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.
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.
The cadherin-catenin complex is important for mediating homotypic, calcium-dependent cell-cell interactions in diverse tissue types. Although proteins of this complex have been identified, little is known about their interactions. Using a genetic assay in yeast and an in vitro protein-binding assay, we demonstrate that 13-catenin is the linker protein between E-cadherin and c-catenin and that E-cadherin does not bind directly to a-catenin. We show that a 25-amino acid sequence in the cytoplasmic domain of E-cadherin and the amino-terminal domain of a-catenin are independent binding sites for 8-catenin. In addition to 13-catenin and plakoglobin, another member of the armadillo family, p120 binds to E-cadherin. However, unlike 13-catenin, p120does not bind a-catenin in vitro, although a complex of p120 and endogenous oi-catenin could be immunoprecipitated from cell extracts. In vitro protein-binding assays using recombinant E-cadherin cytoplasmic domain and x-catenin revealed two catenin pools in cell lysates: an -1000-to '2000-kDa complex bound to E-cadherin and an -220-kDa pool that did not contain E-cadherin. Only 18-catenin in the -220-kDa pool bound exogenous E-cadherin. Delineation of these molecular linkages and the demonstration of separate pools of catenins in different cell lines provide a foundation for examining regulatory mechanisms involved in the assembly and function of the cadherin-catenin complex.The cadherin superfamily comprises glycoproteins responsible for calcium-dependent, homotypic cell interactions (1). Ecadherin is generally expressed in epithelial tissues and has been shown to regulate cell-cell adhesion (2), cell migration (3), morphogenesis (4), and the establishment of membrane polarity (5).Homotypic interactions between extracellular domains of cadherins are necessary but not sufficient for cell-cell adhesion (2). Linkage of the cadherin cytoplasmic domain to three cytosolic proteins, named a-catenin, ,B-catenin, and plakoglobin (y-catenin), is required (6-8). Although a-catenin, 3-catenin, and plakoglobin can be coimmunoprecipitated in a complex with E-cadherin (1, 7), the binding order of proteinprotein interactions has not been resolved. Insight into this problem is important for understanding functions of the cadherin-catenin complex and the regulation of cadherincatenin complex assembly. Here, we define the binding order of protein-protein interactions in the cadherin-catenin complex using genetic and biochemical approaches. MATERIALS AND METHODSStrain and Microbiological Techniques. All cloning procedures and bacterial transformation were performed by standard procedures outlined by Sambrook et at (9). Yeast strain Y190 (MATa gal4 gal80 his3-200 trpl-901 ade2-101 ura3-52
-Catenin, a member of the Armadillo repeat protein family, binds directly to the cytoplasmic domain of Ecadherin, linking it via ␣-catenin to the actin cytoskeleton. A 30-amino acid region within the cytoplasmic domain of E-cadherin, conserved among all classical cadherins, has been shown to be essential for -catenin binding. This region harbors several putative casein kinase II (CKII) and glycogen synthase kinase-3 (GSK-3) phosphorylation sites and is highly phosphorylated. Here we report that in vitro this region is indeed phosphorylated by CKII and GSK-3, which results in an increased binding of -catenin to E-cadherin. Additionally, in mouse NIH3T3 fibroblasts expression of E-cadherin with mutations in putative CKII sites resulted in reduced cell-cell contacts. Thus, phosphorylation of the E-cadherin cytoplasmic domain by CKII and GSK-3 appears to modulate the affinity between -catenin and E-cadherin, ultimately modifying the strength of cellcell adhesion.E-cadherin belongs to the group of classical cadherins, Ca 2ϩ -dependent adhesion molecules, that mediate cell-cell adhesion in many different tissues and various species (1). E-cadherin is a type I transmembrane protein, making mostly homophilic cell-cell interactions via its extracellular domain, whereas the cytoplasmic domain is anchored to actin microfilaments via three cytoplasmic proteins ␣-, -, and ␥-catenin (plakoglobin) (2, 3). Another catenin p120 ctn is less tightly associated to E-cadherin, and its function is presently less well understood (4). Biochemical studies on cultured cells as well as the use of recombinant proteins have provided a detailed picture of how the different E-cadherin-associated components interact (2, 3, 5-7). Whereas -catenin or plakoglobin bind directly to the cytoplasmic domain of E-cadherin, the linkage of the whole complex to the cytoskeleton is mediated by ␣-catenin, which binds to -catenin or plakoglobin on one side and to actin on the other side (8 -10). Plakoglobin, originally found as a major component of desmosomal junctions, and -catenin form mutually exclusive complexes with E-cadherin, but the function of these two different complexes is not yet known (7, 11). Both proteins belong to the so-called Arm-repeat protein family, a class of proteins characterized by a repeating motif originally identified in the product of the Drosophila segment polarity gene armadillo (12). Armadillo, as well as -catenin, have been shown to be part of the Wingless cascade in Drosophila (Wnt in vertebrates) (for review, see Ref. 13).Cell-cell adhesion is a rather dynamic process for which interactions between cell adhesion molecules and the cytoskeleton must be continually modified. Thus E-cadherin and especially catenins are major target sites for posttranslational modifications, primarily phosphorylation and dephosphorylation events. Tyrosine phosphorylation of -catenin and plakoglobin decreases cell-cell adhesion (14 -17), whereas ectopic expression of tyrosine phosphatases strengthens it (18), but the exact underlyi...
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