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. 2019 Jul 23;28(4):1074-1089.e5.
doi: 10.1016/j.celrep.2019.06.083.

USP9X Deubiquitylates DVL2 to Regulate WNT Pathway Specification

Casey P Nielsen  1 , Kristin K Jernigan  1 , Nicole L Diggins  2 , Donna J Webb  2 , Jason A MacGurn  3
Affiliations

USP9X Deubiquitylates DVL2 to Regulate WNT Pathway Specification

Casey P Nielsen et al. Cell Rep. .

Abstract

The WNT signaling network is comprised of multiple receptors that relay various input signals via distinct transduction pathways to execute multiple complex and context-specific output processes. Integrity of the WNT signaling network relies on proper specification between canonical and noncanonical pathways, which presents a regulatory challenge given that several signal transducing elements are shared between pathways. Here, we report that USP9X, a deubiquitylase, and WWP1, an E3 ubiquitin ligase, regulate a ubiquitin rheostat on DVL2, a WNT signaling protein. Our findings indicate that USP9X-mediated deubiquitylation of DVL2 is required for canonical WNT activation, while increased DVL2 ubiquitylation is associated with localization to actin-rich projections and activation of the planar cell polarity (PCP) pathway. We propose that a WWP1-USP9X axis regulates a ubiquitin rheostat on DVL2 that specifies its participation in either canonical WNT or WNT-PCP pathways. These findings have important implications for therapeutic targeting of USP9X in human cancer.

Keywords: DUB-E3 interactions; E3 ubiquitin ligases; USP9X; WNT signaling; WNT-PCP signaling; WWP1; deubiquitylases; non-canonical WNT signaling; ubiquitin rheostat.

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Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. WWP1 Interacts with USP9X and DVL2 in MDA-MB-231 Human Breast Cancer Cells
(A) Co-immunoprecipitation analysis testing hits from SILAC-MS analysis in Figure S1A. FLAG-WWP1 was affinity purified from MDA-MB-231 cell lysates. Input and immunoprecipitated (IP) fractions were blotted for the indicated species. (B) Schematic of known domains in WWP1, DVL2, and USP9X. (C) Analysis of co-purification of DVL2 and USP9X with WWP1 variants. The indicated WWP1 variants were FLAG affinity purified, and quantitative immunoblots were performed to assess co-purification of interacting factors. The wwp1–4ww mutant indicates that all four WW domains have been mutated to disrupt the ability to bind PY motifs. (D) Quantification of USP9X co-purification with FLAG-WWP1 variants (normalized to wild-type [WT]) over multiple experiments (n = 3). Red asterisk indicates a significant difference based on Student’s t test analysis. (E) Analysis of co-purification of DVL2 and USP9X with WWP1 variants. The indicated WWP1 variants were FLAG affinity purified, and quantitative immunoblots were performed to assess co-purification of interacting factors. Each mutant variant of WWP1 contains a single intact WW domain (indicated), while the remaining three are mutated to disrupt PY motif binding. (F) Quantification of DVL2 co-purification with FLAG-WWP1 variants (normalized to WT) over multiple experiments (n = 3). (G) Analysis of co-purification of WWP1 and USP9X with DVL2 variants. The indicated mutant DVL2 variants were FLAG affinity purified, and quantitative immunoblots were performed to assess co-purification of interacting factors. Mutant variants of DVL2 included point mutations disrupting the ability of its PY motifs to interact with WW domains. (H) Analysis of co-purification of WWP1 and DVL2 with USP9X variants. The indicated USP9X variants were FLAG affinity purified, and quantitative immunoblots were performed to assess co-purification of interacting factors. Mutant variants of USP9X included a small C-terminal truncation, which results in deletion of both C-terminal PY motifs. (I) A schematic model for how USP9× and DVL2 both interact with WWP1 via PY motif interactions. The data also suggest an interaction between USP9X andDVL2 that is independent of WWP1 (dotted red line). (D and F) Error bars represent SD of the mean.
Figure 2.
Figure 2.. WWP1 and USP9X Operate on DVL2 to Establish a Ubiquitylation Rheostat
(A) In vitro ubiquitylation reactions were performed using recombinant purified FLAG-WWP1 (60 nM) and HA-DVL2 purified from cultured cells. E3 conjugation reactions were allowed to proceed for 60 min before the conjugation reaction was terminated. (B) Deconjugation assays were performed using HA-DVL2 ubiquitylated in vitro by WWP1 (as generated in A). Equivalent molar ratios of USP9X, OTUB1, and AMSH (100 nM) were added and each reaction was allowed to proceed for 60 min before the deconjugation reaction was terminated and HA-DVL2 was resolved by SDS-PAGE and immunoblot (bottom panel). For each sample, a ratio of modified:unmodified HA-DVL2 was measured using ImageJ. The modified:unmodified HA-DVL2 ratio was averaged over multiple replicate experiments (n = 3). The red asterisk indicates a statistically significant difference (p < 0.05) compared to DVL2 that has been modified by WWP1 and subsequently treated with OTUB1 or AMSH. Error bars represent SD of the mean. (C) Representative immunoblot of in vitro ubiquitylation and/or deubiquitylation reactions (replicate experiments shown in Figures S2D and S2E). Experiments were performed using indicated combinations of purified FLAG-WWP1 and 6xHIS-USP9X with HA-DVL2 purified from cultured cells (see Figures S2A and S2B). Reactions were allowed to proceed for 60 min before being terminated and HA-DVL2 was resolved by SDS-PAGE and immunoblot. (D) Line density plots from the HA-DVL2 immunoblot data in (C) illustrate differences in extent of ubiquitylation in each sample. Replicate experiments are shown in Figures S2D and S2E.
Figure 3.
Figure 3.. USP9X Promotes Canonical WNT Activation
(A) Analysis of ligand-stimulated WNT activation in MDA-MB-231 cells (blue bars) and usp9x knockout equivalents (gray bars). TopFLASH luciferase assays were used to measure WNT activation (top panel), and immunoblotting was performed to assess stabilization of nuclear β-catenin. The asterisk (*) indicates WNT activation in usp9x knockout cells is significantly decreased (p < 0.05) compared to sibling MDA-MB-231 cells. (B) MDA-MB-231 cells (WT and usp9x knockout) were stimulated with WNT3a ligand and R-spondin, and WNT activation was measured by TopFLASH luciferase assays. Prior to activation, cells were transiently transfected with empty vector (EV) or vector expression USP9X (WT or catalytic dead [CD; C1566V]). (C) Ligand-stimulated WNT activation was measured in the presence of indicated concentrations of WP1130. Based on the data, an IC50 was estimated. (D) MDA-MB-231 cells were transfected with empty vector or plasmids for CMV-driven expression of WT or CD USP9X. (E) Ligand-stimulated WNT activation was measured using TopFLASH luciferase assays for MDA-MB-231 cells and usp9x knockout equivalents. Cells were transfected with control siRNA or siRNA targeting knockdown of NEDD4L, WWP1, or WWP2. (A, B, D, and E) Error bars represent SD of the mean.
Figure 4.
Figure 4.. USP9X Promotes Canonical WNT Activation via Deubiquitylation of DVL2
(A) Immunoblotting analysis of FLAG-DVL2 mobility by SDS-PAGE from lysates of MDA-MB-231 cells or usp9x knockout equivalents. A low-exposure α-FLAG blot is shown to illustrate differences in DVL2 abundance, while the higher-exposure α-FLAG is shown to illustrate the higher molecular weight (MW) species evident in usp9X knockout cells. (B) SILAC-MS profiling of post-translational modification sites (phosphorylation and ubiquitylation) on DVL2. In this experiment, FLAG-DVL2 purified from MDA-MB-231 cells (heavy) was compared to usp9x knockout equivalents (light). A schematic illustrating the domain structure of DVL2 is shown at the top with asterisks to indicate the position of detected ubiquitylation events. (C) Analysis of ligand-stimulated WNT activation was measured using TopFLASH reporter assays in STF-293 cells transfected with the indicated expression vectors. UL36 is the DUB domain fused to DVL2 in these experiments. (D) Following siRNA knockdown of USP9X, ligand-stimulated WNT activation was measured using TopFLASH reporter assays in STF-293 cells transfected with the indicated HA-DVL2 expression vectors. DVL2-K0 is a variant where all encoded Lys residues are substituted with Arg residues. The asterisk (*) represents a statistically significant difference when compared to indicated sample (p < 0.05). n.s. represents no significant difference relative to indicated comparison. (C and D) Error bars represent SD of the mean.
Figure 5.
Figure 5.. DVL2 Ubiquitylation State Specifies Association with Canonical WNT or WNT-PCP Pathway Factors
(A) SILAC-MS was used to resolve the DVL2 interaction network in MDA-MB-231 human breast cancer cells. Near-stoichiometric interactions (solid lines) were detected with the AP-2 clathrin adaptor complex and VANGL1-cofilin, which comprise a subcomplex of the WNT-PCP pathway. Sub-stoichiometric interactions (dotted lines) were identified for NEDD4 family E3 ubiquitin ligases and proteasome subunits. (B) Interactions detected by SILAC-MS were confirmed by co-immunoprecipitation. (C and D) SILAC-MS analysis was performed to measure how DVL2 interactions are impacted by catalytic active versus CD DUB fusion (C) or by loss of USP9X (D). Scatterplots depict individual heavy:light (H:L) ratio measurements (LOG2 transformed) for each peptide resolved for the indicated proteins. The mean value and standard deviation for each protein is indicated. For the experiment shown in (C), several peptides with extremely low H:L ratios are not depicted here but are shown in Figure S5B. These outlier peptides correspond to sites of ubiquitin modification (Figure S5C) shown in Figure 4B. Differences between DVL2 (bait) and all interacting proteins (AP2 complex, VANGL1, CFL1, and proteasome subunits) are statistically significant (p < 0.005). (C and D) Error bars represent SD of the mean.
Figure 6.
Figure 6.. USP9X Regulates DVL2 Localization and Antagonizes WNT-PCP Activation
(A) Immunofluorescence imaging of FLAG-DVL2 (green) and actin (phalloidin, blue) in MDA-MB-231 human breast cancer cells transfected with control siRNA (top panels), siRNA targeting USP9X (middle panels), or a combination of siRNA targeting USP9X and siRNA targeting WWP1 (bottom panels). Scale bars are 25 μm. (B) Quantification of DVL2 cellular distribution over a population of MDA-MB-231 cells (n > 100). (C) WT MDA-MB-231 and usp9x knockout (KO) variant cell lysates were assayed for active Rho by pull-down assay. Addition of GDP or non-hydrolyzable GTP (GTPƔS) to cell lysates served as negative and positive controls, respectively. (D) Quantification of Rho activation shown in Figure 6C (n = 3) was performed by measuring the amount of active Rho detected in parent and usp9x knockout cells and normalizing to the GTPγS (maximal activation) control. The red asterisk indicates a significant difference when compared to WT cells (p < 0.05). (E) MDA-MB-231 cells were plated at low density, and cell migration (μm/h) speed was measured. Red asterisk indicates a significant difference between the tested populations (n > 30). (F) Rose plots showing trajectories of individual MDA-MB-231 cells (left) and usp9x knockout equivalents (right). (D and E) Error bars represent SD of the mean.
Figure 7.
Figure 7.. USP9X-Mediated Regulation of Cell Motility Requires DVL2 Ubiquitylation
(A) Migration speed of MDA-MB-231 usp9x KO cells treated with either control siRNA or siRNA targeting WWP1 was measured after plating at low density for the indicated conditions. The red asterisk indicates a significant difference compared to control siRNA-treated cells (p < 0.005). (B) MDA-MB-231 cell lines stably expressing DVL2-DUB fusions (catalytic active and CD) were treated with control siRNA or siRNA targeting USP9X, and cell speed was measured for 25 cells. (C) MDA-MB-231 cell lines stably expressing DVL2-DUB fusions (catalytic active and CD) underwent mock or WNT5a treatment, and cell speed was measured for 25 cells. The red asterisk indicates a statistically significant difference (p < 0.005) compared to MDA-MB-231 cells stably expressing the CD DUB-DVL2 fusion treated with WNT5a. (D) Model for DVL2 regulation by the USP9X-WWP1 axis to establish a ubiquitin rheostat on DVL2 that influences interaction preference with either the clathrin adaptor AP-2 complex, which is important for canonical WNT signaling, or the VANGL1-cofilin complex, which is important for the WNT-PCP pathway. Thus, the USP9X-WWP1 axis regulates DVL2 ubiquitylation to determine WNT pathway specification. (A–C) Error bars represent SD of the mean.
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