The Novel Catenin p120casBinds Classical Cadherins and Induces an Unusual Morphological Phenotype in NIH3T3 Fibroblasts

EXPERIMENTAL CELL RESEARCH ARTICLE NO.

225, 328–337 (1996)

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The Novel Catenin p120cas Binds Classical Cadherins and Induces an Unusual Morphological Phenotype in NIH3T3 Fibroblasts ALBERT B. REYNOLDS,1 JULIET M. DANIEL, YIN-YUAN MO, JING WU,

AND

ZHI ZHANG

Department of Tumor Cell Biology, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, Tennessee 38105

INTRODUCTION p120cas (CAS) is a tyrosine kinase substrate whose phosphorylation has been implicated in cell transformation by Src and in ligand-induced signaling through the EGF, PDGF, and CSF-1 receptors. More recently, CAS has been shown to associate with E-cadherin and its cofactors (catenins), molecules that are involved in cell adhesion. Although both CAS and b-catenin contain armadillo repeat domains (Arm domains), the amino acid identity between these proteins in this region is only 22%, and it is not yet clear whether CAS will emulate other catenins by associating with other members of the cadherin family. Here we report that in addition to binding E-cadherin, wild-type CAS associated with N-cadherin and P-cadherin. Transient transfection of cloned CAS isoforms into MDCK epithelial cells indicated that CAS1 and CAS2 isoforms are equally capable of binding to E-cadherin even though these cells preferentially express CAS2 isoforms. In addition, CAS colocalized with N-cadherin in NIH3T3 cells and analysis of CAS mutants in vivo indicated that the CAS–N-cadherin interaction requires an intact CAS Arm domain. The data suggest that CAS– cadherin interactions in general are dictated by the conserved armadillo repeats and are not heavily influenced by sequences added outside the Arm domain by alternative splicing. Interestingly, overexpression of CAS in NIH3T3 cells induced a striking morphological phenotype characterized by the presence of long dendrite-like processes. This branching phenotype was specific for CAS, since (i) overexpression of the stucturally similar b-catenin had little effect on cell morphology, and (ii) the branching was abolished by deletions in the CAS Arm domain. Our data indicate that, like other catenins, CAS is a cofactor for multiple members of the cadherin family. However, the dramatically distinct phenotype exhibited by fibroblasts overexpressing CAS, versus b-catenin, support recent data suggesting that these catenins have fundamentally different and possibly opposing roles in cadherin complexes. q 1996 Academic Press, Inc.

1 To whom correspondence and reprint requests should be addressed. Fax: 901-495-2381.

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0014-4827/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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p120cas (CAS) is a tyrosine kinase substrate whose phosphorylation correlates by genetic analysis with transformation by Src [1]. CAS is also phosphorylated on tyrosine in cells stimulated by epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and colony-stimulating factor-1 (CSF-1) [2, 3], implying that tyrosine phosphorylation of CAS plays a role in both ligand-induced signaling and cell transformation. cDNA cloning of cas revealed that the protein contains 11 armadillo repeats and is structurally similar to the cell adhesion proteins b-catenin and plakoglobin [4, 5]. Recently, we and others found that CAS associates and colocalizes in vivo with the transmembrane cell–cell adhesion molecule, E-cadherin, and its cytoplasmic cofactors, the catenins (a-, b-, and g-/plakoglobin) [6–8]. CAS binds directly to E-cadherin and this interaction is mediated by the CAS armadillo repeats [9]. CAS therefore appears to be a new catenin and presumably plays a role in E-cadherin mediated functions. Cadherins comprise a superfamily of transmembrane cell adhesion molecules with important roles in cell – cell adhesion, development, morphogenesis, and metastasis (for reviews, see [10 – 12]). Their extracellular domains mediate cell – cell contact by Ca2/-dependent homophilic interactions, while their cytoplasmic domains interact with the actin cytoskeleton via the catenins. E-cadherin binds directly to bcatenin (or plakoglobin) [13, 14], which in turn directly associates with a-catenin [13]. a-catenin shares significant homology with the actin-binding protein vinculin [15, 16] and is thought to link cadherin complexes to the actin cytoskeleton by direct interaction [15 – 17] or indirectly via a-actinin [18]. Several lines of evidence indicate that defects in any of these proteins alter the protein – protein interactions of the complex, leading to decreased cell – cell adhesiveness and hence metastasis [19 – 26]. Despite the above-mentioned similarities, several recent observations suggest that the role of CAS in the cadherin complex is fundamentally different from that of b-catenin and plakoglobin. First, although all three

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proteins interact with the same 37-amino-acid catenin binding region of E-cadherin [7], b-catenin and plakoglobin form mutually exclusive complexes with E-cadherin [6, 13, 27, 28] while CAS forms complexes of either CAS-E-cadherin– b-catenin or CAS-E-cadherin– plakoglobin [6, 29]. Thus, b-catenin and plakoglobin appear to bind identical or overlapping sites on E-cadherin whereas CAS binds independently to a different but closely juxtaposed epitope [9]. Second, CAS does not bind to a-catenin [9] and therefore, unlike b-catenin and plakoblobin, is not likely to be involved in the direct linkage of E-cadherin to the cytoskeleton. Third, although both b-catenin and plakoglobin interact with APC via their armadillo repeat domains (Arm domains) [13, 28], CAS does not interact with APC [9]. Another significant departure from the b-catenin/ plakoglobin paradigm is that CAS is expressed as at least four distinct isoforms which vary from one cell type to another. For example, NIH3T3 fibroblasts (which make N-cadherin) primarily express CAS 1 isoforms while MDCK epithelial cells (which make Ecadherin) express mainly CAS 2 isoforms [6]. Possible cell type-specific roles for the different isoforms include directing the binding of CAS to different cadherins, or alternatively, recruiting distinct factors into cadherin complexes. The murine CAS isoforms have been partially characterized and are thought to occur by alternative splicing [6]. CAS1A (Ç115 kDa) contains a 21amino-acid sequence near the carboxy terminus which is absent from CAS1B (Ç112 kDa). CAS2A (Ç100 kDa) and CAS2B (Ç96 kDa) differ by the same carboxy-terminal 21-amino-acid sequence, and in addition lack an amino-terminal sequence defined by the Mab 2B12 epitope. The functional significance of these multiple isoforms and whether the alternative splicing affects interactions with specific cadherins is unknown. The classical cadherins (LCAM, E-, N-, and P-cadherin) have highly conserved cytoplasmic domains, and all associate with b-catenin and plakoglobin [10]. Presumably, the roles of the different cadherins are tailored to the adhesive requirements of specific cell types, and selective association with various isoforms of CAS could mediate some of these roles. Here, we have demonstrated that CAS is not restricted to binding E-cadherin, but also interacts with N-cadherin and P-cadherin. Furthermore, four different CAS isoforms were equally capable of interacting with E-cadherin in vivo, suggesting that cadherins do not preferentially bind to one isoform over another. In addition, we overexpressed a panel of CAS mutants in NIH3T3 cells and examined their effects on cell morphology as compared to the effects of overexpressed b-catenin. Interestingly, CAS, but not b-catenin, induced a striking dendritelike morphology which required the presence of the majority of its armadillo repeats. Hence, like other cate-

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nins, CAS associates with multiple cadherins; however, CAS and b-catenin are likely to have quite different and possibly opposing roles in cadherin complexes. MATERIALS AND METHODS Cell culture and plasmids. The murine fibroblast cell lines NIH3T3, Swiss3T3, CH310T1/2, and NIH(pMcsrc527/Foc)B1 were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml). The Src-transformed NIH3T3 cell line NIH(pMcsrc527/Foc)B1 [30] was a kind gift from Dr. David Shalloway. Culture conditions for the human colon tumor cell lines HCT116 and SW480 and the human prostate tumor cell line PC3 have been described previously [9]. HCT116/Src is a Src-transformed clonal cell line derived by cotransfection with pSVneo and a Moloney long terminal repeat-based plasmid containing v-src (pMvsrc) [31], followed by selection in G418. The eukaryotic expression vectors pRc/CMV and pRc/RSV were purchased from Invitrogen (San Diego, CA). The CMV promoterbased CAS construct (pRc/CMVp120) and its epitope-tagged counterpart (pRc/CMVp120tag) have been described previously [6]. The cloning and characterization of the CAS isoforms CAS1A, CAS1B, CAS2A, and CAS2B are described elsewhere (Mo and Reynolds, submitted for publication). All four isoforms were epitope tagged by subcloning into the pRc/CMVp120tag vector. The RSV promoter-based CAS construct (pRc/RSVp120) and its tagged counterpart (pRc/ RSVp120tag) are identical to the pRc/CMVp120-based plasmids with the exception that the CMV promoter has been replaced by the RSV promoter (Invitrogen, San Diego, CA). These RSV-based plasmids were constructed by ligation of an EcoRI to XmaI fragment from tagged or untagged pRc/CMVp120 (containing the full-length coding sequence for CAS1A and some 3* vector sequence) into the identical sites in pRc/RSV. To generate a b-catenin expression construct, the full-length murine b-catenin gene [32], a generous gift from Dr. Rolf Kemler, was subcloned into the pRc/CMV polylinker yielding the plasmid pRc/ CMV-b-cat. Mutations in CAS were made either by deleting in-frame segments between restriction sites or by site-directed mutagenesis. The nomenclature we have adopted uses ‘‘D’’ for deletion and ‘‘R’’ for repeat. The amino acids deleted and the nomenclature associated with these mutants are as follows: (i) 1–158, DN-term; (ii) 28–233, DN2; (iii) 233–387, DR1-3; (iv) 365–495, DR3-5; (v) 390–820, DR3-11; (vi) 657–789, DR8-10; (vii) 789–911, DR11DC. Nucleotides flanking the deletions were verified by sequencing. cDNA constructs were transfected into cells using Lipofectamine (BRL, Gaithersburg, MD) according to the manufacturer’s instructions, and cells were analyzed 12–18 h later by immunoprecipitation and Western blotting, or by immunofluorescence. Immunoprecipitation, Western blotting, and immunofluorescence. Cells were lysed at 07C in a buffer containing 0.5% Nonidet P-40 (NP40), 10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 0.1 mM sodium vanadate, 0.1 trypsin inhibitor units/ml of aprotinin, and 5 mg/ml of leupeptin (protease inhibitors from Sigma, St. Louis, MO). Immunoprecipitation, Western blotting, and immunofluorescent labeling methods, and most of the reagents used in these experiments have been previously described [6]. The monoclonal antibody to N-cadherin (Mab 13A9) was a generous gift from Dr. Margaret Wheelock and has been characterized previously [18]. Monoclonal antibodies to human E- and P-cadherin were purchased from Transduction Laboratories (Lexington, KY). The caninespecific E-cadherin Mab rr1 [33] was kindly provided by Dr. Barry Gumbiner or obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA) under Contract No. N01-

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FIG. 1. CAS associates with several distinct cadherins. Cell lysates from the cell lines indicated at the top of the panel were immunoprecipitated with Mab pp120 and then Western blotted with (A) anti-CAS Mab pp120, (B) anti-N-cadherin Mab 13A9, (C) anti-Pcadherin Mab, or (D) anti-E-cadherin Mab. The positions of CAS1 isoforms (112–115 kDa) and CAS2 isoforms (96–100 kDa) are indicated. The CAS bands migrating faster than 96 kDa are due to degradation. N-, P-, and E-cadherins migrated at 135, 120, and 120 kDa, respectively. The minor upper band in the N-cadherin blot is an Ncadherin precursor.

and all of these cadherins coprecipitated with CAS. Consistent with a trend noted previously [6], the epithelial cell lines (lanes 1–4) expressed mostly CAS2 isoforms, whereas the fibroblast cell lines (lanes 5 and 6) expressed mostly CAS1 isoforms. These data raised the question as to whether cadherins have specific binding preferences for one CAS isoform over another. The abundance of CAS1 isoforms in the fibroblast lines (Fig. 1, lanes 5 and 6, slow migrating bands) suggests that most of the N-cadherin in these CAS immunoprecipitates was associated with CAS1. However, in the PC3 cell line, N-cadherin was abundant in CAS immunoprecipitates even though CAS2 isoforms were the major isoforms expressed (Fig. 1, panel B, lane 4). To directly compare the ability of each isoform to interact with a specific cadherin, expression plasmids encoding the epitope-tagged CAS isoform genes were transiently transfected into MDCK cells (Fig. 2) and the amount of E-cadherin that coprecipitated with each CAS isoform was measured by Western blotting. E-cadherin associated equally with all of the exogenously expressed isoforms, suggesting that E-cadherin does not have a strong preference for one isoform over another (Fig. 2, compare panels A and B, lanes 2–5). Although the transfection efficiency of the CAS2B isoform (as determined by immunofluorescence) was consistently low, E-cadherin nevertheless coprecipitated with this isoform in proportion to its presence in the cell lysates (Fig. 2, lane 5). No proteins

HD-2-3144 from the National Institute of Child Health and Human Development.

RESULTS

CAS Associates with Multiple Cadherins To determine whether CAS can associate with cadherins other than E-cadherin, cell lysates from a variety of cell types were immunoprecipitated with Mab pp120 and then Western blotted with antibodies specific for N-, P-, or E-cadherin (Fig. 1). In each of the cell lines, the major cadherin(s) expressed coprecipitated efficiently with CAS. In HCT116 cells, both E-cadherin and P-cadherin coprecipitated with CAS (Fig. 1, panels C and D, lane 1). Expression of v-src in these cells caused morphological changes typically associated with Src-induced transformation, but had no effect on the association between the cadherins and CAS, nor did it affect the isoforms of CAS expressed in these cells (lane 2). Whereas the SW480 colon carcinoma cell line expressed only minor amounts of E-cadherin, the prostate tumor cell line PC3 expressed N-, P-, and E-cadherin

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FIG. 2. Similar association of the four murine isoforms with Ecadherin. MDCK cells were transfected with the epitope-tagged CAS isoforms listed at the top of the figure. The tagged CAS proteins were immunoprecipitated with anti-tag Mab 12CA5 and then Western blotted with Mab 12CA5 (A) or with the E-cadherin Mab rr1 (B). Although normally CAS2 isoforms predominate in MDCK cells, when individual isoforms were introduced by transfection, the ability of the exogenous CAS1 isoforms to associate with E-cadherin in these cells was equivalent to that of the exogenous CAS2 isoforms. The transfection efficiency for the CAS2B construct was consistently lower than that of the other isoforms in MDCK cells, and the amount of E-cadherin detected in the CAS2B immunoprecipitates was correspondingly decreased. The estimated molecular weights of the proteins are shown on the right.

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Immunofluorescent localization of CAS in NIH3T3 cells revealed that CAS was concentrated at sites of cell–cell contact (Fig. 4, arrows). In relatively isolated cells CAS was often diffusely distributed over the cell surface and absent from the cell boundaries, whereas

FIG. 3. Characterization of CAS complexes in NIH3T3 cells. NIH3T3 cell lysates were precipitated with antibodies to cadherin complex proteins or with the control Mab 12CA5, as indicated at the top of the figure. Immunoprecipitates were separated on 7% polyacrylamide gels and then Western blotted with Mab pp120. The p120 isoforms CAS1A (115 kDa), CAS1B (112 kDa), CAS2A (100 kDa), and CAS2B (96 kDa) are indicated. All four isoforms can be detected in N-cadherin and b-catenin immunoprecipitates.

were immunoprecipitated from cells transfected with empty vectors (Fig. 2, lane 1). Together, Figs. 1 and 2 indicate that CAS interacts with multiple cadherins and that the differences between CAS isoforms are unlikely to affect the ability of an isoform to interact with a given cadherin. CAS Associates with N-Cadherin Complexes in NIH3T3 Cells Because transient transfection of CAS into fibroblasts, but not epithelial cells, resulted in marked morphological changes (see below), we characterized the association between CAS and N-cadherin in greater detail using NIH3T3 fibroblasts (Fig. 3). The CAS1 isoforms were the predominant species detected in complexes precipitated with antibodies to N-cadherin or the catenins (Fig. 3, lanes 3–6), but in the N-cadherin and b-catenin immunoprecipitates low levels of the CAS2 isoforms were also detected in longer exposures (Fig. 3, lanes 3 and 5). The relative proportions of the isoforms in these complexes closely correlated with their relative expression levels in NIH3T3 cells. The low level of CAS in the plakoglobin immunoprecipitate (Fig. 3, lane 6) is probably due to the lower affinity of plakoglobin for N-cadherin (relative to b-catenin) and to the low expression level of plakoglobin in NIH3T3 cells (data not shown). Although our a-catenin antibodies function poorly under these conditions, they nevertheless precipitated detectable levels of CAS (Fig. 3, lane 4). CAS was not immunoprecipitated by the control antibody, Mab 12CA5 (lane 7).

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FIG. 4. Colocalization of CAS with N-cadherin. NIH3T3 cells were double immunolabeled with antibodies to CAS (PabF1; A) and N-cadherin (Mab 15F11; B). No staining was detected after immunolabelling with control antibodies (Mab 12CA5; C). Endogenous CAS is concentrated along with N-cadherin at sites of cell–cell contact (arrows). Magnification, 160 (all panels).

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upon cell crowding, staining was intense where cells came into contact with one another. Double immunofluorescent labeling of these cells with antibodies to CAS (Fig. 4A), and to N-cadherin (Fig. 4B) indicated that, as with E-cadherin and CAS [6], these proteins colocalized and were heavily concentrated at cell–cell contacts (Fig. 4, arrows). Staining was not detected with control antibodies (Fig. 4C). The Arm Domain of CAS Mediates Binding to NCadherin Complexes To map the regions of CAS required for binding to N-cadherin, we generated a panel of deletion mutants. Collectively, the deletions span the entire protein coding sequence of CAS (Fig. 5A). Mutant proteins of the predicted size could be immunoprecipitated and Western blotted by anti-tag Mab 12CA5 (Fig. 5B, lanes 3– 9) or in the case of DR11DC (which is not tagged) with Mab 2B12 (Fig. 5B, lane 10). Deletions in the Arm domain reduced or blocked the interactions between CAS and N-cadherin or b-catenin (Fig. 6, panels B and C). N-cadherin and b-catenin behaved similarly in these experiments, which is consistent with our hypothesis that N-cadherin, like E-cadherin, interacts simultaneously and directly with both CAS and b-catenin [9]. The interactions between these proteins were abolished by deletion of repeats 1–5 in CAS (Fig. 6, lanes 4 and 5) and severly reduced by deletion of repeats 8–10 (Fig. 6, lane 7) or deletion of amino acids 28–233 (DN2, Fig. 6, lane 3). N-cadherin and b-catenin did not coprecipitate with DR1-3 even though its colocalization in cells was similar to that of overexpressed wild-type CAS and it retained some ability to induce the branching phenotype (described later). Although the DN-term mutant retained a nearly wild-type ability to bind to N-cadherin, the N-cadherin observed in this precipitate migrated slightly faster than wild-type N-cadherin, suggesting that overexpression of DN-term might affect posttranslational modification (e.g., glycosylation) of N-cadherin. Enforced CAS Expression Causes a Dendrite-like Morphology in NIH3T3 Cells Attempts to obtain clonal cell lines that stably overexpress CAS have thus far been unsuccessful. However, immunofluorescent observation of transfected NIH3T3 cell colonies grown from originally positive clones revealed occasional CAS-overexpressing cells which were significantly elongated relative to cells which had lost the exogenous CAS gene. We therefore cloned CAS1A into the Rc/CMV expression vector and analyzed the phenotype in more detail by transient transfection methods (Fig. 7A). NIH 3T3 cells expressing high levels of wild-type CAS, as assessed by immu-

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FIG. 5. Characterization of CAS deletion mutants. (A) Schematic representation of CAS deletion mutants illustrating the amino acid residues deleted and their positions relative to the armadillo repeats in CAS. Collectively, the deletions span the entire protein coding region. The presence (/) or absence (0) of branching activity and Ncadherin (and b-catenin) binding for each mutant is indicated on the right. (B) Electrophoretic mobilities and expression levels of the CAS variants. Vector alone (lane 1), untagged CAS and DR11DC (lanes 2 and 10, respectively), and tagged versions of wild-type CAS and CAS mutants (lanes 3–9), as indicated at the top of the figure, were transiently transfected into NIH3T3 cells. Immunoprecipitates obtained with the epitope tag Mab 12CA5 (lanes 1–9) or anti-CAS Mab 2B12 (lane 10) were separated by SDS–PAGE and Western blotted with Mab 12CA5 (lanes 1–9) or Mab 2B12 (lane 10). Epitope-tagged CAS, endogenous CAS, and DR11DC are indicated. Molecular weight markers are indicated on the left.

nofluorescence, displayed a highly unusual morphology characterized by either abnormal cellular elongation or the presence of dendrite-like branching structures (Fig. 7A, arrows). The phenotype was unaffected by addition

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catenin were often slightly withdrawn or rounded rather than extended and branching. To determine if tyrosine phosphorylation of CAS might positively regulate (enhance) the dendritic phenotype, we examined the effects of CAS overexpression in Src-transformed cells (data not shown). CAS overexpression in these cells did not induce any obvious morphologic changes beyond those already induced by Src, suggesting either that tyrosine phosphorylation of CAS inhibits the branching activity or, more likely, that the disruption of the actin cytoskeleton associated with Src-induced transformation is incompatible with formation of the dendrite-like structures. Intact Armadillo Repeats Are Required for CASInduced Branching

FIG. 6. Association of CAS with N-cadherin– b-catenin complexes requires armadillo repeats 3–10. NIH3T3 cells were transiently transfected with the constructs indicated at the top of the figure. After 18 h, cells were lysed and immunoprecipitated with the epitope tag Mab 12CA5. Immunoprecipitates were then immunoblotted with Mab 12CA5 (A), the N-cadherin Mab 13A9, or polyclonal anti-b-catenin, as indicated. N-cadherin migrated at Ç135 kDa and b-catenin migrated at 95 kDa. Molecular weight standards relative to the CAS mutants is shown in Fig. 5B.

of the C-terminal tag (data not shown) and epitopetagged CAS was therefore used for subsequent experiments. The abnormal morphology correlated with the CAS expression level, as judged by the intensity of the fluorescence in individual cells; transfected cells that were not significantly altered expressed relatively low levels of CAS (Fig. 7A, filled arrowheads), whereas cells that were elongated or had dendrite-like extensions expressed high levels of CAS (Fig. 7A, arrows). In similar transfection experiments using several other fibroblast cell lines (e.g., Swiss 3T3, C3H10T1/2, and COS cells), CAS overexpression induced a similar dendrite-like phenotype indicating that the effect is a general phenomenon not restricted to NIH3T3 cells. However, only minor changes were observed after transient transfection of CAS into MDCK epithelial cells, indicating that the magnitude of the morphological changes may be cell type-dependent (data not shown). To determine if the branching phenotype was CAS specific, we performed analogous experiments using the pRc/CMV-b-catenin construct to induce a high level expression of the CAS-related protein, b-catenin (Fig. 7B). Only minor morphological changes were observed in cells overexpressing b-catenin (Fig. 7B, unfilled arrowheads). In fact, cells expressing high levels of b-

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To map the regions of CAS required for the branching phenotype, NIH3T3 cells were transfected with CAS and CAS mutant constructs and then examined by immunofluorescence for morphological changes (Fig. 8). Deletion of the N-terminal 158 amino acids (DN-term), or an internal segment spanning amino acids 28–233 (DN2), did not disrupt branching activity (Figs. 8B and 8C). In contrast, a deletion spanning amino acids 233– 387 (DR1-3), which removed part of the N-terminal domain and the first two and a half armadillo repeats, reduced both the proportion of cells that displayed branching and the severity of the branching phenotype (Fig. 8D). Virtually no branching activity was induced by any of the other CAS deletion mutants including DR11DC, whose last armadillo repeat and C-terminal tail were deleted (Figs. 8E–8H). Therefore, armadillo repeats 3–11, and possibly the C-terminus of CAS, are necessary for the branching phenotype, but the N-terminal third of CAS, including repeats 1 and 2, are dispensable. DISCUSSION

The presence of CAS in cadherin complexes implies that its physiological role is relevant to cadherin function. CAS shares several characteristics with the classical catenins b-catenin and plakoglobin; these include structural similarity [4, 5], cellular location, Src-induced tyrosine phosphorylation [6], and the ability to bind directly to E-cadherin [9]. However, unlike these proteins, CAS does not bind to a-catenin or APC [9] and appears to occupy its own nonoverlapping binding site on E-cadherin [6–8]. Hence, there are substantial differences between CAS and the classical catenins, and it was not clear, a priori, that CAS would interact with other cadherins. Here, we demonstrate that CAS binds not only to E-cadherin, but also to N- and Pcadherin. Thus, like the classical catenins, CAS is a

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FIG. 7. Overexpression of CAS induces dendrite-like cell extensions. Epitope-tagged CAS1A (A) or b-catenin (B) were overexpressed in NIH3T3 fibroblasts by transient transfection, and immunolabeled with the epitope tag Mab12CA5 (A) or antibodies to b-catenin (B). Photographs of representative fields were taken at low power (magnification, 120). (A) Approximately half of the CAS-transfected cells expressed high levels of CAS and exhibited either abnormal elongation or dendrite-like branching (arrows). Low levels of CAS overexpression did not induce this phenotype (solid arrowheads). (B) No extensions or branching resulted from high level transient overexpression of bcatenin (open arrowheads), but some cells were smaller and slightly rounded compared to the normal untransfected cells.

cofactor for multiple members of the cadherin family and presumably contributes a function common to different cadherins. An important difference between CAS and b-catenin

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(or plakoglobin) is the potential diversity generated by the alternatively spliced isoforms of CAS. Recent evidence suggests that the isoforms interchange with one another and exist in mutually exclusive complexes akin

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FIG. 8. CAS-induced branching activity requires armadillo repeats 3–11. NIH3T3 cells were transiently transfected with epitope-tagged constructs encoding CAS or individual CAS mutants, and then immunolabeled 18 h later with the epitope tag Mab 12CA5 (A–G) or antiCAS Pab F1 (H). The anti-CAS polyclonal antibody was used to label the C-terminal deletion mutant DR11DC because this mutant lacked the C-terminal epitope tag. Representative cells expressing the CAS constructs are illustrated: (A) wild-type CAS1A, (B) DN-term, (C) DN2, (D) DR1-3, (E) DR3-5, (F) DR3-11, (G) DR8-10, and (H) DR11DC. Magnification, 140 (all panels).

to b-catenin and plakoglobin [8]. Hence, differential expression of the isoforms could represent a novel mechanism of regulating or modulating cadherin function in different cell types. For example, the CAS1 isoforms are the predominant isoforms in fibroblasts and macrophages, cells which are highly motile, while more static cells such as epithelial cells tend to express high levels of the CAS2 isoforms (unpublished data). Previously, we postulated that different isoforms might interact with different cadherins within a cell or bind distinct effectors involved in cell adhesion or mitogenic signaling. Several lines of new evidence favor the second alternative. First, specific cadherins do not always coprecipitate with the same CAS isoforms, and vice versa. For example, N-cadherin was abundant in CAS immunoprecipitates irrespective of whether the cells expressed primarily CAS1 (e.g., fibroblasts) or CAS2 (e.g., PC3 epithelial cells) (Fig. 1, panel B, lanes 4, 5, and 6). Similarly, in N-cadherin immunoprecipitates from NIH 3T3 fibroblasts, all four isoforms were detected in proportion to their abundance in cell lysates.

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Second, in our transient transfection experiments, all four CAS isoforms were equally capable of interacting with E-cadherin (Fig. 2) even though CAS1 isoforms are normally expressed at extremely low levels in MDCK cells. This suggests that the alternatively spliced sequences do not affect the binding to cadherins. Consistent with this model, the splice inserts are located at the N- and C-terminal ends of CAS where they do not interrupt the central Arm domain. Third, a potential role for the unique N-terminal end of CAS1 (defined by the Mab 2B12 epitope) has surfaced recently in a report by Kim and Wong [34], which suggests that CAS1 isoforms specifically interact with the FER tyrosine kinase, which in turn interacts with activated PDGF receptors. Hence, an exciting unexplored possibility is that CAS1 also recruits FER kinase into a complex with N-cadherin. The striking phenotype observed after transfection of CAS into NIH3T3 cells and other fibroblast cell lines was surprising because, despite the developmental roles attributed to b-catenin and plakoglobin, overex-

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pression of these proteins in cultured cell lines has not been associated with morphological changes. Several lines of evidence indicate that this phenotype is specifically associated with CAS. First, to our knowledge the dramatic arborization induced by CAS has not been reported for other proteins. The failure of b-catenin to generate branching under the same transient conditions is particularly interesting because it demonstrates a clear selective effect of CAS that cannot be duplicated even by a protein that is related to CAS in many respects. Second, mutations in the Arm domain abolished the phenotype even though some of the mutant proteins were expressed at wild-type levels. Third, the transfected cells survived for at least 48 h posttransfection even though the phenotype was fully developed within 12 h. Thus, if toxicity is a factor, it is likely to be secondary to the morphological changes induced by CAS. Although several epithelial cell lines were mildly affected by forced expression of CAS, the phenotype was unremarkable compared to the fibroblast cell lines that were tested. The reason for this difference is not clear. Although both types of cells express high levels of CAS, none of the four isoforms induced a strong branching phenotype when individually transfected into MDCK cells, whereas all four isoforms were highly active in fibroblasts (data not shown). It is noteworthy that so far, all cell lines that give a phenotype upon CAS transfection contain N-cadherin. It is possible that cotransfection of CAS and N-cadherin might induce the branching phenotype in epithelial cells. Recent data indicate that CAS does not associate with truncated E-cadherin molecules that lack the carboxy-terminal 37 amino acids necessary for catenin binding [7]. The conservation of this region in multiple cadherins probably reflects a functional requirement for the binding of both CAS and b-catenin (or plakoglobin) which brings them into close proximity in the cadherin complex. We postulate that CAS and b-catenin (or plakoglobin) have opposing roles which together mediate a crucial dynamic interaction between cadherins and the actin cytoskeleton. Although it has been previously proposed that b-catenin and plakoglobin play opposing roles [35], we find this less likely since these proteins do not exist simultaneously within the same cadherin complex. One possible explanation for the phenotype is that overexpression of CAS leads to a chronic imbalance in the effects of CAS and bcatenin at the cadherin–actin filament interface. To fully understand the phenotype and the role of CAS in adhesion and signaling, it will be important to identify CAS-specific binding partners and to develop stable CAS-expressing cell lines that are amenable to biochemical analysis.

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We thank Rolf Kemler for the plasmid containing the murine bcatenin cDNA, Margaret J. Wheelock for the N-cadherin Mab 13A9, Pierre D. McCrea for the polyclonal antisera to b-catenin, and David Shalloway for the Src-transformed NIH3T3 cells. This work was supported in part by NIH Grant CA55724 (A.B.R.), the NIH Cancer Center CORE Grant P30 CA21756, and the American Lebanese Syrian Associated Charities of St. Jude Children’s Research Hospital.

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Received January 12, 1996 Revised version received January 18, 1996

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