The desmosome is a highly organized plasma membrane domain that couples intermediate filaments to the plasma membrane at regions of cell–cell adhesion. Desmosomes contain two classes of cadherins, desmogleins, and desmocollins, that bind to the cytoplasmic protein plakoglobin. Desmoplakin is a desmosomal component that plays a critical role in linking intermediate filament networks to the desmosomal plaque, and the amino-terminal domain of desmoplakin targets desmoplakin to the desmosome. However, the desmosomal protein(s) that bind the amino-terminal domain of desmoplakin have not been identified. To determine if the desmosomal cadherins and plakoglobin interact with the amino-terminal domain of desmoplakin, these proteins were co-expressed in L-cell fibroblasts, cells that do not normally express desmosomal components. When expressed in L-cells, the desmosomal cadherins and plakoglobin exhibited a diffuse distribution. However, in the presence of an amino-terminal desmoplakin polypeptide (DP-NTP), the desmosomal cadherins and plakoglobin were observed in punctate clusters that also contained DP-NTP. In addition, plakoglobin and DP-NTP were recruited to cell–cell interfaces in L-cells co-expressing a chimeric cadherin with the E-cadherin extracellular domain and the desmoglein-1 cytoplasmic domain, and these cells formed structures that were ultrastructurally similar to the outer plaque of the desmosome. In transient expression experiments in COS cells, the recruitment of DP-NTP to cell borders by the chimera required co-expression of plakoglobin. Plakoglobin and DP-NTP co-immunoprecipitated when extracted from L-cells, and yeast two hybrid analysis indicated that DP-NTP binds directly to plakoglobin but not Dsg1. These results identify a role for desmoplakin in organizing the desmosomal cadherin–plakoglobin complex and provide new insights into the hierarchy of protein interactions that occur in the desmosomal plaque.

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The receptor protein-tyrosine phosphatase (PTP) DEP-1 (CD148/PTP-) has been implicated in the regulation of cell growth, differentiation, and transformation, and most recently has been identified as a potential tumor suppressor gene mutated in colon, lung, and breast cancers. We have generated constructs comprising the cytoplasmic segment of DEP-1 fused to the maltose-binding protein to identify potential substrates and thereby suggest a physiological function for DEP-1. We have shown that the substrate-trapping mutant form of DEP-1 interacted with a small subset of tyrosine-phosphorylated proteins from lysates of the human breast tumor cell lines MDA-MB-231, T-47D, and T-47D/Met and have identified the hepatocyte growth factor/scatter factor receptor Met, the adapter protein Gab1, and the junctional component p120 catenin as potential substrates. Following ligand stimulation, phosphorylation of specific tyrosyl residues in Met induces mitogenic, motogenic, and morphogenic responses. When co-expressed in 293 cells, the full-length substrate-trapping mutant form of DEP-1 formed a stable complex with the chimeric receptor colony stimulating factor 1 (CSF)-Met and wild type DEP-1 dephosphorylated CSF-Met. Furthermore, we observed that DEP-1 preferentially dephosphorylated a Gab1 binding site (Tyr 1349 ) and a COOH-terminal tyrosine implicated in morphogenesis (Tyr 1365 ), whereas tyrosine residues in the activation loop of Met (Tyr 1230 , Tyr 1234 , and Tyr 1235 ) were not preferred targets of the PTP. The ability of DEP-1 preferentially to dephosphorylate particular tyrosine residues that are required for Met-induced signaling suggests that DEP-1 may function in controlling the specificity of signals induced by this PTK, rather than as a simple "off-switch" to counteract PTK activity.A variety of ligands trigger the reversible phosphorylation of tyrosyl residues in cellular proteins, a process that underlies the control of such fundamental cellular functions as growth and proliferation, migration, and morphogenesis. Tyrosine phosphorylation is regulated by the coordinated action of protein-tyrosine kinases (PTKs) 1 and protein-tyrosine phosphatases (PTPs). Classically it was thought that the PTKs provided the "on-switch" to initiate a physiological response, whereas the PTPs functioned to counteract the PTKs and to return the system to its basal state. However, it was soon shown that PTPs may themselves function positively to promote signaling, for example, by promoting the dephosphorylation and activation of PTKs, thus coordinating with, rather than antagonizing PTK function (reviewed in Ref. 1). A further level of complexity has been introduced with the realization that whether a defined PTP functions positively or negatively may depend upon the signaling context. Thus, SHP-2 is an activator of signaling through the HGF/SF receptor Met (2) and the epidermal growth factor receptor (3), but is an inhibitor of signaling through the platelet-derived growth factor receptor (4). Following ligand binding, a receptor PT...

Tyrosine phosphorylation of junctional components has been proposed as a mechanism for modulating cellcell adhesion. Although a correlation exists between the tyrosine phosphorylation of the adherens junction protein ␤-catenin and loss of classical cadherin-mediated adhesion, the effects of tyrosine phosphorylation on the function of the adherens junction and desmosome-associated protein plakoglobin is unknown. In the present study, we investigated the effects of epidermal growth factor receptor (EGFR) tyrosine kinase activation on the subcellular distribution of plakoglobin and its association with its junctional binding partners. Long term epidermal growth factor (EGF) treatment of A431 cells revealed a modest decrease in the cytoskeleton-associated pool of plakoglobin (Pg) and a corresponding increase in the cytosolic pool of Pg. After short term EGF treatment, plakoglobin was rapidly phosphorylated, and tyrosine-phosphorylated Pg was distributed predominantly in a membrane-associated Triton X-100-soluble pool, along with a co-precipitating high molecular weight tyrosine-phosphorylated protein identified as desmoglein 2. Analysis of deletion and point mutants defined the primary EGFR-dependent targets as one or more of three C-terminal tyrosine residues. Whereas phosphorylated Pg remained associated with the desmoglein tail after both short and long term EGFR activation, no phosphorylated Pg was found associated with the N-terminal Pg-binding domain (DPNTP) of the intermediate filament-associated protein, desmoplakin. Together these results are consistent with the possibility that EGF-dependent tyrosine phosphorylation of Pg may modulate cell-cell adhesion by compromising the link between desmosomal cadherins and the intermediate filament cytoskeleton.Modulation of adhesive junctions plays a critical role during development and wound healing (1-3). Moreover, the ability of cells to down-regulate or alter the adhesive capabilities of their cell-cell junctions is a contributing factor during tumor metastasis (4). However, the exact mechanisms that regulate intercellular adhesion during these processes are not well understood. Tyrosine phosphorylation of junctional components, and in particular phosphorylation of members of the armadillo family of proteins, has been proposed as a critical step in modulating cell-cell adhesion. Several lines of evidence suggest that tyrosine phosphorylation of the cadherin-catenin complex down-regulates cell adhesion and possibly the association of cadherin-catenin complexes with the cytoskeleton (5-11). In addition, plakoglobin (Pg) 1 and ␤-catenin have been reported to associate with the tyrosine kinases c-ErbB-2, the epidermal growth factor receptor (EGFR) (12-13), and protein-tyrosine phosphatases PTP (14) and LAR (15). These data suggest that these members of the armadillo family of proteins may function as modulatable components within complex adhesive structures allowing for rapid responses to intracellular or extracellular signals.In certain cell types, tyrosine phosphory...

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