EGFR signaling to p120-catenin through phosphorylation at Y228.
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Epidermal growth factor receptor (EGFR) signals to p120(ctn) (p120), implying a role for EGFR in modulating cell-cell adhesion in epithelial tissues. p120 controls cadherin turnover, and may have other roles that modulate cadherin adhesiveness. To clarify the role for EGFR and other tyrosine kinases in regulating p120 function, we have generated and characterized a new phosphospecific antibody to p120 Y228, as well as a novel siRNA-based reconstitution system for analyzing roles of individual p120 phosphorylation events. In A431 cells, epidermal growth factor induced striking p120 phosphorylation at Y228. Y228-phosphorylated p120 localized to adherens junctions and lamellipodia, and was significantly enhanced in cells around the colony periphery. A screen of carcinoma cell lines revealed that some contain unusually high steady state levels of Y228 phosphorylation, suggesting that disregulated kinase activity in tumors may affect adhesion by constitutive cross talk to cadherin complexes. Despite these observations, mutation of Y228 and other prominent Src-associated p120 phosphorylation sites did not noticeably reduce the ability of E-cadherin to assemble junctions and induce compaction of cultured cells. Although A431 cells display significant activation of both EGFR and Src kinases, our data suggest that these account for only a fraction of the steady state activity that targets p120 Y228, and that Src family kinases are not necessary intermediates for epidermal growth factor-induced signaling to p120 Y228. (+info)
Prognostic impact of alterations in P-cadherin expression and related cell adhesion markers in endometrial cancer.
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PURPOSE: Reduced tumor cell adhesion is associated with invasive growth and unfavorable prognosis. In endometrial carcinoma, the prognostic impact of adhesion markers (E-cadherin, beta-catenin [beta-catenin], P-cadherin, and p120(ctn)) is partly unknown. We wanted to examine the expression pattern and prognostic value of these molecules in a population-based series of endometrial carcinoma patients. PATIENTS AND METHODS: All patients diagnosed with endometrial carcinoma between 1981 and 1990 in Hordaland County, Norway, were included. Paraffin-embedded tumor tissue was available for 96% of the patients (n = 286), and was studied immunohistochemically for expression of E-cadherin, beta-catenin, P-cadherin, and p120(ctn). The tissue microarray technique was used for P-cadherin and p120(ctn). Median follow-up time for survivors was 9 years (range, 4 to 16 years) and follow-up was complete. RESULTS: Pathologic expression of P-cadherin, E-cadherin, and beta-catenin was associated with a majority of the clinicopathologic variables. In univariate survival analyses, all adhesion markers influenced survival significantly (P <.05). Tumors with pathologic expression of both E-cadherin (low expression) and P-cadherin (high expression; 19%), and beta-catenin (low expression) and P-cadherin (high expression; 16%), had significantly reduced survival compared with the remaining samples (P <.001 for both). In multivariate models, all markers except E-cadherin showed independent prognostic significance in addition to the traditional tumor features. CONCLUSION: Differential expression of P-cadherin and beta-catenin seems to be important in endometrial carcinoma and is associated with aggressive subgroups. Our findings also indicate that a shift from E-cadherin to P-cadherin expression (cadherin switch) is an important prognostic feature in these tumors. (+info)
E-cadherin-mediated adhesion inhibits ligand-dependent activation of diverse receptor tyrosine kinases.
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E-cadherin is an essential adhesion protein as well as a tumor suppressor that is silenced in many cancers. Its adhesion-dependent regulation of signaling has not been elucidated. We report that E-cadherin can negatively regulate, in an adhesion-dependent manner, the ligand-dependent activation of divergent classes of receptor tyrosine kinases (RTKs), by inhibiting their ligand-dependent activation in association with decreases in receptor mobility and in ligand-binding affinity. E-cadherin did not regulate a constitutively active mutant RTK (Neu*) or the ligand-dependent activation of LPA receptors or muscarinic receptors, which are two classes of G protein-coupled receptors. EGFR regulation by E-cadherin was associated with complex formation between EGFR and E-cadherin that depended on the extracellular domain of E-cadherin but was independent of beta-catenin binding or p120-catenin binding. Transfection of E-cadherin conferred negative RTK regulation to human melanoma and breast cancer lines with downregulated endogenous E-cadherin. Abrogation of E-cadherin regulation may contribute to the frequent ligand-dependent activation of RTK in tumors. (+info)
Vertebrate development requires ARVCF and p120 catenins and their interplay with RhoA and Rac.
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Using an animal model system and depletion-rescue strategies, we have addressed the requirement and functions of armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF) and p120 catenins in early vertebrate embryogenesis. We find that xARVCF and Xp120 are essential to development given that depletion of either results in disrupted gastrulation and axial elongation, which are specific phenotypes based on self-rescue analysis and further criteria. Exogenous xARVCF or Xp120 cross-rescued depletion of the other, and each depletion was additionally rescued with (carefully titrated) dominant-negative RhoA or dominant-active Rac. Although xARVCF or Xp120 depletion did not appear to reduce the adhesive function of C-cadherin in standard cell reaggregation and additional assays, C-cadherin levels were somewhat reduced after xARVCF or Xp120 depletion, and rescue analysis using partial or full-length C-cadherin constructs suggested contributory effects on altered adhesion and signaling functions. This work indicates the required functions of both p120 and ARVCF in vertebrate embryogenesis and their shared functional interplay with RhoA, Rac, and cadherin in a developmental context. (+info)
The transcription factor Snail downregulates the tight junction components independently of E-cadherin downregulation.
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Snail, a transcriptional repressor of E-cadherin expression, is involved in epithelial-mesenchymal transitions during development. We demonstrate that Snail activity is not restricted to E-cadherin downregulation. Expression of tight junction proteins, including claudin-1, occludin and ZO-1, was downregulated in MDCK cells exogenously expressing Snail protein. Although occludin mRNA levels were downregulated by Snail expression, the transcription of claudin-1 and ZO-1 were unaffected. Reporter assays using the claudin-1 promoter region revealed that promoter activity was not affected by Snail overexpression. Decreased synthesis of claudin-1 protein was observed, however, suggesting that Snail may act in translation initiation. Snail expression also altered the splicing pattern of p120. The levels of mRNA encoding the epithelial variant decreased, while the fibroblastic mRNA form increased. Although ectopic E-cadherin expression resulted in a downregulation of Snail-induced fibronectin expression, fibroblastic morphology was affected only minimally; the expression of tight junctional proteins remained at low levels. These results indicate that Snail is involved in both the direct transcriptional repression of genes, such as E-cadherin and occludin, and post-transcriptional events, including downregulation of claudin-1. These data support the idea that Snail is a transcription factor possessing pleiotropic activities. (+info)
Disruption of cadherin/catenin expression, localization, and interactions during HgCl2-induced nephrotoxicity.
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The cadherin/catenin complex is an essential regulator of intercellular adhesion and is critical for the establishment of epithelial cell polarity. The purpose of this study was to (1) determine the spatial pattern of cadherin and catenin expression, colocalization, and interaction along the mouse nephron, and (2) investigate the expression, localization, and interaction of proximal tubular cadherins and catenins during mercuric chloride-induced nephrotoxicity. Using a combination of Western blot analysis, colocalization studies, and coimmunoprecipitation, we conclude that two distinct cadherin/catenin complexes exist in adult mouse kidney proximal tubules: N-cadherin/beta-catenin/alpha-catenin and E-cadherin/beta-catenin/alpha-catenin/p120-catenin. In the distal tubule, E-cadherin/beta-catenin/alpha-catenin and E-cadherin/gamma-catenin/alpha-catenin complexes are present. Male C3H mice were challenged with 0-25 micromol/kg mercuric chloride i.p. (6-48 h) to assess the impact of nephrotoxicity on cadherin/catenin complexes. Plasma creatinine and blood urea nitrogen were increased between 6 and 48 h, indicating the onset of renal failure. In addition, histological evaluation demonstrated alterations in the proximal tubules. At 24 h, we observed decreases in Ksp- and N-cadherin, but not in E-cadherin. Additionally, alpha-catenin expression was decreased, in the absence of changes in beta-, gamma-, and p120-catenin. The early stages (6 h) of mercuric chloride-induced nephrotoxicity were associated with disruption of complex integrity. N-cadherin and alpha-catenin localizations were disrupted at 6 h. These changes in cadherin and catenin localization corresponded with a decrease in the coimmunoprecipitation of alpha-catenin with both beta-catenin and N-cadherin. Interestingly, these changes occurred at the same time that aberrant staining of Na+/K(+)-ATPase staining was seen. Taken together, these data suggest that alterations in cadherin and catenin expression, localization, and interaction are associated with nephrotoxicity. (+info)
NLS-dependent nuclear localization of p120ctn is necessary to relieve Kaiso-mediated transcriptional repression.
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The Armadillo catenin p120(ctn) regulates cadherin adhesive strength at the plasma membrane and interacts with the novel BTB/POZ transcriptional repressor Kaiso in the nucleus. The dual localization of p120(ctn) at cell-cell junctions and in the nucleus suggests that its nucleocytoplasmic trafficking is tightly regulated. Here we report on the identification of a specific and highly basic nuclear localization signal (NLS) in p120(ctn). The functionality of the NLS was validated by its ability to direct the nuclear localization of a heterologous beta-galactosidase-GFP fusion protein. Mutating two key positively charged lysines to neutral alanines in the NLS of full-length p120(ctn) inhibited both p120(ctn) nuclear localization as well as the characteristic p120(ctn)-induced branching phenotype that correlates with increased cell migration. However, while these findings and others suggested that nuclear localization of p120(ctn) was crucial for the p120(ctn)-induced branching phenotype, we found that forced nuclear localization of both wild-type and NLS-mutated p120(ctn) did not induce branching. Recently, we also found that one role of p120(ctn) was to regulate Kaiso-mediated transcriptional repression. However, it remained unclear whether p120(ctn) sequestered Kaiso in the cytosol or directly inhibited Kaiso transcriptional activity in the nucleus. Using minimal promoter assays, we show here that the regulatory effect of p120(ctn) on Kaiso transcriptional activity requires the nuclear translocation of p120(ctn). Therefore, an intact NLS in p120(ctn) is requisite for its first identified regulatory role of the transcriptional repressor Kaiso. (+info)
R-cadherin influences cell motility via Rho family GTPases.
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Classical cadherins are the transmembrane proteins of the adherens junction and mediate cell-cell adhesion via homotypic interactions in the extracellular space. In addition, they mediate connections to the cytoskeleton by means of their association with catenins. Decreased cadherin-mediated adhesion has been implicated as an important component of tumorigenesis. Cadherin switching is central to the epithelial to mesenchymal transitions that drive normal developmental processes. Cadherin switching has also been implicated in tumorigenesis, particularly in metastasis. Recently, cadherins have been shown to be engaged in cellular activities other than adhesion, including motility, invasion, and signaling. In this study, we show that inappropriate expression of R-cadherin in tumor cells results in decreased expression of endogenous cadherins (cadherin switching) and sustained signaling through Rho GTPases. In addition, we show that R-cadherin induces cell motility when expressed in epithelial cells and that this increased motility is dependent upon Rho GTPase activity. (+info)