Cadherin Sequences That Inhibit β-Catenin Signaling: A Study in Yeast and Mammalian Cells

Abstract

Drosophila Armadillo and its mammalian homologue ␤-catenin are scaffolding proteins involved in the assembly of multiprotein complexes with diverse biological roles. They mediate adherens junction assembly, thus determining tissue architecture, and also transduce Wnt/Wingless intercellular signals, which regulate embryonic cell fates and, if inappropriately activated, contribute to tumorigenesis. To learn more about Armadillo/␤-catenin's scaffolding function, we examined in detail its interaction with one of its protein targets, cadherin. We utilized two assay systems: the yeast two-hybrid system to study cadherin binding in the absence of Armadillo/␤-catenin's other protein partners, and mammalian cells where interactions were assessed in their presence. We found that segments of the cadherin cytoplasmic tail as small as 23 amino acids bind Armadillo or ␤-catenin in yeast, whereas a slightly longer region is required for binding in mammalian cells. We used mutagenesis to identify critical amino acids required for cadherin interaction with Armadillo/␤-catenin. Expression of such short cadherin sequences in mammalian cells did not affect adherens junctions but effectively inhibited ␤-catenin-mediated signaling. This suggests that the interaction between ␤-catenin and T cell factor family transcription factors is a sensitive target for disruption, making the use of analogues of these cadherin derivatives a potentially useful means to suppress tumor progression.

Systematic Peptide Array-based Delineation of the Differential β-Catenin Interaction with Tcf4, E-Cadherin, and Adenomatous Polyposis Coli

“…After affinity chromatography on reduced glutathioneSepharose (Amersham Biosciences), ␤-catenin was cleaved off the column by thrombin and further purified by size exclusion chromatography on Superdex 75 (Amersham Biosciences). For fluorescent ␤-catenin 41 and Leu 48 as a potential drug target site are shown in their binding pocket on ␤-catenin. Although E-cadherin binding to ␤-catenin shows two leucines in the same position, these interactions are not important for the interaction (see "Results").…”

“…2B). 15-mers starting from residue 17 or higher did not show any binding by themselves, although some residues in the C-terminal part of the CBD such as Leu 41 and Leu 48 did contribute to the binding energy (see Fig. 2A and Table I).…”

“…It comprises the complete minimal binding region of 23-27 residues that has been identified for Drosophila E-cadherin (41) and covers a similar surface of ␤-catenin as the Tcf4-CBD. (41). As for Tcf, variants of the minimal CBD of E-cadherin (residues 652-700) with all possible single alanine mutations were synthesized on a membrane, and ␤-catenin binding was quantified as described above.…”

Turn‐off, drop‐out: Functional state switching of cadherins

“…The site is adjacent to and partially overlaps the core-binding domain for ␤-catenin determined both by using in vitro binding assays (Kaplan et al, 2001; see Fig. 2) and in situ binding assays (Simcha et al, 2001; see Fig. 2).…”

“…␤-catenin and PTP1B binding domains on N-cadherin and conservation of the PTP1B site. The core ␤-catenin binding sequence defined by deletions (Kaplan et al, 2001 andSimcha et al 2001) and that defined by peptide competition (Arregui et al, 2000) are compared in Figure 2. In this figure we use the region defined by the peptide competitor.…”