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Crk-associated substrate (p130(Cas), Cas) is a docking protein first recognized as having elevated phosphotyrosine content in mammalian cells transformed by v-Src and v-Crk oncoproteins. Subsequent studies have implicated Cas in the control of normal cell behavior through its roles in integrin-mediated signal transduction and organization of the actin cytoskeleton at sites of cell adhesion. In this study, we sought to gain new insight into normal Cas function by identifying previously unrecognized interacting proteins. A yeast two-hybrid screen using the C-terminal region of Cas as a bait identified the Src homology 3 (SH3) domain of the mouse "nephrocystin" protein-orthologous to a human protein whose loss of function leads to the cystic kidney disease familial juvenile nephronophthisis. The putative full-length mouse and partial canine nephrocystin sequences were deduced from cDNA clones. Additional studies using epitope-tagged mouse nephrocystin indicated that nephrocystin and Cas can interact in mammalian cells and revealed that both proteins prominently localize at or near sites of cell-cell contact in polarized Madin-Darby canine kidney epithelial cells. Our findings provide novel insight into the normal cellular activities regulated by both Cas and nephrocystin, and raise the possibility that these proteins have a related function in polarized epithelial cells.
Copyright 2000 Academic Press.
ARVCF is a novel Armadillo repeat domain protein that is closely related to the catenin p120(ctn). Using new ARVCF monoclonal antibodies, we have found that ARVCF associates with E-cadherin and competes with p120 for interaction with the E-cadherin juxtamembrane domain. ARVCF also localized to the nucleus in some cell types, however, and was significantly more nucleophilic than p120. Surprisingly, despite apparently ubiquitous expression, ARVCF was at least tenfold less abundant than p120 in a wide variety of cell types, and was difficult to detect by immunofluorescence unless overexpressed. Consequently, it is not likely to be abundant enough in adult tissues to functionally compete with p120. ARVCF also completely lacked the ability to induce the cell-branching phenotype associated with overexpression of p120. Expression of ARVCF/p120 chimeras confirmed previous results indicating that the branching activity of p120 maps to its Armadillo repeat domain. Surprisingly, the preferential localization of ARVCF to the nucleus required sequences in the amino-terminal end of ARVCF, suggesting that the sequences directing nuclear translocation of ARVCF are distinct from the predicted bipartite nuclear localization signal located between repeats 6 and 7. The dual localization of ARVCF to junctions and to nuclei suggests activities in different cellular compartments, as is the case for several other Armadillo repeat proteins including beta-catenin, p120 and the plakophilins.
p120(ctn) is a catenin whose direct binding to the juxtamembrane domain of classical cadherins suggests a role in regulating cell-cell adhesion. The juxtamembrane domain has been implicated in a variety of roles including cadherin clustering, cell motility, and neuronal outgrowth, raising the possibility that p120 mediates these activities. We have generated minimal mutations in this region that uncouple the E-cadherin-p120 interaction, but do not affect interactions with other catenins. By stable transfection into E-cadherin-deficient cell lines, we show that cadherins are both necessary and sufficient for recruitment of p120 to junctions. Detergent-free subcellular fractionation studies indicated that, in contrast to previous reports, the stoichiometry of the interaction is extremely high. Unlike alpha- and beta-catenins, p120 was metabolically stable in cadherin-deficient cells, and was present at high levels in the cytoplasm. Analysis of cells expressing E-cadherin mutant constructs indicated that p120 is required for the E-cadherin-mediated transition from weak to strong adhesion. In aggregation assays, cells expressing p120-uncoupled E-cadherin formed only weak cell aggregates, which immediately dispersed into single cells upon pipetting. As an apparent consequence, the actin cytoskeleton failed to insert properly into peripheral E-cadherin plaques, resulting in the inability to form a continuous circumferential ring around cell colonies. Our data suggest that p120 directly or indirectly regulates the E-cadherin-mediated transition to tight cell-cell adhesion, possibly blocking subsequent events necessary for reorganization of the actin cytoskeleton and compaction.
We have generated the first monoclonal antibodies (MAbs) to Armadillo repeat gene deleted in velo-cardiofacial syndrome (ARVCF), a recently identified Armadillo repeat-containing protein closely related to the catenin p120ctn. Six ARVCF-specific MAbs were characterized for isotype, species cross-reactivity, and utility in assays including immunofluorescence, immunoprecipitation, and Western blotting. All six antibodies were isotyped as IgG1 and several cross-reacted with ARVCF from a variety of species including human, rat, dog, and monkey, but not mouse. Importantly, none of the ARVCF MAbs cross-reacted with p120ctn, despite the high homology between these proteins. MAbs 3B2 and 4B1 were consistently the best in all applications and will provide valuable tools for further study of the role of ARVCF in cells.
To investigate the trafficking of transforming growth factor-alpha precursor (pro-TGF-alpha) in polarized epithelial cells, wild type and membrane-fixed human pro-TGF-alpha were introduced into Madin-Darby canine kidney (MDCK) cells. We show that wild type pro-TGF-alpha was synthesized and processed normally to release mature 5.6-kDa TGF-alpha into the basal medium while membrane-fixed pro-TGF-alpha remained cell-associated. Antibody (mAb-528) receptor blockade experiments demonstrated the efficient consumption of basally released TGF-alpha by basolateral epidermal growth factor receptors, indicating that TGF-alpha can act in an autocrine manner in these polarized epithelial cells. Biochemical analysis showed pro-TGF-alpha was expressed on the basolateral surface as either a 17- or 30-kDa species; the 17-kDa forms of both pro-TGF-alpha constructs had basolateral/apical ratios of > 20:1. By confocal microscopy, membrane-fixed pro-TGF-alpha was immunolocalized to lateral membrane surfaces. In pulse-chase experiments combined with cell surface immunoprecipitation, we demonstrated that newly synthesized wild type and membrane-fixed pro-TGF-alpha are delivered directly to the basolateral surface with 94 and 96% efficiency, respectively. These results also provide direct evidence for sequential cleavage of pro-TGF-alpha at the basolateral membrane surface. Thus, pro-TGF-alpha is sorted intracellularly and vectorially targeted to the basolateral membrane domain in these polarized epithelial cells. The MDCK cell line provides an ideal in vitro model to examine the molecular basis for trafficking of pro-TGF-alpha and other epidermal growth factor-like growth factors in polarized epithelial cells and their potential interactions with basolateral epidermal growth factor receptors.
HaCaT cells, an immortalized keratinocyte line, incubated in plastic wells in the presence of conditioned medium from 804G cells adhered and spread rapidly in less than 30 min. In contrast, cells plated in fibroblast or keratinocyte conditioned medium adhered poorly and remained rounded at 30 min. Immunodepletion of 804G conditioned medium with polyclonal antisera to laminin-5r, but not control antisera, abolished rapid cell spreading. Electron microscopy of HaCaT cells spread by incubation in 804G conditioned medium, but not control medium, revealed mature hemidesmosomes after 24 h. Rapid spreading was also observed in wells precoated with 804G conditioned medium or 804G cell-deposited matrix, but not with fibronectin, vitronectin, or laminin-1. Immunoblotting of 804G conditioned medium with anti-laminin-5r antibodies unveiled polypeptides of 150, 140, 135, and 100 kDa, identical by electrophoretic mobility to immunoreactive polypeptides in 804G deposited matrix. Our results suggest that addition of laminin-5r in a soluble form is sufficient to promote rapid spreading and hemidesmosome assembly in keratinocytes. The mechanism of soluble laminin-5r action may include efficient surface "priming" for cell adhesion. Soluble laminin-5r may have a physiologic role in morphogenesis and repair of the epidermis and may be of use for therapeutic applications.