Herr P, Basler KPorcupine-mediated lipidation is required for Wnt recognition by Wls. Dev Biol 361:392-402

Institute of Molecular Life Sciences, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
Developmental Biology (Impact Factor: 3.55). 11/2011; 361(2):392-402. DOI: 10.1016/j.ydbio.2011.11.003
Source: PubMed


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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    • "Wnts to be recognized and transported by Wntless (Wls) from the Golgi to the plasma membrane 388 (Coombs et al., 2010; Herr and Basler, 2012). Therefore, Porc inhibition causes intracellular 389 accumulation of Wnt3EGFP and a reduction of the membrane-bound fraction. "
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    ABSTRACT: The boundaries of brain regions are associated with the tissue-specific secretion of ligands from different signalling pathways. The dynamics of these ligands in vivo and the impact of its disruption remain largely unknown. We used light and fluorescence microscopy for the overall imaging of the specimen and fluorescence correlation spectroscopy (FCS) to determine Wnt3 dynamics and demonstrated that Wnt3 regulates cerebellum development during embryogenesis using zebrafish Wnt3 transgenics with either tissue-specific expression of an EGFP reporter or a functionally active fusion protein, Wnt3EGFP. The results suggest a state of dynamic equilibrium of Wnt3EGFP mobility in polarized neuroepithelial-like progenitors in the dorsal midline and cerebellar progenitors on the lateral side. Wnt3EGFP secretes from the cerebellum as shown by measurements of its mobility in the ventricular cavity. The importance of Wnt secretion in brain patterning was validated with the Porc inhibitor Wnt-C59 (C59), which applied early reduced membrane-bound and secreted fractions of Wnt3EGFP and led to a malformed brain characterized by the absence of epithalamus, optic tectum and cerebellum. Likewise, interference with Wnt secretion later on during cerebellar development negatively impacted cerebellar growth and patterning. Our work supported by quantitative analysis of protein dynamics in vivo, highlights the importance of membrane localized and secreted Wnt3 during cerebellum development.
    Development 09/2015; DOI:10.1242/dev.127589 · 6.46 Impact Factor
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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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