Fig 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.

Fig 3: 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 a-FLAG blot is shown to illustrate differences in DVL2 abundance, while the higher-exposure a-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.

Fig 4: DUB knockout library kit-based screening for USPs regulating MAST1 protein level by Western blot analysis. (A) Schematic representation of secondary screening with a CRISPR/Cas9-based sgRNA library to find DUBs that regulate MAST1 protein level. Steps 1-2: The designed DUB knockout sgRNA library, which consists of an entire set of genes encoding USPs, was co-transfected with Cas9 into HeLa-cisR cells (day 1). Step 3: The cells were placed under puromycin selection (2 µg/mL) and incubated for 3 days (days 2-5). Step 4: The transfected cells were harvested and lysed, and protein was isolated. Steps 5-6: Protein concentration was estimated by Bradford reagent, and equal concentrations of all DUBKO cell lysates were loaded on SDS-PAGE and screened for DUB candidates regulating endogenous expression pattern of MAST1 using Western blot (WB) analysis. (B) Equal protein concentrations from the cell lysates from (A) were subjected to Western blotting to determine the endogenous MAST1 protein level. For each blot, HeLa-cisR cells co-transfected with scrambled sgRNA and Cas9 served as the mock control. GAPDH was used as a loading control. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control for each individual sgRNA (MAST1/GAPDH) and presented below the blot. (C) The effects of the targeting the putative DUB candidates on the MAST1 protein level were estimated by Western blotting. The protein band intensities were estimated using ImageJ software with reference to the GAPDH control band for each individual sgRNA (MAST1/GAPDH) ) and presented below the blot. (D) The interactions between putative DUB candidates and MAST1 by co-immunoprecipitation analysis. Myc-MAST1 and DUBs (Flag-USP1, Flag-USP28, and Flag-USP44) were transfected into HEK293 cells. (E) The interaction between endogenous USP9X and MAST1 by co-immunoprecipitation analysis. Cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (F) A Venn diagram showing the overlapping DUB candidate based on cisplatin cytotoxicity, loss-of-function effect on MAST1 protein level, and interaction analysis with MAST1.

Fig 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.