The USP46 deubiquitylase complex increases Wingless/Wnt signaling strength by stabilizing Arrow/LRP6

doi:10.1038/s41467-023-41843-0

. 2023 Oct 5;14(1):6174.

doi: 10.1038/s41467-023-41843-0.

The USP46 deubiquitylase complex increases Wingless/Wnt signaling strength by stabilizing Arrow/LRP6

Zachary T Spencer^#¹, Victoria H Ng^#², Hassina Benchabane^#¹, Ghalia Saad Siddiqui¹, Deepesh Duwadi¹, Ben Maines¹, Jamal M Bryant², Anna Schwarzkopf², Kai Yuan¹, Sara N Kassel², Anant Mishra¹, Ashley Pimentel¹, Andres M Lebensohn³, Rajat Rohatgi⁴, Scott A Gerber⁵, David J Robbins⁶, Ethan Lee⁷, Yashi Ahmed⁸

Affiliations

¹ Department of Molecular and Systems Biology and the Dartmouth Cancer Center, Geisel School of Medicine, Dartmouth College, Hanover, NH, 03755, USA.
² Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, 37232, USA.
³ Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
⁴ Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305, USA.
⁵ Department of Molecular and Systems Biology and the Dartmouth Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH, 03766, USA.
⁶ Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, 20057, USA.
⁷ Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, 37232, USA. ethan.lee@vanderbilt.edu.
⁸ Department of Molecular and Systems Biology and the Dartmouth Cancer Center, Geisel School of Medicine, Dartmouth College, Hanover, NH, 03755, USA. yfa@dartmouth.edu.

^# Contributed equally.

PMID: 37798281
PMCID: PMC10556106
DOI: 10.1038/s41467-023-41843-0

The USP46 deubiquitylase complex increases Wingless/Wnt signaling strength by stabilizing Arrow/LRP6

Zachary T Spencer et al. Nat Commun. 2023.

. 2023 Oct 5;14(1):6174.

doi: 10.1038/s41467-023-41843-0.

Authors

Affiliations

¹ Department of Molecular and Systems Biology and the Dartmouth Cancer Center, Geisel School of Medicine, Dartmouth College, Hanover, NH, 03755, USA.
² Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, 37232, USA.
³ Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892, USA.
⁴ Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305, USA.
⁵ Department of Molecular and Systems Biology and the Dartmouth Cancer Center, Geisel School of Medicine at Dartmouth, Lebanon, NH, 03766, USA.
⁶ Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC, 20057, USA.
⁷ Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, 37232, USA. ethan.lee@vanderbilt.edu.
⁸ Department of Molecular and Systems Biology and the Dartmouth Cancer Center, Geisel School of Medicine, Dartmouth College, Hanover, NH, 03755, USA. yfa@dartmouth.edu.

^# Contributed equally.

PMID: 37798281
PMCID: PMC10556106
DOI: 10.1038/s41467-023-41843-0

Abstract

The control of Wnt receptor abundance is critical for animal development and to prevent tumorigenesis, but the mechanisms that mediate receptor stabilization remain uncertain. We demonstrate that stabilization of the essential Wingless/Wnt receptor Arrow/LRP6 by the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex controls signaling strength in Drosophila. By reducing Arrow ubiquitylation and turnover, the Usp46 complex increases cell surface levels of Arrow and enhances the sensitivity of target cells to stimulation by the Wingless morphogen, thereby increasing the amplitude and spatial range of signaling responses. Usp46 inactivation in Wingless-responding cells destabilizes Arrow, reduces cytoplasmic accumulation of the transcriptional coactivator Armadillo/β-catenin, and attenuates or abolishes Wingless target gene activation, which prevents the concentration-dependent regulation of signaling strength. Consequently, Wingless-dependent developmental patterning and tissue homeostasis are disrupted. These results reveal an evolutionarily conserved mechanism that mediates Wnt/Wingless receptor stabilization and underlies the precise activation of signaling throughout the spatial range of the morphogen gradient.

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Conflict of interest statement

D.J.R. and E.L. are founders of StemSynergy Therapeutics Inc., a company commercializing small-molecule cell signaling inhibitors, including Wnt inhibitors. The remaining authors declare no competing interests.

Figures

**Fig. 1. The Usp46 complex promotes expression of the Wingless target gene *senseless* (*sens*) in the larval wing disc.**
A–L RNAi constructs targeting each Usp46 complex component or the *yellow* negative control were expressed in the posterior compartment (marked by Engrailed (En, green)) of the third instar larval wing discs using the *hedgehog (hh)-Gal4* driver. Senseless (Sens, magenta). DAPI (blue) marks nuclei. Scale bar (A–L): 20 µM. Dorsal, top and posterior, right. A–C *hh-Gal4*-driven expression of a control RNAi construct targeting the *yellow (y)* gene. No loss of Sens was observed. D–L *hh-Gal4-*driven expression of RNAi constructs targeting *Usp46* (D, F), *Wdr20* (G, I) and *Uaf1* (J, L) results in decreased Sens in the posterior compartment. Only one RNAi line targeting *Uaf1* was available. M Quantification is shown as percentage of discs of each genotype with decreased Sens. N is the number of discs analyzed. ****p < 0.0001 (0 for all genotypes, one-tailed t-test). Source data are provided in the Source Data file.

**Fig. 2. Usp46 promotes expression of the Wingless target gene *fz3* in the posterior midgut.**
A–C *Usp46*¹ null mutant clones (magenta) and *frizzled3-GFP* (*fz3-GFP)* expression (green) in the adult intestinal epithelium of the posterior midgut. The midgut-hindgut boundary (MHB) is delineated (M | H). DAPI (blue) marks nuclei. Posterior, right. D–F Higher magnification view of box D in panel A showing a region distant from the MHB. *Usp46*¹ null mutant cells in this region showed complete loss of *fz3-GFP* (white arrows), whereas those slightly closer to the MHB showed a nearly complete reduction in *fz3-GFP* (yellow arrows). G–I Higher magnification view of box G in panel A showing a region near the MHB. *Usp46*¹ null mutant cells in this region display either a partial decrease (yellow arrows) or no decrease in *fz3-GFP*. Scale bars (A–C) and (D–I): 50 µM. J Quantification is shown as percentage of clones of each genotype with decreased *fz3-GFP* (yellow) or the absence of *fz3-GFP* (gray) expression. Clones (n=number) close and far from the MHB were analyzed. ****p < 0.0001 (0 for all genotypes, one-tailed t-test). Source data are provided in the Source Data file.

**Fig. 3. The Usp46 complex is required for expression of the Wingless target gene *naked* in the posterior midgut.**
A–I Null mutant MARCM clones (green) of *Usp46* (A, C), *Wdr20* (D, F), and *Uaf1* (G, I) in the posterior midgut resulted in decreased expression of *nkd-lacZ* (magenta) in a cell-autonomous manner (orange arrows). For inactivation of all three Usp46 components, the largest reduction in *nkd-lacZ* was displayed in mutant clones far from the MHB. DAPI (blue) marks nuclei. J–L *nkd-lacZ* does not decrease in wild-type (*FRT82B*) clones. Scale bar (A, L): 20 µM. M Quantification is shown as percentage of clones of each genotype with decreased *nkd-lacZ* expression. N is the number of clones analyzed. ****p < 0.0001 (0 for all genotypes, one-tailed t-test). Source data are provided in the Source Data file.

**Fig. 4. Usp46 and Wdr20 regulate intestinal stem cell proliferation in the adult midgut.**
A, B Wild-type adult posterior midgut epithelium stained for *escargot (esg)>GFP* (green) to mark intestinal stem and progenitor cells, Prospero (Pros, magenta) to mark enteroendocrine cells, and Armadillo (Arm, magenta) to mark the plasma membrane of all epithelial cells. Panel B is a higher magnification view of A. **C–F** Posterior midgut from transheterozygote null mutants of *Usp46* (C, D) or *Wdr20* (E, F) are shown. Overproliferation of adult midgut epithelial cells, revealed by *esg* > *GFP* (green), is observed following inactivation of *Usp46* or *Wdr20*. Panels D and F are higher magnification views of C and E, respectively. Armadillo (Arm, magenta), Prospero (Pros, magenta). Df refers to a chromosomal deficiency that eliminates *Wdr20* (see Methods). Scale bars (A, C, E) and (B, D, F): 20 µM. G Quantification of *esg* > *GFP* positive cells is shown as mean (red line). Each point represents an individual posterior midgut (n = 15 for WT, n = 15 for *Usp46*^21/MiMIC, n = 15 for *Usp46*^1/MiMIC, n = 15 for *Usp46*^1/21, n = 9 for *Wdr20*^13/34, n = 13 for *Wdr20*^13/Df, n = 13 for *Wdr20*^34/Df). A 0.051mm² field was measured in the R5 region of each posterior midgut. ****p < 0.0001 (p values in order: < 1E-8, 7E-8, 1.5E-5, 1.5E-7, 2E-8 and 3.6E-7, two-tailed t-test). *Usp46*^MiMIC contains an insertion of a *Minos*-mediated integration cassette in the *Usp46* gene (see Methods). Source data are provided in the Source Data file.

**Fig. 5. RNAi-mediated depletion of Usp46 complex components reduces Arrow-induced ectopic Wingless signaling.**
A Wild-type wing. Higher magnification of the posterior wing margin is shown on the right. B Overexpression of *arrow* with the *hh-Gal4* driver activates Wingless signaling, resulting in the formation of ectopic sensory bristles within the adult wing blade. Formation of ectopic bristles is not rescued by RNAi-mediated depletion of the *ebony* control. C–F Expression of two different *Usp46* RNAi (C, D) or *Wdr20* RNAi (E, F) constructs with *hh-Gal4* reduced the number of ectopic bristles induced by Arrow overexpression. G Quantification of percentage of flies with severe ectopic wing bristles resulting from Arrow overexpression coupled with RNAi-mediated knockdown of *Usp46*, *Wdr20*, or *ebony*. N is the number of flies analyzed. ****p < 0.0001 (0 for all genotypes, one-tailed t-test). Source data are provided in the Source Data file.

**Fig. 6. Arrow expression rescues loss of Sens resulting from RNAi-mediated depletion of Usp46 complex components.**
A–H *hh-Gal4-*driven expression in third instar larval wing discs of RNAi constructs targeting *Usp46* (A, C) and *Wdr20* (E, G) results in decreased Sens (magenta) in the posterior compartment (marked by Engrailed (En, green)). Sens expression is rescued by co-expression of Arrow (B, D, F, H). *GFP-lacZ* is expressed in control discs (A, C, E and G). DAPI (blue) marks nuclei. Scale bar: 50 µM. Dorsal, top and posterior, right. I Quantification is shown as percentage of discs of each genotype with decreased Sens. N is the number of discs analyzed. *p < 0.05 (0.022), **p < 0.01 (p values in order: 0.006, 0.0041, and 0.002, one-tailed t-test). Source data are provided in the Source Data file.

**Fig. 7. The Usp46 complex interacts with Arrow and regulates steady-state Arrow levels.**
A V5-tagged Usp46 complex components co-immunoprecipitate with FLAG-tagged Arrow. HEK293 cells were transfected with V5-tagged Usp46 complex components and FLAG-tagged Arrow as indicated; FLAG-Arrow was immunoprecipitated (IP) with anti-FLAG conjugated beads. Co-immunoprecipitated V5-tagged Usp46 complex components were detected by immunoblotting. WCL = whole cell lysates. A representative immunoblot (n = 3 independent experiments) is shown. B Arrow antibody specificity and knockdown efficiency. RNAi-mediated knockdown with dsRNAs targeting Ctrl (*white* negative control) or *arrow* demonstrate efficient Arrow knockdown and the specificity of the Arrow antibody. A representative immunoblot (n = 3 independent experiments) is shown. C Efficient RNAi-mediated knockdown of the Usp46 complex. Drosophila S2R+ cells were transfected with HA-tagged Usp46 complex components, followed by knockdown with indicated dsRNAs. A representative immunoblot (n = 3 independent experiments) is shown. D RNAi-mediated knockdown of the Usp46 complex decreases steady-state levels of Arrow. *Drosophila* S2R+ cells were treated with Ctrl or Usp46 complex dsRNAs, followed by immunoblotting with Arrow antibody. RNAi-mediated knockdown of the Usp46 complex resulted in decreased Arrow levels. Tubulin was used as a loading control. Dashed line: lanes between Ctrl and Usp46 were removed. A representative immunoblot (n = 3 independent experiments) is shown. E Quantification of Arrow levels normalized to tubulin, mean ± SD, n = 3. ****p < 0.0001 (6E-5 for *Usp46* and 7.8E-5 *for Wdr20*), ***p < 0.001 (8E-4 for *Uaf1*) (two tailed t-test). Source data are provided in the Source Data file.

**Fig. 8. The Usp46 complex deubiquitylates and stabilizes Arrow at the cell surface.**
A The Usp46 complex deubiquitylates Arrow. HEK293 cells were transfected with FLAG-tagged Arrow, V5-tagged Usp46 complex, and HA-tagged Ubiquitin. Lysates were immunoprecipitated with HA antibody and blotted with FLAG antibody. Co-transfection of V5-Usp46 complex components with FLAG-Arrow decreased the levels of ubiquitylated Arrow. Treatment with bafilomycin A (BFA) increased ubiquitylated Arrow, which decreased upon co-transfection of V5-Usp46 complex components (Tri46-V5). A representative immunoblot (n = 3 independent experiments) is shown. B The Usp46 complex (Tri46-HA) decreased ubiquitylation of endogenous Arrow. *Drosophila* S2R+ cells were transfected with the indicated plasmids. Lysates were immunoprecipitated with Ubiquitin antibody or control IgG and analyzed by immunoblot with Arrow antibody. A representative immunoblot (n = 3 independent experiments) is shown. C The Usp46 complex (Tri46-HA) increases cell surface levels of Arrow. S2R+ cells were transfected with Usp46 complex components as indicated. Following cell surface biotinylation, lysates were subjected to neutravidin pull down and immunoblotted for endogenous Arrow. Tubulin was used as a loading control. WCL whole cell lysates. Dashed line: A lane between plus biotin and minus biotin that contained the protein ladder was removed. A representative immunoblot (n = 3 independent experiments) is shown. For (A–C) source data are provided in the Source Data file. D Model for the regulation of Arrow/LRP6 by the Usp46 complex in Wingless/Wnt signaling. Regulation of cell surface Arrow abundance is mediated by a tightly controlled balance in the opposing processes of ubiquitylation and deubiquitylation that is essential for achieving concentration-dependent signaling responses in the morphogen gradient.

**Fig. 9. Usp46 complex inactivation reduces Arrow levels in a cell autonomous manner.**
A–D *arr*² null mutant clones (green) in the adult posterior midgut. Upon Arrow loss, the Arrow signal intensity is decreased at the cell membrane and in the cytoplasm, confirming the specificity of the Arrow antisera. Additionally, cytoplasmic Arm (yellow) is reduced in Wingless-responding cells upon Arrow loss. E–G *Usp46*¹ null mutant clones (magenta) in the adult posterior midgut. When Usp46 is inactivated, the levels of cell membrane-associated and cytoplasmic Arrow (green) are decreased cell-autonomously, as is cytoplasmic Arm (yellow). H–J *Wdr20*³³ null mutant clones (magenta) in the adult posterior midgut. Both Arrow (green) and cytoplasmic Arm (yellow) are decreased cell autonomously when Wdr20 is inactivated. Some cells also display a non-autonomous decrease in cell membrane-associated Arm. K–M *Uaf1*⁴ null mutant clones (magenta) in the adult posterior midgut. When Uaf1 is inactivated, the levels of cell membrane-associated and cytoplasmic Arrow (green) are decreased cell-autonomously. Some cells also display a reduction in membrane-associated Arm (yellow). DAPI (blue) marks nuclei. Scale bars (A-D) and (E-M): 20 µM (N, O) Quantification is shown as percentage of clones of each genotype with decreased Arrow (N) or Arm (O). N is the number of clones analyzed, *p < 0.05 (0.043 for *Uaf1*⁴ in O), ****p < 0.0001 (0 for all genotypes in N, 0 for *Wdr20*³³ and 1E-5 for *Usp46*¹ in O, one-tailed t-test). Source data are provided in the Source Data file.

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References

1. Zhong Z, Virshup DM. Wnt signaling and drug resistance in cancer. Mol. Pharmacol. 2020;97:72–89. doi: 10.1124/mol.119.117978. - DOI - PubMed
1. Zecca M, Basler K, Struhl G. Direct and long-range action of a wingless morphogen gradient. Cell. 1996;87:833–844. doi: 10.1016/S0092-8674(00)81991-1. - DOI - PubMed
1. Neumann CJ, Cohen SM. Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development. 1997;124:871–880. doi: 10.1242/dev.124.4.871. - DOI - PubMed
1. Alexandre C, Baena-Lopez A, Vincent J-P. Patterning and growth control by membrane-tethered wingless. Nature. 2014;505:180–185. doi: 10.1038/nature12879. - DOI - PMC - PubMed
1. Apitz H, Salecker I. Spatio-temporal relays control layer identity of direction-selective neuron subtypes in Drosophila. Nat. Commun. 2018;9:1–16. doi: 10.1038/s41467-018-04592-z. - DOI - PMC - PubMed

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[1] Zhong Z, Virshup DM. Wnt signaling and drug resistance in cancer. Mol. Pharmacol. 2020;97:72–89. doi: 10.1124/mol.119.117978. - DOI - PubMed

[2] Zhong Z, Virshup DM. Wnt signaling and drug resistance in cancer. Mol. Pharmacol. 2020;97:72–89. doi: 10.1124/mol.119.117978. - DOI - PubMed

[3] Zecca M, Basler K, Struhl G. Direct and long-range action of a wingless morphogen gradient. Cell. 1996;87:833–844. doi: 10.1016/S0092-8674(00)81991-1. - DOI - PubMed

[4] Zecca M, Basler K, Struhl G. Direct and long-range action of a wingless morphogen gradient. Cell. 1996;87:833–844. doi: 10.1016/S0092-8674(00)81991-1. - DOI - PubMed

[5] Neumann CJ, Cohen SM. Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development. 1997;124:871–880. doi: 10.1242/dev.124.4.871. - DOI - PubMed

[6] Neumann CJ, Cohen SM. Long-range action of Wingless organizes the dorsal-ventral axis of the Drosophila wing. Development. 1997;124:871–880. doi: 10.1242/dev.124.4.871. - DOI - PubMed

[7] Alexandre C, Baena-Lopez A, Vincent J-P. Patterning and growth control by membrane-tethered wingless. Nature. 2014;505:180–185. doi: 10.1038/nature12879. - DOI - PMC - PubMed

[8] Alexandre C, Baena-Lopez A, Vincent J-P. Patterning and growth control by membrane-tethered wingless. Nature. 2014;505:180–185. doi: 10.1038/nature12879. - DOI - PMC - PubMed

[9] Apitz H, Salecker I. Spatio-temporal relays control layer identity of direction-selective neuron subtypes in Drosophila. Nat. Commun. 2018;9:1–16. doi: 10.1038/s41467-018-04592-z. - DOI - PMC - PubMed

[10] Apitz H, Salecker I. Spatio-temporal relays control layer identity of direction-selective neuron subtypes in Drosophila. Nat. Commun. 2018;9:1–16. doi: 10.1038/s41467-018-04592-z. - DOI - PMC - PubMed

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The USP46 deubiquitylase complex increases Wingless/Wnt signaling strength by stabilizing Arrow/LRP6

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The USP46 deubiquitylase complex increases Wingless/Wnt signaling strength by stabilizing Arrow/LRP6

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