Abstract
beta-Catenin is a bifunctional protein participating in both cell adhesion and canonical Wnt signaling. In cell adhesion, it bridges the transmembrane cadherin and the actin-binding protein alpha-catenin and is essential for adherens junction formation, whereas in canonical Wnt signaling, it shuttles between the cytosol and nucleus and functions as an essential transcriptional activator. Schmidtea mediterranea beta-catenin-1 was identified as a determinant of antero-posterior polarity during body regeneration by mediating Wnt signaling. Here we show that S. mediterranea beta-catenin-2 is specifically expressed in epithelial cells in the gut and pharynx, where it has a putative role in mediating cell adhesion. We show evidence that planarian beta-catenin-1 and -2 have distinct biochemical properties. beta-Catenin-1 can interact with the components of the canonical Wnt signaling pathway but not with alpha-catenin, whereas beta-catenin-2 interacts with cell adhesion molecules, including E-cadherin and alpha-catenin, but not with Wnt signaling components. Consistent with their specific function, beta-catenin-1 is a potent transcriptional activator, whereas beta-catenin-2 has no transcriptional activity. Protein sequence alignment also indicates that the planarian beta-catenin-1 and -2 retain distinct critical residues and motifs, which are in agreement with the differences in their biochemical properties. At last, phylogenetic analysis reveals a probable Platyhelminthes- specific structural and functional segregation from which the monofunctional beta-catenins evolved. Our results thus identify the first two monofunctional beta-catenins in metazoans.
Highlights
-Catenin was originally identified as a component of cell adhesion complexes, linking the cytoplasmic tail of cadherins with ␣-catenin [1]. ␣-Catenin, in turn, binds and bundles actin filaments through its C-terminal domain [2, 3]
We show evidence that planarian -catenin-1 and -2 have distinct biochemical properties. -Catenin-1 can interact with the components of the canonical Wnt signaling pathway but not with ␣-catenin, whereas -catenin-2 interacts with cell adhesion molecules, including E-cadherin and ␣-catenin, but not with Wnt signaling components
RNAi of Smed-dishevelled, Smed-evi/wls, and Smed-WntP-1 caused a similar phenotype [30, 32, 33], and RNAi of apc, the negative regulator of the canonical Wnt signaling, caused the opposite phenotype, the double-tail worms [30]. These results demonstrated that, similar to antero-posterior (A-P) axis determination in vertebrate embryos, A-P patterning in planarians is regulated by canonical Wnt signaling and that Smed--catenin-1 is involved in transducing the Wnt signal
Summary
Planarians—The planarians used belong to an asexual race of S. mediterranea collected from Montjuïc (Barcelona, Spain) and maintained as described elsewhere [34]. The polyclonal antibody against Smed-catenin-2 was produced against the last 14 amino acids of Smed--catenin-2 protein (AC EU082825) by Invitrogen. It was used at a 1:2000 dilution. The remaining supernatant was incubated with FLAG-M2 beads (Sigma) or other antibodies (antiMyc or anti-hemagglutinin) at 4 °C for 4 h and followed by incubation with protein A/G-Sepharose (Santa Cruz Biotechnology, Inc.) overnight. When testing the interactions between -catenins with -Trcp (-transducin repeat-containing protein), 25 mM ALLN (Sigma) was added to the culture medium 4 h before harvesting the cells, and phosphatase inhibitors (50 mM NaF, 1 mM sodium vanadate) were added to the lysis buffer. After washing, staining with 4Ј,6-diamidino-2-phenylindole (Roche Applied Science) was performed overnight, and animals were mounted in Prolong Gold antifade reagent (Invitrogen) and stored at 4 °C before imaging. Accession numbers were as follows: Homo sapiens, NP_002221.1, NP_001895.1; Mus musculus, NP_034723.1, AAA37280.1; Xenopus laevis, AAA49931.1; Danio rerio, AAH47815.1, NP_571252.1; Xenopus tropicalis, AAI35470.1; D. melanogaster, NP_476666.1; Nasonia vitripennis, XP_001603109.1; Pharhyale hawaiensis, ACH92925; Aplysia californica, AAY29569.1; Cerebratulus lacteus, ABY21456.1; Platynereis dumerilii, ABQ85061.1; Chaetopterus variopedatus, AAL49497.1; Dugesia japonica, BAD93243.1; Hydra magnipapillata, AAC47137.1; S. mediterranea, ABW79875.1, ABW79874.1; S. mansoni, XM_ 002573746.1 (Sm--catenin-1; see supplemental Fig. 2 for Sm--catenin-2); Dictyostelium discoideum, XP_646234
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