A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation

Abstract

Signalling by the Wnt family of secreted lipoproteins has essential functions in development and disease. The canonical Wnt/beta-catenin pathway requires a single-span transmembrane receptor, low-density lipoprotein (LDL)-receptor-related protein 6 (LRP6), whose phosphorylation at multiple PPPSP motifs is induced upon stimulation by Wnt and is critical for signal transduction. The kinase responsible for LRP6 phosphorylation has not been identified. Here we provide biochemical and genetic evidence for a 'dual-kinase' mechanism for LRP6 phosphorylation and activation. Glycogen synthase kinase 3 (GSK3), which is known for its inhibitory role in Wnt signalling through the promotion of beta-catenin phosphorylation and degradation, mediates the phosphorylation and activation of LRP6. We show that Wnt induces sequential phosphorylation of LRP6 by GSK3 and casein kinase 1, and this dual phosphorylation promotes the engagement of LRP6 with the scaffolding protein Axin. We show further that a membrane-associated form of GSK3, in contrast with cytosolic GSK3, stimulates Wnt signalling and Xenopus axis duplication. Our results identify two key kinases mediating Wnt co-receptor activation, reveal an unexpected and intricate logic of Wnt/beta-catenin signalling, and illustrate GSK3 as a genuine switch that dictates both on and off states of a pivotal regulatory pathway.

Figure 1. GSK3 involvement in PPPSP phosphorylation

a. Five PPPSPxS motifs (motifs a-e) in LRP5/6. Site I, site II and another potential phosphorylation site are highlighted. b. GSK3β, but not the kinase-dead GSK3βKM, promoted LRP6 phosphorylation. LRP6 PPPSP phosphorylation in vivo was examined in 293T cells or MEFs that stably express LRP6 (VSVG-tagged) in this and other figures. c. Wnt3a-induced LRP6 PPPSP phosphorylation in MEFs was inhibited by LiCl (50 mM) or SB216763 (3 or 30 μM). d. Wnt3a-induced LRP6 PPPSP phosphorylation was abolished in MEFs that lack Gsk3α and Gsk3β. Gsk3α alleles were deleted via Cre expression in Gsk3β^(−/−); Gsk3α^(flox/flox) MEFs. β-actin: loading control. e. GSK3β(R96A) phosphorylated LRP6 PPPSP motif, but not β–catenin S33/S37/T41, detected via a phosphorylation-specific antibody. GSK3βKM phosphorylated neither LRP6 nor β–catenin.

Figure 2. Sequential phosphorylation of the PPPSPxS motif is induced by Wnt3a and required for LRP6 signalling

a. Scheme of five PPPSPxS motifs in LRP6 and of LDLRΔN-PPPSP. Site I, site II, and their Alanine substitutions are illustrated. b-e. TCF/β–catenin reporter assays. b, c. Alanine substitution of site II or site I diminished the activity of LDLRΔN-PPPSP (b) and LRP6 (c). Protein expression level was examined via the VSVG tag. The site II mutation did not affect the mobility shift, indicating normal site I phosphorylation (b). d, e. LRP6m5 and LRP6m10 did not synergize with Wnt3a (d) but inhibited signalling by Wnt3a (e) or by Wnt8 in Xenopus embryos (not shown). LRP6m5′ had diminished ability to synergize with Wnt3a (d) but did not inhibit Wnt3a signalling. f. Sequential phosphorylation of site I and site II in LDLRΔN-PPPSP and LRP6 (see Supplementary Fig. 4). Left: LDLRΔN-PPPSP/derivatives. Ab1490 and Ab1493 specifically recognized the phosphorylated/slower-migrating band. Right: GSK3β promoted LRP6 phosphorylation at site I and site II. g, h. LRP6 phosphorylation at site I and site II was induced by Wnt3a CM in MEFs (g), but not in MEFs lacking Gsk3α and Gsk3β (h).

Figure 3. Site I and site II phosphorylation by GSK3 and CK1 promotes LRP6 recruitment of Axin

a. DN-CK1α plus DN-CK1δ, but neither alone, prevented Wnt3a-induced site II, but not site I, phosphorylation. Ck1ε-/- MEFs (Supplementary Fig. 5) were used. b. Alanine substitution at site I or site II in the PPPSPxS motif prevented Axin-binding. Axin was co-expressed with LDLRΔN-PPPSP or mutants. Axin co-precipitated the phosphorylated motif, but neither the unphosphorylated one (fast migrating) nor the site I or site II mutant, which had normal site I phosphorylation. c. GST-LRP6C binding to Axin in vitro required phosphorylation by recombinant GSK3β plus CK1ε. Phosphorylated GST-LRP6C was examined for phosphorylation and used to precipitate cell extracts expressing Flag-tagged Axin. d. Axin binding to phosphorylated GST-LRP6C required site I and site II. Axin exhibited much diminished binding to GST-LRP6Cm5, GST-LRP6Cm5′, or GST-LRP6Cm10.

Figure 4. Membrane-associated GSK3 phosphorylates LRP6 and activates LRP6 and TCF/β-catenin signalling

a. Membrane-associated GSK3α/β and CK1α/ε is independent of Wnt3a stimulation. 10% GSK3α/β and 20% CK1α/ε were detected in the membrane fraction, which was marked by the transferrin receptor (TfR) and a lack of α-tubulin. Wnt3a stabilized cytosolic β-catenin. b. mGSK3β, like GSK3β, promoted LRP6 PPPSP phosphorylation. mGSK3β (VSVG-tagged) and GSK3β (HA-tagged) were detected via the two antibodies. c, d. TCF/β–catenin reporter assays. mGSK3β activated, whereas GSK3β inhibited, LRP6 signalling (c). mGSK3β did not synergize with LRP6m5; the slight stimulation seen might be due to the endogenous LRP5/6 (d). e, f. mGSK3β synergized with LRP6 to induce axis duplication (e) and Xnr3 expression (f) in Xenopus embryos. RNA doses injected are per embryo. e. The number above each bar indicates embryos injected. f. RT-PCR assay. EF-1α: loading control; -RT: without reverse transcriptase; con: un-injected embryos.