Stability elements in the LRP6 cytoplasmic tail confer efficient signalling upon DIX-dependent polymerization

Abstract

Wnt/beta-catenin signalling controls cell fates in development, tissue homeostasis and cancer. Wnt binding to Frizzled receptors triggers recruitment of Dishevelled to the plasma membrane and formation of a signalosome containing the LRP5/6 co-receptor, whose cytoplasmic tail (ctail) thus becomes phosphorylated at multiple PPP(S/T)Px(S/T) motifs. These then directly inhibit GSK3beta, which results in beta-catenin accumulation and signalling. Here, we revisit previous epistasis experiments, and show that Dishevelled signals through LRP5/6 in human cells and Drosophila embryos. To recapitulate this signalling event, and to define its functional elements, we fused the Dishevelled DIX domain to the LRP6 ctail, which forms cytoplasmic signalosomes with potent signalling activity mediated by its PPP(S/T)Px(S/T) motifs. Their phosphorylation and activity depends critically on DIX-mediated polymerization, and on multiple stability elements in the LRP6 ctail, including the T1479 epitope upstream of the membrane-proximal PPP(S/T)Px(S/T) motif. Thus, stable polymerization emerges as a key principle underlying the function of Dishevelled-dependent signalosomes.

Fig. 1.

Overexpressed Dvl signals through LRP6/Arrow. (A-F) Ventral cuticles of wt or mutant Drosophila embryos, with or without Dsh overexpressed in alternate segments (by prd-GAL4), as indicated; naked cuticle (arrowheads) reflects the signalling activity of overexpressed Dsh. Note that 50% of the arr² null mutants (selected by absence of RFP) should express Dsh, although a denticle-lawn phenotype is observed in 62% of the embryos (D), suggesting that this class contains embryos overexpressing Dsh without any activity; occasional naked patches are visible in the remaining 38% (arrowheads in E), reflecting residual activity of the prd-GAL4-driven Dsh (see also text). By contrast, the Dsh-overexpressing embryos are readily identifiable amongst the wg^cx4 homozygotes (at the expected frequency of 25%) by their four wide naked zones interspersed between their denticle bands (F), reflecting strong and 100% penetrant activity of overexpressed Dsh. (G) TOPFLASH assays from lysates of LRP6- and mock-depleted HEK293 cells, with or without overexpressed FLAG-Dvl2. Error bars indicate standard deviations from three independent experiments. Inset, relative protein expression levels.

Fig. 2.

Potent signalling activity of a minimal DIX>ctail module in human cell lines. (A) Schematic representation of LRP6, wt and mutant DIX>ctail derivatives; red and green bars within the DIX domain (hatched) indicate positions of M4 and M2 point mutations, respectively, in its complementary self-interacting surfaces; black bars mark PPPSPxS motifs. (B-E) Confocal images of fixed HeLa cells overexpressing various GFP-tagged constructs, as indicated. (F) TOPFLASH assays (as in Fig. 1G), monitoring the signalling activity of DIX>ctail compared with its controls in HEK293 cells; inset, relative expression levels (arrowheads point to breakdown products of the DIX domain, which in some constructs appears prone to degradation during cell lysis). (G,H) Immunostaining of endogenous β-catenin (red) in HeLa cells transfected with DIX>ctail or M2>ctail (green), co-stained with DAPI (blue) to mark the nuclei. Note the increased level of cytoplasmic and nuclear β-catenin in the DIX>ctail-expressing cell, compared with the non-transfected or M2>ctail-expressing cells; arrows indicate recruitment of β-catenin into DIX>ctail puncta. Scale bars: 10 μm.

Fig. 3.

Potent signalling activity of dDIX>ctail in Drosophila embryos. (A,B) Confocal sections of the lateral epidermis of 5- to 7-hour-old fixed wt and mutant embryos, ubiquitously expressing dDIX>ctail or the non-polymerizing M4 mutant, respectively, and (C,D) resulting ventral cuticles (as in Fig. 1). (E-I) Ventral cuticles of dDIX>ctail-expressing wt and mutant embryos, as indicated; null mutants (E,F) were selected by absence of RFP-marked chromosomes. Naked cuticles (C,E,F,I) reflect strong signalling activity of dDIX>ctail, independent of (E) dsh and (F) arrow, while dM4>ctail is completely inactive, allowing 100% of the dM4>ctail-expressing embryos to hatch into normal larvae (D). The denticle-lawn phenotype in H was observed at the frequency expected for dTCF³ homozygotes (in 25% of the dDIX>ctail-expressing embryos), and is indistinguishable from that of the dTCF³ mutants (G), indicating that dDIX>ctail is completely inactive in dTCF³ mutants.

Fig. 4.

Elements flanking the membrane-proximal PPPSPAT motif are required for full DIX>ctail activity. (A) Key features of the LRP6 ctail (top) in relation to DIX>ctail deletion mutants (underneath), with sequences of PPPSPxS motifs (vertical black bars) shown on the right (*, membrane-proximal motif; see also Fig. 2A); blue, Ser/Thr cluster; red, phosphorylation sites; red arrow points to GSK3β-mediated priming at the −3 positions for site II phosphorylation by CK1, and blue arrow to putative −3 priming for phospho-T1479 (see also text). (B) TOPFLASH assays in HEK293 cells, revealing loss of activity of m10 and polymerization-defective mutants, and residual activity of the ΔAB double deletion. (C-E) Confocal images of HeLa cells expressing deletion mutants, as indicated, immunostained for endogenous β-catenin. Cytoplasmic and nuclear accumulation of β-catenin, and recruitment into the puncta (marked by arrows), are observed in cells expressing the single mutants, but not in those expressing the double-deletion DIX>ctailΔAB. Scale bars: 10 μm.

Fig. 5.

Phosphorylation of DIX>ctail depends on a polymerization-competent DIX domain. (A) Western blots of total lysates of HEK293 cells transfected with wt and mutant DIX>ctail, showing phospho-S1490 levels (top) relative to total expression levels of wt and mutant DIX>ctail (bottom panels; re-probing of the blots shown above). Note the reductions of phospho-S1490 (indicated by arrows) in the polymerization-deficient mutants (M4>ctail and M4>ctailΔA behave the same as their M2-mutant counterparts; not shown), and the lack of a signal in m1 (bearing an S1490A substitution). (B-E) HeLa cells expressing DIX>ctail wt and deletion mutants, immunostained with the phospho-specific Tp1479 antibody, revealing strong punctate staining for DIX>ctail and DIX>ctailΔB, but complete absence of staining for M2>ctail and M2>ctailΔB. Scale bars: 10 μm.

Fig. 6.

Stability elements in the LRP6 ctail are essential for its efficient signalling activity. (A-E) Live-cell imaging of HeLa cells expressing (A) Dvl2-GFP or (C) DIX>ctail, subjected to FRAP analysis; relative fluorescence intensities were measured after bleaching of >10 single puncta (boxed in red) by short laser pulses (at T=4 seconds), and plotted against time (B,D,E). Fluorescence recovery is observed for every single Dvl2-GFP punctum (A,B), but not for any of the puncta in wt DIX>ctail or single deletion mutants (B,C). (D,E) FRAP analysis of multiple DIX>ctailΔAB puncta (D), exhibiting variable recovery (see also text), and of DIX>ctailΔABplus puncta (E), exhibiting complete stability, restored by the KGTYFP epitope. (F) TOPFLASH assays in HEK293 cells, revealing restoration of signalling activity of the double-deletion by the KGTYFP epitope (values are directly comparable to those in Fig. 2F and Fig. 4B). Inset: protein expression levels. (G) HeLa cells expressing DIX>ctailΔABplus, immunostained with anti-Tp1479, showing that T1479 within the KGTYFP epitope remains unphosphorylated in the DIX>ctailΔABplus puncta (see also Fig. 5B-E), probably because of the lack of a −3 priming residue.

Fig. 7.

Efficient recruitment of Axin into DIX>ctail puncta. (A-D) Confocal images of HeLa cells, transfected and immunostained as indicated (a catalytically inactive mutant was used to monitor CK1γ). Note the apparent stoichiomeric recruitment of HA-Axin into DIX>ctail puncta (A), in contrast to the kinase recruitment (arrows in B-D) resembling the sub-stoichiomeric recruitment of β-catenin (Fig. 2G). (E-G) Confocal images of 5- to 7-hour-old wt and sgg^M11 null mutant embryos, coexpressing dDIX>ctail and FLAG-Axin, fixed and stained as indicated. (E) Low-magnification overview of multiple parasegments of the lateral epidermis (note that arm-GAL4-mediated expression is somewhat stochastic, with individual cells expressing variable levels of FLAG-Axin and/or dDIX>ctail). (F,G) High-magnification views, revealing a high incidence of dDIX>ctail-associated FLAG-Axin puncta (arrows in F) in wt cells that coexpress both proteins, but largely separate dDIX>ctail and FLAG-Axin puncta in the sgg null mutants (G). Scale bars: 10 μm (A-D), 25 μm (E), and 5 μm (F,G).