Induction of a secondary body axis in Xenopus by antibodies to beta-catenin.

We have obtained evidence that a known intracellular component of the cadherin cell-cell adhesion machinery, beta-catenin, contributes to the development of the body axis in the frog Xenopus laevis. Vertebrate beta-catenin is homologous to the Drosophila segment polarity gene product armadillo, and to vertebrate plakoglobin (McCrea, P. D., C. W. Turck, and B. Gumbiner. 1991. Science (Wash. DC). 254: 1359-1361.). Beta-Catenin was found present in all Xenopus embryonic stages examined, and associated with C-cadherin, the major cadherin present in early Xenopus embryos. To test beta-catenin's function, affinity purified Fab fragments were injected into ventral blastomeres of developing four-cell Xenopus embryos. A dramatic phenotype, the duplication of the dorsoanterior embryonic axis, was observed. Furthermore, Fab injections were capable of rescuing dorsal features in UV-ventralized embryos. Similar phenotypes have been observed in misexpression studies of the Wnt and other gene products, suggesting that beta-catenin participates in a signaling pathway which specifies embryonic patterning.

The vertebrate adhesive junction proteins beta-catenin and plakoglobin and the Drosophila segment polarity gene armadillo form a multigene family with similar properties.

Three proteins identified by quite different criteria in three different systems, the Drosophila segment polarity gene armadillo, the human desmosomal protein plakoglobin, and the Xenopus E-cadherin-associated protein beta-catenin, share amino acid sequence similarity. These findings raise questions about the relationship among the three molecules and their roles in different cell-cell adhesive junctions. We have found that antibodies against the Drosophila segment polarity gene armadillo cross react with a conserved vertebrate protein. This protein is membrane associated, probably via its interaction with a cadherin-like molecule. This cross-reacting protein is the cadherin-associated protein beta-catenin. Using anti-armadillo and antiplakoglobin antibodies, it was shown that beta-catenin and plakoglobin are distinct molecules, which can coexist in the same cell type. Plakoglobin interacts with the desmosomal glycoprotein desmoglein I, and weakly with E-cadherin. Although beta-catenin interacts tightly with E-cadherin, it does not seem to be associated with either desmoglein I or with isolated desmosomes. Anti-armadillo antibodies have been further used to determine the intracellular localization of beta-catenin, and to examine its tissue distribution. The implications of these results for the structure and function of different cell-cell adhesive junctions are discussed.

beta-Catenin associates with the actin-bundling protein fascin in a noncadherin complex.

Catenins were first characterized as linking the cytoplasmic domains of cadherin cell-cell adhesion molecules to the cortical actin cytoskeleton. In addition to their essential role in modulating cadherin adhesivity, catenins have more recently been indicated to participate in cell and developmental signaling pathways. beta-Catenin, for example, associates directly with at least two receptor tyrosine kinases and transduces developmental signals within the Wnt pathway. Catenins also complex with the tumor suppressor protein adenomatous polyposis coli (APC), which appears to have a role in regulating cell proliferation. We have used the yeast two-hybrid method to reveal that fascin, a bundler of actin filaments, binds to beta-catenin's central Armadillo repeat domain. Western blotting of immunoprecipitates from cell line and mouse and rat brain extracts indicate that this interaction exists in vivo. Fascin and beta-catenin's association was further substantiated in vitro using purified proteins isolated from recombinant bacterial and baculoviral sources. Immunoprecipitation analysis indicates that fascin additionally binds to plakoglobin, which is highly homologous to beta-catenin but not to p120cas, a newly described catenin which contains a more divergent Armadillo-repeat domain. Immunoprecipitation, in vitro competition, and domain-mapping experiments demonstrate that fascin and E-cadherin utilize a similar binding site within beta-catenin, such that they form mutually exclusive complexes with beta-catenin. Immunofluorescence microscopy reveals that fascin and beta-catenin colocalize at cell-cell borders and dynamic cell leading edges of epithelial and endothelial cells. In addition to cell-cell borders, cadherins were unexpectedly observed to colocalize with fascin and beta-catenin at cell leading edges. It is conceivable that beta-catenin participates in modulating cytoskeletal dynamics in association with the microfilament-bundling protein fascin, perhaps in a coordinate manner with its functions in cadherin and APC complexes.

A homolog of the armadillo protein in Drosophila (plakoglobin) associated with E-cadherin.

Three cytoplasmic proteins, called catenins, bind to the cytoplasmic tail of the epithelial cell-cell adhesion molecule E-cadherin. The complementary DNA sequence was determined for the 92-kilodalton beta catenin of Xenopus laevis. The sequence is homologous to mammalian plakoglobin, a protein of desmosomal and zonula adherens cell junctions, and to the plakoglobin homolog in Drosophila melanogaster, the product of the segment polarity gene armadillo. A monoclonal antibody to bovine plakoglobin recognizes the analogous beta catenin in the Madin-Darby canine kidney (MDCK) cell line. Armadillo plakoglobin may link E-cadherin to the underlying actin cytoskeleton at cell-cell junctions; the E-cadherin-catenin protein complex may also participate in the transmission of developmental information.

Cortical stabilization of beta-catenin contributes to NHERF1/EBP50 tumor suppressor function.

Anchorage-independent growth is a hallmark of tumor growth and results from enhanced proliferation and altered cell-cell and cell-matrix interactions. By using gene-deficient mouse embryonic fibroblasts (MEFs), we showed for the first time that NHERF1/EBP50 (Na/H exchanger regulator factor 1/ezrin-radixin-moesin binding phosphoprotein 50), an adapter protein with membrane localization under physiological conditions, inhibits cell motility and is required to suppress anchorage-independent growth. Both NHERF1 PDZ domains are necessary for the tumor suppressor effect. NHERF1 associates directly through the PDZ2 domain with beta-catenin and is required for beta-catenin localization at the cell-cell junctions in MEFs. Mechanistically, the absence of NHERF1 selectively decreased the interaction of beta-catenin with E-cadherin, but not with N-cadherin. The ensuing disorganization of E-cadherin-mediated adherens junctions as well as the observed moderate increase in beta-catenin transcriptional activity contributed most likely to the anchorage-independent growth of NHERF1-deficient MEFs. In vivo, NHERF1 is specifically localized at the apical brush-border membrane in intestinal epithelial cells and is required to maintain a fraction of the cortical beta-catenin at this level. Thus, NHERF1 emerges as a cofactor essential for the integrity of epithelial tissues by maintaining the proper localization and complex assembly of beta-catenin.

Identification of a new catenin: the tyrosine kinase substrate p120cas associates with E-cadherin complexes.

p120cas is a tyrosine kinase substrate implicated in ligand-induced receptor signaling through the epidermal growth factor, platelet-derived growth factor, and colony-stimulating factor receptors and in cell transformation by Src. Here we report that p120 associates with a complex containing E-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Furthermore, p120 precisely colocalizes with E-cadherin and catenins in vivo in both normal and Src-transformed MDCK cells. Unlike beta-catenin and plakoglobin, p120 has at least four isoforms which are differentially expressed in a variety of cell types, suggesting novel means of modulating cadherin activities in cells. In Src-transformed MDCK cells, p120, beta-catenin, and plakoglobin were heavily phosphorylated on tyrosine, but the physical associations between these proteins were not disrupted. Association of p120 with the cadherin machinery indicates that both Src and receptor tyrosine kinases cross talk with proteins important for cadherin-mediated cell adhesion. These results also strongly suggest a role for p120 in cell adhesion.

E-cadherin transfection down-regulates the epidermal growth factor receptor and reverses the invasive phenotype of human papilloma virus-transfected keratinocytes.

The human papillomavirus type 16 (HPV-16), the type most often associated with cervical cancer, immortalizes primary keratinocytes and inhibits serum/calcium-stimulated differentiation in culture. In this study, we have used a model of keratinocyte immortalization based upon HPV-16 to analyze perturbation of function and expression of E-cadherin, a Ca(2+)-dependent cell-cell adhesion molecule expressed by normal keratinocytes, and its associated proteins. An immortalized keratinocyte cell line generated by cotransfection with HPV-16 E6 and E7 showed decreased membrane E-cadherin expression and redistribution of alpha-, beta-, and gamma-catenin from the undercoat membrane to the cytoplasm. No changes in the level of expression were seen. Selection of the immortalized keratinocyte cell line for resistance to differentiation generated a more transformed cell line with an invasive phenotype, down-regulated E-cadherin and alpha-catenin, and up-regulated the epidermal growth factor receptor (EGFr). Transfection of an E-cadherin expression construct into the differentiation-resistant cell line restored membrane-bound E-cadherin and catenin expression, down-regulated the EGFr, and reversed the invasive phenotype. These results indicate that overexpression of the EGFr correlates with perturbation of the E-cadherin/catenin complex seen in the HPV-16 E6- and E7-transfected keratinocytes and may underlie a functional interaction between growth-regulatory factors and adhesion molecules (E-cadherin/catenin).

C-erbB-2/ HER-2 upregulates fascin, an actin-bundling protein associated with cell motility, in human breast cancer cell lines.

The over-expression of c-erbB-2/ HER-2, a receptor tyrosine kinase, correlates with poor prognosis in patients with breast and ovarian cancer. In the human breast cancer cell line, MDA-MB-435, c-erbB-2 over-expression results in increased chemoinvasion and higher metastatic properties in nude mice. However, the mechanisms by which c-erbB-2 increases the malignant potential of cells remains unclear. We have determined that over-expression of c-erbB-2 in MDA-MB-435 cells, and in some additional breast cancer cell lines, is associated with graphic increases in mRNA and protein levels of the actin bundling protein fascin. Heightened fascin expression has been observed in other systems to result in greatly increased cell motility, and indeed, our work employing semi-automated time-lapse microscopy demonstrates that MDA-MB-435 cells over-expressing c-erbB-2 exhibit significantly heightened cellular dynamics and locomotion, while visualization of bundled microfilaments within fixed cells revealed enhanced formation of dendritic-like processes, microspikes and other dynamic actin based structures. To address the means by which c-erbB-2 over-expression might result in elevated fascin levels, we identified multiple perfect match TCF and NF-kappaB consensus sites in fascin's promoter and first intron, which appeared consistent with the greater endogenous transcriptional activities of TCF and NF-kappaB in c-erbB-2 over-expressing MDA-MB-435 cells. While such transcriptional modulation may occur in the context of the intact gene/chromatin, subsequent tests using reporter constructs did not support involvement of these signaling pathways. In conclusion, highly increased fascin levels were observed in MDA-MB-435 over-expressing c-erbB-2, likely contributing to these cells' altered actin dynamics, and increased cell motility and malignancy. Studies in progress aim to discern the means by which c-erbB-2 over-expression leads to transcriptional activation of the fascin gene.

Assessment of the aggregation state of integral membrane proteins in reconstituted phospholipid vesicles using small angle neutron scattering.

The assessment of the physical size of integral membrane protein complexes has generally been limited to samples solubilized in non-ionic detergent, a process which may introduce artifacts of unknown scope and severity. A system has been developed that allows observation of the small angle scattering profile of an integral membrane protein while incorporated in small unilamellar phospholipid vesicles. Contrast matching of isotopically substituted phospholipid eliminates the contribution of the bilayer to the observed scattering, resulting in a profile dependent only on the structure of the individual membrane protein complexes and their spatial arrangement in the vesicle. After appropriate compensation for their spatial arrangement, information about the molecular mass and radius of gyration of the individual complexes can be obtained. The validity of the approach has been established using monomeric bacteriorhodopsin as a model system.

Increased expression of fascin, motility associated protein, in cell cultures derived from ovarian cancer and in borderline and carcinomatous ovarian tumors.

Fascin bundles actin microfilaments within dynamic cellular structures such as microspikes, stress fibers and membrane ruffles. Fascin overexpression induces membrane protrusions and increased cell motility, and is highly expressed in various transformed cells, and in specialized normal cells including neuronal, endothelial and dendritic cells. In breast cancer, fascin expression correlates with high-grade tumors. To investigate whether fascin might be a predictor factor for ovarian cancer progression, eighteen cell cultures derived from ovarian cancer, and thirty four archival paraffin-embedded material of normal versus borderline and carcinomatous ovaries were stained by immunocytochemistry and immunohistochemistry with fascin Mab 55K-2. Overall expression of the fascin protein was found in 50% (9/18) of cell cultures derived from original samples of ovarian tumors. Expression of fascin protein was found in 67% (6/9) of cell cultures derived from patients diagnosed with stage IV disease, and 33% (3/9) of cell cultures from patients diagnosed with stage II/III. There was no clear relationship between fascin expression and histologic types, tumor grade, or DNA ploidy. However, 75% of cell cultures, which developed into a xenograft after intraperitoneal inoculation, showed fascin expression, while 86% of non-tumorigenic cell cultures did not show fascin expression. Expression of fascin in these established ovarian tumor cell cultures was significantly associated with the ability for these cells to grow intraperitoneally (P < 0.05). Furthermore, fascin was never expressed in normal epithelial ovarian tissues, but was present in all pathologic ovaries. Both diffuse and focal patterns were observed in borderline ovarian tumors (67% and 33%), advanced primary ovarian cancer (67% and 33%) and metastatic ovarian cancer (89% and 11%). Therefore, our data suggest that fascin could serve as a prognostic factor for abnormal ovarian epithelial pathology and could be a novel target for the treatment of ovarian cancer.

Catenins as mediators of the cytoplasmic functions of cadherins.

The catenins are polypeptides that bind to the conserved cytoplasmic tail of cadherins and are required for cadherin function. alpha-Catenin is related to vinculin and seems to be required for the interaction of cadherins with the actin cytoskeleton. beta-Catenin is homologous to armadillo, a segment polarity gene in Drosophila that participates in developmental signaling. Recent findings indicate that beta-catenin also participates in developmental signaling and embryonic patterning in Xenopus laevis. At least a portion of the electrophoretic band migrating at the position of gamma-catenin consists of plakoglobin, a desmosomal and zonula adherens protein that has high sequence similarity to beta-catenin and armadillo. The catenins may be involved in the regulation of cadherin function during tissue morphogenesis and tumorigenesis.

Purification of a 92-kDa cytoplasmic protein tightly associated with the cell-cell adhesion molecule E-cadherin (uvomorulin). Characterization and extractability of the protein complex from the cell cytostructure.

The transmembrane epithelial cell-cell adhesion protein E-cadherin (uvomorulin) associates via its cytoplasmic domain with three or more proteins whose structure and function are not yet established. The associated proteins, also termed catenins (Ozawa, M., Baribault, H., and Kemler, R. (1989) EMBO J. 8, 1711-1717), are of interest because they may form a link to the cytoskeleton, and/or regulate E-cadherin function. In this report immunoprecipitates of E-cadherin complexes, isolated from Xenopus laevis A6 and Madin-Darby canine kidney cell monolayers, were stringently washed to leave a single very tightly associated protein of approximately 92 kDa. We report on the 92-kDa protein's association with E-cadherin in the presence of various perturbants, its preferential dissociation from the immune complex upon exposure to mixed detergent micelles containing sodium dodecyl sulfate, and its preparative purification to homogeneity. We have additionally found that E-cadherin and its associated proteins may be easily and quantitatively extracted from both subconfluent and fully confluent cells by a variety of mild nonionic detergents made up in isotonic buffers. In contrast, such extractions at short times left most of the cytoskeletal protein fodrin in the insoluble pellet fraction. Western blots of immunoprecipitated E-cadherin complexes failed to detect the presence of fodrin, or that of the cytoskeletal proteins adducin, alpha-actinin, and vinculin. If the E-cadherin-associated protein complex interacts with known proteins of the cell cytoskeleton, such interactions are labile and/or transient.

The E-cadherin complex contains the src substrate p120.

Using normal MDCK cells, and MDCK cells stably transfected with a temperature-sensitive viral src allele (pp60 ts-v-src), we have examined the composition and tyrosine phosphorylation of the E-cadherin complex. E-cadherin is a transmembrane calcium-dependent cell-cell adhesion molecule that is complexed with cytoplasmic proteins including alpha-catenin, beta-catenin, plakoglobin (gamma-catenin), and actin. We have identified two heterodimeric complexes which demonstrate that alpha-catenin interacts directly with beta-catenin, or with plakoglobin, in the absence of E-cadherin. beta-Catenin has previously been shown to bind directly to E-cadherin. We propose that E-cadherin associates with alpha-catenin, and thereby the actin cytoskeleton, via either beta-catenin or plakoglobin. We have further identified three new but related protein components of the E-cadherin complex, which are each cross-reactive by Western blot analysis to antibodies directed against p120, a phosphotyrosine substrate of src, and a phosphotyrosine, phosphoserine, and phosphothreonine substrate of growth factor-stimulated signaling pathways. Greater quantities of the p120-related proteins were found present in the E-cadherin immunoprecipitates of ts-src MDCK cells compared to normal MDCK cells, while two of the p120 cross-reactive species were significantly tyrosine phosphorylated in both normal and ts-src MDCK cells. The association of p120-related species with the E-cadherin complex adds them to our consideration of possible modulators of cadherin function. Likewise, the newly identified alpha-catenin-beta-catenin and alpha-catenin-plakoglobin dimers may have interesting biological properties, conceivably including the titration of catenins between cadherin and APC complexes.

A beta-catenin/engrailed chimera selectively suppresses Wnt signaling.

beta-catenin plays an integral role in cell-cell adhesion by linking the cadherin complex of the adherens junction to the underlying actin cytoskeleton. In addition, beta-catenin transduces intracellular signals within the Wnt developmental pathway that are crucial to the proper establishment of embryonic axes and pattern formation of early mesoderm and ectoderm. For example, in the context of a defined dorsal 'organizer' region of early Xenopus embryos, beta-catenin enters the nucleus and associates with transcription factors of the HMG (High Mobility Group) Lef/Tcf protein family. Consequently, genes such as siamois, a homeobox gene contributing to the specification of the dorsoanterior axis, are activated. To further examine the role that beta-catenin plays in Wnt signaling, we generated a chimeric protein, beta-Engrailed (beta-Eng), in which the C-terminal trans-activation domain of beta-catenin is replaced with the transcriptional repression domain of Drosophila Engrailed. Dorsal overexpression of this mRNA in early Xenopus embryos leads to suppression of organizer-specific molecular markers such as siamois, Xnr-3 and goosecoid, corresponding with the dramatic morphological ventralization of embryos. Ventralized embryos further exhibit reduced activity of the Wnt pathway, as indicated by the loss of the notochord/organizer marker, chordin. Importantly, beta-Eng associates and functions normally with the known components of the cadherin complex, providing the experimental opportunity to repress beta-catenin's signaling function apart from its role in cadherin-mediated cell-cell adhesion.

Transmembrane topography of the nicotinic acetylcholine receptor delta subunit.

Current folding models for the nicotinic acetylcholine receptor (AChR) predict either four or five transmembrane segments per subunit. The N-terminus of each subunit is almost certainly extracellular. We have tested folding models by determining biochemically the cellular location of an intermolecular disulfide bridge thought to lie at the delta subunit C-terminus. Dimers of AChR linked through the delta-delta bridge were prepared from Torpedo marmorata and T.californica electric organ. The disulfide's accessibility to hydrophilic reductants was tested in a reconstituted vesicle system. In right-side-out vesicles (greater than 95% ACh binding sites outwards), the bridge was equally accessible whether or not vesicles had been disrupted by freeze--thawing or by detergents. Control experiments based on the rate of reduction of entrapped diphtheria toxin and measurements of radioactive reductant efflux demonstrated that the vesicles provide an adequate permeability barrier. In reconstituted vesicles containing AChR dimers in scrambled orientations, right-side-out dimers were reduced to monomers three times more rapidly than inside-out dimers, consistent with the measured rate of reductant permeation. These observations indicate that in reconstituted vesicles the delta-delta disulfide bridge lies in the same aqueous space as the ACh binding sites. They are most easily reconciled with folding models that propose an even number of transmembrane crossing per subunit.

Glucocorticoid down-regulation of fascin protein expression is required for the steroid-induced formation of tight junctions and cell-cell interactions in rat mammary epithelial tumor cells.

Glucocorticoid hormones, which are physiological regulators of mammary epithelium development, induce the formation of tight junctions in rat Con8 mammary epithelial tumor cells. We have discovered that, as part of this process, the synthetic glucocorticoid dexamethasone strongly and reversibly down-regulated the expression of fascin, an actin-bundling protein that also interacts with the adherens junction component beta-catenin. Ectopic constitutive expression of full-length mouse fascin containing a Myc epitope tag (Myc-fascin) in Con8 cells inhibited the dexamethasone stimulation of transepithelial electrical resistance, disrupted the induced localization of the tight junction protein occludin and the adherens junction protein beta-catenin to the cell periphery, and prevented the rearrangement of the actin cytoskeleton. Ectopic expression of either the carboxyl-terminal 213 amino acids of fascin, which includes the actin and beta-catenin-binding sites, or the amino-terminal 313 amino acids of fascin failed to disrupt the glucocorticoid induction of tight junction formation. Mammary tumor cells expressing the full-length Myc-fascin remained generally glucocorticoid responsive and displayed no changes in the levels or protein-protein interactions of junctional proteins or the amount of cytoskeletal associated actin filaments. However, a cell aggregation assay demonstrated that the expression of Myc-fascin abrogated the dexamethasone induction of cell-cell adhesion. Our results implicate the down-regulation of fascin as a key intermediate step that directly links glucocorticoid receptor signaling to the coordinate control of junctional complex formation and cell-cell interactions in mammary tumor epithelial cells.

Misexpression of the catenin p120(ctn)1A perturbs Xenopus gastrulation but does not elicit Wnt-directed axis specification.

Modulators of cadherin function are of great interest given that the cadherin complex actively contributes to the morphogenesis of virtually all tissues. The catenin p120(ctn) (formerly p120cas) was first identified as a src- and receptor-protein tyrosine kinase substrate and later shown to interact directly with cadherins. In common with beta-catenin and plakoglobin (gamma-catenin), p120(ctn) contains a central Armadillo repeat region by which it binds cadherin cytoplasmic domains. However, little is known about the function of p120(ctn) within the cadherin complex. We examined the role of p120(ctn)1A in early vertebrate development via its exogenous expression in Xenopus. Ventral overexpression of p120(ctn)1A, in contrast to beta-catenin, did not induce the formation of duplicate axial structures resulting from the activation of the Wnt signaling pathway, nor did p120(ctn) affect mesoderm induction. Rather, dorsal misexpression of p120(ctn) specifically perturbed gastrulation. Lineage tracing of cells expressing exogenous p120(ctn) indicated that cell movements were disrupted, while in vitro studies suggested that this may have been a consequence of reduced adhesion between blastomeres. Thus, while cadherin-binding proteins beta-catenin, plakoglobin, and p120(ctn) are members of the Armadillo protein family, it is clear that these proteins have distinct biological functions in early vertebrate development. This work indicates that p120(ctn) has a role in cadherin function and that heightened expression of p120(ctn) interferes with appropriate cell-cell interactions necessary for morphogenesis.