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, 29 (12), 1271-84

A YAP/TAZ-induced Feedback Mechanism Regulates Hippo Pathway Homeostasis

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A YAP/TAZ-induced Feedback Mechanism Regulates Hippo Pathway Homeostasis

Toshiro Moroishi et al. Genes Dev.

Abstract

YAP (Yes-associated protein) and TAZ (transcriptional coactivator with PDZ-binding motif) are major downstream effectors of the Hippo pathway that influences tissue homeostasis, organ size, and cancer development. Aberrant hyperactivation of YAP/TAZ causes tissue overgrowth and tumorigenesis, whereas their inactivation impairs tissue development and regeneration. Dynamic and precise control of YAP/TAZ activity is thus important to ensure proper physiological regulation and homeostasis of the cells. Here, we show that YAP/TAZ activation results in activation of their negative regulators, LATS1/2 (large tumor suppressor 1/2) kinases, to constitute a negative feedback loop of the Hippo pathway in both cultured cells and mouse tissues. YAP/TAZ in complex with the transcription factor TEAD (TEA domain family member) directly induce LATS2 expression. Furthermore, YAP/TAZ also stimulate the kinase activity of LATS1/2 through inducing NF2 (neurofibromin 2). This feedback regulation is responsible for the transient activation of YAP upon lysophosphatidic acid (LPA) stimulation and the inhibition of YAP-induced cell migration. Thus, this LATS-mediated feedback loop provides an efficient mechanism to establish the robustness and homeostasis of YAP/TAZ regulation.

Keywords: Hippo pathway; TAZ; YAP; cancer; negative feedback; phosphorylation.

Figures

Figure 1.
Figure 1.
YAP and TAZ negatively regulate each other via TEAD-mediated transcription. (A) TAZ accumulates in the livers of YAP knockout mice. Immunoblot (IB) analysis of liver extracts from control and liver-specific YAP cKO mice with antibodies to the indicated proteins is shown. n = 3 mice per group. (B) TAZ accumulates in YAP-deficient tissues. Immunohistochemical (IHC) staining of TAZ was performed on paraffin-embedded liver and intestine tissues from control and liver- or intestine-specific YAP cKO mice. Shown at the bottom right are higher magnifications of the indicated boxed regions. (C) The abundance of TAZ is inversely regulated by YAP expression. MCF10A cells stably expressing Myc-YAP(5SA), shRNA specific for YAP, or control vector were subjected to immunoblot analysis. (D) TEAD-binding of YAP is required for YAP-induced TAZ reduction. MCF10A cells with Myc-YAP(5SA), Myc-YAP(5SA/S94A), or control vector overexpression were subjected to immunoblot analysis. (E) TEADs are required for YAP-induced TAZ reduction. MCF10A cells stably expressing Myc-YAP(5SA) (or control vector) were infected with lentiviruses encoding shRNAs specific for TEAD1/3/4. Immunoblot analysis was performed with the indicated antibodies. (F) Active TAZ decreases YAP. MCF10A cells were infected with retroviruses encoding Flag-TAZ(4SA) or Flag-TAZ(4SA/S51A) or with empty retrovirus (control). Cell lysates were immunoblotted with the indicated antibodies. (G) TAZ facilitates YAP degradation. MCF10A cells infected as in F were exposed to 100 μg/mL cycloheximide for the indicated times and then subjected to immunoblot analysis. The percentage of YAP remaining after the various incubation times was quantitated by image analysis. (l.e.) Long exposure. (H) Inhibition of proteasomal degradation blocks mutual regulation between YAP and TAZ. MCF10A cells stably expressing the indicated constructs were exposed to 10 μM proteasome inhibitor MG132 for 10 h and then subjected to immunoblot analysis. See also Supplemental Figure S1.
Figure 2.
Figure 2.
YAP and TAZ activate LATS1/2 kinases. (A) TAZ increases YAP phosphorylation. Cell lysates from the same experiment shown in Figure 1F were analyzed with the phos-tag electrophoresis for assessment of YAP phosphorylation status. (B) YAP/TAZ increase the protein abundance of LATS2 and phosphorylation levels of LATS1/2 at their HM. Cell lysates from the same experiment shown in Figure 1D (left) or 1F (right) were subjected to immunoblot (IB) analysis. (C) YAP/TAZ increase the activating phosphorylation of LATS1/2 at their HM and activation loop (AL). MCF10A cells were infected with retroviruses encoding Flag-TAZ(4SA) or Flag-TAZ(4SA/S51A) or with empty retrovirus (control). Cell lysates were then subjected to immunoprecipitation (IP) and immunoblot analysis. The asterisk indicates a nonspecific band. (D) TAZ activates LATS1 kinase activity. Endogenous LATS1 was immunoprecipitated from MCF10A cells infected as in C. The resulting immunoprecipitates were subjected to in vitro kinase assay. (E) Angiomotin (AMOT) is phosphorylated and accumulated by TAZ overexpression. MCF10A cells infected as in C were subjected to immunoblot analysis. (F) Active TAZ promotes YAP cytoplasmic localization. MCF10A cells infected as in C were subjected to immunostaining analysis. YAP subcellular localization was determined by immunofluorescence staining for endogenous YAP along with DAPI for DNA. Representative images are presented in the left panel. (Right panel) Cells in five random views (∼100 cells) were selected for the quantification of YAP localization. See also Supplemental Figure S2.
Figure 3.
Figure 3.
YAP and TAZ directly induce LATS2 expression. (A) YAP induces the transcription of LATS2. Total RNA extracted from MCF10A cells stably expressing the indicated constructs were subjected to RT and real-time PCR analysis. Data are means ± SD of triplicates from a representative experiment. (B) Loss of YAP decreases the mRNA abundance of LATS2. Total RNA extracted from MCF10A cells stably expressing shRNA specific for YAP (or control) were subjected to RT and real-time PCR analysis of LATS2 mRNA. Data are means ± SD of triplicates from a representative experiment. (C) Serum stimulation activates YAP and increases LATS2 mRNA levels. MCF10A cells were starved in serum-free medium for 8 h and then stimulated with 5% horse serum for 90 min. (Left) Cell lysates were subjected to immunoblotting (IB) with the indicated antibodies. (Top left) Where indicated, gels containing phos-tag were employed for assessment of YAP phosphorylation status. (Right) Total RNA were subjected to RT and real-time PCR analysis of LATS2 mRNA. Data are means ± SD of triplicates from a representative experiment. (D) Activation of LATS2 reporter by YAP and TEAD1. HEK293A cells were transfected with LATS2 luciferase construct and with (or without) increasing amounts (1× and 2×) of an expression vector for Flag-YAP or Myc-TEAD1. Luciferase activities were assayed 30 h after transfection. Data are means ± SD from three independent experiments. (E) Endogenous YAP and TEAD1 both bind to the LATS2 promoter. MCF10A cells starved in serum-free medium for 18 h were stimulated with 5% horse serum for 2 h. The cells were then subjected to chromatin immunoprecipitation (ChIP) with antibodies to endogenous YAP or TEAD1 (or control IgG), and the precipitated DNA was quantitated by real-time PCR analysis with primers specific for a promoter region or a control region (CR) of the indicated genes. Data are means ± SD of triplicates from a representative experiment. (F) Dot plots showing the positive correlation between LATS2 and CYR61 or CTGF mRNA expression in cell lines from 967 subjects. R-values were calculated for each correlation; P < 0.0001; Pearson's correlation coefficient. See also Supplemental Figure S3.
Figure 4.
Figure 4.
YAP and TAZ stimulate the intrinsic kinase activity of LATS1/2 through NF2. (A) MST1/2 are largely required for YAP-induced LATS1/2 activation. Wild-type (WT) and MST1/2 double-knockout (DKO) HEK293A cells infected with retroviruses encoding Myc-YAP(5SA) or with control empty retrovirus were subjected to immunoblot (IB) analysis. (l.e.) Long exposure. (B) TAZ does not stimulate MST1/2 kinase activity. Endogenous MST1 or MST2 was immunoprecipitated (IP) from MCF10A cells infected with retroviruses encoding Flag-TAZ(4SA) or Flag-TAZ(4SA/S51A) or with the empty retrovirus (control). The resulting immunoprecipitates were subjected to in vitro kinase assay. (C) TAZ increases NF2 abundance. MCF10A cells infected as in B were subjected to immunoblot analysis. (D) TEADs are required for NF2 induction and LATS1/2 activation by YAP. MCF10A cells stably expressing Myc-YAP(5SA) or control vector were infected with lentiviruses encoding shRNAs specific for TEAD1/3/4. Immunoblot analysis was performed with the indicated antibodies. (E) NF2 is required for YAP-induced activating phosphorylation of LATS1/2. Wild-type and NF2 knockout (KO) HEK293A cells infected with retroviruses encoding Myc-YAP(5SA) or with control empty retrovirus were subjected to immunoblot analysis. (F) NF2 is required for TAZ-induced LATS1 activation. Endogenous LATS1 was immunoprecipitated from wild-type or NF2 knockout HEK293A cells stably expressing Flag-TAZ(4SA), Flag-TAZ(4SA/S51A), or control vector. The resulting immunoprecipitates were subjected to in vitro kinase assay. See also Supplemental Figure S4.
Figure 5.
Figure 5.
LATS1/2 mediate negative feedback regulation of YAP/TAZ activity. (A) YAP induces the transcription of Lats2 in vivo. RT and real-time PCR analysis of the indicated mRNA in the livers of 2-mo-old YAP Tg and non-Tg (control) mice fed 0.2 mg/mL doxycycline in drinking water for 2 wk. Normalized data are expressed relative to the corresponding value for non-Tg littermates and are mean ± SD. n = 3 mice per group. (n.s.) Not significant. (*) P < 0.05 (Student's t test). (B) YAP stimulates negative feedback in vivo. Immunoblot (IB) analysis of liver extracts from mice treated as in A with antibodies to the indicated proteins. n = 3 mice per group. The asterisk indicates a nonspecific band. (l.e.) Long exposure. (C) LATS1/2 are required for YAP-induced negative feedback regulation of YAP and TAZ. Wild-type (WT) or Lats1–/–Lats2Δ/Δ (LATS1/2 double-knockout [DKO]) mouse embryonic fibroblasts (MEFs) were infected with retroviruses encoding Myc-YAP(5SA) or Myc-YAP(5SA/S94A) or with empty retrovirus. Cell lysates were immunoblotted with the indicated antibodies. The asterisk indicates a nonspecific band. (D) LATS1/2 are required for YAP-induced cytoplasmic localization of TAZ. MEFs infected as in C were subjected to immunostaining analysis. TAZ subcellular localization was determined by immunofluorescence staining for endogenous TAZ along with DAPI for DNA. Representative images are presented in the left panel. (Right panel) Cells in five random views (∼100 cells) were quantified for TAZ localization. See also Supplemental Figure S5.
Figure 6.
Figure 6.
YAP/TEAD-induced feedback mechanism maintains the transient YAP activation on stimulation. (A) YAP induction promptly stimulates the LATS-mediated feedback loop. Tetracycline repressor-expressing MCF10A cells were infected with retroviruses encoding YAP(S127A) or with empty retrovirus, incubated with 1 μg/mL doxycycline for the indicated times, and then subjected to immunoblot (IB) analysis. (B) YAP/TEAD-induced negative feedback is required for the transient activation of YAP upon LPA stimulation. MCF10A cells infected with lentiviruses encoding shRNA specific for TEAD1/3/4 (or control) were starved in serum-free medium for 15 h and then stimulated with 5 μM LPA for the indicated times. Cell lysates were subjected to immunoblotting with the indicated antibodies. (C) LPA induces LATS2 transcription in a TEAD-dependent manner. MCF10A cells infected and starved as in B were stimulated with 5 μM LPA for 2 h. Total RNA were subjected to RT and real-time PCR analysis of LATS2 mRNA. Data are means ± SD of triplicates from a representative experiment. See also Supplemental Figure S6.
Figure 7.
Figure 7.
The LATS-mediated negative feedback limits actin stress fiber formation and cell migration upon YAP activation. (A) Inducible expression of YAP triggers the negative feedback machinery via LATS1/2 and MST1/2. Tetracycline repressor-expressing HEK293A cells were infected with retroviruses encoding Myc-YAP(5SA) or with control empty retrovirus, incubated with 200 ng/mL doxycycline for 24 h, and then subjected to immunoblot (IB) analysis. (B) Loss of LATS1/2 or MST1/2 enhances actin stress fiber formation by YAP(5SA) overexpression. HEK293A cells infected and induced as in A were seeded on fibronectin-coated coverslips for 1 h in serum-free medium. F-actin was stained with Alexa fluor 488 phalloidin (green) along with DAPI for DNA (blue). (C) LATS1/2 or MST1/2 depletion enhances cell migration by YAP(5SA) overexpression. HEK293A cells infected and induced as in A were subjected to transwell migration assay. Cells on the bottom sides of the transwells were stained with crystal violet (top) and quantified (bottom). (D) A proposed model of negative feedback regulation in the mammalian Hippo pathway. In response to YAP/TAZ-activating stimulations, YAP/TAZ in complex with TEAD directly induces LATS2 expression. YAP/TAZ also stimulate the kinase activity of LATS1/2 through NF2 induction. YAP/TAZ activation therefore results in activation of their negative regulators, LATS1/2, to constitute a negative feedback loop of the Hippo pathway. See also Supplemental Figure S7.

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