Porcupine-mediated lipidation is required for Wnt recognition by Wls

Institute of Molecular Life Sciences, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Developmental Biology (Impact Factor: 3.64). 11/2011; 361(2):392-402. DOI: 10.1016/j.ydbio.2011.11.003
Source: PubMed

ABSTRACT Wnt proteins are members of a conserved family of secreted signaling ligands and play crucial roles during development and in tissue homeostasis. There is increasing evidence that aberrant Wnt production is an underlying cause of dysregulated Wnt signaling, however little is known about this process. One protein known to play a role in secretion is the transmembrane protein Wntless (Wls). However, the mechanism by which Wls promotes Wnt secretion is a riddle. It is not known which Wnt family members require Wls and what the structural requirements are that make some of them reliant on Wls for secretion. Here we present a systematic analysis of all known Drosophila Wnt family members with respect to their dependence on Wls function for secretion. We first show that the glycosylation status of Wg at conserved sites does not determine its dependence on Wls. Moreover, in apparent contrast to murine wls, Drosophila wls is not a target gene of canonical Wnt signaling. We then show that all Wnts, with the exception of WntD, require Wls for secretion. All Wnts, with the exception of WntD, also contain a conserved Serine residue (in Wg S239), which we show to be essential for their functional and physical interaction with Wls. Finally, all Wnts, with the exception of WntD, require the acyltransferase Porcupine for activity and for functionally interacting with Wls. Together, these findings indicate that Por-mediated lipidation of the S239-equivalent residue is essential for the interaction with, and secretion by, Wls.

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    • "Porcupine catalyzes the palmitoylation of Wnts which facilitates their secretion to become functionally active. In mice, Wnt3a porcupine allows for lipidation at the cysteine 77 and serine 209 [6], [7]. The lipidated Wnts are then transported to the Golgi where they are bound by the transmembrane protein Wntless. "
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    ABSTRACT: Wnts are small secreted glycoproteins that are highly conserved among species. To date, 19 Wnts have been described, which initiate a signal transduction cascade that is either β-catenin dependent or independent, culminating in the regulation of hundreds of target genes. Extracellular release of Wnts is dependent on lipidation of Wnts by porcupine, a membrane-bound-O-acyltransferase protein in the endoplasmic reticulum. Studies demonstrating the requirement of porcupine for Wnts production are based on cell line and non-human primary cells. We evaluated the requirement for porcupine for Wnts production in human primary astrocytes and CD8+ T cells. Using IWP-2, an inhibitor of porcupine, or siRNA targeting porcupine, we demonstrate that porcupine is not required for the release of Wnt 1, 3, 5b, 6,7a, 10b, and 16a. While IWP had no effect on Wnt 2b release, knockdown of porcupine by siRNA reduced Wnt 2b release by 60%. These data indicate that porcupine-mediated production of Wnts is context dependent and is not required for all Wnts production, suggesting that alternative mechanisms exist for Wnts production.
    PLoS ONE 03/2014; 9(3):e92159. DOI:10.1371/journal.pone.0092159 · 3.23 Impact Factor
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    • "WNT proteins are highly hydrophobic, a property imparted by the covalent attachment of at least one lipid molecule to a conserved residue [31,32]. This modification is important for proper processing and secretion [32,33]. In addition, as revealed by the Wnt-Fzd co-crystal structure, the lipid moiety is critical for receptor binding [34]. "
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    ABSTRACT: WNT5A, a member of the WNT family of secreted lipid-modified glycoproteins, is a critical regulator of a host of developmental processes, including limb formation, lung morphogenesis, intestinal elongation and mammary gland development. Altered WNT5A expression has been associated with a number of cancers. Interestingly, in certain types of cancers, such as hematological malignancies and colorectal carcinoma, WNT5A is inactivated and exerts a tumor suppressive function, while in other cancers, such as melanoma and gastric carcinoma, WNT5A is overexpressed and promotes tumor progression. The mechanism by which WNT5A achieves these distinct activities in cancers is poorly understood. Here, we provide evidence that the WNT5A gene produces two protein isoforms, WNT5A-long (WNT5A-L) and WNT5A-short (WNT5A-S). Amino-terminal sequencing and a WNT5A-L specific antibody demonstrate that the mature and secreted isoforms are distinct, with WNT5A-L carrying an additional 18 N-terminal amino acids. Biochemical analysis indicates that both purified proteins are similar with respect to their stability, hydrophobicity and WNT/β-catenin signaling activity. Nonetheless, modulation of these two WNT5A isoforms, either through ectopic expression or knockdown, demonstrates that they exert distinct activities in cancer cell lines: while WNT5A-L inhibits proliferation of tumor cell lines, WNT5A-S promotes their growth. Finally, we show that expression of these two WNT5A isoforms is altered in breast and cervix carcinomas, as well as in the most aggressive neuroblastoma tumors. In these cancers, WNT5A-L is frequently down-regulated, whereas WNT5A-S is found overexpressed in a significant fraction of tumors. Altogether, our study provides evidence that the distinct activities of WNT5A in cancer can be attributed to the production of two WNT5A isoforms.
    PLoS ONE 11/2013; 8(11):e80526. DOI:10.1371/journal.pone.0080526 · 3.23 Impact Factor
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    • "The secretion of all mammalian Wnt ligands is highly conserved and controlled by post-translational modifications (Herr et al., 2012; Najdi et al., 2012). Palmitoylation of a conserved serine (S209 in Wnt3a) by Porcn (Kadowaki et al., 1996; Takada et al., 2006), a member of the membrane-bound O-acyl transferase (MBOAT) family (Hofmann, 2000), is required for binding of Wnt ligands to Wls (Coombs et al., 2010; Herr and Basler, 2012), a cargo receptor required for transport from the Golgi to the cell surface, as well as binding to Fzd receptors on signal-receiving cells (Janda et al., 2012; Komekado et al., 2007). These functions place Porcn in a key position in the Wnt signaling network, as Wnt ligands fail to be secreted efficiently from cells in culture or to activate Fzddependent Wnt signaling in the absence of Porcn (Galli and Burrus, 2011; Janda et al., 2012; Najdi et al., 2012; Takada et al., 2006), and result in tight control over Wnt signaling by Porcn function in the secreting cells (Biechele et al., 2011; Proffitt and Virshup, 2012). "
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    ABSTRACT: In mice and humans the X-chromosomal porcupine homolog (Porcn) gene is required for the acylation and secretion of all 19 Wnt ligands and thus represents a bottleneck for all Wnt signaling. We have generated a mouse line carrying a floxed allele for Porcn and used zygotic, oocyte-specific and visceral endoderm-specific deletions to investigate embryonic and extra-embryonic requirements for Wnt ligand secretion. We show that there is no requirement for Porcn-dependent secretion of Wnt ligands during preimplantation development of the mouse embryo. Porcn-dependent Wnts are first required for the initiation of gastrulation, where Porcn function is required in the epiblast but not the visceral endoderm. Heterozygous female embryos, which are mutant in both trophoblast and visceral endoderm due to imprinted X chromosome inactivation, complete gastrulation but display chorio-allantoic fusion defects similar to Wnt7b mutants. Our studies highlight the importance of Wnt3 and Wnt7b for embryonic and placental development but suggest that endogenous Porcn-dependent Wnt secretion does not play an essential role in either implantation or blastocyst lineage specification.
    Development 06/2013; 140(14). DOI:10.1242/dev.094458 · 6.27 Impact Factor
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