Frizzled-Dishevelled signaling specificity outcome can be modulated by Diego in Drosophila

Members of the Frizzled (Fz) family of seven-pass transmembrane receptors are required for the transduction of both Wnt-Fz/beta-catenin and Fz/planar cell polarity (PCP) signals. Although both pathways transduce signals via interactions between Fz and the cytoplasmic protein Dishevelled (Dsh), each pathway has specific and distinct effectors. One explanation for the pathway specificity is that signal-induced conformational changes result in unique Fz-Dsh interactions. Our mutational analyses of Fz-Dsh activities in vivo do however not support this model, since both pathways are affected by all mutations tested. Alternatively, the interaction of Fz or Dsh with other proteins could modulate the signaling outcome. We examined the role of a Dsh-binding PCP molecule, Diego (Dgo), in both Wnt-Fz/beta-catenin and Fz/PCP signaling. Both loss-of-function and gain-of-function results suggest that Dgo promotes Fz-Dsh/PCP signaling at the expense of Wnt-Fz/beta-catenin signaling. Our data suggest that Dgo sequesters Dsh to a functionally distinct Fz/PCP signaling compartment within the cell.     

Dishevelled activates Ca2+ flux,PKC, and CamKII in vertebrate embryos

Wnt ligands and Frizzled (Fz) receptors have been shown to activate multiple intracellular signaling pathways. Activation of the Wnt-beta-catenin pathway has been described in greatest detail, but it has been reported that Wnts and Fzs also activate vertebrate planar cell polarity (PCP) and Wnt-Ca2+ pathways. Although the intracellular protein Dishevelled (Dsh) plays a dual role in both the Wnt-beta-catenin and the PCP pathways, its potential involvement in the Wnt-Ca2+ pathway has not been investigated. Here we show that a Dsh deletion construct, XDshDeltaDIX, which is sufficient for activation of the PCP pathway, is also sufficient for activation of three effectors of the Wnt-Ca2+ pathway: Ca2+ flux, PKC, and calcium/calmodulin-dependent protein kinase II (CamKII). Furthermore, we find that interfering with endogenous Dsh function reduces the activation of PKC by Xfz7 and interferes with normal heart development. These data suggest that the Wnt-Ca2+ pathway utilizes Dsh, thereby implicating Dsh as a component of all reported Fz signaling pathways.     

The casein kinase I family in Wnt signaling.

The canonical Wnt-signaling pathway is critical for many aspects of development, and mutations in components of the Wnt pathway are carcinogenic. Recently, sufficiency tests identified casein kinase Iepsilon (CKIepsilon) as a positive component of the canonical Wnt/beta-catenin pathway, and necessity tests showed that CKIepsilon is required in vertebrates to transduce Wnt signals. In addition to CKIepsilon, the CKI family includes several other isoforms (alpha, beta, gamma, and delta) and their role in Wnt sufficiency tests had not yet been clarified. However, in Caenorhabditis elegans studies, loss-of-function of a CKI isoform most similar to alpha produced the mom phenotype, indicative of loss-of-Wnt signaling. In this report, we examine the ability of the various CKI isoforms to activate Wnt signaling and find that all the wild-type CKI isoforms do so. Dishevelled (Dsh), another positive component of the Wnt pathway, becomes phosphorylated in response to Wnt signals. All the CKI isoforms, with the exception of gamma, increase the phosphorylation of Dsh in vivo. In addition, CKI directly phosphorylates Dsh in vitro. Finally, we find that CKI is required in vivo for the Wnt-dependent phosphorylation of Dsh. These studies advance our understanding of the mechanism of Wnt action and suggest that more than one CKI isoform is capable of transducing Wnt signals in vivo.     

Casein kinase Iepsilon modulates the signaling specificities of dishevelled

Wnt signaling is critical to many aspects of development, and aberrant activation of the Wnt signaling pathway can cause cancer. Dishevelled (Dvl) protein plays a central role in this pathway by transducing the signal from the Wnt receptor complex to the beta-catenin destruction complex. Dvl also plays a pivotal role in the planar cell polarity pathway that involves the c-Jun N-terminal kinase (JNK). How functions of Dvl are regulated in these two distinct pathways is not clear. We show that deleting the C-terminal two-thirds of Dvl, which includes the PDZ and DEP domains and is essential for Dvl-induced JNK activation, rendered the molecule a much more potent activator of the beta-catenin pathway. We also found that casein kinase Iepsilon (CKIepsilon), a previously identified positive regulator of Wnt signaling, stimulated Dvl activity in the Wnt pathway, but dramatically inhibited Dvl activity in the JNK pathway. Consistent with this, overexpression of CKIepsilon in Drosophila melanogaster stimulated Wnt signaling and disrupted planar cell polarity. We also observed a correlation between the localization and the signaling activity of Dvl in the beta-catenin pathway and the JNK pathway. Furthermore, by using RNA interference, we demonstrate that the Drosophila CKIepsilon homologue Double time positively regulates the beta-catenin pathway through Dvl and negatively regulates the Dvl-induced JNK pathway. We suggest that CKIepsilon functions as a molecular switch to direct Dvl from the JNK pathway to the beta-catenin pathway, possibly by altering the conformation of the C terminus of Dvl.     

Planar polarity is positively regulated by casein kinase Iepsilon in Drosophila

Members of the casein kinase I (CKI) family have been implicated in regulating canonical Wnt/Wingless (Wg) signaling by phosphorylating multiple pathway components. Overexpression of CKI in vertebrate embryos activates Wg signaling, and one target is thought to be the cytoplasmic effector Dishevelled (Dsh), which is an in vitro target of CKI phosphorylation. Phosphorylation of Dsh by CKI has also been suggested to switch its activity from noncanonical to canonical Wingless signaling. However, in vivo loss-of-function experiments have failed to identify a clear role for CKI in positive regulation of Wg signaling. By examining hypomorphic mutations of the Drosophila CKIepsilon homolog discs overgrown (dco)/double-time, we now show that it is an essential component of the noncanonical/planar cell polarity pathway. Genetic interactions indicate that dco acts positively in planar polarity signaling, demonstrating that it does not act as a switch between canonical and noncanonical pathways. Mutations in dco result in a reduced level of Dishevelled phosphorylation in vivo. Furthermore, in these mutants, Dishevelled fails to adopt its characteristic asymmetric subcellular localisation at the distal end of pupal wing cells, and the site of hair outgrowth is disrupted. Finally, we also find that dco function in polarity is partially redundant with CKIalpha.     

Dissecting the PCP pathway: One or more pathways?

Planar cell polarity (PCP), the alignment of cells within 2D tissue planes, involves a set of core molecular regulators highly conserved between animals and cell types. These include the transmembrane proteins Frizzled (Fz) and VanGogh and the cytoplasmic regulators Dishevelled (Dsh) and Prickle. It is widely accepted that this core forms part of a 'PCP pathway' for signal transduction, which can affect cell morphology through activation of an evolutionary ancient regulatory module involving Rho family GTPases and Myosin II, and/or the JNK kinase cascade. We have re-examined the evidence for interactions between the proposed PCP pathway components, and question the placing of the cell morphology regulators in the same pathway as the PCP core. While Fz and Dsh are clearly involved in both PCP and Rho-based cell morphology regulation, available evidence cannot currently discriminate whether these processes are linked mechanistically by a shared Fz/Dsh population, or pass by two distinct pathways.     

Rap2 is required for Wnt/beta-catenin signaling pathway in Xenopus early development

The Wnt/beta-catenin signaling pathway is critical for the establishment of organizer and embryonic body axis in Xenopus development. Here, we present evidence that Xenopus Rap2, a member of Ras GTPase family, is implicated in Wnt/beta-catenin signaling during the dorsoventral axis specification. Ectopic expression of XRap2 can lead to neural induction without mesoderm differentiation. XRap2 dorsalizes ventral tissues, inducing axis duplication, organizer-specific gene expression and convergent extension movements. Knockdown of XRap2 causes ventralized phenotypes including shortened body axis and defective dorsoanterior patterning, which are associated with aberrant Wnt signaling. In line with this, XRap2 depletion inhibits beta-catenin stabilization and the induction of ectopic dorsal axis and Wnt-responsive genes caused by XWnt8, Dsh or beta-catenin, but has no effect on the signaling activities of a stabilized beta-catenin. Its knockdown also disrupts the vesicular localization of Dsh, thereby inhibiting Dsh-mediated beta-catenin stabilization and the membrane recruitment and phosphorylation of Dsh by frizzled signaling. Taking together, we suggest that XRap2 is involved in Wnt/beta-catenin signaling as a modulator of the subcellular localization of Dsh.     

Ryk cooperates with Frizzled 7 to promote Wnt11-mediated endocytosis and is essential for Xenopus laevis convergent extension movements

The single-pass transmembrane protein Ryk (atypical receptor related tyrosine kinase) functions as a Wnt receptor. However, Ryk's correlation with Wnt/Frizzled (Fz) signaling is poorly understood. Here, we report that Ryk regulates Xenopus laevis convergent extension (CE) movements via the β-arrestin 2 (βarr2)-dependent endocytic process triggered by noncanonical Wnt signaling. During X. laevis gastrulation, βarr2-mediated endocytosis of Fz7 and dishevelled (Dvl/Dsh) actually occurs in the dorsal marginal zone tissues, which actively participate in noncanonical Wnt signaling. Noncanonical Wnt11/Fz7-mediated endocytosis of Dsh requires the cell-membrane protein Ryk. Ryk interacts with both Wnt11 and βarr2, cooperates with Fz7 to mediate Wnt11-stimulated endocytosis of Dsh, and signals the noncanonical Wnt pathway in CE movements. Conversely, depletion of Ryk and Wnt11 prevents Dsh endocytosis in dorsal marginal zone tissues. Our study suggests that Ryk functions as an essential regulator for noncanonical Wnt/Fz-mediated endocytosis in the regulation of X. laevis CE movements.     

The balance between the novel protein target of wingless and the Drosophila Rho-associated kinase pathway regulates planar cell polarity in the Drosophila wing

Planar cell polarity (PCP) signaling is mediated by the serpentine receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling utilizes Drosophila Frizzled 2 (DFz2) as a receptor and also requires Dsh for transducing signals to regulate cell proliferation and differentiation in many developmental contexts. Distinct pathways are activated downstream of Dsh in Wg- and Fz-signaling pathways. Recently, a number of genes, which have essential roles as downstream components of PCP signaling, have been identified in Drosophila. They include the small GTPase RhoA/Rho1, its downstream effector Drosophila rho-associated kinase (Drok), and a number of genes such as inturned (in) and fuzzy (fy), whose biochemical functions are unclear. RhoA and Drok provide a link from Fz/Dsh signaling to the modulation of actin cytoskeleton. Here we report the identification of the novel gene target of wingless (tow) by enhancer trap screening. tow expression is negatively regulated by Wg signaling in wing imaginal discs, and the balance between tow and the Drok pathway regulates wing-hair morphogenesis. A loss-of-function mutation in tow does not result in a distinct phenotype. Genetic interaction and gain-of-function studies provide evidence that Tow acts downstream of Fz/Dsh and plays a role in restricting the number of hairs that wing cells form.     

Identification and comparative expression analyses of Daam genes in mouse and Xenopus

During vertebrate embryogenesis, secreted Wnt molecules regulate cell fates by signaling through the canonical pathway mediated by beta-catenin, and regulate planar cell polarity (PCP) and convergent extension movements through alternative pathways. The phosphoprotein Dishevelled (Dsh/Dvl) is a Wnt signal transducer thought to function in all Wnt signaling pathways. A recently identified member of the Formin family, Daam (Dishevelled--associated activator of morphogenesis), regulates the morphogenetic movements of vertebrate gastrulation in a Wnt-dependent manner through direct interactions with Dsh/Dvl and RhoA. We describe two mouse Daam cDNAs, mDaam1 and mDaam2, which encode proteins characterized by highly conserved formin homology domains and which are expressed in complementary patterns during mouse development. Cross-species comparisons indicate that the expression domains of Xenopus Daam1 (XDaam1) mirror mDaam1 expression. Our results demonstrate that Daams are expressed in tissues known to require Wnts and are consistent with Daams being effectors of Wnt signaling during vertebrate development.     

Role of glypican 4 in the regulation of convergent extension movements during gastrulation in Xenopus laevis

Coordinated morphogenetic cell movements during gastrulation are crucial for establishing embryonic axes in animals. Most recently, the non-canonical Wnt signaling cascade (PCP pathway) has been shown to regulate convergent extension movements in Xenopus and zebrafish. Heparan sulfate proteoglycans (HSPGs) are known as modulators of intercellular signaling, and are required for gastrulation movements in vertebrates. However, the function of HSPGs is poorly understood. We analyze the function of Xenopus glypican 4 (Xgly4), which is a member of membrane-associated HSPG family. In situ hybridization revealed that Xgly4 is expressed in the dorsal mesoderm and ectoderm during gastrulation. Reducing the levels of Xgly4 inhibits cell-membrane accumulation of Dishevelled (Dsh), which is a transducer of the Wnt signaling cascade, and thereby disturbs cell movements during gastrulation. Rescue analysis with different Dsh mutants and Wnt11 demonstrated that Xgly4 functions in the non-canonical Wnt/PCP pathway, but not in the canonical Wnt/beta-catenin pathway, to regulate gastrulation movements. We also provide evidence that the Xgly4 protein physically binds Wnt ligands. Therefore, our results suggest that Xgly4 functions as positive regulator in non-canonical Wnt/PCP signaling during gastrulation.     

Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions

Canonical Wnt/beta-catenin signaling has central roles in development and diseases, and is initiated by the action of the frizzled (Fz) receptor, its coreceptor LDL receptor-related protein 6 (Lrp6), and the cytoplasmic dishevelled (Dvl) protein. The functional relationships among Fz, Lrp6 and Dvl have long been enigmatic. We demonstrated previously that Wnt-induced Lrp6 phosphorylation via glycogen synthase kinase 3 (Gsk3) initiates Wnt/beta-catenin signaling. Here we show that both Fz and Dvl functions are critical for Wnt-induced Lrp6 phosphorylation through Fz-Lrp6 interaction. We also show that axin, a key scaffolding protein in the Wnt pathway, is required for Lrp6 phosphorylation via its ability to recruit Gsk3, and inhibition of Gsk3 at the plasma membrane blocks Wnt/beta-catenin signaling. Our results suggest a model that upon Wnt-induced Fz-Lrp6 complex formation, Fz recruitment of Dvl in turn recruits the axin-Gsk3 complex, thereby promoting Lrp6 phosphorylation to initiate beta-catenin signaling. We discuss the dual roles of the axin-Gsk3 complex and signal amplification by Lrp6-axin interaction during Wnt/beta-catenin signaling.     

The prickle-related gene in vertebrates is essential for gastrulation cell movements

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis [1,2]. Recently, it was reported that a noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila [3,4], participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo [5-8]. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal [6-8]. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK [9,10] and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, we show that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene [11-13], is an essential component for gastrulation cell movement. Both gain-of-function and loss-of-function of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrate that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation.     

The mitogen-activated protein kinase kinase kinase kinase GCKR positively regulates canonical and noncanonical Wnt signaling in B lymphocytes

Wnt ligands bind receptors of the Frizzled (Fz) family to control cell fate, proliferation, and polarity. Canonical Wnt/Fz signaling stabilizes beta-catenin by inactivating GSK3beta, leading to the translocation of beta-catenin to the nucleus and the activation of Wnt target genes. Noncanonical Wnt/Fz signaling activates RhoA and Rac, and the latter triggers the activation of c-Jun N-terminal kinase (JNK). Here, we show that exposure of B-lymphocytes to Wnt3a-conditioned media activates JNK and raises cytosolic beta-catenin levels. Both the Rac guanine nucleotide exchange factor Asef and the mitogen-activated protein kinase kinase kinase kinase germinal center kinase-related enzyme (GCKR) are required for Wnt-mediated JNK activation in B cells. In addition, we show that GCKR positively affects the beta-catenin pathway in B cells. Reduction of GCKR expression inhibits Wnt3a-induced phosphorylation of GSK3beta at serine 9 and decreases the accumulation of cytosolic beta-catenin. Furthermore, Wnt signaling induces an interaction between GCKR and GSK3beta. Our findings demonstrate that GCKR facilitates both canonical and noncanonical Wnt signaling in B lymphocytes.     

Functional dissection of phosphorylation of Disheveled in Drosophila

Disheveled/Dsh proteins (Dvl in mammals) are core components of both Wnt/Wg-signaling pathways: canonical β-catenin signaling and Frizzled (Fz)-planar cell polarity (PCP) signaling. Although Dsh is a key cytoplasmic component of both Wnt/Fz-pathways, regulation of its signaling specificity is not well understood. Dsh is phosphorylated, but the functional significance of its phosphorylation remains unclear. We have systematically investigated the phosphorylation of Dsh by combining mass-spectrometry analyses, biochemical studies, and in vivo genetic methods in Drosophila. Our approaches identified multiple phospho-residues of Dsh in vivo. Our data define three novel and unexpected conclusions: (1) strikingly and in contrast to common assumptions, all conserved serines/threonines are non-essential for Dsh function in either pathway; (2) phosphorylation of conserved Tyrosine473 in the DEP domain is critical for PCP-signaling — Dsh^Y473F behaves like a PCP-specific allele; and (3) defects associated with the PCP specific dsh¹ allele, Dsh^K417M, located within a putative Protein Kinase C consensus site, are likely due to a post-translational modification requirement of Lys417, rather than phosphorylation nearby. In summary, our combined data indicate that while many Ser/Thr and Tyr residues are indeed phosphorylated in vivo, strikingly most of these phosphorylation events are not critical for Dsh function with the exception of DshY473.     

Prickle and Strabismus form a functional complex to generate a correct axis during planar cell polarity signaling

Frizzled (Fz) signaling regulates the establishment of planar cell polarity (PCP). The PCP genes prickle (pk) and strabismus (stbm) are thought to antagonize Fz signaling. We show that they act in the same cell, R4, adjacent to that in which the Fz/PCP pathway is required in the Drosophila eye. We demonstrate that Stbm and Pk interact physically and that Stbm recruits Pk to the cell membrane. Through this interaction, Pk affects Stbm membrane localization and can cause clustering of Stbm. Pk is also known to interact with Dsh and is thought to antagonize Dsh by affecting its membrane localization. Thus our data suggest that the Stbm/Pk complex modulates Fz/Dsh activity, resulting in a symmetry-breaking step during polarity signaling.     

The effect of pleiotrophin signaling on adipogenesis

Pleiotrophin (PTN) plays diverse roles in cell growth and differentiation. In this investigation, we demonstrate that PTN plays a negative role in adipogensis and that glycogen synthase kinase 3beta (GSK-3beta) and beta-catenin are involved in the regulation of PTN-mediated preadipocyte differentiation. Knocking down the expression of PTN using siRNA resulted in an increase in phospho-GSK-3beta expression, and the accumulation of nuclear beta-catenin, which are critical downstream signaling proteins for both the PTN and Wnt signaling pathways. Our investigation suggests that there is a PTN/PI3K/AKT/GSK-3beta/beta-catenin signaling pathway, which cross-talks with the Wnt/Fz/GSK-3beta/beta-catenin pathway and negatively regulates adipogenesis.