Jörg Stappert

β‐catenin is a target for the ubiquitin–proteasome pathway

β‐catenin is a central component of the cadherin cell adhesion complex and plays an essential role in the Wingless/Wnt signaling pathway. In the current model of this pathway, the amount of β‐catenin (or its invertebrate homolog Armadillo) is tightly regulated and its steady‐state level outside the cadherin–catenin complex is low in the absence of Wingless/Wnt signal. Here we show that the ubiquitin‐dependent proteolysis system is involved in the regulation of β‐catenin turnover. β‐catenin, but not E‐cadherin, p120cas or α‐catenin, becomes stabilized when proteasome‐mediated proteolysis is inhibited and this leads to the accumulation of multi‐ubiquitinated forms of β‐catenin. Mutagenesis experiments demonstrate that substitution of the serine residues in the glycogen synthase kinase 3β (GSK3β) phosphorylation consensus motif of β‐catenin inhibits ubiquitination and results in stabilization of the protein. This motif in β‐catenin resembles a motif in IκB (inhibitor of NFκB) which is required for the phosphorylation‐dependent degradation of IκB via the ubiquitin–proteasome pathway. We show that ubiquitination of β‐catenin is greatly reduced in Wnt‐expressing cells, providing the first evidence that the ubiquitin–proteasome degradation pathway may act downstream of GSK3β in the regulation of β‐catenin.

Brachyury is a target gene of the Wnt/β-catenin signaling pathway

To identify target genes of the Wnt/beta-catenin signaling pathway in early mouse embryonic development we have established a co-culture system consisting of NIH3T3 fibroblasts expressing different Wnts as feeder layer cells and embryonic stem (ES) cells expressing a green fluorescent protein (GFP) reporter gene transcriptionally regulated by the TCF/beta-catenin complex. ES cells specifically respond to Wnt signal as monitored by GFP expression. In GFP-positive ES cells we observe expression of Brachyury. Two TCF binding sites located in a 500 bp Brachyury promoter fragment bind the LEF-1/beta-catenin complex and respond specifically to beta-catenin-dependent transactivation. From these results we conclude that Brachyury is a target gene for Wnt/beta-catenin signaling.

A Short Core Region of E-cadherin is Essential for Catenin Binding and is Highly Phosphorylated

Classical cadherins associate with three cytoplasmic proteins, termed alpha, -beta- and gamma-catenin. This association mediates the attachment of cadherins to the microfilament network, which is believed to be of major importance for cadherin function. Deletion of the carboxyterminal 72-amino acid residues of E-cadherin had been previously shown to prevent catenin binding. Here we have analyzed additional mutants of E-cadherin with deletions within this region and identified a core region of 30 amino acids (E-cadherin pos. 832-862) essential for the interaction with catenins. Phosphorylation analysis of wild-type and mutant E-cadherin indicates that the catenin-binding domain is highly phosphorylated. In particular, the 30 amino acid region contains 8 serine residues which are well conserved among cadherins. To elucidate whether phosphorylation might be important for cadherin-catenin complex formation, site-directed mutagenesis experiments were performed. Partial substitutions of up to 5 of the 8 serine residues in the cluster had no influence on E-cadherin-catenin complex formation and E-cadherin mediated cell adhesion, although phosphorylation of E-cadherin was reduced. In contrast, substitution of the whole serine cluster completely abolished phosphorylation and affected complex formation with catenins. These results suggest that E-cadherin-catenin interaction may be regulated by phosphorylation of the catenin-binding domain, which might represent one molecular mechanism to regulate cadherin mediated cell adhesion.

MODIFICATION OF THE E-CADHERIN-CATENIN COMPLEX IN MITOTIC MADIN-DARBY CANINE KIDNEY EPITHELIAL CELLS

One of the hallmarks of polarized epithelial cells undergoing mitosis is their rounded morphology. This phenotype correlates with a reduced cell-substratum adhesion, apparently caused by a modulation of integrin function. However, it is still unclear whether the cadherin-mediated cell-cell adhesion is affected as well. To address this question, the cadherin complex was analyzed in different cell cycle stages of Madin-Darby canine kidney cells. By immunofluorescence, mitotic Madin-Darby canine kidney cells showed an increased staining of E-cadherin and the catenins (α-catenin, β-catenin, plakoglobin, p120ctn) in the cytosol, suggesting a reorganization of the cadherin-catenin complex during mitosis. Biochemical analysis revealed that the overall amount of these components, as well as the proportion of the complex associated with the actin cytoskeleton, remained unchanged in mitotic cells. However, we found evidence for an internalization of E-cadherin during mitosis. In addition, the cadherin-catenin complex was analyzed for mitosis-specific changes in phosphorylation. We report a decrease in the tyrosine phosphorylation of β-catenin, plakoglobin, and p120ctn during mitosis. Moreover, we observed a mitosis-specific Ser/Thr-phosphorylation of p120ctn, as detected by the MPM-2 antibody. Hence, the cadherin/catenin complex is a target for different posttranslational modifications during mitosis, which may also have a profound impact on cadherin-mediated cell-cell adhesion.

Intracellular associations of adhesion molecules

Significant advances have recently been made in our understanding of the cytoplasmic anchorage of adhesion molecules. The identification of catenins, a new class of proteins involved in the cytoplasmic anchorage of cadherins that are structurally homologous to other peripheral cytoplasmic proteins, emphasizes the existence of protein families that modulate the function of cell-substrate and cell-cell adhesion molecules.

The Cadherin Superfamily

Publisher Summary The structure and functions of cadherins have been found to be much more complex and diverse than previously assumed. Cadherins are involved in the regulation of morphogenesis in many organisms, including invertebrate species, thus indicating the general importance of this family in organizing multicellular structures. In the classical view, cadherins function mainly in the adhesion of solid tissues, but they have been found also in mesenchymal tissues and on migrating cells as well, indicating that they may be involved in cell rearrangement of most if not all tissues. This seems to be achieved at least, in part, by the expression of a large number of cadherin subtypes with distinct adhesion specificities. Crystal structures from the parts of the extracellular domains of E- and N-cadherin reveal new insights in the interaction mechanisms of these proteins. However, it is still unclear whether each cadherin varies in its three-dimensional structure and what the consequences of its cytoplasmic anchorage are. Thus, a major area for future studies is to resolve the 3D structure of other cadherins and the complex cytoplasmic network underlying cadherin clusters. The knowledge about the proteins being differentially recruited into junctions mediated by different cadherins may answer the question about the signals cadherins are able to transmit.