SNX3-retromer requires an evolutionary conserved MON2:DOPEY2:ATP9A complex to mediate Wntless sorting and Wnt secretion.

Wntless transports Wnt morphogens to the cell surface and is required for Wnt secretion and morphogenic gradients formation. Recycling of endocytosed Wntless requires the sorting nexin-3 (SNX3)-retromer-dependent endosome-to-Golgi transport pathway. Here we demonstrate the essential role of SNX3-retromer assembly for Wntless transport and report that SNX3 associates with an evolutionary conserved endosome-associated membrane re-modelling complex composed of MON2, DOPEY2 and the putative aminophospholipid translocase, ATP9A. In vivo suppression of Ce-mon-2, Ce-pad-1 or Ce-tat-5 (respective MON2, DOPEY2 and ATP9A orthologues) phenocopy a loss of SNX3-retromer function, leading to enhanced lysosomal degradation of Wntless and a Wnt phenotype. Perturbed Wnt signalling is also observed upon overexpression of an ATPase-inhibited TAT-5(E246Q) mutant, suggesting a role for phospholipid flippase activity during SNX3-retromer-mediated Wntless sorting. Together, these findings provide in vitro and in vivo mechanistic details to describe SNX3-retromer-mediated transport during Wnt secretion and the formation of Wnt-morphogenic gradients.

Protein kinase CK2 is required for Wntless internalization and Wnt secretion.

Wnt proteins are lipid modified signaling molecules that have essential functions in development and adult tissue homeostasis. Secretion of Wnt is mediated by the transmembrane protein Wntless, which binds Wnt and transports it from the endoplasmic reticulum to the cell surface for release. To maintain efficient Wnt secretion, Wntless is recycled back to the Golgi and the endoplasmic reticulum through endocytosis and retromer dependent endosome to Golgi transport. We have previously identified protein kinase CK2 (CK2) in a genome-wide screen for regulators of Wnt signaling in Caenorhabditis elegans. Here, we show that CK2 function is required in Wnt producing cells for Wnt secretion. This function is evolutionarily conserved, as inhibition of CK2 activity interferes with Wnt5a secretion from mammalian cells. Mechanistically, we show that inhibition of CK2 function results in enhanced plasma membrane localization of Wls in C. elegans and mammalian cells, consistent with the notion that CK2 is involved in the regulation of Wls internalization.

Huwe1-mediated ubiquitylation of dishevelled defines a negative feedback loop in the Wnt signaling pathway.

Wnt signaling plays a central role in development, adult tissue homeostasis, and cancer. Several steps in the canonical Wnt/ß-catenin signaling cascade are regulated by ubiquitylation, a protein modification that influences the stability, subcellular localization, or interactions of target proteins. To identify regulators of the Wnt/ß-catenin pathway, we performed an RNA interference screen in Caenorhabditis elegans and identified the HECT domain-containing ubiquitin ligase EEL-1 as an inhibitor of Wnt signaling. In human embryonic kidney 293T cells, knockdown of the EEL-1 homolog Huwe1 enhanced the activity of a Wnt reporter in cells stimulated with Wnt3a or in cells that overexpressed casein kinase 1 (CK1) or a constitutively active mutant of the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6). However, knockdown of Huwe1 had no effect on reporter gene expression in cells expressing constitutively active ß-catenin, suggesting that Huwe1 inhibited Wnt signaling upstream of ß-catenin and downstream of CK1 and LRP6. Huwe1 bound to and ubiquitylated the cytoplasmic Wnt pathway component Dishevelled (Dvl) in a Wnt3a- and CK1ε-dependent manner. Mass spectrometric analysis showed that Huwe1 promoted K63-linked, but not K48-linked, polyubiquitination of Dvl. Instead of targeting Dvl for degradation, ubiquitylation of the DIX domain of Dvl by Huwe1 inhibited Dvl multimerization, which is necessary for its function. Our findings indicate that Huwe1 is part of an evolutionarily conserved negative feedback loop in the Wnt/ß-catenin pathway.

Retromer dependent recycling of the Wnt secretion factor Wls is dispensable for stem cell maintenance in the mammalian intestinal epithelium.

In C. elegans and Drosophila, retromer mediated retrograde transport of Wntless (Wls) from endosomes to the trans-Golgi network (TGN) is required for Wnt secretion. When this retrograde transport pathway is blocked, Wls is missorted to lysosomes and degraded, resulting in reduced Wnt secretion and various Wnt related phenotypes. In the mammalian intestine, Wnt signaling is essential to maintain stem cells. This prompted us to ask if retromer mediated Wls recycling is also important for Wnt signaling and stem cell maintenance in this system. To answer this question, we generated a conditional Vps35 (fl) allele. As Vps35 is an essential subunit of the retromer complex, this genetic tool allowed us to inducibly interfere with retromer function in the intestinal epithelium. Using a pan-intestinal epithelial Cre line (Villin-CreERT2), we did not observe defects in crypt or villus morphology after deletion of Vps35 from the intestinal epithelium. Wnt secreted from the mesenchyme of the intestine may compensate for a reduction in epithelial Wnt secretion. To exclude the effect of the mesenchyme, we generated intestinal organoid cultures. Loss of Vps35 in intestinal organoids did not affect the overall morphology of the organoids. We were able to culture Vps35 (∆/∆) organoids for many passages without Wnt supplementation in the growth medium. However, Wls protein levels were reduced and we observed a subtle growth defect in the Vps35 (∆/∆) organoids. These results confirm the role of retromer in the retrograde trafficking of Wls in the intestine, but show that retromer mediated Wls recycling is not essential to maintain Wnt signaling or stem cell proliferation in the intestinal epithelium.

RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-ß-catenin signaling.

Casein kinase 1 (CK1) members play key roles in numerous biological processes. They are considered "rogue" kinases, because their enzymatic activity appears unregulated. Contrary to this notion, we have identified the DEAD-box RNA helicase DDX3 as a regulator of the Wnt-ß-catenin network, where it acts as a regulatory subunit of CK1ε: In a Wnt-dependent manner, DDX3 binds CK1ε and directly stimulates its kinase activity, and promotes phosphorylation of the scaffold protein dishevelled. DDX3 is required for Wnt-ß-catenin signaling in mammalian cells and during Xenopus and Caenorhabditis elegans development. The results also suggest that the kinase-stimulatory function extends to other DDX and CK1 members, opening fresh perspectives for one of the longest-studied protein kinase families.

Reduced expression of ATP7B affected by Wilson disease-causing mutations is rescued by pharmacological folding chaperones 4-phenylbutyrate and curcumin.

UNLABELLED: Wilson disease (WD) is an autosomal recessive copper overload disorder of the liver and basal ganglia. WD is caused by mutations in the gene encoding ATP7B, a protein localized to the trans-Golgi network that primarily facilitates hepatic copper excretion. Current treatment comprises reduction of circulating copper by zinc supplementation or copper chelation. Despite treatment, a significant number of patients have neurological deterioration. The aim of this study was to investigate the possibility that defects arising from some WD mutations are ameliorated by drug treatment aimed at improvement of protein folding and restoration of protein function. This necessitated systematic characterization of the molecular consequences of distinct ATP7B missense mutations associated with WD. With the exception of p.S1363F, all mutations tested (p.G85V, p.R778L, p.H1069Q, p.C1104F, p.V1262F, p.G1343V, and p.S1363F) resulted in reduced ATP7B protein expression, whereas messenger RNA abundance was unaffected. Retention of mutant ATP7B in the endoplasmic reticulum, increased protein expression, and normalization of localization after culturing cells at 30 degrees C, and homology modeling suggested that these proteins were misfolded. Four distinct mutations exhibited residual copper export capacity, whereas other mutations resulted in complete disruption of copper export by ATP7B. Treatment with pharmacological chaperones 4-phenylbutyrate (4-PBA) and curcumin, a clinically approved compound, partially restored protein expression of most ATP7B mutants. CONCLUSION: These findings might enable novel treatment strategies in WD by directly enhancing the protein expression of mutant ATP7B with residual copper export activity. 1795.).