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Wnt Induces LRP6 Signalosomes and Promotes Dishevelled-Dependent LRP6 Phosphorylation

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Science  15 Jun 2007:
Vol. 316, Issue 5831, pp. 1619-1622
DOI: 10.1126/science.1137065

Abstract

Multiple signaling pathways, including Wnt signaling, participate in animal development, stem cell biology, and human cancer. Although many components of the Wnt pathway have been identified, unresolved questions remain as to the mechanism by which Wnt binding to its receptors Frizzled and Low-density lipoprotein receptor–related protein 6 (LRP6) triggers downstream signaling events. With live imaging of vertebrate cells, we show that Wnt treatment quickly induces plasma membrane–associated LRP6 aggregates. LRP6 aggregates are phosphorylated and can be detergent-solubilized as ribosome-sized multiprotein complexes. Phospho-LRP6 aggregates contain Wnt-pathway components but no common vesicular traffic markers except caveolin. The scaffold protein Dishevelled (Dvl) is required for LRP6 phosphorylation and aggregation. We propose that Wnts induce coclustering of receptors and Dvl in LRP6-signalosomes, which in turn triggers LRP6 phosphorylation to promote Axin recruitment and β-catenin stabilization.

Wnt signaling plays diverse roles during embryonic development such as axis formation and nervous system patterning, and it is implicated in human disease, including cancer (14). Wnt ligands are thought to form a ternary complex with low-density lipoprotein receptor–related proteins 5 and 6 (LRP5/6) and Frizzled (Fz) receptors (5). A key step after Wnt stimulation is the phosphorylation of the LRP6 intracellular domain by Casein kinase 1γ (CK1 γ). This phosphorylation event activates LRP6 and promotes recruitment of the negative regulator Axin (68), which, in turn, stabilizes the Wnt signaling transducer β-catenin (5). Many aspects of Wnt signal transduction at the plasma membrane remain elusive. For example, the mechanism that triggers LRP6 phosphorylation by CK1γ is unknown, and it is unclear how Fz and the scaffold protein Dishevelled (Dvl) (911) fit in.

To analyze LRP6 activation upon Wnt stimulation, we used real-time confocal microscopy. Live cell imaging allows a dynamic view of receptor activation to reveal spatiotemporal aspects that can not be visualized by methods such as biochemical analysis or immunofluorescence of fixed samples. The negative Wnt regulator Axin is recruited to the phosphorylated cytoplasmic domain of LRP6 (12). We therefore transfected HeLa cells with enhanced cyan fluorescent protein (ECFP)–Axin (green signal) and LRP6–enhanced yellow fluorescent protein (EYFP) (red signal) and monitored their localization simultaneously. When the proteins overlap, yellow signals appear (Fig. 1A and movie S1). In unstimulated cells, Axin was localized in intracellular punctae, and LRP6 showed continuous staining at the plasma membrane. No yellow signals were visible, indicating that the two proteins resided in different compartments. However, within 15 min of Wnt treatment, a small fraction of LRP6 protein started coalescing into punctate structures at or below the plasma membrane (fig. S1A), which colocalized with Axin and were therefore yellow. As soon as yellow signals were observed, they were punctate in nature, without transiting through a continuous plasma membrane staining. This suggests that Axin is recruited to LRP6 when the receptor is aggregated. This is supported by biochemical analyses (see below). The number of the LRP6-Axin aggregates plateaued after 1 to 2 hours, and once they formed they were quite stable over the course of 30 min. As seen in time-space plots, neither LRP6 nor Axin showed extensive movements or further coalescence into higher-order structures (fig. S1, B and C).

Fig. 1.

Wnt induces phospho-LRP6 aggregates. (A) Live cell imaging of HeLa cells transfected with ECFP-Axin (green) and LRP6-EYFP (red) treated at t0 with Wnt-conditioned medium. Arrows point to membrane-associated aggregates of both proteins (yellow) forming after 13 min of Wnt treatment. Individual frames from a movie (movie S1) are shown. (B) Immunofluorescence of HeLa cells transfected with LRP6-EYFP and treated for 3 hours with control (Con) or Wnt3a-conditioned medium. Scale bars indicate 5 μm. tot., total. (C) Tp1479 immunofluorescence staining of endogenous phospho-LRP6 and Dvl proteins in Xenopus animal caps treated for 30 min with control (Con) or Wnt3a-conditioned medium. “Magnified” shows unmerged and merged pictures of the white-boxed area. Hoechst-stained nuclei are blue; scale bar, 50 μm.

Because Axin recruitment requires CK1γ-mediated phosphorylation of LRP6, we carried out immunofluorescence with a phosphospecific antibody recognizing phosphothreonine 1479 (Ab-Tp1479) in the intracellular domain (7) of LRP6. Ab-Tp1479 detected LRP6 phosphorylation within 10 min of Wnt stimulation in Western blots, indicating that this is an immediate response (7). In unstimulated cells, Ab-Tp1479 immunoreactivity was undetectable (Fig. 1B top). In contrast, after Wnt3a treatment, Ab-Tp1479 detected discrete punctate signals at the plasma membrane, which colocalized with a small fraction of total LRP6 (Fig. 1B bottom).

To test whether phospho-LRP6 aggregates are also observed under physiological conditions in untransfected cells and in vivo, we analyzed animal caps of Xenopus embryos, which are Wnt-responsive. Robust LRP6-Tp1479 immunoreactivity was reproducibly induced within 30 min of Wnt treatment in the form of punctate structures (Fig. 1C). These phospho-LRP6 aggregates colocalized with Dvl2 (see below). We conclude that phospho-LRP6 aggregates occur under physiological conditions as a rapid response to Wnt stimulation.

The results indicated that Wnt signaling induces large plasma membrane–associated aggregates enriched in phosphorylated LRP6, Axin, and Dvl. To further characterize phospho-LRP6 aggregates, we performed colocalization analyses with markers for vesicular traffic compartments. Occasional colocalization of Wnt-induced phospho-LRP6 aggregates was observed with the fluid phase marker dextran Texas red (fig. S2A), whereas no colocalization was seen with other markers of vesicular transport: TGN 38 (Golgi), Calnexin (endoplasmic reticulum), EEA1 (early endosome), or Clathrin (endocytic vesicles) (fig. S2). Partial colocalization of phospho-LRP6 aggregates was observed with Caveolin within 1 hour but not with 3.5-hour Wnt treatment (Fig. 2A and fig. S2, F and G). Caveolin—which is a marker of caveolae, cholesterol-rich invaginations of the plasma membrane—was recently shown to colocalize with LRP6 and to be required for Wnt-mediated LRP6 endocytosis (13).

Fig. 2.

Colocalization of phospho-LRP6 aggregates with Caveolin and Wnt pathway members. (A to E) HeLa cells were transfected with LRP6 and the indicated protein (green) and stained for phospho-LRP6 (red). Cells were treated with Wnt3a-conditioned medium for 1 hour (A) or 3 hours (B to E). (F) HeLa cells transfected with membrane-bound EYFP (green) and constitutively active LRP6(ΔE1-4) and stained for phospho-LRP6 (red). Note large cytoplasmic aggregates. Scale bar, 10 μm.

In contrast to most trafficking markers, colocalization of phospho-LRP6 aggregates was observed with other components of the Wnt/β-catenin network, including Fz8, glycogen synthesis kinase 3β (GSK3β), Dvl, and Axin (Fig. 2, B to E). Furthermore, very pronounced phosphorylated aggregates were observed in the cytoplasm of cells transfected with constitutively active LRP6 (LRP6ΔE1-4, lacking the ligand binding domain) (fig. S2H) without Wnt stimulation (Fig. 2F). This suggests that LRP6ΔE1-4 clusters even without Wnt. Wnt-dependent colocalization was also observed for endogenous LRP6 and Dvl2 and Axin in P19 cells (fig. S3A), thus corroborating co-aggregation observed for Xenopus LRP6 and Dvl2 (Fig. 1C). We conclude that phospho-LRP6 aggregates represent specialized compartments that assemble components of the Wnt and β-catenin pathway. This compartment is associated with the plasma membrane, partially positive for Caveolin, and largely devoid of fluid-phase marker.

To analyze phospho-LRP6 aggregates biochemically, we solubilized the complexes in detergent and separated them by sucrose density gradient centrifugation (Fig. 3). In Wnt-stimulated and unstimulated cells, total LRP6 showed a relatively discrete peak, cosedimenting approximately with thyroglobulin (670 kD, 19S) (Fig. 3A). In contrast, in Wnt-stimulated cells about half of phospho-LRP6 was “tailing” into much heavier fractions (fractions 1 to 7), overlapping the ribosome peaks, whereas only little phospho-LRP6 was detectable in unstimulated cell lysates. Constitutively active LRP6ΔE1-4 showed even greater high–molecular weight (MW) enrichment than phospho-LRP6, despite its four times lower MW (Fig. 3B). This indicates that LRP6ΔE1-4 undergoes strong self-aggregation without Wnt stimulation. In contrast, cotransfected green fluorescent protein (GFP) was never detected in high MW fractions, even in a deliberately overexposed immunoblot (Fig. 3B).

Fig. 3.

LRP6 analysis in sucrose gradient sedimentation. (A to C) Sucrose gradient sedimentation analysis of Triton X-100 lysates. Total and phosphorylated (Tp1479) LRP6 amounts were analyzed by immunoblot. (A) HEK 293T cells were transfected with FLAG-hLRP6, Mesd, mFz8, hAxin, and GSK3β and treated with control (Con) or Wnt3a-conditioned medium for 3 hours. (B) HEK 293T cells were transfected with FLAG-LRP6ΔE1-4 and GFP. (C) MEFs were treated with control (Con) or Wnt3a-conditioned medium for 1 hour. (D) Immunoprecipitation (IP) of FLAG-LRP6 from the indicated pooled sucrose gradient fractions [experiment in (A)] and detection of coimmunoprecipitated Axin and GSK3β by immunoblot. Asterisk marks unspecific band; white asterisk indicates a staining artifact.

Endogenous phospho-LRP6 in nontransfected mouse embryonic fibroblasts (MEFs) was also detectable in fractions 3 to 7 in a Wnt-dependent manner (Fig. 3C), corroborating that this is not an overexpression artefact. The weaker signal in this case, as well as the observed heterogeneity of phospho-LRP6 aggregates, may be due to their instability, for example, because of partial dephosphorylation and/or degradation during the biochemical analysis.

Axin and GSK3β were cosedimenting with phospho-LRP6 in heavy fractions independent of Wnt treatment (fig. S4). Dvl2, despite its ability to readily form large intracellular polymers (14, 15), was found in the light fractions (fig. S4), supporting the idea that these polymers are highly dynamic, short-lived, and hence unstable in lysates [see below and (16)].

To confirm that LRP6, Axin, and GSK3β not only cosediment but are in a complex, we carried out immunoprecipitations from pooled fractions of the sucrose gradient (Fig. 3D). This analysis showed that, whereas Axin is present in all phospho-LRP6–containing fractions (fig. S4), it is complexed only to the aggregated LRP6 form (fractions 3 to 5) and only after Wnt stimulation (compare lane 6 to lane 7). Also, GSK3β is bound preferentially to aggregated LRP6.

These data with detergent-solubilized cell lysates indicate (i) that LRP6 aggregation is accompanied by its phosphorylation at T1479, (ii) that the microscopically observed phospho-LRP6 aggregates are not the result of cotrapping of Wnt pathway components in a cargo vesicle (17, 18, 13) but that they represent high-MW protein complexes, (iii) that constitutive activation of LRP6 (ΔE1-4) is accompanied by spontaneous aggregation, and (iv) that Axin and GSK3β are preferentially associated with aggregated LRP6.

Dvl is a component of the Wnt pathway that is also known to oligomerize and to occur in punctate structures (14, 19, 15). Recent studies (20, 16) demonstrated that Dvl can polymerize and form large protein assemblies that are highly dynamic, short-lived, and reversible, reminiscent of the LRP6 aggregates described here. We observed that endogenous Dvl2 forms membrane-associated aggregates in response to Wnt stimulation (fig. S3B), similar to LRP6. This is an important observation, because Dvl punctae tend to be dismissed as an unphysiological response due to overexpression (21).

In light of these and previous findings (22) and our observation that endogenous Dvl2 and activated LRP6 reside in a common protein assembly, we asked whether Dvl may be required for LRP6 phosphorylation and aggregation. Indeed, knockdown of dsh/dvl by RNA interference (RNAi) in Drosophila and small interfering RNAs (siRNAs) in MEFs, respectively, inhibited Wnt-induced LRP6 phosphorylation at the CK1γ site T1479 (Fig. 4A). Similarly, dominant negative Dvl mutants M1 and M2, which block both self-aggregation and signaling of wild-type Dvl (16), also blocked LRP6 phosphorylation (fig. S5A). Concomitantly, phospho-LRP6 aggregates were reduced, as determined by immunofluorescence (Fig. 4B).

Fig. 4.

Dsh/Dvl is required for LRP6 phosphorylation and aggregate formation. (A) Immunoblot of transfected LRP6 from Drosophila S2R+ cells and endogenous LRP6 and Dvl2 from MEF, treated with dsh dsRNA and dvl1/2/3 siRNAs, respectively. (B) HeLa cells were transfected with LRP6-EYFP, dominant negative dvl mutants M1 and M2, or dominant negative CK1γ1K73R and analyzed by immunofluorescence microscopy for phospho-LRP6 aggregates. Cells positive for EYFP were scored for phospho-LRP6 staining. Error bars indicate standard deviation of the mean; n = 3 experiments. (C) Immunofluorescence of HeLa cells transfected with LRP6-EYFP and dominant negative CK1γ1K73R, treated with Wnt3a-conditioned medium and stained with Ab Tp1479. LRP6-EYFP (tot. LRP6) and phospho-LRP6 are shown. Note Wnt-induced LRP6 aggregates (arrowheads and higher-magnification insets), which are nonphosphorylated. (D) LRP6-signalosome model. Wnts bridge LRP6 and Fz transmembrane receptors and promote recruitment of polymers of Dvl, which binds Fz. Dvl and Axin co-polymerize LRP6, which induces receptor phosphorylation by CK1γ. Axin sequestration in LRP6-signalosomes may block GSK3β phosphorylation of β-catenin, leading to β-catenin accumulation. Other components of the Wnt pathway, such as GSK3β and adenomatous polyposis coli (APC), are likely components of the LRP6-signalosome but not shown for clarity.

The scaffold protein Dvl was previously thought to act downstream of LRP6 because dsh overexpression activates ß-catenin signaling in Drosophila LRP6 (arrow) mutants (23) and because the constitutively active Dfz2-Arrow fusion protein is inactive in dsh mutants (24). The explanation for this discrepancy may be that overexpressing Dsh/Dvl leads to artificial sequestration of Axin or that the protein has multiple functions in the Wnt pathway.

Taken together, the results suggest that Dvl-mediated co-aggregation triggers LRP6 phosphorylation by CK1γ. In this model (Fig. 4D), upon Wnt signaling Dvl aggregates form at the plasma membrane, where they co-cluster LRP6 with other pathway components including Fz, Axin, and GSK3β, in a “LRP6-signalosome.” The role of Wnt would be to bridge LRP6 and Fz (25, 5), which copolymerize on a Dvl platform. Clustering of LRP6 then provides a high local receptor concentration that triggers phosphorylation by CK1γ and Axin recruitment.

Predictions of this model are as follows: (i) artificial oligomerization of LRP6 should activate the receptor and (ii) oligomerized LRP6 should signal independent of Dvl. Indeed, forced oligomerization of LRP6 using a synthetic multimerizer is sufficient to induce Wnt signaling, and this oligomerization bypasses the need for Dvl (25). (iii) Constitutively active LRP6 should signal independently of Dvl because its self-aggregation should bypass the need for Dvl polymers. This is also the case as shown in reporter assays with Dvl siRNA knockdown (fig. S5, B and C), which supports previous findings (26, 25). (iv) If LRP6 aggregation is a prerequisite for phosphorylation by CK1γ rather than its consequence, LRP6 aggregates should form even when the kinase is blocked. This is the case: Nonphosphorylated LRP6 aggregates were observed in response to Wnt treatment in cells transfected with dominant-negative CK1γ (Fig. 4C). The model of LRP6-signalosomes not only provides a mechanism for Wnt signal transduction but may also be relevant for the understanding of intracellular transport of maternal Wnt determinants in the fertilized Xenopus egg (27).

Supporting Online Material

www.sciencemag.org/cgi/content/full/316/5831/1619/DC1

Materials and Methods

Figs. S1 to S5

Movie S1

References and Notes

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