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Fig. 1Major functions of β-catenin in bilaterians. In signal transduction, in the presence of Wnt ligands, the β-catenin degradation complex is inhibited by Dishevelled. β-catenin accumulates in the cytoplasm and translocates to the nucleus, where it binds TCF and displaces the transcriptional repressor TLE, resulting in gene transcription of canonical Wnt signaling target genes. In structural roles, β-catenin contributes to E-cadherin-mediated cell-cell adhesion by linking E-cadherin to α-catenin, and reinforces cellular structure by binding α-catenin to the actin cytoskeleton
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Fig. 2Primary and 3D structures of nonbilaterian β-catenins. (A) Overview of phylogeny and structure of β-catenins of early-branching (nonbilaterian) metazoans and ARM6 protein of the unicellular sister taxon, Choanoflagellata. (B) Domain organizations of β-catenin proteins. Metazoan β-catenins show a high degree of conservation of the central region, GSK3β/CK1α phosphorylation sites, and αE-catenin binding sites. At the C-terminus, motif A is conserved in only Bilateria and Cnidaria, whereas motif B is shared among all metazoan lineages, except for Ctenophora. In the choanoflagellate, Salpingoeca rosetta, possible GSK3β phosphorylation sites (S33/T41) were identified. (B) Surface depictions of β-catenin homology structural models were compared with mouse β-catenin. This confirmed that the three crucial arginine residues (dark blue) of nonbilaterians were localized similarly to mouse and Xenopus β-catenins. K556, R551, and K590 of B. mikado β-catenin were located at positions corresponding to mouse β-catenin R474, R469, and K508, but the R582, H578, R612, and Y654 assembly of mouse β-catenin is completely absent in B. mikado β-catenin. Structures visualized using UCSF Chimera software
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Fig. 2Primary and 3D structures of nonbilaterian β-catenins. (A) Overview of phylogeny and structure of β-catenins of early-branching (nonbilaterian) metazoans and ARM6 protein of the unicellular sister taxon, Choanoflagellata. (B) Domain organizations of β-catenin proteins. Metazoan β-catenins show a high degree of conservation of the central region, GSK3β/CK1α phosphorylation sites, and αE-catenin binding sites. At the C-terminus, motif A is conserved in only Bilateria and Cnidaria, whereas motif B is shared among all metazoan lineages, except for Ctenophora. In the choanoflagellate, Salpingoeca rosetta, possible GSK3β phosphorylation sites (S33/T41) were identified. (B) Surface depictions of β-catenin homology structural models were compared with mouse β-catenin. This confirmed that the three crucial arginine residues (dark blue) of nonbilaterians were localized similarly to mouse and Xenopus β-catenins. K556, R551, and K590 of B. mikado β-catenin were located at positions corresponding to mouse β-catenin R474, R469, and K508, but the R582, H578, R612, and Y654 assembly of mouse β-catenin is completely absent in B. mikado β-catenin. Structures visualized using UCSF Chimera software
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Fig. 3Xenopus secondary axis induction by nonbilaterian β-catenins. (A) Cnidarian (N. vectensis) and poriferan (E. fluviatilis) β-catenins (100 pg mRNA) induced a secondary body axis similar to that induced by Xenopus β-catenin. Ctenophore (B. mikado) β-catenin showed no inductive activity. (B) Western blot analysis of Flag-tagged β-catenins confirmed the expression of nonbilaterian β-catenin proteins in Xenopus embryos. (C) The majority of secondary body axes induced by injection of X. laevis (n = 24), N. vectensis (n = 20), and E. fluviatilis (n = 18) β-catenin mRNAs (100 pg) were complete (the second axis had a pair of eyes and a cement gland). This was followed by incomplete axis inductions in which head features were not fully developed. Secondary axes were not observed in embryos injected with B. mikado β-catenin mRNA (100 pg) (n = 19). (D) Expression of X. laevis, N. vectensis, and E. fluviatilis FLAG-tagged β-catenin mRNA (100 pg) in Xenopus embryos resulted in a significant increase in β-catenin TOPflash activity. No detectable level of TOPflash activation was observed by expression of B. mikado β-catenin. Asterisks denote statistical significance P < 0.0001 (Two-way ANOVA). This result was reproduced in two independent experiments
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Fig. 4Precipitation of β-catenin interacting proteins. (A) Scheme of our IP-MS analysis of β-catenin protein complexes. (B) Mass spectrometry volcano plots resulting from analysis of enriched metazoan FLAG-tagged β-catenins expressed in Xenopus embryos. High confidence proteins (1% FDR), with a fold abundance ratio ≥ 2 and P < 0.05, were considered “true” interactions
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Fig. 4Precipitation of β-catenin interacting proteins. (A) Scheme of our IP-MS analysis of β-catenin protein complexes. (B) Mass spectrometry volcano plots resulting from analysis of enriched metazoan FLAG-tagged β-catenins expressed in Xenopus embryos. High confidence proteins (1% FDR), with a fold abundance ratio ≥ 2 and P < 0.05, were considered “true” interactions
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Fig. 5Potential evolutionarily conserved β-catenin interactions. Several proteins, e.g., CDH1, ARVCF, CTNNA1, that interact with β-catenin mainly in bilaterian models, formed complexes with basal metazoan β-catenins. Furthermore, new interactions were identified that may shed additional light on functional evolution of β-catenin protein machinery in metazoans
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