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: Post-translational stabilization of beta-catenin is a key step in Wnt signaling, but the features of beta-catenin required for stabilization are incompletely understood. We show that forms of beta-catenin lacking the unstructured C-terminal domain (CTD) show faster turnover than full-length or minimally truncated beta-catenins. Mutants that exhibit faster turnover show enhanced association with axin in co-transfected cells, and excess CTD polypeptide can compete binding of the beta-catenin armadillo (arm) repeat domain to axin in vitro, indicating that the CTD may restrict beta-catenin binding to the axin-scaffold complex. Fluorescent resonance energy transmission (FRET) analysis of cyan fluorescent protein (CFP)-arm-CTD-yellow fluorescent protein beta-catenin reveals that the CTD of beta-catenin can become spatially close to the N-terminal arm repeat region of beta-catenin. FRET activity is strongly diminished by the coexpression of beta-catenin binding partners, indicating that an unliganded groove is absolutely required for an orientation that allows FRET. Amino acids 733-759 are critical for beta-catenin FRET activity and stability. These data indicate that an N-terminal orientation of the CTD is required for beta-catenin stabilization and suggest a model where the CTD extends toward the N-terminal arm repeats, shielding these repeats from the beta-catenin destruction complex.Journal of Biological Chemistry 09/2009; 284(41):28222-31. · 4.65 Impact Factor
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ABSTRACT: Wnt/β-catenin signaling is a branch of a functional network that dates back to the first metazoans, and is involved in a broad range of biological systems; including stem cells, embryonic development and adult organs. De-regulation of components involved in Wnt/β-catenin signaling has been implicated in a wide spectrum of diseases including, a number of cancers and degenerative diseases. The key mediator of Wnt signaling, β-catenin, serves several cellular functions. It functions in a dynamic mode at multiple cellular locations, including the plasma membrane, where β-catenin contributes to the stabilization of intercellular adhesive complexes, the cytoplasm where β-catenin levels are regulated and the nucleus where β-catenin is involved in transcriptional regulation and chromatin interactions. Central effectors of β-catenin levels are a family of cysteine-rich secreted glycoproteins, known as Wnt morphogens. Through the LRP5/6-Frizzled receptor complex, Wnts regulate the location and activity of the destruction complex and consequently intracellular β-catenin levels. However, β-catenin levels and their effects on transcriptional programs are also influenced by multiple other factors including hypoxia, inflammation, hepatocyte growth factor-mediated signaling, and the cell adhesion molecule E-cadherin. The broad implications of Wnt/β-catenin signaling in development, in the adult body and in disease render the pathway a prime target for pharmacological research and development. The intricate regulation of β-catenin at its various locations provides alternative points for therapeutic interventions.Current pharmaceutical design 09/2012; · 4.41 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).