Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press A YAP/TAZ-induced feedback mechanism regulates Hippo pathway homeostasis Toshiro Moroishi,1 Hyun Woo Park,1 Baodong Qin,1,2 Qian Chen,3 Zhipeng Meng,1 Steven W. Plouffe,1 Koji Taniguchi,4,5 Fa-Xing Yu,6,7 Michael Karin,4,5 Duojia Pan,3 and Kun-Liang Guan1 1Department of Pharmacology, Moores Cancer Center, University of California at San Diego, La Jolla, California 92093, USA; 2Department of Laboratory Medicine, Shanghai Changzheng Hospital, Second Military Medical University, Shanghai 200003, China; 3Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA; 4Department of Pharmacology, 5Department of Pathology, University of California at San Diego, La Jolla, California 92093, USA; 6Children’s Hospital, 7Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China 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; YAP; TAZ; negative feedback; phosphorylation; cancer] Supplemental material is available for this article. Received March 26, 2015; revised version accepted May 28, 2015. Yes-associated protein (YAP) and transcriptional coacti- Hippo pathway, which was initially identified through vator with PDZ-binding motif (TAZ; also known as genetic mutant screens for tumor suppressors in Droso- WWTR1) play pivotal roles in regulating cell proliferation, phila melanogaster (Pan 2010). In mammals, the core survival, and differentiation; organ development; regener- components of the Hippo pathway consist of mammalian ation; and stem cell biology (Barry and Camargo 2013; STE20-like protein kinase 1 (MST1; also known as STK4) Johnson and Halder 2014; Mo et al. 2014; Piccolo et al. and MST2 (also known as STK3)—the mammalian homo- 2014). YAP and TAZ share ∼50% amino acid sequence logs of Hippo in D. melanogaster—as well as large tumor identity with a similar domain organization, each con- suppressor 1 (LATS1) and LATS2. When the pathway is ac- taining a TEAD (TEA domain family member)-binding tivated, MST1/2 phosphorylate and activate the LATS1/2 domain, a WW domain, and a C-terminal transactivation kinases, which in turn directly phosphorylate YAP/TAZ domain. YAP and TAZ are transcriptional coactivators on multiple serine residues, leading to cytoplasmic reten- that shuttle between the cytoplasm and the nucleus, tion of YAP/TAZ via a 14-3-3 interaction (Oh and Irvine where they associate with several transcription factors— 2010). Furthermore, phosphorylation of YAP/TAZ by mainly TEAD transcription factors (Zhao et al. 2008). LATS1/2 primes YAP/TAZ for subsequent phosphory- The YAP/TAZ–TEAD complex activates expression of lation events by casein kinase 1 (CK1), resulting in recruit- target genes involved in cell proliferation and survival ment of β-transducin repeat-containing proteins (β-TRCP; (Varelas 2014). the F-box protein subunit of the SCFβ-TRCP ubiquitin– The activity of YAP/TAZ is largely regulated through a phosphorylation-dependent inhibition mechanism by the © 2015 Moroishi et al. This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). Corresponding author: [email protected] After six months, it is available under a Creative Commons License (Attri- Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.262816. bution-NonCommercial 4.0 International), as described at http:// 115. creativecommons.org/licenses/by-nc/4.0/. GENES & DEVELOPMENT 29:1271–1284 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/15; www.genesdev.org 1271 Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Moroishi et al. ligase complex) and consequent degradation by the ubiq- Results uitin–proteasome pathway (Zhao et al. 2010). Inhibition of YAP/TAZ represents the major functional output of Mutual inhibition between YAP and TAZ by the Hippo pathway. a mechanism dependent on TEAD-mediated Although the core components of the Hippo pathway transcription are well characterized, the upstream regulators of this pathway are just beginning to be delineated. Previous Because the Hippo pathway controls tissue homeostasis studies revealed a general role of G-protein-coupled recep- and organ size, it must be tightly regulated by extracel- tors (GPCRs) as prime regulators of Hippo signaling, lular signals as well as internal feedback mechanisms. where LATS1/2 kinases are acutely inhibited by the extra- Convincing studies have revealed that this pathway is reg- cellular hormones, such as lysophosphatidic acid (LPA) ulated by cell density, extracellular signaling, and mecha- and sphingosine-1-phosphate (S1P) (Park and Guan 2013; notransduction. We hypothesized that there may be a Wackerhage et al. 2014). In addition to hormonal regula- mechanism that monitors the total output of YAP/TAZ tion, several properties of tissue architecture, such as api- activity with a feedback control for the pathway. Interest- cal–basal polarity, planar cell polarity, and various types ingly, we found that YAP negatively regulates its paralog, of cell–cell junctions, have been implicated in Hippo path- TAZ, in vivo. TAZ accumulated in the liver of YAP con- way regulation (Enderle and McNeill 2013; Thompson ditional knockout (cKO) mice (Fig. 1A). We observed sig- et al. 2013). Moreover, studies have revealed that YAP/ nificant accumulation as well as nuclear localization of TAZ are regulated by mechanical cues, such as extracellu- TAZ in the livers and intestines of YAP cKO mice (Fig. lar matrix (ECM) stiffness and traction forces exerted by 1B). In MCF10A mammary epithelial cells, depletion of neighboring cells (Halder et al. 2012; Low et al. 2014). endogenous YAP by shRNA increased the amount of Aberrant hyperactivation of YAP/TAZ causes tissue endogenous TAZ, whereas overexpression of YAP(5SA) overgrowth and confers principal cancer features, such (an active mutant of YAP with all five LATS phosphoryla- as epithelial–mesenchymal transition (EMT), increased tion sites mutated to alanine, thereby unresponsive to migration and potential for metastasis, and cancer stem inhibition by the LATS kinase) decreased TAZ levels cell properties (Harvey et al. 2013; Moroishi et al. 2015). (Fig. 1C). We confirmed the specificity of YAP shRNA, In contrast, inactivation of YAP/TAZ impairs tissue de- as re-expression of YAP prevented TAZ accumulation velopment, stem cell function, and regeneration (Barry in YAP knockdown MCF10A cells (Supplemental Fig. and Camargo 2013; Mo et al. 2014). Therefore, strict and S1A). These observations suggest that cells have an intrin- dynamic control of YAP/TAZ activity is essential for sic mechanism to maintain total output/activity of the proper proliferative cellular response to ensure tissue ho- Hippo pathway. meostasis. However, regulatory mechanisms involved in Next, we investigated whether YAP regulates TAZ in a the dynamic control of YAP/TAZ activity following sig- manner dependent on its transcriptional activity. TEAD nal perturbation have been poorly understood. is the major YAP-associated transcription factor, and its Cell-intrinsic negative feedback loops generally play binding to YAP requires the Ser94 residue in YAP (Zhao important roles in establishing robustness to bring sys- et al. 2008). Mutating Ser94 abolished the ability of YAP tems back to their initial equilibrium states. In D. mela- (5SA) to suppress TAZ (Fig. 1D). Consistently, shRNA- nogaster, Yorkie (the fly homolog of YAP/TAZ) was mediated depletion of endogenous TEAD1/3/4 (Supple- found to induce its negative regulators, including ex- mental Fig. S1B) attenuated the effect of YAP(5SA) on panded, merlin, four-jointed, and kibra, to provide a pos- TAZ (Fig. 1E), suggesting that the protein abundance of sible negative feedback onto itself during developmental TAZ is negatively regulated by YAP-induced TEAD tran- growth control (Cho et al. 2006; Hamaratoglu et al. scriptional activity rather than YAP protein abundance. 2006; Genevet et al. 2010). However, the biological signif- We confirmed the specificity of TEAD1/3/4 shRNA, icance of this potential negative feedback