DUB3 Deubiquitylating Enzymes Regulate Hippo Pathway Activity by Regulating the Stability of ITCH, LATS and AMOT Proteins

<div><p>The YAP and TAZ transcriptional coactivators promote oncogenic transformation. Elevated YAP/TAZ activity has been documented in human tumors. YAP and TAZ are negatively regulated by the Hippo tumor suppressor pathway. The activity and stability of several Hippo pathway components, including YAP/TAZ, is regulated by ubiquitin mediated protein turnover and several ubiquitin ligase complexes have been implicated in human cancer. However, little is known about the deubiquitylating enzymes that counteract these ubiquitin ligases in regulation of the Hippo pathway. Here we identify the DUB3 family deubiquitylating enzymes as regulators of Hippo pathway activity. We provide evidence that DUB3 proteins regulate YAP/TAZ activity by controlling the stability of the E3 ligase ITCH, the LATS kinases and the AMOT family proteins. As a novel Hippo pathway regulator, DUB3 has the potential to act a tumor suppressor by limiting YAP activity.</p></div

DUB3 mediates ITCH, LATS1/2 and AMOT proteins to regulate Hippo activity.

<p>(A) A schematic view of DUB3-mediated regulation of Hippo signaling. DUB3 de-ubiquitylates ITCH, LATS1/2 and AMOT to promote their stability. In the presence of stabilized AMOT, ITCH promotes YAP degradation. (B) Luciferase reporter assays showing the effects of ITCH overexpression on YAP/TAZ activity. HEK293T cells were transfected to express the luciferase reporters together with a control or ITCH expression vector in the presence of a mixture of siRNAs targeting AMOT, AMOTL1 and AMOTL2, a mixture siRNAs targeting LATS1 and LATS2 or a scrambled siRNA control. Data represent the average of three independent transfection experiments ± SD. (C) Luciferase reporter assays showing the effects of DUB3 siRNAs on YAP/TAZ activity. HEK293T cells were transfected to express the luciferase reporters together with a control or DUB3 siRNA in the presence of ITCH siRNA, a mixture of siRNAs targeting ITCH and NEDD4 or a scrambled siRNA control. Data represent the average of three independent transfection experiments ± SD.</p

LATS and AMOT proteins are required for DUB3-mediated regulation of Hippo signaling.

<p>(A) Immunoblots showing the effect of DUB3 expression on LATS kinases, AMOT and YAP. HEK293T cells were transfected with a vector expressing Flag-DUB3 or a control vector together with a mixture of siRNAs targeting AMOT, AMOTL1 and AMOTL2 or a scrambled siRNA control. Blots were probed with antibodies against Flag, AMOT, AMOTL1, LATS1, LATS2 and YAP. Anti-Actin was used to control for loading. Samples were run on the same SDS-acrylamide gels with intervening lanes removed. (B) Immunoblots showing the effect of DUB3 depletion on LATS kinases, AMOT and YAP. HEK293T cells were transfected with a vector expressing Flag-DUB3 or a control vector together with a mixture of siRNAs targeting AMOT, AMOTL1 and AMOTL2 or a scrambled siRNA control. Blots were probed with antibodies against DUB3, AMOT, LATS1, LATS2 and YAP. Anti-Actin was used to control for loading. Samples were run on the same gels with intervening lanes removed. (C) Immunoblots showing the effect of DUB3 depletion on ITCH, LATS kinases and AMOT. HEK293T cells were transfected with a control or DUB3 siRNA in the presence of LATS1 and LATS2 siRNAs or a scrambled siRNA control. Blots were probed with antibodies against DUB3, ITCH, AMOT, LATS1, LATS2 and YAP. Anti-Actin was used to control for loading. (D) Luciferase reporter assays showing the effects of DUB3 siRNAs on YAP/TAZ activity. HEK293T cells were transfected to express the luciferase reporters together with a control or DUB3 siRNA in the presence of a mixture of siRNAs targeting AMOT, AMOTL1 and AMOTL2, a mixture of siRNAs targeting LATS1 and LATS2 or a scrambled siRNA control. Data represent the average of three independent transfection experiments ± SD. P values were determined using Student’s T-test (2-tailed, unequal variance).</p

DUB3 interacts with ITCH and mediates its stability.

<p>(A) Immunoblots showing the effect of DUB3 expression on ITCH. HEK293T cells were transfected with a vector expressing Flag-DUB3, its C89S DUB3 or a control vector. Blots were probed with anti-Flag, anti-ITCH, anti-NEDD4 and anti-SMURF1 antibodies. Anti-Actin was used to control for loading. (B) Immunoblots showing the effect of DUB3 siRNAs on ITCH. HEK293T cells were transfected with independent siRNAs against DUB3 or a scrambled siRNA. Blots were probed with antibodies against ITCH or Actin for loading control. (C) Immunoprecipitation assays showing interaction between DUB3 and ITCH. HEK293T cells were transfected to express Flag-tagged DUB3 and Myc-tagged ITCH as indicated. Transfected cells were treated with MG132 5μM overnight before being harvested for immunoprecipitation with anti-Flag or anti-Myc-conjugated beads. Blots were probed with anti- Flag to detect DUB3 or anti-Myc to detect ITCH. (D) Ubiquitylation assay showing the effect of DUB3 on ITCH ubiquitylation. HEK293T cells were co-transfected with a vector expressing Myc-ITCH and a vector expressing Flag-tagged DUB3, Flag-tagged DUB3 C89S or a control vector. Transfected cells were treated with MG132 5μM overnight before being harvested for immunoprecipitation with anti-Myc-or isotype IgG-conjugated beads in PLC buffer freshly supplemented with 10mM of NEM. Immunoblots were probed with antibodies against HA, Flag and Myc.</p

DUB3 regulates Hippo activity by mediating YAP turnover.

<p>(A) Luciferase reporter assays showing the effects of DUB3 shRNAs on YAP/TAZ activity. HEK293T cells were transfected to express the 8XGTIIC_luc YAP/TAZ reporter, which contains 8 TEAD binding sites to control the expression of firefly luciferase and a vector expressing CMV-Renilla luciferase to normalize for transfection efficiency, together with independent shRNA vectors to deplete DUB3 or with a control shRNA. shRNA to deplete LATS2 was used as a positive control. Data represent the average of three independent transfection experiments ± SD. P values were determined using Student’s T-test (2-tailed, unequal variance). (B) Luciferase reporter assays showing the effects of DUB3 siRNAs on YAP/TAZ activity. HEK293T cells were transfected to express the luciferase reporters together with independent siRNAs to deplete DUB3 or with a control scrambled siRNA. Data represent the average of three independent transfection experiments ± SD. P values were determined using Student’s T-test (2-tailed, unequal variance). (C) Luciferase reporter assays showing the effect of DUB3 on YAP/TAZ activity. HEK293T cells were transfected to express the luciferase reporters together with a vector expressing Flag-DUB3, the C89S mutant form of DUB3 or relevant controls vectors. shRNA to deplete LATS2 was used as a positive control. Data represent the average of three independent replicates ± SD. P values were determined using Student’s T-test (2-tailed, unequal variance). (D) Immunoblots showing the effect of DUB3 expression on YAP protein. HEK293T cells were transfected with a vector expressing Flag-DUB3, the C89S mutant form of DUB3 or a control vector. Blots were probed with anti-YAP antibody and anti-Flag. Anti-Actin was used to control for loading. (E) Immunoblots showing the effect of DUB3 depletion on the YAP expression level. HEK293T cells were transfected with independent DUB3 siRNAs or a scrambled siRNA control. Blots were probed with anti-YAP antibody and anti-Flag. Anti-Actin was used to control for loading. (F) Effect of DUB3 siRNAs on the expression of YAP transcriptional targets. HEK293T cells were transfected with DUB3 or control siRNAs. mRNA expression of DUB3, Cyr61, ANKRD1 was measured by RT-PCR. GAPDH mRNA was used for normalization and TBP was used as an additional control gene. Data represent the average of 3 independent experiments ± SD. * indicates p <0.01, compared to the relevant controls (Student’s T-test; 2-tailed, unequal variance). (G) Effect of DUB3 on cell growth. Human primary fibroblast BJ cells were engineered to express hTert, H-Ras^G12V and to deplete p53 and p16, or with the addition or YAP^S127A/S397A. BJ^{p53kd/p16kd/HRas} and BJ^{p53kd/p16kd/HRas/YAPS127A/S397A} cells were virally transduced and selected to stably express DUB3, its inactive C89S mutant form or with an empty vector as a control. Cells were counted at 24-hour intervals for a period of 72h. The assay was performed in triplicate. Data represent the average ± SD. * indicate p <0.05, compared to the relevant controls (Student’s T-test; 2-tailed, unequal variance).</p

Morphology of mid-oogenesis egg chambers and border cell migration.

<p>Mid-oogenesis egg chambers labelled with Phalloidin (green) and border cell marker α-Slbo (white). The germ line derived nurse cell (NC) cluster and oocyte (O) as well as the somatic follicular epithelium (FE), which encapsulates the germ line cells, are identified. A Stage S8 egg chamber. Slbo-positive border cells form in the FE anterior to the NC cluster (arrow). B Stage S9 egg chamber. The FE migrates towards the oocyte where it forms a columnar epithelium. Follicle cells stretch over the NC cluster to form a flat epithelium. The border cells (arrow) migrate through the NC cluster, roughly in parallel to the leading edge of the migrating external follicle cell sheet (arrowheads). C Stage S10A egg chamber. Migration of the border cell cluster and the migrating FE have essentially completed. D Stage S10B egg chamber. The centripetal follicle cells migrate over the anterior face of the oocyte (arrowheads).</p

A miRNA sensor suggest somatic miR-<i>989</i> activity.

<p>Stage 8 (A, B) and Stage 10A (C, D) eggchambers expressing a <i>miR-989</i> GFP sensor in a wild type (A, C) or in a <i>miR-989</i> mutant background (B, D). While the GFP sensor was expressed at similar levels in the germ line cells in both genetic backgrounds, it was not detectable in the somatic follicle cells in the wild-type egg chambers. However, follicle cells were positively labelled by the sensor in the absence of <i>miR-989</i>. Note that the sensor also labels the border cells in (D) (arrow).</p

The border cell migration phenotype was rescued by transgenic expression of <i>miR-989.</i>

<p>A–D Expression of <i>miR-989</i> in border cells in a <i>miR-989</i> mutant background suppressed the delayed border cell migration phenotype. Presence of GFP indicates transgenic <i>miR-989</i> expression. A Example of a mosaic egg chamber in which <i>miR-989</i> and GFP were expressed in border cells and border cell migration was normal. B Example of a mosaic egg chamber in which <i>miR-989</i> and GFP were not expressed in border cells, and border cell migration was drastically delayed. C Quantification of border cell migration progress in mosaic stage 9 and stage 10A egg chambers. Mosaic egg chambers in which border cells were mutant for <i>miR-989</i> were strongly delayed in their migration. Transgenic expression of <i>miR-989</i> in border cells of sibling mosaics partially suppressed the delayed border cell phenotype. D Quantification of border cell migration progression in mosaic stage 10B egg chambers. Border cells transgenically expressing <i>miR-989</i> in a <i>miR-989</i> mutant background most often reached the oocyte by this stage. In contrast, <i>miR-989</i> and GFP negative border cells in sibling mosaics were strongly delayed.</p

Clonal analysis demonstrates a somatic requirement for <i>miR-989</i> for normal border cell migration.

<p>A Genetic mosaics: wild type cells are labelled by nuclear GFP (upper panel) while <i>miR-989</i> mutant cells do not express GFP. The middle panels are labelled with phalloidin to highlight the F-actin cytoskeleton. The two egg chambers shown represent extreme cases of mosaicism. In the stage 10A egg chamber on the left all somatic cells are wild-type (GFP positive), while all germ line cells are mutant for <i>miR-989</i> (GFP negative). The border cells were not delayed in their migration. In the stage 10A egg chamber shown on the right all germ line cells are wild type (GFP positive) while all somatic cells are mutant for <i>miR-989</i> (GFP negative). Border cell migration was strongly delayed. B Quantification of the migration defects in migrating border cells in late stage 9 and stage 10A egg chambers. GL denotes germ line, and BC border cells. Migration was strongly delayed if the border cells were mutant, but not if the germ line cells lacked <i>miR-989</i>. We observed a population of border cell clusters that were partially wild-type and partially mutant for <i>miR-989</i>. In these cases, border cell migration was also delayed in comparison to controls. *** indicates p<0.001. C Quantification of the migration defects in stage 10B egg chambers. Wild type border cells migrated normally when the germ line was mutant for <i>miR-989.</i> Conversely, <i>miR-989</i> mutant border cells were delayed when the germ line was wild type.</p

Border cell migration is delayed in <i>miR-989</i> mutant egg chambers.

<p>A Late stage 9 egg chambers, labelled with α-Slbo (white) and Phalloidin (green). Border cell clusters are highlighted by arrows. In <i>miR-989</i> mutant egg chambers, border cells were frequently delayed relative to the migrating main body follicular epithelium. B Quantification of the border cell migration phenotype in stage 9 and stage 10A egg chambers. Border cell migration is delayed in <i>miR-989</i> mutant egg chambers compared to heterozygous control egg chambers (*** p<0.001). X-Axis labels are in μm, error bars denote standard deviation. C Stage 10B egg chambers, labelled with α-Slbo (white) and Phalloidin (green). Border cell clusters are highlighted by arrows. Frequently, border cells had not reached the oocyte by this stage in <i>miR-989</i> mutant egg chambers. D Quantification of the border cell migration phenotype in stage 10B egg chambers.</p