Proc Natl Acad Sci U S A
June 10, 2008;
LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3''s phosphorylation of beta-catenin.
Wnt/beta-catenin signaling controls various cell fates in metazoan development and is misregulated in several cancers and developmental disorders. Binding of a Wnt ligand to its transmembrane coreceptors inhibits phosphorylation and degradation of the transcriptional coactivator beta-catenin, which then translocates to the nucleus
to regulate target gene expression. To understand how Wnt signaling prevents beta-catenin degradation, we focused on the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6
), which is required for signal transduction and is sufficient to activate Wnt signaling when overexpressed. LRP6
has been proposed to stabilize beta-catenin by stimulating degradation of Axin, a scaffold protein required for beta-catenin degradation. In certain systems, however, Wnt-mediated Axin turnover is not detected until after beta-catenin has been stabilized. Thus, LRP6
may also signal through a mechanism distinct from Axin degradation. To establish a biochemically tractable system to test this hypothesis, we expressed and purified the LRP6
intracellular domain from bacteria and show that it promotes beta-catenin stabilization and Axin degradation in Xenopus egg
extract. Using an Axin mutant that does not degrade in response to LRP6
, we demonstrate that LRP6
can stabilize beta-catenin in the absence of Axin turnover. Through experiments in egg
extract and reconstitution with purified proteins, we identify a mechanism whereby LRP6
stabilizes beta-catenin independently of Axin degradation by directly inhibiting GSK3''s phosphorylation of beta-catenin.
Proc Natl Acad Sci U S A
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Fig. 1. Recombinant LRP6ICD activates Wnt signaling in vivo and in Xenopus egg extract. (A) LRP6ICD spans the intracellular domain of mouse LRP6 (amino acids 1397–1614) and does not include its transmembrane domain. ECD, extracellular domain; ICD, intracellular domain; TM, transmembrane domain; HT, 6Xhistidine tag. (B) Coomassie-stained gel of recombinant LRP6ICD (1 μg) purified from bacteria. (C) Injection of LRP6ICD protein (33 nM) into each ventral blastomere of 4-cell Xenopus embryos promotes development of a complete ectopic axis (Bottom Left, embryo side view; Bottom Right, embryo ventral view) in 73% of embryos (n = 15). A lower dose of LRP6ICD protein (20 nM) promotes axis duplication in 46% of embryos (n = 15). (D) Injection of LRP6ICD (33 nM) at the four-cell stage promotes ectopic transcription of Wnt/β-catenin targets Xnr3 and siamois in animal caps as assayed by RT-PCR. WE, whole embryos; Caps, animal caps; WE-RT, no reverse transcriptase added; ODC, ornithine decarboxylase (loading control). (E) Addition of LRP6ICD (1.6 μM) to Xenopus egg extract prevents degradation of radiolabeled, IVT β-catenin and promotes degradation of radiolabeled, IVT Axin and Axin2. (F) Unlike LRP6ICD, LRP6ICD(PPPAPX5) (1.6 μM) does not inhibit β-catenin degradation or promote Axin degradation.
Fig. 2. LRP6ICD mediates Axin degradation independently of GSK3 inhibition. (A) LRP6ICD stimulates addition of GST-ubiquitin to radiolabeled, IVT Axin in egg extract. GST-ubiquitin conjugates were pulled down with glutathione beads at indicated times and analyzed by using SDS/PAGE and autoradiography. Asterisk indicates full-length Axin. (B) Addition of the proteasome inhibitor MG132 (1 mM) to egg extract inhibits LRP6ICD-mediated Axin degradation. (C) Degradation of Axin mutants in egg extract in the presence of LRP6ICD. The indicated LRP5/6-binding region on Axin is based on previous Axin-LRP5 and Axin-Arr yeast two-hybrid studies (6, 7); dotted lines represent large deletions of Axin that were not further mapped, and the borders of Axin-LRP5/6 interaction likely reside within the dotted lines. RGS, RGS domain; GBS, GSK3 binding site; βBS, β-catenin binding site; DIX, DIX domain. (D) LRP6ICD promotes degradation of Axin and Axin375–869, whereas the GSK3 inhibitor BIO (50 μg/ml, Calbiochem) promotes degradation of Axin but not Axin375–869. (E) Inhibition of GSK3-mediated Axin phosphorylation [by LiCl (50 mM) or mutagenesis (AxinSA)], but not incubation with LRP6ICD, alters the trypsin proteolysis pattern of IVT Axin after incubation in egg extract for 30 min (note bands at level of asterisk). All experiments used equal concentrations of IVT Axin. An SDS/PAGE autoradiograph of IVT Axin and AxinSA before trypsin treatment is shown at right.
Fig. 3. LRP6ICD's inhibition of GSK3-mediated β-catenin phosphorylation is sufficient to promote β-catenin stabilization in the absence of Axin degradation. (A) LRP6ICD inhibits β-catenin degradation in extract where endogenous Axin is replaced by nondegradable Axin1-713. Addition of IVT Axin or Axin1-713 restores the ability of Axin-depleted extract to degrade radiolabeled β-catenin. LRP6ICD inhibits both IVT Axin and Axin1-713-induced β-catenin-degradation. (B) LRP6ICD does not affect the ability of Axin to bind GSK3 or β-catenin in egg extract. Endogenous Axin was immunoprecipitated from extract and immunoblotted for GSK3, β-catenin, and Axin. (C) Incubation of LiCl (50 mM) or LRP6ICD [but not LRP6ICD(PPPAPX5)] in egg extract (30 min) inhibits phosphorylation of endogenous β-catenin at GSK3 target sites P33/37/41. Immunoblot of LRP6ICD from the same gel reveals that LRP6ICD, but not LRP6ICD(PPPAPX5), is phosphorylated at the PPPSP Ser-1490. All samples were blotted from a single gel. (D) LRP6ICD inhibits GSK3-mediated β-catenin phosphorylation in extract in which endogenous Axin is replaced by nondegradable Axin1-713. Axin depletion did not affect total β-catenin levels as assayed by immunoblot (Fig. S7). Depletion of endogenous Axin prevents β-catenin P33/37/41 phosphorylation. Addition of IVT Axin1-713 restores β-catenin phosphorylation in Axin-depleted extract. LRP6ICD inhibits IVT Axin1-713-induced β-catenin phosphorylation. Extracts were analyzed after 2-h incubation. All samples were blotted from a single gel; intervening lanes were removed for clarity.
Fig. 4. LRP6ICD directly and specifically inhibits GSK3's phosphorylation of β-catenin. (A) LiCl (50 mM) inhibits phosphorylation of β-catenin P33/37/41 and exogenous Tau P396 by GSK3, whereas LRP6ICD inhibits phosphorylation of β-catenin, but not Tau, by GSK3. Extract was supplemented with GSK3 to enhance detection of phosphorylated Tau as previously described (32). β-catenin and Tau from the same reaction sample were immunoblotted from a single gel; intervening lanes were removed for clarity. (B) In an in vitro kinase assay containing purified, recombinant Axin (0.1 μM), GSK3 (0.79 μM), CK1 (1.37 μM), Tau (0.34 μM), and β-catenin (0.22 μM), LRP6ICD (4.1 μM) inhibits phosphorylation of β-catenin P33/37/41 by GSK3 without inhibiting the phosphorylation of Tau P396 by GSK3. (C) In a kinase reaction in which recombinant Axin is absent, phosphorylation of β-catenin by GSK3 is inhibited by LRP6ICD but not by LRP6ICD(PPPAPX5) (D). For (B), (C), and (D), β-catenin and Tau were incubated in the same reaction and immunoblotted from a single gel.
Bilic, Wnt induces LRP6 signalosomes and promotes dishevelled-dependent LRP6 phosphorylation. 2007, Pubmed