Kremen2 modulates Dickkopf2 activity during Wnt/LRP6 signaling

Dickkopf1 (Dkk1) is a secreted antagonist of the Wnt/beta-catenin signaling pathway that acts by direct binding to and inhibiting the Wnt co-receptor LRP6. The related Dkk2, however, can function either as LRP6 agonist or antagonist, depending on the cellular context, suggesting that its activity is modulated by unknown co-factors. We have recently identified the transmembrane proteins Kremen1 and -2 as additional Dkk receptors, which bind to both Dkk1 and Dkk2 with high affinity. Here we show that Kremen2 (Krm2) regulates Dkk2 activity during Wnt signaling. In human 293 fibroblasts transfected dkk2 activates LRP6 signaling. However, co-transfection of krm2 blocks the ability of Dkk2 to activate LRP6 and enhances inhibition of Wnt/Frizzled signaling. Krm2 also co-operates with Dkk4 to inhibit Wnt signaling, but not with Dkk3, which has no effect on Wnt signaling. Likewise, in Xenopus embryos, Dkk2 and Krm2 co-operate in Wnt inhibition leading to anteriorized embryos. Finally, we show that interaction with Krm2 is mediated by the second cysteine-rich domain of Dkks. These results suggest that Krm2 can function as a switch that turns Dkk2 from an activator into an inhibitor of Wnt/lRP6 signaling.     

Clathrin and AP2 are required for PtdIns(4,5)P2-mediated formation of LRP6 signalosomes

Canonical Wnt signaling is initiated by the binding of Wnt proteins to their receptors, low-density lipoprotein-related protein 5 and 6 (LRP5/6) and frizzled proteins, leading to phosphatidylinositol (4,5)bisphosphate (PtdIns(4,5)P₂) production, signalosome formation, and LRP phosphorylation. However, the mechanism by which PtdIns(4,5)P₂ regulates the signalosome formation remains unclear. Here we show that clathrin and adaptor protein 2 (AP2) were part of the LRP6 signalosomes. The presence of clathrin and AP2 in the LRP6 signalosomes depended on PtdIns(4,5)P₂, and both clathrin and AP2 were required for the formation of LRP6 signalosomes. In addition, WNT3A-induced LRP6 signalosomes were primarily localized at cell surfaces, and WNT3A did not induce marked LRP6 internalization. However, rapid PtdIns(4,5)P₂ hydrolysis induced artificially after WNT3A stimulation could lead to marked LRP6 internalization. Moreover, we observed WNT3A-induced LRP6 and clathrin clustering at cell surfaces using super-resolution fluorescence microscopy. Therefore, we conclude that PtdIns(4,5)P₂ promotes the assembly of LRP6 signalosomes via the recruitment of AP2 and clathrin and that LRP6 internalization may not be a prerequisite for Wnt signaling to β-catenin stabilization.     

Dkk1 Stabilizes Wnt Co-Receptor LRP6: Implication for Wnt Ligand-Induced LRP6 Down-Regulation

Background

The low density lipoprotein receptor-related protein-6 (LRP6) is an essential co-receptor for canonical Wnt signaling. Dickkopf 1 (Dkk1), a major secreted Wnt signaling antagonist, binds to LRP6 with high affinity and prevents the Frizzled-Wnt-LRP6 complex formation in response to Wnts. Previous studies have demonstrated that Dkk1 promotes LRP6 internalization and degradation when it forms a ternary complex with the cell surface receptor Kremen.

Methodology/Principal Findings

In the present study, we found that transfected Dkk1 induces LRP6 accumulation while inhibiting Wnt/LRP6 signaling. Treatment with Dkk1-conditioned medium or recombinant Dkk1 protein stabilized LRP6 with a prolonged half-life and induces LRP6 accumulation both at the cell surface and in endosomes. We also demonstrated that Kremen2 co-expression abrogated the effect of Dkk1 on LRP6 accumulation, indicating that the effect of Kremen2 is dominant over Dkk1 regulation of LRP6. Furthermore, we found that Wnt3A treatment induces LRP6 down-regulation, an effect paralleled with a Wnt/LRP6 signaling decay, and that Dkk1 treatment blocked Wnt3A-induced LRP6 down-regulation. Finally, we found that LRP6 turnover was blocked by an inhibitor of caveolae-mediated endocytosis.

Conclusions/Significance

Our results reveal a novel role for Dkk1 in preventing Wnt ligand-induced LRP6 down-regulation and contribute significantly to our understanding of Dkk1 function in Wnt/LRP6 signaling.     

Wnt proteins induce dishevelled phosphorylation via an LRP5/6- independent mechanism, irrespective of their ability to stabilize beta-catenin

Wnt glycoproteins play essential roles in the development of metazoan organisms. Many Wnt proteins, such as Wnt1, activate the well-conserved canonical Wnt signaling pathway, which results in accumulation of beta-catenin in the cytosol and nucleus. Other Wnts, such as Wnt5a, activate signaling mechanisms which do not involve beta-catenin and are less well characterized. Dishevelled (Dvl) is a key component of Wnt/beta-catenin signaling and becomes phosphorylated upon activation of this pathway. In addition to Wnt1, we show that several Wnt proteins, including Wnt5a, trigger phosphorylation of mammalian Dvl proteins and that this occurs within 20 to 30 min. Unlike the effects of Wnt1, phosphorylation of Dvl in response to Wnt5a is not concomitant with beta-catenin stabilization, indicating that Dvl phosphorylation is not sufficient to activate canonical Wnt/beta-catenin signaling. Moreover, neither Dickkopf1, which inhibits Wnt/beta-catenin signaling by binding the Wnt coreceptors LRP5 and -6, nor dominant-negative LRP5/6 constructs could block Wnt-mediated Dvl phosphorylation. We conclude that Wnt-induced phosphorylation of Dvl is independent of LRP5/6 receptors and that canonical Wnts can elicit both LRP-dependent (to beta-catenin) and LRP-independent (to Dvl) signals. Our data also present Dvl phosphorylation as a general biochemical assay for Wnt protein function, including those Wnts that do not activate the Wnt/beta-catenin pathway.     

Wnt-3a and Dickkopf-1 stimulate neurite outgrowth in Ewing tumor cells via a Frizzled3- and c-Jun N-terminal kinase-dependent mechanism

Recombinant Wnt-3a stimulated the rapid formation of elongated processes in Ewing sarcoma family tumor (ESFT) cells that were identified as neurites. The processes stained positively for polymerized actin and microtubules as well as synapsin I and growth-associated protein 43. Inhibition of the Wnt receptor, Frizzled3 (Fzd3), with antiserum or by short interfering RNA (siRNA) markedly reduced neurite extension. Knockdown of Dishevelled-2 (Dvl-2) and Dvl-3 also suppressed neurite outgrowth. Surprisingly, disruption of the Wnt/Fzd/lipoprotein receptor-related protein (LRP) complex and the associated beta-catenin signaling by treating cells either with the Wnt antagonist Dickkopf-1 (Dkk1) or LRP5/LRP6 siRNA enhanced neuritogenesis. Neurite outgrowth induced by Dkk1 or with LRP5/LRP6 siRNA was inhibited by secreted Fzd-related protein 1, a Wnt antagonist that binds directly to Wnt. Moreover, Dkk1 stimulation of neurite outgrowth was blocked by Fzd3 siRNA. These results suggested that Dkk1 shifted endogenous Wnt activity from the beta-catenin pathway to Fzd3-mediated, noncanonical signaling that is responsible for neurite formation. In particular, c-Jun amino-terminal kinase (JNK) was important for neurite outgrowth stimulated by both Wnt-3a and Dkk1. Our data demonstrate that Fzd3, Dvl, and JNK activity mediate Wnt-dependent neurite outgrowth and that ESFT cell lines will be useful experimental models for the study of Wnt-dependent neurite extension.     

High-Affinity Dkk1 Receptor Kremen1 Is Internalized by Clathrin-Mediated Endocytosis

Kremens are high-affinity receptors for Dickkopf 1 (Dkk1) and regulate the Wnt/β-catenin signaling pathway by down-regulating the low-density lipoprotein receptor-related protein 6 (LRP6). Dkk1 competes with Wnt for binding to LRP6; binding of Dkk1 inhibits canonical signaling through formation of a ternary complex with Kremen. The majority of down-regulated clathrin-mediated endocytic receptors contain short conserved regions that recognize tyrosine or dileucine sorting motifs. In this study, we found that Kremen1 is internalized from the cell surface in a clathrin-dependent manner. Kremen1 contains an atypical dileucine motif with the sequence DXXXLV. Mutation of LV to AA in this motif blocked Kremen1 internalization; as reported previously for other proteins, the aspartic acid residue in Kremen1 is not critical. Inhibition of expression of the adaptor protein 2 (AP-2) or inhibition of clathrin by pitstop 2 also blocked Kremen1 internalization. The novel amino acid sequence identified in Kremen1 is similar to the motif previously identified in hydra, yeast, and other organisms known to signal from the trans-Golgi network to the endosomal compartment.     

Caveolin is necessary for Wnt-3a-dependent internalization of LRP6 and accumulation of beta-catenin

beta-catenin-mediated Wnt signaling is critical in animal development and tumor progression. The single-span transmembrane Wnt receptor, low-density lipoprotein receptor-related protein 6 (LRP6), interacts with Axin to promote the Wnt-dependent accumulation of beta-catenin. However, the molecular mechanism of receptor internalization and its impact on signaling are unclear. Here, we present evidence that LRP6 is internalized with caveolin and that the components of this endocytic pathway are required not only for Wnt-3a-induced internalization of LRP6 but also for accumulation of beta-catenin. Overall, our data suggest that Wnt-3a triggers the interaction of LRP6 with caveolin and promotes recruitment of Axin to LRP6 phosphorylated by glycogen synthase kinase-3beta and that caveolin thereby inhibits the binding of beta-catenin to Axin. Thus, caveolin plays critical roles in inducing the internalization of LRP6 and activating the Wnt/beta-catenin pathway. We also discuss the idea that distinct endocytic pathways correlate with the specificity of Wnt signaling events.     

Wnt-independent activation of beta-catenin mediated by a Dkk1-Fz5 fusion protein

An XWnt8-Fz5 fusion protein synergizes with LRP6 to potently activate beta-catenin-dependent signaling. Here, we generated a fusion in which XWnt8 was fused to the N-terminus of LRP6 and show it synergizes with both Fz4 and Fz5 to potently transactivate beta-catenin-dependent Wnt signaling. Based on this, we hypothesized that the main function of Wnt is to nucleate the formation of a physical complex between LRP6 and a Frizzled. Dkk1, but not the related Dkk3, binds LRP6 and inhibits canonical Wnt signaling by blocking the interaction of Wnt and LRP6. Therefore, we reasoned that a covalent fusion of Dkk1 to Fz5 (Dkk1-Fz5) would mimic Wnt ligand by nucleating the formation of a complex containing Fz5 and LRP6, while Dkk3 (Dkk3-Fz5) would not. We found that Dkk1-Fz5, but not Dkk3-Fz5, potently synergized with LRP6 to activate signaling in a dishevelled-dependent manner.     

Regulation of Wnt/LRP signaling by distinct domains of Dickkopf proteins

Dickkopfs (Dkks) are secreted developmental regulators composed of two cysteine-rich domains. We report that the effects of Dkks depend on molecular context. Although Wnt8 signaling is inhibited by both Dkk1 and Dkk2 in Xenopus embryos, the same pathway is activated upon interaction of Dkk2 with the Wnt coreceptor LRP6. Analysis of individual Dkk domains and chimeric Dkks shows that the carboxy-terminal domains of both Dkks associate with LRP6 and are necessary and sufficient for Wnt8 inhibition, whereas the amino-terminal domain of Dkk1 plays an inhibitory role in Dkk-LRP interactions. Our study illustrates how an inhibitor of a pathway may be converted into an activator and is the first study to suggest a molecular mechanism for how a ligand other than Wnt can positively regulate beta-catenin signaling.     

Disabled-2 (Dab2) inhibits Wnt/β-catenin signalling by binding LRP6 and promoting its internalization through clathrin

Canonical Wnt signalling requires caveolin-dependent internalization of low-density lipoprotein receptor-related protein 6 (LRP6). Here we report that the tumour suppressor and endocytic adaptor disabled-2 (Dab2), previously described as an inhibitor of Wnt/β-catenin signalling, selectively recruits LRP6 to the clathrin-dependent endocytic route, thereby sequestering it from caveolin-mediated endocytosis. Wnt stimulation induces the casein kinase 2 (CK2)-dependent phosphorylation of LRP6 at S1579, promoting its binding to Dab2 and internalization with clathrin. LRP6 receptor mutant (S1579A), deficient in CK2-mediated phosphorylation and Dab2 binding, fails to associate with clathrin, and thus escapes the inhibitory effects of Dab2 on Wnt/β-catenin signalling. Our data suggest that the S1579 site of LRP6 is a negative regulatory point during LRP6-mediated dorsoventral patterning in zebrafish and in allograft mouse tumour models. We conclude that the tumour suppressor functions of Dab2 involve modulation of canonical Wnt signalling by regulating the endocytic fate of the LRP6 receptor.     

Wnt5a regulates distinct signalling pathways by binding to Frizzled2

Wnt5a regulates multiple intracellular signalling cascades, but how Wnt5a determines the specificity of these pathways is not well understood. This study examined whether the internalization of Wnt receptors affects the ability of Wnt5a to regulate its signalling pathways. Wnt5a activated Rac in the β‐catenin‐independent pathway, and Frizzled2 (Fz2) and Ror1 or Ror2 were required for this action. Fz2 was internalized through a clathrin‐mediated route in response to Wnt5a, and inhibition of clathrin‐dependent internalization suppressed the ability of Wnt5a to activate Rac. As another action of Wnt5a, it inhibited Wnt3a‐dependent lipoprotein receptor‐related protein 6 (LRP6) phosphorylation and β‐catenin accumulation. Wnt3a‐dependent phosphorylation of LRP6 was enhanced in Wnt5a knockout embryonic fibroblasts. Fz2 was also required for the Wnt3a‐dependent accumulation of β‐catenin, and Wnt5a competed with Wnt3a for binding to Fz2 in vitro and in intact cells, thereby inhibiting the β‐catenin pathway. This inhibitory action of Wnt5a was not affected by the impairment of clathrin‐dependent internalization. These results suggest that Wnt5a regulates distinct pathways through receptor internalization‐dependent and ‐independent mechanisms.     

Characterization of the Kremen-binding site on Dkk1 and elucidation of the role of Kremen in Dkk-mediated Wnt antagonism

Wnt signaling is involved in a wide range of developmental, physiological, and pathophysiological processes and is negatively regulated by Dickkopf1 (Dkk1). Dkk1 has been shown to bind to two transmembrane proteins, the low density lipoprotein receptor-related proteins (LRP) 5/6 and Kremen. Here, we show that Dkk1 residues Arg(197), Ser(198), and Lys(232) are specifically involved in its binding to Kremen rather than to LRP6. These residues are localized at a surface that is at the opposite side of the LRP6-binding surface based on a three-dimensional structure of Dkk1 deduced from that of Dkk2. We were surprised to find that the Dkk1 mutants carrying a mutation at Arg(197), Ser(198), or Lys(232), the key Kremen-binding residues, could antagonize Wnt signaling as well as the wild-type Dkk1. These mutations only affected their ability to antagonize Wnt signaling when both LRP6 and Kremen were coexpressed. These results suggest that Kremen may not be essential for Dkk1-mediated Wnt antagonism and that Kremen may only play a role when cells express a high level of LRP5/6.     

Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation

Wnt signaling acts in part through the low density lipoprotein receptor-related transmembrane proteins LRP5 and LRP6 to regulate embryonic development and stem cell proliferation. Up-regulated signaling is associated with many forms of cancer. Casein kinase I epsilon (CKIepsilon) is a known component of the Wnt-beta-catenin signaling pathway. We find that CKIepsilon binds to LRP5 and LRP6 in vitro and in vivo and identify three CKIepsilon-specific phosphorylation sites in LRP6. Two of the identified phosphorylation sites, Ser1420 and Ser1430, influence Wnt signaling in vivo, since LRP6 with mutation of these sites is a more potent activator of both beta-catenin accumulation and Lef-1 reporter activity. Whereas Wnt3a regulates CKIepsilon kinase activity, LRP6 does not, placing CKIepsilon upstream of LRP6. Mutation of LRP6 Ser1420 and Ser1430 to alanine strengthens its interaction with axin, suggesting a mechanism by which CKIepsilon may negatively regulate Wnt signaling. The role of CKIepsilon is therefore more complex than was previously appreciated. Generation of active CKIepsilon may induce a negative feedback loop by phosphorylation of sites on LRP5/6 that modulate axin binding and hence beta-catenin degradation.     

Kremen is required for neural crest induction in Xenopus and promotes LRP6-mediated Wnt signaling

Kremen 1 and 2 (Krm1/2) are transmembrane receptors for Wnt antagonists of the Dickkopf (Dkk) family and function by inhibiting the Wnt co-receptors LRP5/6. Here we show that Krm2 functions independently from Dkks during neural crest (NC) induction in Xenopus. Krm2 is co-expressed with, and regulated by, canonical Wnts. Krm2 is differentially expressed in the NC, and morpholino-mediated Krm2 knockdown inhibits NC induction, which is mimicked by LRP6 depletion. Conversely, krm2 overexpression induces ectopic NC. Kremens bind to LRP6, promote its cell-surface localization and stimulate LRP6 signaling. Furthermore, Krm2 knockdown specifically reduces LRP6 protein levels in NC explants. The results indicate that in the absence of Dkks, Kremens activate Wnt/beta-catenin signaling through LRP6.     

Reconstitution of a Frizzled8��Wnt3a��LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Wnt/β-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/β-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD·Wnt3a·LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth β-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/β-catenin signaling by Dkk1.     

R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and beta-catenin signaling

R-spondin proteins are newly identified secreted molecules that activate beta-catenin signaling. However, the mechanism of R-spondin action and its relationship with Wnt signaling remain unclear. Here we show that human R-spondin1 (hRspo1) is a high affinity ligand for the Wnt co-receptor LRP6 (K(d) = 1.2 nm). hRspo1 induces glycogen synthase kinase 3-dependent phosphorylation and activation of LRP6. DKK1, an LRP6 antagonist, inhibits hRspo1-induced LRP6 phosphorylation. We further demonstrate that hRspo1 synergizes with Frizzled5 in Xenopus axis induction assays and induces the phosphorylation of Dishevelled, a cytoplasmic component downstream of Frizzled function. Our study reveals interesting similarity and distinction between Wnt and R-spondin signaling.     

The mechanism of endogenous receptor activation functionally distinguishes prototype canonical and noncanonical Wnts

Wnt glycoproteins are developmentally essential signaling molecules, and lesions afflicting Wnt pathways play important roles in human diseases. Some Wnts signal to the canonical pathway by stabilizing beta-catenin, while others lack this activity. Frizzled serpentine receptors mediate distinct signaling pathways by both classes of Wnts. Here, we tandemly linked noncanonical Wnt5a with the C-terminal half of Dickkopf-2 (Dkk2C), a distinct ligand of the Wnt coreceptor LRP5/6. Whereas Wnt5a, Dkk2C, or both together were incapable of stimulating endogenous canonical signaling, the Wnt5a/Dkk2C chimera efficiently activated this pathway in a manner inhibitable by specific antagonists of either frizzled or LRP receptors. Thus, activation of the canonical pathway requires ligand coupling of an endogenous frizzled/LRP coreceptor complex, rather than Wnt triggering each receptor independently. Moreover, fusion of Wnt5a with Dkk2C unmasked its ability to signal to Dishevelled through multiple frizzleds, indicating that the lack of functional interaction with LRP distinguishes noncanonical Wnt5a from canonical Wnts in mammalian cells. These findings provide a novel mechanism by which the same receptor can be switched between distinct signaling pathways depending on the differential recruitment of a coreceptor by members of the same ligand family.     

Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6

LDL receptor-related proteins 5 and 6 (LRP5/6) are coreceptors for Wnt growth factors, and also bind Dkk proteins, secreted inhibitors of Wnt signaling. The LRP5/6 ectodomain contains four β-propeller/EGF-like domain repeats. The first two repeats, LRP6(1-2), bind to several Wnt variants, whereas LRP6(3-4) binds other Wnts. We present the crystal structure of the Dkk1 C-terminal domain bound to LRP6(3-4), and show that the Dkk1 N-terminal domain binds to LRP6(1-2), demonstrating that a single Dkk1 molecule can bind to both portions of the LRP6 ectodomain and thereby inhibit different Wnts. Small-angle X-ray scattering analysis of LRP6(1-4) bound to a noninhibitory antibody fragment or to full-length Dkk1 shows that in both cases the ectodomain adopts a curved conformation that places the first three repeats at a similar height relative to the membrane. Thus, Wnts bound to either portion of the LRP6 ectodomain likely bear a similar spatial relationship to Frizzled coreceptors.     

Wnt signal amplification via activity, cooperativity, and regulation of multiple intracellular PPPSP motifs in the Wnt co-receptor LRP6

Low density lipoprotein receptor-related protein 6 (LRP6) and its homologue LRP5 serve as Wnt co-receptors that are essential for the Wnt/beta-catenin pathway. Wnt activation of LRP6 leads to recruitment of the scaffolding protein Axin and inhibition of Axin-mediated phosphorylation/destruction of beta-catenin. We showed that five conserved PPPSP motifs in the LRP6 intracellular domain are required for LRP6 function, and mutation of these motifs together abolishes LRP6 signaling activity. We further showed that Wnt induces the phosphorylation of a prototypic PPPSP motif, which provides a docking site for Axin and is sufficient to transfer signaling activity to a heterologous receptor. However, the activity, regulation, and functionality of multiple PPPSP motifs in LRP6 have not been characterized. Here we provide a comprehensive analysis of all five PPPSP motifs in LRP6. We define the core amino acid residues of a prototypic PPPSP motif via alanine scanning mutagenesis and demonstrate that each of the five PPPSP motifs exhibits signaling and Axin binding activity in isolation. We generated two novel phosphorylation-specific antibodies to additional PPPSP motifs and show that Wnt induces phosphorylation of these motifs in the endogenous LRP6 through glycogen synthase kinase 3. Finally, we uncover the critical cooperativity of PPPSP motifs in the full-length LRP6 by demonstrating that LRP6 mutants lacking a single PPPSP motif display compromised function, whereas LRP6 mutants lacking two of the five PPPSP motifs are mostly inactive. This cooperativity appears to reflect the ability of PPPSP motifs to promote the phosphorylation of one another and to interact with Axin synergistically. These results establish the critical role and a common phosphorylation/activation mechanism for the PPPSP motifs in LRP6 and suggest that the conserved multiplicity and cooperativity of the PPPSP motifs represents a built-in amplifier for Wnt signaling by the LRP6 family of receptors.