Fig 2: YAP/YAPdeltaC are co-factors of RORa. a Luciferase assays with COS-7 cells showing that RORa but not mutant RORa (RORa Y507A) lacking the consensus motif for the WW domain of YAP, acts with YAP or YAPdeltaC to transactivate luciferase gene expression via the RORa-responsive element. Single transfection of YAP or YAPdeltaC expression vector yielded insufficient transactivation. Double asterisks indicate statistical significance (p < 0.01, N = 5) in one-way ANOVA with post hoc Tukey’s HSD test. b Protein expression levels of RORa, YAP, and YAPdeltaC in the luciferase assays shown in Fig. 5a, determined by western blot. Because YAP is expressed endogenously in COS-7 cells, the amount of YAP was quantified (N = 5). c Left: effect of YAP knockdown and/or Tip60 knockdown on RORa-mediated transcription, evaluated by luciferase assays in 293T cells expressing RORa. Asterisks indicate statistical significance (p < 0.05, N = 8) in one-way ANOVA with post hoc Tukey’s HSD test. Right: levels of RORa, YAP, YAPdeltaC, and Tip60 are shown in lower panels. Double asterisks indicate statistical significance (p < 0.01, N = 5) in one-way ANOVA with post hoc Tukey’s HSD test. d Pull-down assay showing that YAP and Tip60 compete for interaction with RORa. A GST fusion protein of a part of RORa (a.a. 424–523) was used in this assay, as described in Methods. e Effect of Atxn1 knockdown on RORa-mediated transcription, evaluated by luciferase assays in 293T cells expressing RORa. Expression levels of RORa and Atxn1 are shown in right panel. Asterisks indicate statistical significance (p < 0.05, N = 6) in one-way ANOVA with post hoc Tukey’s HSD test

Fig 3: K63-linked ubiquitination of YAP induces its growth-promoting function. a Left panel: immunoblotting of YAP and GAPDH in HMLE cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). Right panel: growth curves. n = 5 biological replicates. b Images (upper panel) and quantification (lower panel) of soft agar colony formation by the cells described in (a). n = 3 biological replicates. c Endpoint images of tumors dissected from NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. d, e Tumor growth curves (d) and tumor weight (at the endpoint, e) of NSG mice with mammary fat pad injection of MDA-MB-231 cells transduced with wild-type (WT) YAP or the K321R/K497R mutant (2KR). n = 10 (mock), 10 (YAP WT) and 9 (YAP 2KR) mice per group. f Left panel: immunoblotting of YAP, SKP2, and GAPDH in BT549 cells transduced with MYC-SKP2 with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. g Left panel: immunoblotting of YAP, OTUD1 and GAPDH in BT549 cells transduced with OTUD1 shRNA with or without YAP shRNA. Right panel: growth curves. n = 5 biological replicates. h, i Kaplan–Meier curves of recurrence-free survival of patients with breast cancer, stratified by SKP2 (n = 3951 patients, probe: 203625_x_at, (h) or OTUD1 (n = 1764 patients, probe: 226140_s_at, (i) expression levels. Data were obtained from http://kmplot.com/analysis/. Auto-select best cutoff was used in the analysis. j Model for the regulation of YAP localization and activity by non-proteolytic ubiquitination. Error bars in a, b, and d–g are s.e.m. Statistical significance was determined by a two-tailed, unpaired Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 4: SKP2 and K63-linked ubiquitination enhance the YAP-TEAD interaction. a HEK293A cells were stably transfected with SFB-YAP and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against YAP-interacting proteins. H: high density; L: low density. b The HEK293A SFB-YAP stable cell line was transfected with MYC-SKP2 and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against YAP-interacting proteins. c The HEK293A SFB-YAP stable cell line was transfected with MYC-SKP2. SFB-YAP was pulled down with S-protein beads and then incubated with His-TEAD1 protein purified from bacteria. After in vitro binding, the proteins bound to S-protein beads were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against His and FLAG tags. d HEK293T cells were transfected with SFB-YAP (WT or the 2KR mutant). SFB-YAP was pulled down with S-protein beads and then incubated with His-TEAD1 protein purified from bacteria. After in vitro binding, the proteins bound to S-protein beads were eluted by boiling in Laemmli sample buffer and immunoblotted with antibodies against His and FLAG tags. e SKP2 forms a complex with YAP and TEAD1. HA-TEAD1 and MYC-SKP2 were co-transfected with or without SFB-YAP into HEK293T cells. SFB-YAP was pulled down with streptavidin beads and then eluted with biotin buffer. The eluded proteins were subjected to immunoprecipitation with a MYC-specific antibody to pull down MYC-SKP2 and immunoblotting with antibodies against FLAG, HA, and MYC

Fig 5: K63-linked ubiquitination of YAP is independent of Hippo signaling-mediated YAP phosphorylation. a Immunoblotting of p-YAP (S127), FLAG-YAP, and GAPDH in HEK293T cells transfected with SFB-YAP (wild-type, K321R, K497R, or K321R/K497R). b YAP knockout HEK293A cells (generated by CRISPR-Cas9) were transfected with SFB-YAP (S127A or S127A/K321R/K497R) and immunostained with a YAP-specific antibody (green). DAPI (blue) was used to stain DNA. Two different fields are shown. Scale bar, 20 µm. c Immunoblotting of p-YAP (S127), YAP, and SKP2 in HEK293A cells stably expressing MYC-SKP2 (upper panel) or SKP2 shRNA ( lower panel). Scr: a scramble control. d Immunoblotting of p-YAP (S127), YAP, OTUD1, and GAPDH in HEK293A cells stably transfected with HA-OTUD1 (upper panel) or OTUD1 shRNA (lower panel). Scr: a scramble control. e HEK293T cells were co-transfected with SFB-YAP (wild-type or S127A), MYC-SKP2 and the K63-specific mutant of HA-ubiquitin and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against HA and FLAG. f HEK293T cells were co-transfected SFB-YAP (wild-type or S127A), FLAG-OTUD1 and the K63-specific mutant of HA-ubiquitin and then subjected to a pulldown assay with S-protein beads and immunoblotting with antibodies against HA and FLAG. g YAP knockout HEK293A cells (generated by CRISPR-Cas9) were transfected with SFB-YAP (WT or S127A) with or without SKP2 shRNA (on a GFP-positive pGIPZ vector), and immunostained with a YAP-specific antibody (red). DAPI (blue) was used to stain DNA. Scr: a scramble control. Scale bar, 20 µm