Sailing with the Wnt: Charting the Wnt processing and secretion route

Wnt proteins are members of a highly conserved family of signalling molecules that play a central role in development and disease. During the past years, the different signalling pathways that are triggered by Wnt proteins have been studied in detail, but it is still largely unknown how a functional Wnt protein is produced and secreted. The recent finding that Wnt proteins are post-translationally modified and the discovery of the Wnt binding protein Wntless and its trafficking by the retromer complex show that Wnt secretion is a complex and highly regulated process. In this review, we will give an overview of the Wnt maturation and secretion pathway and discuss how this process may influence the spreading and signalling activity of Wnt.     

WNT secretion and signalling in human disease

Wnt signalling, a key pathway involved in various aspects of embryonic development, also underlies many human diseases, in particular, cancer. Research focused on signal transduction within signal-receiving cells led to the discovery of many Wnt pathway components, but study of the secretion of Wnt ligands themselves was neglected until recently. Attention was drawn to this highly regulated process by the association of aberrant Wnt levels with an increasing number of diseases. Studying the biogenesis and processing of active Wnt ligands will open new avenues for generating therapeutics to specifically target aberrant Wnt signalling. Here we review the proteins required for Wnt secretion and signalling at the plasma membrane, ending with a discussion on potential therapeutic approaches to treat Wnt-induced diseases.     

Lipid-independent secretion of a Drosophila Wnt protein

Wnt proteins comprise a large class of secreted signaling molecules with key roles during embryonic development and throughout adult life. Recently, much effort has been focused on understanding the factors that regulate Wnt signal production. For example, Porcupine and Wntless/Evi/Sprinter have been identified as being required in Wnt-producing cells for the processing and secretion of many Wnt proteins. Interestingly, in this study we find that WntD, a recently characterized Drosophila Wnt family member, does not require Porcupine or Wntless/Evi/Sprinter for its secretion or signaling activity. Because Porcupine is involved in post-translational lipid modification of Wnt proteins, we used a novel labeling method and mass spectrometry to ask whether WntD undergoes lipid modification and found that it does not. Although lipid modification is also hypothesized to be required for Wnt secretion, we find that WntD is secreted very efficiently. WntD secretion does, however, maintain a requirement for the secretory pathway component Rab1. Our results show that not all Wnt family members require lipid modification, Porcupine, or Wntless/Evi/Sprinter for secretion and suggest that different modes of secretion may exist for different Wnt proteins.     

Organising cells into tissues: new roles for cell adhesion molecules in planar cell polarity

Planar cell polarity (PCP) is the coordinated organization of cells within the plane of the epithelium, first described in Drosophila. A Frizzled signalling pathway dedicated to PCP (the non-canonical Frizzled pathway) acts through Dishevelled and small G proteins, as does the classical Wnt pathway, but then diverges downstream of Dishevelled. Recent studies have demonstrated a crucial role for several atypical cadherin molecules (Fat, Dachsous and Flamingo) in controlling PCP signalling. Recent work has also indicated that the first sign of PCP during development is the polarized localization of PCP proteins (Frizzled, Flamingo, Dishevelled, etc). Exciting new data reveal that this PCP pathway is conserved to man.     

A genome-wide RNA interference screen uncovers two p24 proteins as regulators of Wingless secretion

Wnt proteins are secreted, lipid-modified glycoproteins that control animal development and adult tissue homeostasis. Secretion of Wnt proteins is at least partly regulated by a dedicated machinery. Here, we report a genome-wide RNA interference screen for genes involved in the secretion of Wingless (Wg), a Drosophila Wnt. We identify three new genes required for Wg secretion. Of these, Emp24 and Eclair are required for proper export of Wg from the endoplasmic reticulum (ER). We propose that Emp24 and Eca act as specific cargo receptors for Wg to concentrate it in forming vesicles at sites of ER export.     

Updating the Wnt pathways

In the three decades since the discovery of the Wnt1 proto-oncogene in virus-induced mouse mammary tumours, our understanding of the signalling pathways that are regulated by the Wnt proteins has progressively expanded. Wnts are involved in an complex signalling network that governs multiple biological processes and cross-talk with multiple additional signalling cascades, including the Notch, FGF (fibroblast growth factor), SHH (Sonic hedgehog), EGF (epidermal growth factor) and Hippo pathways. The Wnt signalling pathway also illustrates the link between abnormal regulation of the developmental processes and disease manifestation. Here we provide an overview of Wnt-regulated signalling cascades and highlight recent advances. We focus on new findings regarding the dedicated Wnt production and secretion pathway with potential therapeutic targets that might be beneficial for patients with Wnt-related diseases.     

Wnts need a p(assport)24 to leave the ER

Wnts are secreted through a dedicated exocytic pathway, which has been only partly characterized. Here, Palmer and colleagues comment on two recent reports by the groups of K. Basler and M. Boutros, respectively, in which they show that p24 proteins take part in this exocytic route and are required for Wnt exit from the ER to the Golgi.EMBO Rep (2011) advance online publication. doi:10.1038/embor.2011.212Wnt signalling proteins regulate diverse cellular processes during development and homeostasis. Since misregulation of Wnt signalling is associated with cancer, most research has been aimed at characterizing the signal transduction pathway. Recently, attention has focused on Wnt production due to the identification of factors required specifically for Wnt secretion. For instance, the specific requirement of Evi, also known as Wntless or Sprinter, suggested that Wnts might follow a specialized secretory route (Banziger et al, 2006; Bartscherer et al, 2006). Two recent papers published in EMBO reports by the groups of Konrad Basler in last month''s issue, and Michael Boutros in this issue, show that Wnt secretion requires the activity of p24 family members (Buechling et al, 2011; Port et al, 2011). Therefore, the specialized route might begin at endoplasmic reticulum (ER) exit sites.Wnts are secreted glycoproteins that can act many cell diameters from their source of production. Most, but not all Wnt proteins, are acylated and thus associate tightly with cellular membranes. Despite this association, acylated Wnts can be released from secreting cells and spread in the extracellular space (Bartscherer & Boutros, 2008; Port & Basler, 2010). Acylation of Wnts, which occurs in the ER, is thought to be mediated by the N-acetyl transferase encoded by porcupine (porc; van den Heuvel et al, 1993). After acylation, Wnt proteins associate with Evi, a multipass transmembrane protein found mostly at the Golgi and the plasma membrane (Fig 1A). This association is essential for the secretion of the Wnts that are acylated since, in the absence of Evi, they accumulate on internal membranes (Banziger et al, 2006; Bartscherer et al, 2006). It is therefore thought that acylated Wnts require Evi to exit the Golgi and progress to the cell surface (Port et al, 2008). This does not seem to be a requirement for non-lipidated Wnts, such as Drosophila WntD, which are secreted in the absence of Evi (Ching et al, 2008). Similarly, secretion of other signalling proteins proceeds normally without Evi. After reaching the cell surface, Evi is likely to have a choice between various routes. One such route, which involves the retromer complex, takes it back to the Golgi where it can participate in another round of Wnt secretion (Fig 1A). Alternatively, Evi can be targeted to lysosomes (Bartscherer & Boutros, 2008; Port & Basler, 2010). The factors that determine Evi transport remain poorly understood. Nevertheless, these studies highlight the essential and specific role of Evi for the secretion of lipidated Wnts.Open in a separate windowFigure 1Model summarizing the suggested roles of p24 proteins in endoplasmic reticulum to Golgi transport of Wg. (A) Schematic of a cell showing the current model of the Wg secretory pathway. Wg is produced in the ER where it is lipid-modified by Porc, and moved to the Golgi with the assistance of p24 proteins. In the Golgi, Wg joins Evi, which facilitates Wg transport to the cell surface. Evi is then recycled back to the Golgi in a path mediated by the retromer complex. (B)(i) In the absence of p24 proteins, there is no recruitment of Wg to COPII-coated vesicles and therefore a block of secretion in the ER. (ii) Port et al (2011) propose a similar model in which CHOp24/Emp24 and Éclair are involved in Wg recruitment, although only CHOp24/Emp24 binds to Wg. (iii) According to Buechling et al (2011), Opm recruits Wg to COPII-coated vesicles for movement to the Golgi. CHOp24 and p24-1 are also required for this process. ER, endoplasmic reticulum.Two groups have now reported the use of cell-based RNA interference (RNAi) screens to identify further proteins required for the secretion of Wingless (Wg), the main Drosophila Wnt (Buechling et al, 2011; Port et al, 2011). They found that p24 family members, a group of proteins previously implicated in both retrograde and anterograde transport between the ER and Golgi (Strating & Martens, 2009), are required for Wg secretion by S2 cells. Similarly, knockdown by transgenic RNAi shows that p24 proteins are required for normal levels of Wg secretion in Drosophila wing imaginal discs (Buechling et al, 2011; Port et al, 2011). As with Evi, this requirement seems to be relatively specific, since general secretion and the secretion of other signalling proteins, including the lipid-modified morphogen Hedgehog, are unaffected by p24 knockdown. Buechling et al also assessed the role of p24 proteins in WntD secretion. They found that RNAi against opossum (opm), one of the p24 members, prevents WntD secretion in cultured cells. They also show that the phenotypes of opm mutants and WntD mutant embryos resemble each other (Buechling et al, 2011). Therefore, while Evi is specifically required for the secretion of acylated Wnts, p24 proteins could contribute to the secretion of all Wnts. This function is likely to be conserved since the mammalian homologue of Opm, TMED5, is required for Wnt1 signalling, at least in a mammalian cell culture assay (Buechling et al, 2011).To gain understanding of the role of p24 proteins in Wnt secretion, both groups analysed the subcellular localization of Wg following p24 knockdown. They found accumulation in the ER and concomitant depletion in the Golgi, as indicated by reduced co-localization with Golgi markers (Fig 1Bi). They also found that p24 knockdown prevents Wg from stabilizing Evi in producing cells, suggesting that the stabilizing influence of Wg requires its exit from the ER (Buechling et al, 2011; Port et al, 2011). These results lead the authors to propose that the loss of p24 prevents the transport of Wg from the ER to the Golgi. Importantly, immunoprecipitation experiments suggest that Wg might interact physically with Opm and Emp24 (also known as CHOp24). This led both sets of authors to postulate a model whereby p24 proteins act as cargo receptors to escort Wnt proteins from the ER to the Golgi, whereupon they can bind to Evi, which will escort them to the plasma membrane. Thus, in this context, p24 proteins seem to have an anterograde function.Although both studies highlight the role of p24 proteins in Wnt secretion, they disagree on the relative importance of the various family members. Among the nine predicted p24 proteins encoded by the Drosophila genome, only Éclair and Emp24/CHOp24 were found to be required for Wg secretion by Port et al (2011; Fig 1Bii). By contrast, Buechling et al found that Opm, Emp24/CHOp24 and p24-1 all play a role in Wg secretion (fig 1Biii). Thus, only Emp24/CHOp24 is found by both groups to be essential for Wg secretion. Although functional redundancy among p24 proteins could explain why the removal of a single p24 protein has a relatively weak phenotype, there is no simple explanation as to why the very similar assays used by the two groups do not lead to identical conclusions. These differences could be worked out by the exchange of reagents and protocols.Regardless of the discrepancies, the two studies provide an important step in our understanding of Wnt secretion by demonstrating that Wnts engage with specialized components of the secretory machinery as early as in the ER. It might be relevant that the anterograde function of p24 proteins is directed at glycophosphatidylinositol (GPI)-anchored proteins, which have been shown to partition in raft-like microdomains (Strating & Martens, 2009). It is conceivable that GPI-anchored proteins, as well as Wnts, gather in a subdomain of the ER where they could both interact with p24 proteins and set off along their specialized secretory pathways. Wnt targeting to specialized membrane domains could in principle be mediated by their lipid moieties (Bartscherer & Boutros, 2008; Port & Basler, 2010). However, the process might turn out to be more complex if it is confirmed that p24 proteins are also required for the secretion of non-acylated Wnts (for example, WntD), as suggested by Buechling et al. In any case, it will be interesting to determine the precise molecular mechanism underlying the functional interaction between Wnts and p24 proteins as it is likely to explain how Wnts are allowed to exit the ER and start their journey out of the cell.     

Fatty acid modification of Wnt1 and Wnt3a at serine is prerequisite for lipidation at cysteine and is essential for Wnt signalling

The Wnt family of proteins is a group of extracellular signalling molecules that regulate cell-fate decisions in developing and adult tissues. It is presumed that all 19 mammalian Wnt family members contain two types of post-translational modification: the covalent attachment of fatty acids at two distinct positions, and the N-glycosylation of multiple asparagines. We examined how these modifications contribute to the secretion, extracellular movement and signalling activity of mouse Wnt1 and Wnt3a ligands. We revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. As such, mutant proteins that lack the crucial serine residue are not lipidated. Interestingly, although double-acylation of Wnt1 was indispensable for signalling in mammalian cells, in Xenopus embryos the S-palmitoyl-deficient form retained the signalling activity. In the case of Wnt3a, the functional duality of the attached acyls was less prominent, since the ligand lacking S-linked palmitate was still capable of signalling in various cellular contexts. Finally, we show that the signalling competency of both Wnt1 and Wnt3a is related to their ability to associate with the extracellular matrix.     

PTK7/Otk interacts with Wnts and inhibits canonical Wnt signalling

Wnt signalling is an evolutionarily conserved pathway that directs cell-fate determination and morphogenesis during metazoan development. Wnt ligands are secreted glycoproteins that act at a distance causing a wide range of cellular responses from stem cell maintenance to cell death and cell proliferation. How Wnt ligands cause such disparate responses is not known, but one possibility is that different outcomes are due to different receptors. Here, we examine PTK7/Otk, a transmembrane receptor that controls a variety of developmental and physiological processes including the regulation of cell polarity, cell migration and invasion. PTK7/Otk co-precipitates canonical Wnt3a and Wnt8, indicating a role in Wnt signalling, but PTK7 inhibits rather than activates canonical Wnt activity in Xenopus, Drosophila and luciferase reporter assays. Loss of PTK7 function activates canonical Wnt signalling and epistasis experiments place PTK7 at the level of the Frizzled receptor. In Drosophila, Otk interacts with Wnt4 and opposes canonical Wnt signalling in embryonic patterning. We propose a model where PTK7/Otk functions in non-canonical Wnt signalling by turning off the canonical signalling branch.     

Wnt signalling: pathway or network?

Members of the Wnt family of secreted glycoproteins participate in many signalling events during development. Recent findings suggest that Wnt signals can sometimes play a permissive role during cell-fate assignment. Wnt proteins have been shown to interact with a number of extracellular and cell-surface proteins, whereas many intracellular components of the Wnt-signalling pathway are also involved in other cellular functions. The consequences of Wnt signalling can be affected by members of the MAP kinase family. These observations suggest that the future understanding of Wnt signalling may require models that are based on a signalling network rather than a single linear pathway.     

The C-terminal cytoplasmic Lys-thr-X-X-X-Trp motif in frizzled receptors mediates Wnt/beta-catenin signalling

Frizzled receptors are components of the Wnt signalling pathway, but how they activate the canonical Wnt/beta-catenin pathway is not clear. Here we use three distinct vertebrate frizzled receptors (Xfz3, Xfz4 and Xfz7) and describe whether and how their C-terminal cytoplasmic regions transduce the Wnt/beta-catenin signal. We show that Xfz3 activates this pathway in the absence of exogenous ligands, while Xfz4 and Xfz7 interact with Xwnt5A to activate this pathway. Analysis using chimeric receptors reveals that their C-terminal cytoplasmic regions are functionally equivalent in Wnt/beta-catenin signalling. Furthermore, a conserved motif (Lys-Thr-X-X-X-Trp) located two amino acids after the seventh transmembrane domain is required for activation of the Wnt/beta-catenin pathway and for membrane relocalization and phosphorylation of Dishevelled. Frizzled receptors with point mutations affecting either of the three conserved residues are defective in Wnt/beta-catenin signalling. These findings provide functional evidence supporting a role of this conserved motif in the modulation of Wnt signalling. They are consistent with the genetic features exhibited by Drosophila Dfz3 and Caenorhabditis elegans mom-5 in which the tryptophan is substituted by a tyrosine.     

The retromer complex influences Wnt secretion by recycling wntless from endosomes to the trans-Golgi network

Secreted Wnt proteins play essential roles in many biological processes during development and diseases. However, little is known about the mechanism(s) controlling Wnt secretion. Recent studies have identified Wntless (Wls) and the retromer complex as essential components involved in Wnt signaling. While Wls has been shown to be essential for Wnt secretion, the function(s) of the retromer complex in Wnt signaling is unknown. Here, we have examined a role of Vps35, an essential retromer subunit, in Wnt signaling in Drosophila and mammalian cells. We provide compelling evidence that the retromer complex is required for Wnt secretion. Importantly, Vps35 colocalizes in endosomes and interacts with Wls. Wls becomes unstable in the absence of retromer activity. Our findings link Wls and retromer functions in the same conserved Wnt secretion pathway. We propose that retromer influences Wnt secretion by recycling Wntless from endosomes to the trans-Golgi network (TGN).     

Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells

Cell-cell communication via Wnt signals represents a fundamental means by which animal development and homeostasis are controlled. The identification of components of the Wnt pathway is reaching saturation for the transduction process in receiving cells but is incomplete concerning the events occurring in Wnt-secreting cells. Here, we describe the discovery of a novel Wnt pathway component, Wntless (Wls/Evi), and show that it is required for Wingless-dependent patterning processes in Drosophila, for MOM-2-governed polarization of blastomeres in C. elegans, and for Wnt3a-mediated communication between cultured human cells. In each of these cases, Wls is acting in the Wnt-sending cells to promote the secretion of Wnt proteins. Since loss of Wls function has no effect on other signaling pathways yet appears to impede all the Wnt signals we analyzed, we propose that Wls represents an ancient partner for Wnts dedicated to promoting their secretion into the extracellular milieu.     

Wnt signalling: antagonistic Dickkopfs

Dickkopf proteins are secreted antagonists of the Wnt cell signalling molecules, which have a novel mode of action. Dickkopf1 binds to the LRP5/6 Wnt co-receptor and prevents the formation of active Wnt--Frizzled--LRP5/6 receptor complexes, thus blocking the canonical Wnt--beta-catenin pathway.     

Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface

Glypicans are heparan sulfate proteoglycans that are attached to the cell surface by a GPI (glycosylphosphatidylinositol)anchor. Glypicans regulate the activity of Wnts, Hedgehogs,bone morphogenetic proteins and fibroblast growth factors. In the particular case of Wnts, it has been proposed that GPI-anchored glypicans stimulate Wnt signalling by facilitating and/or stabilizing the interaction between Wnts and their cell surface receptors. On the other hand, when glypicans are secreted to the extracellular environment, they can act as competitive inhibitors of Wnt. Genetic screens in Drosophila have recently identified a novel inhibitor of Wnt signalling named Notum. The Wnt inhibiting activity of Notum was associated with its ability to release Dlp [Dally (Division abnormally delayed)-like protein; a Drosophila glypican] from the cell surface by cleaving the GPI anchor. Because these studies showed that the other Drosophila glypican Dally was not released from the cell surface by Notum,it remains unclear whether this enzyme is able to cleave glypicans from mammalian cells. Furthermore, it is also not known whether Notum cleaves GPI-anchored proteins that are not members of the glypican family. Here, we show that mammalian Notum can cleave several mammalian glypicans. Moreover, we demonstrate that Notum is able to release GPI-anchored proteins other than glypicans. Another important finding of the present study is that,unlike GPI-phospholipase D, the other mammalian enzyme that cleaves GPI-anchored proteins, Notum is active in the extracellular environment. Finally, by using a cellular system in which GPC3 (glypican-3) stimulates Wnt signalling, we show that Notum can act as a negative regulator of this growth factor.     

The C-terminal domain of armadillo binds to hypophosphorylated teashirt to modulate wingless signalling in Drosophila

Wnt signalling is a key pathway for tissue patterning during animal development. In Drosophila, the Wnt protein Wingless acts to stabilize Armadillo inside cells where it binds to at least two DNA-binding factors which regulate specific target genes. One Armadillo-binding protein in Drosophila is the zinc finger protein Teashirt. Here we show that Wingless signalling promotes the phosphorylation and the nuclear accumulation of Teashirt. This process requires the binding of Teashirt to the C-terminal end of Armadillo. Finally, we present evidence that the serine/threonine kinase Shaggy is associated with Teashirt in a complex. We discuss these results with respect to current models of Armadillo/beta-catenin action for the transmission of the Wingless/Wnt pathway.     

Wnt signalling requires MTM-6 and MTM-9 myotubularin lipid-phosphatase function in Wnt-producing cells

Wnt proteins are lipid-modified glycoproteins that have important roles in development, adult tissue homeostasis and disease. Secretion of Wnt proteins from producing cells is mediated by the Wnt-binding protein MIG-14/Wls, which binds Wnt in the Golgi network and transports it to the cell surface for release. It has recently been shown that recycling of MIG-14/Wls from the plasma membrane to the trans-Golgi network is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is still poorly understood. In this study, we report the identification of MTM-6 and MTM-9 as novel regulators of MIG-14/Wls trafficking in Caenorhabditis elegans. MTM-6 and MTM-9 are myotubularin lipid phosphatases that function as a complex to dephosphorylate phosphatidylinositol-3-phosphate, a central regulator of endosomal trafficking. We show that mutation of mtm-6 or mtm-9 leads to defects in several Wnt-dependent processes and demonstrate that MTM-6 is required in Wnt-producing cells as part of the MIG-14/Wls-recycling pathway. This function is evolutionarily conserved, as the MTM-6 orthologue DMtm6 is required for Wls stability and Wg secretion in Drosophila. We conclude that regulation of endosomal trafficking by the MTM-6/MTM-9 myotubularin complex is required for the retromer-dependent recycling of MIG-14/Wls and Wnt secretion.     

Wnt signaling requires retromer-dependent recycling of MIG-14/Wntless in Wnt-producing cells

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. We have previously shown that Wnt signaling requires retromer function in Wnt-producing cells. The retromer is a multiprotein complex that mediates endosome-to-Golgi transport of specific sorting receptors. MIG-14/Wls is a conserved transmembrane protein that binds Wnt and is required in Wnt-producing cells for Wnt secretion. Here, we demonstrate that in the absence of retromer function, MIG-14/Wls is degraded in lysosomes and becomes limiting for Wnt signaling. We show that retromer-dependent recycling of MIG-14/Wls is part of a trafficking pathway that retrieves MIG-14/Wls from the plasma membrane. We propose that MIG-14/Wls cycles between the Golgi and the plasma membrane to mediate Wnt secretion. Regulation of this transport pathway may enable Wnt-producing cells to control the range of Wnt signaling in the tissue.