A GSK3beta phosphorylation site in axin modulates interaction with beta-catenin and Tcf-mediated gene expression.
ABSTRACT Upon binding of a Wnt to its receptor, GSK3beta is inhibited through an unknown mechanism involving Dishevelled (Dsh), resulting in the dephosphorylation and stabilization of beta-catenin, which translocates to the nucleus and interacts with Lef/Tcf transcription factors to activate target gene expression. Axin is a scaffold protein which binds beta-catenin and GSK3beta (as well as several other proteins) and thus promotes the phosphorylation of beta-catenin. Here we report that Axin is phosphorylated on Ser and Thr residues in several regions in vivo, while only one region (amino acids 600-672) is efficiently phosphorylated by GSK3beta in vitro. Site-directed mutagenesis, together with in vitro and in vivo phosphorylation assays, demonstrates that Axin residues T609 and S614 are physiological GSK3beta targets. Substitutions for one or more of these residues, which lie within a beta-catenin binding site, reduce the ability of Axin to modulate Wnt-induced signaling in a Lef/Tcf reporter assay. These amino acid substitutions also reduce the binding between Axin and beta-catenin. We propose a model in which inhibition of GSK3beta activity upon Wnt signaling leads to the dephosphorylation of GSK3beta sites in Axin, resulting in the release of beta-catenin from the phosphorylation complex.
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ABSTRACT: The precise orchestration of two opposing protein complexes - one in the cytoplasm (β-catenin destruction complex) and the other at the plasma membrane (LRP6 signaling complex) - is critical for controlling levels of the transcriptional co-factor β-catenin, and subsequent activation of the Wnt/β-catenin signal transduction pathway. The Wnt pathway component Axin acts as an essential scaffold for the assembly of both complexes. How the β-catenin destruction and LRP6 signaling complexes are modulated following Wnt stimulation remains controversial. A recent study in Science by He and coworkers reveals an underlying logic for Wnt pathway control in which Axin phosphorylation toggles a switch between the active and inactive states. This mini-review focuses on this and two other recent studies that provide insight into the initial signaling events triggered by Wnt exposure. We emphasize regulation of the β-catenin destruction and LRP6 signaling complexes and propose a framework for future work in this area.BioEssays 09/2013; · 5.42 Impact Factor
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ABSTRACT: Wnt signaling stabilizes β-catenin through the LRP6 receptor signaling complex, which antagonizes the β-catenin destruction complex. The Axin scaffold and associated glycogen synthase kinase-3 (GSK3) have central roles in both assemblies, but the transduction mechanism from the receptor to the destruction complex is contentious. We report that Wnt signaling is governed by phosphorylation regulation of Axin scaffolding function. Phosphorylation by GSK3 kept Axin activated ("open") for β-catenin interaction and poised for engagement of LRP6. Formation of the Wnt-induced LRP6-Axin signaling complex promoted Axin dephosphorylation by protein phosphatase-1 and inactivated ("closed") Axin through an intramolecular interaction. Inactivation of Axin diminished its association with β-catenin and LRP6, thereby inhibiting β-catenin phosphorylation and enabling activated LRP6 to selectively recruit active Axin for inactivation reiteratively. Our findings reveal mechanisms for scaffold regulation and morphogen signaling.Science 04/2013; · 31.20 Impact Factor
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ABSTRACT: Reports Signaling by secreted Wnt morphogens governs developmental, ho-meostatic, and pathological processes by regulating β-catenin stability, and represents a critical target for cancer and disease therapeutics (1, 2). Without Wnt stimulation, cytosolic β-catenin concentrations are kept low because a "destruction complex" assembled by the Axin scaffold binds to β-catenin, Adenomatosis polyposis coli (APC), casein kinase-1α (CK1α), and glycogen synthase kinase-3 (GSK3), and promotes phos-phorylation of β-catenin by CK1α and GSK3 thus ensuring β-catenin ubiquitination and degradation (1–3). Upon Wnt stimulation, a receptor complex on the cell surface is formed between Frizzled (Fz) and LDL receptor-related protein 6 (LRP6), resulting in phosphorylation and acti-vation of LRP6 and its recruitment of Axin (4–7). Assembly of the Fz-LRP6 complex and associated Dishevelled (Dvl) and the Axin destruc-tion complex, referred to collectively as the "LRP6 signaling complex (signalosome)," inhibits phosphorylation of β-catenin thereby causing its stabilization (6–10). The mechanism by which LRP6 activation leads to β-catenin stabilization remains enigmatic (1, 2, 11). Axin is a phospho-protein and central to assemblies of both destruc-tion (12–15) and signaling complexes (4–10), and becomes dephosphor-ylated upon Wnt stimulation (16, 17). We generated an antibody, Ab-pS497/500 (fig. S1, A to C), for Axin phosphorylated at serines 497 and 500, which are GSK3 phosphorylation sites in vitro (18). Axin phos-phorylation at S497/S500 was decreased within 15-30 min of Wnt3a treatment of mouse L fibroblasts (Fig. 1A), embryonic fibroblasts (fig. S1D), and human embryonic kidney (HEK) 293T cells (Fig. 1, C and D). Wnt-induced dephosphorylation of Axin likely reflects the counterbalance between GSK3 and a protein phospha-tase (PP) such as PP1, whose catalytic subunit, PP1c, was identified in an RNAi screen in Drosophila cells as a requirement for Wnt/β-catenin signal-ing (19). Through a functional cDNA overexpression screen in HEK293T cells, we identified PP1cγ, one of the three PP1c genes in the human genome (20), as an activator of the Wnt/β-catenin signaling reporter TOP-Flash (fig. S2A). PP1cγ overexpression de-creased phosphorylation of Axin but not of LRP6 (Fig. 1B); a pharmacologi-cal PP1 inhibitor, Tautomycin (TM), prevented Wnt-induced dephosphorylation of Axin without affecting LRP6 phosphorylation (Fig. 1C and fig. S3). PP1 has pleiotropic roles and its specificity is conferred by hundreds of PP1c-binding proteins (20). Inhibitor-2 (I2, or PPP1R2) is a specific inhibitor of PP1c (20). Overexpression of I2 countered Wnt3a-induced Axin dephosphorylation (without affecting LRP6 phosphorylation) and β-catenin stabilization (Fig. 1D and fig. S2B), inhibited Wnt3a-or PP1cγ-activated TOP-Flash (fig. S2, C and D), and an-tagonized β-catenin stabilization by an activated LRP6 (fig. S2E). Depletion of the endogenous I2 with shRNAs result-ed in accumulation of β-catenin and increased TOP-Flash (Fig. 1E and fig. S2F). A morpholino antisense-oligonucleotide (MO) that targets Xenopus I2 mRNA and blocked I2 protein synthesis caused deficiency in Xenopus head development and reduced anterior marker expression, which were restored by human I2 mRNA injection or knockdown of β-catenin (Fig. 1F and fig. S4). Thus I2 antagonizes Wnt/β-catenin signal-ing and participates in vertebrate anteriorization, which requires Wnt pathway inhibition (21).