title: Analysis of the signaling activities of localization mutants of beta-catenin during axis specification in Xenopus.
abstract: In Xenopus embryos, beta-catenin has been shown to be both necessary and sufficient for the establishment of dorsal cell fates. This signaling activity is thought to depend on the binding of beta-catenin to members of the Lef/Tcf family of transcription factors and the regulation of gene expression by this complex. To test whether beta-catenin must accumulate in nuclei to establish dorsal cell fate, we constructed various localization mutants that restrict beta-catenin to either the plasma membrane, the cytosol, or the nucleus. When overexpressed in Xenopus embryos, the proteins localize as predicted, but surprisingly all forms induce an ectopic axis, indicative of inducing dorsal cell fates. Given this unexpected result, we focused on the membrane-tethered form of beta-catenin to resolve the apparent discrepancy between its membrane localization and the hypothesized role of nuclear beta-catenin in establishing dorsal cell fate. We demonstrate that overexpression of membrane-tethered beta-catenin elevates the level of free endogenous beta-catenin, which subsequently accumulates in nuclei. Consistent with the hypothesis that it is this pool of non-membrane-associated beta-catenin that signals in the presence of membrane-tethered beta-catenin, overexpression of cadherin, which binds free beta-catenin, blocks the axis-inducing activity of membrane- tethered beta-catenin. The mechanism by which ectopic membrane-tethered beta-catenin increases the level of endogenous beta-catenin likely involves competition for the adenomatous polyposis coli (APC) protein, which in other systems has been shown to play a role in degradation of beta-catenin. Consistent with this hypothesis, membrane-tethered beta-catenin coimmunoprecipitates with APC and relocalizes APC to the membrane in cells. Similar results are observed with ectopic plakoglobin, casting doubt on a normal role for plakoglobin in axis specification and indicating that ectopic proteins that interact with APC can artifactually elevate the level of endogenous beta-catenin, likely by interfering with its degradation. These results highlight the difficulty in interpreting the activity of an ectopic protein when it is assayed in a background containing the endogenous protein. We next investigated whether the ability of beta-catenin to interact with potential protein partners in the cell may normally be regulated by phosphorylation. Compared with nonphosphorylated beta-catenin, beta-catenin phosphorylated by glycogen synthase kinase-3 preferentially associates with microsomal fractions expressing the cytoplasmic region of N-cadherin. These results suggest that protein-protein interactions of beta-catenin can be influenced by its state of phosphorylation, in addition to prior evidence that this phosphorylation modulates the stability of beta-catenin.
authors.affiliation: Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle 98195, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Miller
authors.forename: R T
authors.initials: RT
authors.lastname: Moon
mesh_terms: D025601:Adenomatous Polyposis Coli Protein; D000818:Animals; D001667:Binding, Competitive; D015820:Cadherins / Q000096:biosynthesis; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D051180:Desmoplakins; D003896:Desmosomes / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000378:metabolism / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology; D017491:Genes, APC; D049452:Green Fluorescent Proteins; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism / Q000502:physiology; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology; D016296:Mutagenesis; D010766:Phosphorylation; D015398:Signal Transduction / Q000235:genetics*; D015534:Trans-Activators; D014981:Xenopus / Q000235:genetics / Q000502:physiology*; D029867:Xenopus Proteins; D051176:beta Catenin; D051185:gamma Catenin
chemical_list: D025601:Adenomatous Polyposis Coli Protein; C495266:CTNNB1 protein, Xenopus; C495270:CTNNB1 protein, human; D015820:Cadherins; D003598:Cytoskeletal Proteins; D051180:Desmoplakins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D015534:Trans-Activators; D029867:Xenopus Proteins; D051176:beta Catenin; D051185:gamma Catenin; D049452:Green Fluorescent Proteins