Wnt stabilization of β-catenin reveals principles for morphogen receptor-scaffold assemblies

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 the 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.

Fig. 1

Wnt-induced Axin dephosphorylation by PP1 and effects of I2 on Wnt signaling and Xenopus anteriorization. (A) Wnt3a-induced Axin dephosphorylation, LRP6 phosphorylation and β-catenin stabilization in L cells. Protein detections were performed by immunoblotting throughout the paper unless specified otherwise. actin: a loading control. (B) Effect of PP1cγ overexpression on phosphorylation of Axin but not LRP6 in HEK293T cells. (C and D) Effects of TM (C) or I2 overexpression (D) on Wnt3a-induced Axin dephosphorylation and β-catenin stabilization in HEK293T cells. (E) Effects of I2 depletion with shRNAs (sh-I2) on β-catenin stabilization (top) and TOP-Flash (bottom) in HEK293T cells. shGFP: an shRNA against GFP. Error bars represent SD of triplicates. (F) Effect of I2 depletion with an I2MO in embryos and its rescue by human I2 mRNA. CoMo/Uninj: control MO-injected/uninjected.

Fig. 2

Wnt regulation of Axin-β-catenin and Axin-LRP6 association, and of β-catenin phosphorylation in the Axin complex. (A and B) Quantifications of Wnt3a effects on ratios of Axin-associated β-catenin versus input β-catenin (left), Axin-associated phospho-β-catenin versus Axin-associated β-catenin (middle), and Axin-associated LRP6 versus total LRP6 (right) in L (A) and HEK293T (B) cells. Error bars represent SEM of triplicates.

Fig. 3

Effects of Wnt-regulated Axin phosphorylation on its binding to β-catenin and LRP6. (A) HEK293T cells overexpressing I2 (or control) were treated with Wnt3a. Lysates were incubated with GST-β-catenin or GST-phospho-LRP6C in vitro. Bound or input Axin, and phospho-Axin were examined. (B and C) L cells treated with TM (B) and HEK293T cells overexpressing I2 (or control) (C) were stimulated with Wnt3a for 0.5 hours. Lysates were immunoprecipitated with an Axin antibody, and Axin-associated β-catenin and LRP6, and input proteins were examined. Quantifications show Axin-associated β-catenin versus cytosolic β-catenin (top), and Axin-associated LRP6 versus total LRP6 (bottom). Note that TM or I2 reduced Wnt3a-induced β-catenin levels (Input). Error bars represent SEM of triplicates. *P<0.05 and **P<0.01 with student-Newman-Keuls test. (D) HEK293T cells expressing an I2 (or control) shRNA were treated with Wnt3a for 0.5 hours. Lysates were immunoprecipitated with an Axin antibody, and Axin-associated LRP6 and input lysates were examined. (E) In vitro binding to GST-phospho-LRP6C by Axin from lysates of WT or Gsk3α−/−;Gsk3β−/− mouse ES cells. Bound or input Axin was examined.

Fig. 4

A phosphorylation-regulated Axin intra-molecular interaction and a Wnt signaling model. (A) Association of Axin-DIX with GST-Axin-BCD and its inhibition by GSK3 phosphorylation of Axin-BCD. (B) Competition of β-catenin association with GST-Axin-BCD by Axin-DIX (lanes 1–6) and vice versa (lanes 7–12). Purified recombinant proteins were used in these in vitro assays. Axin-DIX or β-catenin was detected by immunoblotting and GST or GST-Axin-BCD by Ponceau staining (A and B). The lower band of β-catenin (B) was a proteolytic fragment. (C) Comparisons of Axin(SD4), Axin(SA4), and Axin(DA) with Axin in antagonizing Wnt-induced TOP-Flash in HEK293T cells. X-axes represent DNA doses transfected. Note larger TOP-Flash differences at lower overexpression doses. Insets show levels of overexpressed Axin at the 1ng dose and that of endogenous Axin (con). (D) An “Axin inactivation” model for Wnt stabilization of β-catenin. APC and CK1α were omitted for clarity. See text and fig. S18.