcium for 60 min as described under "Experimental Procedures." Although control cells could form small aggregates, expression of E-cadherin or N-cadherin greatly increased the extent of cell aggregation. Bar = 100 µm.

 
A New Assay Confirms E- and N-cadherin Differ in Their Ability to Suppress Motility of Mammary Carcinoma Cells— First, we examined the effects of E- and N-cadherin on movement of MDA-MB-435 cells. When observed 18–24 h after wounding, control cells or N-cadherin-expressing cells generally had migrated further into the wound than E-cadherin-expressing cells. This difference in distance moved by the edge of the wound was not always significant or reproducible, but the assays could be reliably scored based on consistent and striking differences in the appearance of cells at the wound edge. E-cadherin-expressing cells appeared to move as a sheet, resulting in a smooth edge of tightly adherent cells oriented toward the wound. In contrast, N-cadherin or control cells appeared to move as individuals, resulting in a ragged edge with cells oriented in different directions (Fig. 2). The difference in movement of E- and N-cadherin or control cells reflected in appearance of the wound edges was placed on an objective footing by analyzing cell tracks determined from time-lapse video recording. Representative tracks illustrate that E-cadherin cells generally move in a straight line perpendicular to the wound, along the y axis, whereas N-cadherin and control cells often move parallel to the wound, along the x axis (Fig. 2). The cell track data were quantified, revealing that x-axis movement and total movement of the E-cadherin cells were significantly lower than control and N-cadherin cells. In contrast, y axis movement was not significantly different between E-cadherin and control cells (Table II), which confirmed the lack of significant differences when the distance moved by the monolayer edge was measured. Similar results were observed with a second mammary carcinoma cell line, MDA-MB-231 (not shown). Expression of N-cadherin in the MDA-MB-435 cells resulted in a modest but statistically significant increase in cell movement, as previously observed (9, 10). The wound-filling assay was used in all subsequent assays to evaluate the effects of mutant cadherins, scored either by comparing the morphology of the wound edges to that of control and E-cadherin-expressing cells or by quantification of cell tracks and comparison to results from control or E-cadherin-expressing cells. Because expression of E-cadherin was found to decrease x axis movement by 45% compared with control, suppression was defined as the ability to decrease x axis movement by a comparable amount.



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  		FIG. 2.
E-cadherin, but not N-cadherin, suppresses movement of MDA-MB-435 cells. The panels illustrate the movement of MDAMB-435 control cells and cells expressing E- or N-cadherin into wounds. Each panel shows two overlapping images of sheets of cells. In each case, the darker image represents the sheet of cells 1 h after the wound was produced. The lighter image shows the same field 14 h later. Tracks of selected cells, determined from video recordings, are superimposed on the pictures. Bar = 100 µm.

 


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  		TABLE II
E-cadherin, but not N-cadherin, suppresses movement of MDA-MB-435 mammary carcinoma cells

 
To evaluate the effect of wounding on the differences in movement induced by E- and N-cadherin, we also examined the movement of these cells in unwounded monolayers. Here also, E-cadherin-expressing cells were less motile than N-cadherin or control cells. Examples of selected cell tracks determined from video recordings are shown in Fig. 3, and quantification of movement calculated from these tracks indicated the difference in movement of E-cadherin cells, and control or N-cadherin cells was statistically significant. In contrast to its effect in wounding assays, N-cadherin did not promote motility relative to control cells in intact monolayers (Fig. 3).



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  		FIG. 3.
E-cadherin, but not N-cadherin, suppresses movement of individual cells in an unwounded, confluent monolayer. Micrographs of MDA-MB-435 control cells and E- and N-cadherin-expressing cells are shown beside tracks of selected control and E- and N-cadherin cells as determined from time-lapse video recordings. The histogram summarizes the mean rate of total movement over 15 h by 16 individual MDA-MB-435 cells transfected with expression vectors for E-cadherin, N-cadherin, or a control vector and grown to confluency. The asterisk indicates significant difference from control with p < 0.0001. Error bars indicate the S.D. of the mean.

 
The Transmembrane Sequence and Juxtamembrane Domain Determine Ability to Suppress Motility—To investigate the basis for the differing effects on motility of E- and N-cadherin, we constructed and tested chimeric cadherins (Fig. 4) (for convenience, these chimeras are referred to by a three-letter designation, such as NEE, signifying the derivation of the extracellular, transmembrane, and cytoplasmic segments from E- or N-cadherin). The overall structure and function of these chimeric molecules was not disrupted, as in each case we verified that the chimeric proteins were correctly localized to the cell surface, and adhesive activity was preserved. This strategy had the potential to reveal regions within E-cadherin that mediate suppression or regions within N-cadherin that overcome or inhibit suppression. An NEE chimera suppressed the motility of MDA-MB-435 cells, but an ENN chimera did not (Fig. 4), as shown both by the morphology of wound edges and by quantitative analysis of video recordings (x axis movement of NEE was significantly less than control, p < 0.0001). Therefore, the ability to suppress motility is dictated by the transmembrane and/or cytoplasmic domains of these cadherins.



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  		FIG. 4.
Analysis of chimeric cadherins defines a region responsible for the difference between E- and N-cadherin in suppression of motility. The diagram shows the structures of chimeric cadherins in which the extracellular domain, TMS, or portions of the cytoplasmic domain of mouse E- or N-cadherin were fused in various combinations ("Experimental Procedures" and Table I). Each construct was transfected into MDA-MB-435 cells, and the resultant lines were characterized and assayed for the ability to suppress motility by the qualitative assessment of the morphology of wound edges 18–24 h after wounding. The motility of cells expressing the first four constructs listed was also assayed by quantitative analysis of individual cell tracks determined from time-lapse video recording. Aggregation was also assayed as described under "Experimental Procedures" and scored positive or negative by comparison to aggregation of control or E-cadherin-expressing cells as shown in Fig. 1.

 
To further define the region involved in controlling motility, the three segments derived from E- or N-cadherin were linked in all possible combinations, introduced into MDA-MB-435 cells, and analyzed for effects on motility by comparing morphology of the wound edges at 18–24 h to that of E-cadherinand N-cadherin-expressing cells as shown in Fig. 2. In each case, at least two independent lines expressing similar amounts of cadherin were examined, with consistent results. An EEN construct suppressed motility, in contrast to the ENN construct discussed previously, narrowing the sequences that define the difference in suppression ability of E- and N-cadherin to the transmembrane segment (TMS). As this finding would predict, the chimeric cadherin produced by replacing the TMS of N-cadherin with E-cadherin sequences (NEN) also suppressed movement. Unexpectedly, however, two constructs with TMS sequences derived from N-cadherin, an ENE construct and an NNE construct, also suppressed. In short, when results from all eight chimeric constructs were compared, we found that any construct with an E-cadherin TMS or an E-cadherin cytoplasmic domain suppressed, whereas only constructs in which both the TMS and cytoplasmic domains are derived from N-cadherin failed to suppress (Fig. 4). One interpretation of these findings is that the TMS and cytoplasmic regions of E-cadherin possess redundant activities, either one of which is sufficient to suppress motility.

N-cadherin Can Suppress Motility, but This Ability Is Inhibited—A simpler explanation is that N-cadherin actually has the ability to suppress motility, but the TMS and adjacent cytoplasmic sequences comprise a binding site for a component that restricts or prevents this ability. This possibility was tested by introducing a mutation that might be expected to disrupt the binding of such a regulatory component. Amino acids 591 through 599 of mature N-cadherin were replaced with a 9-amino acid HA tag (Fig. 5A) (32). This construct, termed N-HA, was transfected into MDA-MB-435 and MDAMB-231 cells, and several stable lines were isolated. All N-HA lines isolated displayed good aggregation similar to that of N-cadherin-expressing cells (not shown). Immunoprecipitation experiments showed that the HA insertion did not affect the ability of N-HA to associate with p120ctn or {beta}-catenin (Fig. 5C). Motility was suppressed in all three independent MDA-MB-435 lines tested (significantly different from N-cadherin, p < 0.0001, but not different from E-cadherin) (Fig. 5B) and two MDA-MB-231 transfected lines tested (not shown), supporting the hypothesis that N-cadherin possesses the ability to suppress motility but that this ability is latent, suppressed by a mechanism that involves amino acids 591–599. This region is referred to subsequently as the M domain, for modulation of movement.



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  		FIG. 5.
A mutation within the cytoplasmic domain of N-cadherin enables it to suppress motility. A, the panel shows the last 4 amino acids of the TMS, first 35 amino acids of the cytoplasmic domain of mouse E-cadherin (E-cad), the first 38 amino acids of the cytoplasmic domain of mouse N-cadherin (N-cad), and a mutant (N-HA) in which 9 amino acids were replaced by an HA tag as indicated. The p120ctn binding region is also indicated. B, comparison of the motility of MDAMB-435 cells expressing E-, N-, and mutant N-cadherins, determined by quantitative analysis of cell tracks. The histogram shows the mean rate of x axis movement over 14 h of 16 individual cells each from control cells and lines expressing E-cadherin (E-cad), N-cadherin (N-cad), three separate isolates of the HA-tagged insertional mutant of N-cadherin (N-HA1, -2, and -3), and a double insertion/deletion mutant of N-cadherin (N-HA{Delta}720–731). All of these cell lines exhibited good aggregation except control and N-HA{Delta}720–731 lines. The asterisk indicates a significant difference from N-cadherin, with p < 0.0001. Error bars indicate the S.D. of the mean. C, association of the cadherin-binding proteins p120ctn and {beta}-catenin with N-cadherin and N-HA. These cadherins were immunoprecipitated (IP) from transfected lines of MDA-MB-435 cells and blotted for p120ctn (left panel), {beta}-catenin (middle panel), and N-cadherin (right panel).

 
Identification of a Cadherin Cytoplasmic Domain Region That Is Required for Suppression of Cell Motility—Although we focused initially on differences between E- and N-cadherin, the finding that N-cadherin shares with E-cadherin the ability to suppress mammary carcinoma cell motility caused us to examine conserved regions in the two cadherins that might be essential. We, therefore, produced and characterized permanently transfected lines of MDA-MB-435 cells expressing the E-cadherin deletion mutants shown in Fig. 6A. These mutant forms, which centered around the p120ctn and {beta}-catenin binding regions, were tested by co-immunoprecipitation for their ability to associate with these two known cadherin binding partners. Full-length E-cadherin and {Delta}699–710 co-immunoprecipitate both p120ctn and {beta}-catenin, {Delta}581–593 and {Delta}594–618 co-immunoprecipitate {beta}-catenin but not p120ctn, and 692T co-immunoprecipitates p120ctn but not {beta}-catenin (Fig. 6C). The {Delta}699–710 and 692T molecules did not mediate effective aggregation, whereas cells expressing the other mutant molecules aggregated similarly to full-length E-cadherin (Fig. 6A).



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  		FIG. 6.
Definition of a region of E-cadherin required for suppression of motility. A, diagrams of E-cadherin and five mutant constructs showing the p120ctn and {beta}-catenin binding domains (open boxes) and summaries of motility phenotype and adhesion phenotype. Deletions are designated by the amino acids that were deleted, numbered from the N-terminal amino acid of the mature protein, and truncations are designated by the final amino acid translated. A summary of the ability to suppress motility is taken from data in panel B. Aggregation was assayed as described under "Experimental Procedures" and assessed by comparison to control and E-cadherin-expressing cells as shown in Fig. 1. TMS, transmembrane segment. B, motility of MDA-MB-435 cells expressing various forms of E-cadherin, determined by quantitative analysis of cell tracks. The histogram shows the mean rate of x axis movement over 14 h of 16 individual cells from each of 7 cell lines. The asterisks indicate motility significantly different from that of E-cadherin cells with p < 0.0001. Error bars indicate the S.D. of the mean. C, association of the cadherin-binding proteins p120ctn and {beta}-catenin with wild type and mutant E-cadherins. E-cadherin forms were immunoprecipitated (IP) from different transfected lines of MDAMB-435 cells and blotted for p120ctn (top panel), {beta}-catenin (middle panel), and E-cadherin (bottom panel).

 
The motility of the MDA-MB-435 cell lines expressing mutant forms of cadherin was then assayed by time lapse recording of wound-filling (Fig. 6B). Deletion of the entire cytoplasmic domain abolished suppression, as did a truncation that deleted the catenin binding domain and remaining C-terminal sequences. Deletion of amino acids 699–710, a small region that is just C-terminal to the catenin binding domain, also abolished the ability to suppress (Fig. 6A), as the x axis motility of all three of these mutant forms differed significantly from that of E-cadherin with p < 0.0001. We termed the region containing these essential amino acids the S domain, for suppression of motility. Deletion of the S domain had no effect on association of {beta}-catenin or p120ctn, and two deletions ({Delta}581–593 and {Delta}594–618) that abolish p120ctn binding had no effect on suppression, suggesting {beta}-catenin is not sufficient, and p120ctn plays little, if any, role in suppression in these cells.

Recently, cytoplasmic but not membrane-associated p120ctn has been found to induce a dendritic morphology in several cell types and to affect the activity of small GTPases, leading to the suggestion that E-cadherin suppresses motility by sequestering p120ctn at the membrane (18–20). This does not appear to be the case in MDA-MB-435 cells, however, because there was no correlation between the cellular localization of endogenous p120ctn and effects on movement in several of our cadherintransfected cell lines (Fig. 7A) even though overexpression of p120ctn induced a dendritic phenotype in these cells (Fig. 7B). For example, motility was suppressed in the {Delta}594–618 cell line, but not in control MDA-MB-435 cells, even though these cells expressed similar, relatively high amounts of p120ctn in the cytoplasm. Furthermore, the motility of E-cadherin and N-cadherin-expressing cells differed even though the cytoplasm of these lines contained comparable, low amounts of p120ctn (Fig. 7A).



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  		FIG. 7.
Localization of endogenous p120ctn does not correlate with suppression of motility, although p120ctn overexpression does induce morphological changes in MDA-MB-435 cells. A, subcellular localization of p120ctn. Cytosolic (C) and membrane (M) fractions were prepared from MDA-MB-435 cells expressing a control vector (Control), E-cadherin (E-cad), the p120ctn binding domain deletion ({Delta}594–618), or N-cadherin (N-cad). 15 µg of cytosolic protein was loaded per well. For the membrane fractions the volume loaded represented the same proportion of the total sample as the volume of cytosolic sample loaded. Samples were separated by SDS-PAGE and blotted for p120ctn. B, dendritic phenotype induced by p120ctn overexpression. MDA-MB-435 cells grown on coverslips were transfected with either a p120ctn-GFP construct (p120ctn-GFP) or an enhanced GFP control vector (GFP). After 72 h the cells were fixed with 4% paraformaldehyde, mounted, and observed for dendritic morphology. Bar = 100 µm.

 
Because the amino acid sequence of the S domain is highly conserved among cadherins, including N-cadherin (Fig. 8), we investigated whether this region in N-cadherin performs the same function as in E-cadherin. We concluded that the N-cadherin region can substitute for the E-cadherin region, as the chimeric molecules EEN, E618N, and E657N, in which C-terminal sequences of the E-cadherin cytoplasmic domain were replaced with N-cadherin sequences, did suppress motility (Fig. 4). Furthermore, when the region (amino acids 720–731) that is homologous to the E-cadherin S domain was deleted from the N-HA mutant of N-cadherin, which is capable of suppressing, its ability to suppress was abolished (Fig. 5B).



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  		FIG. 8.
The S domain is highly conserved among cadherins. The figure shows C-terminal regions of mouse cadherins aligned with residues 690–728 of E-cadherin. Identical amino residues in the S domain (defined by deletion 699–710) are shaded. The sequences of all cadherins end at their C terminus (COOH), except cadherin 15, which extends 17 additional amino acids beyond those shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our results indicate that two regions of the cadherin cytoplasmic domain are involved in suppressing the movement of mammary carcinoma cells. One region, defined by a mutant that lacks amino acids 699–710 of E-cadherin, was absolutely required for suppression of movement in these cells. The closely related sequence in N-cadherin was capable of substituting for this deleted region in E-cadherin, indicating both cadherins contain functionally equivalent domains, which we termed the S domain, for suppression of movement. These findings suggested that the failure of N-cadherin to suppress motility is not because it lacks the capability to do so but, rather, because this ability is masked or latent. Consistent with this interpretation, we identified a region within N-cadherin, consisting of the transmembrane segment and portion of the adjacent cytoplasmic segment that is required to restrict or repress the ability of N-cadherin to suppress movement. We termed this region the M domain, for modulation of movement.

An important step in these studies was the development of a reliable, quantitative assay. We evaluated suppression of movement by cadherins using in vitro wound-filling assays but found that the way these assays are usually scored (measuring the distance moved by the sheet of cells at the edge of the wound) did not give significant or reproducible results. Instead, we developed two alternative, more reliable scoring methods that entailed qualitative evaluation of wound edge morphology or quantitative analysis of individual cell tracks obtained from video recordings. The quantitative analyses confirmed that the degree of side-to-side movement of E-cadherin-expressing cells was consistently suppressed compared with control or N-cadherin-expressing cells, but these cells did not differ significantly in movement into the wound.

Other investigators (9, 10) report that N-cadherin promotes movement compared with control cells and link this effect to activation of the fibroblast growth factor receptor (9, 10). We also observed a modest enhancement of movement promoted by N-cadherin during wound filling but not in intact, unwounded monolayers. Several differences in cells and procedures may account for the disparity between our results and those of others (9, 10). Different MDA-MB-435 cell isolates vary in expression of endogenous cadherins and EphA22 and may differ in expression of fibroblast growth factor receptor or other components involved in this effect. Additionally, different assays were used to assess cell movement in these studies. Nieman et al. (9) used a transwell assay with conditioned medium as a chemoattractant, which is likely to assess directional migration. The mechanisms controlling directional migration are known to differ from those controlling motility (33, 34).

To define the region(s) required for suppressing the motility of these mammary carcinoma cells, we used the wound-filling assay to test the effect of deletion mutants. Surprisingly, the p120ctn binding region was not required, which differed from results we obtained previously with rat astrocyte-like cells (4). Rather, a region near the C terminus of cadherin, the S domain, was found to be required. The specific reason for this variation in mechanism of suppression remains unclear, but it extends cell type-specific differences in cadherin function seen in other studies (14, 17).

p120ctn has recently been suggested to play a key role in suppression of motility (18–20). Cytoplasmic, but not membrane-associated p120ctn induces a dendritic morphology in several cell types and affects the activity of small GTPases, leading to the suggestion that E-cadherin suppresses motility by sequestering p120ctn at the membrane. This does not appear to be the case in MDA-MB-435 cells, however, because the cellular localization of endogenous p120ctn did not correlate with its effects on movement in several of our cadherin-transfected cell lines even though a dendritic phenotype was induced in these cells by overexpression of p120ctn as in other cell types (18–20).

Deletion of the S domain abolished suppression and compromised cell aggregation, but several results indicated that adhesion alone does not suppress movement. For example, N-cadherin, the ENN chimera, and the 584T mutant all mediate effective adhesion but are unable to suppress motility, and insertion of an HA tag into the M domain of N-cadherin enabled suppression of movement without discernable effects on adhesion. It is likely, therefore, that cell movement is affected by signaling events initiated by cadherins rather than adhesion per se.

Four components, Shc (35), the heterotrimeric G-protein subunit G{alpha}12 (36), and the protein-tyrosine phosphatases PTP1B (37) and PTPµ (38), have been reported to interact with cadherins within the region of the S domain, although the generality and significance of these interactions has not yet been firmly established. Suppression is abolished by overexpression of activated G{alpha}12 (23), suggesting this molecule could play a significant role, possibly by modulating release of {beta}-catenin. {beta}-Catenin has been suggested to induce invasion (21) and motility (22, 39). Substantial amounts of {beta}-catenin remain bound to cadherins lacking the S domain, however, suggesting that sequestering {beta}-catenin is not sufficient to suppress motility. Alternatively, the S domain might be required to generate or modulate tyrosine phosphorylation cascades triggered by E-cadherin (40), which could be regulated by PTP1B or PTPµ. Shc is unlikely to be involved because mutation of tyrosine 705, which is necessary for Shc binding (41), does not affect the ability of E-cadherin to suppress motility.2

The S domain sequences of E- and N-cadherin are nearly identical, and motility was suppressed by several chimeric cadherins in which this portion of E-cadherin was replaced by N-cadherin sequences, indicating that N-cadherin possesses a fully competent S domain. Mutational analysis of N-cadherin showed that the region we termed the M domain disrupts the N-cadherin ability to suppress. The studies of Horikawa and Takeichi (42) also suggest a new function for this region of N-cadherin. They found that overexpression of N-cadherin had no effect on the spreading of myotome precursor cells, but expression of N-cadherin with a deletion of the p120ctn binding domain prevented this spread. This was not due to disruption of p120ctn binding, however, as point mutations that specifically abolish p120ctn binding did not produce the same effects as the deletion, suggesting the spread of myotome cells is affected by some other component that binds to this region (42). Similarly, Ozawa (43) recently observed that this membraneproximal region differentially regulates adhesion of E- and N-cadherin, but again, p120 is not involved. It is possible that the HA-tag mutation we introduced disrupts binding of a component(s) involved in these activities.

Our findings suggest several ways in which diversity in cadherin effects might arise in different cell types. If the M domain and S domain each act by generating intracellular signals, different cells might respond to the same signal in different ways. Variations also would be expected if cells differ in expression of molecules that bind to the S and M domains. For example, N-cadherin does not suppress the movement of MDA-MB-435 cells but would be expected to suppress the movement of any cells that do not express key M domain binding partners. One example may be LM8 mouse osteosarcoma cells, as migration and metastasis of these cells is inhibited by expression of N-cadherin (44). Furthermore, expression of E-cadherin induces robust adhesion in some cells without suppressing movement (4, 45), raising the intriguing possibility that these cells may express regulators that bind to E-cadherin, permitting adhesion but preventing suppression.


   FOOTNOTES
 
* This work was supported by NIAMS, National Institutes of Health Grant AR44713 and United States Army Medical Research and Materiel Command Grant DAMD17-98-1-8292 (to R. B.). 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 U.S.C. Section 1734 solely to indicate this fact. Back

|| Supported by National Institutes of Health Grant CA91159. Present address: Dept. of Biology, University of North Carolina, One University Heights, CPO 2440, Asheville, NC 28804. Back

** A Howard Hughes Medical Institute Predoctoral Fellow. Back

§ To whom correspondence should be addressed: Dept. of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Vontz Center for Molecular Studies, 3125 Eden Ave., Cincinnati, OH 45267-0521. Tel.: 513-558-8192; Fax: 513-558-4454; E-mail: mary.chaiken{at}uc.edu.

1 The abbreviations used are: HA, hemagglutinin; PIPES, 1,4-piperazinediethanesulfonic acid; TMS, transmembrane segment; GFP, green fluorescent protein. Back

2 M. Fedor-Chaiken, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Rakesh Shukla and the Center for Biostatistical Services at the University of Cincinnati for excellent assistance with the statistical calculations and to Amy Koshoffer for excellent technical assistance.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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