Hippo signaling is intrinsically regulated during cell cycle progression by APC/CCdh1 Wantae Kima,b,c, Yong Suk Chod, Xiaohui Wanga, Ogyi Parke, Xueyan Maf, Hanjun Kima, Wenjian Gang, Eek-hoon Jhoh, Boksik Chai, Yun-ji Jeungb, Lei Zhangf, Bin Gaoe, Wenyi Weig, Jin Jiangd, Kyung-Sook Chungb,1, and Yingzi Yanga,1 aDepartment of Developmental Biology, Harvard Stem Cell Institute, Harvard School of Dental Medicine, Boston, MA 02215; bBiomedical Translational Research Center, Korea Research Institute of Bioscience and Biotechnology, 34141 Daejeon, Republic of Korea; cDepartment of Biochemistry, Chungnam National University, 34134 Daejeon, Republic of Korea; dDepartment of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390; eSection on Liver Biology, National Institute on Alcohol Abuse and Alcoholism, National Institute of Health, Bethesda, MD 20892; fShanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, 200031 Shanghai, China; gDepartment of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215; hDepartment of Life Science, University of Seoul, 02504 Seoul, South Korea; and iCardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 Edited by Kun-Liang Guan, University of California, San Diego, La Jolla, CA, and accepted by Editorial Board Member Kathryn V. Anderson March 22, 2019 (received for review December 14, 2018) The Hippo-YAP/TAZ signaling pathway plays a pivotal role in plasm via interaction with 14-3-3 and subsequently degraded growth control during development and regeneration and its through β-TrCP–dependent ubiquitination (20, 21). dysregulation is widely implicated in various cancers. To further As cell proliferation is regulated by proper cell cycle pro- understand the cellular and molecular mechanisms underlying Hippo gression and Hippo-YAP/TAZ signaling is key to ensure precise signaling regulation, we have found that activities of core Hippo growth control, apart from promoting cell proliferation, Hippo- signaling components, large tumor suppressor (LATS) kinases and YAP/TAZ signaling may also sense changes in cell proliferation YAP/TAZ transcription factors, oscillate during mitotic cell cycle. We and tissue growth and constantly modify cell cycle progression further identified that the anaphase-promoting complex/cyclosome accordingly. YAP/TAZ are likely critical factors that bridge in- Cdh1 (APC/C) E3 ubiquitin ligase complex, which plays a key role gov- trinsic and extrinsic changes with cell cycle progression, because erning eukaryotic cell cycle progression, intrinsically regulates Hippo YAP/TAZ can be controlled by both intrinsic and extrinsic stimuli CELL BIOLOGY signaling activities. CDH1 recognizes LATS kinases to promote their that interact with MST1/2 and/or LATS1/2 kinases (1, 19, 22, 23). degradation and, hence, YAP/TAZ regulation by LATS phosphoryla- Cell cycle progression is tightly controlled by periodic expression of tion is under cell cycle control. As a result, YAP/TAZ activities peak in key components of cell cycle machinery (24). The anaphase-promoting Drosophila G1 phase. Furthermore, we show in eye and wing de- complex/cyclosome (APC/C) is a multisubunit E3 ubiquitin ligase velopment that Cdh1 is required in vivo to regulate the LATS homo- complex that governs cell cycle progression by regulating cyclic log Warts with a conserved mechanism. Cdh1 reduction increased degradation of key cell cycle regulators via two adaptor proteins, Warts levels, which resulted in reduction of the eye and wing CDH1 or CDC20 (24–26). Recent reports have suggested critical sizes in a Yorkie dependent manner. Therefore, LATS degradation by APC/CCdh1 represents a previously unappreciated and evolution- arily conserved layer of Hippo signaling regulation. Significance LATS1/2 | YAP/TAZ | Hippo signaling | mitotic cell cycle | APC/CCdh1 The Hippo signaling pathway is evolutionarily conserved in the animal kingdom and plays essential roles in regulating tissue growth during development and regeneration. We have iden- recise growth control in embryonic development and adult Cdh1 tissue regeneration requires tightly regulated cell division and tified APC/C , a core component of cell cycle control ma- P chinery, as an evolutionarily conserved and previously unknown cell loss in response to various changes (1, 2). The Hippo signaling regulator of large tumor suppressor (LATS) kinases, which criti- pathway is evolutionarily conserved from nematodes to human cally inhibit the YAP/TAZ transcription factors in transducing and plays essential roles in regulating tissue growth during de- Cdh1 – Hippo signaling. Our results suggest a model that APC/C velopment and regeneration (1, 3 5). Disruption in Hippo sig- destabilizes LATS1/2 kinases in G1 phase of the cell cycle, lead- naling leads to cancer and other devastating diseases (1, 6, 7). ing to increased YAP/TAZ activities that promote G1/S transition Originally identified in Drosophila as one required to maintain by upregulating downstream gene expression, including E2F1. precise organ sizes of the eye and wing by controlling both cell Our findings have important implications for a link between cell proliferation and survival, the Hippo signaling pathway contains proliferation and LATS-regulated YAP/TAZ activities. Hippo, Salvador, Warts, and Yorkie as core components (8–12) and receives inputs from extracellular environment as well as Author contributions: W.K., Y.S.C., X.W., X.M., L.Z., W.W., J.J., K.-S.C., and Y.Y. designed intracellular pathways to regulate a number of biological pro- research; W.K., Y.S.C., X.W., O.P., X.M., H.K., Y.-j.J., J.J., K.-S.C., and Y.Y. performed re- – search; W.K., Y.S.C., X.W., X.M., W.G., E.-h.J., B.C., L.Z., B.G., W.W., J.J., K.-S.C., and Y.Y. cesses (13 18). Central to the Hippo signaling cascade is the contributed new reagents/analytic tools; W.K., Y.S.C., X.W., O.P., X.M., H.K., W.G., E.-h.J., regulation of the transcription factor Yorkie by Warts-mediated B.C., Y.-j.J., B.G., W.W., J.J., K.-S.C., and Y.Y. analyzed data; and W.K., J.J., and Y.Y. wrote phosphorylation. Increased Yorkie protein levels and nuclear the paper. localization due to Warts inactivation result in dramatic tissue The authors declare no conflict of interest. overgrowth by activating downstream gene expression to promote This article is a PNAS Direct Submission. K.-L.G. is a guest editor invited by the cell survival and proliferation. The mammalian Hippo pathway Editorial Board. consists of Hippo homologs Ste20-like kinase MST1 and MST2, Published under the PNAS license. the scaffolding protein Salvador (SAV, also known as WW45), Data deposition: Data related to this paper have been deposited in the Gene Expression Omnibus (accession no. GSE95463). Warts homologs large tumor suppressor kinase 1/2 (LATS1/2), 1To whom correspondence may be addressed. Email: [email protected] or Yorkie homologs transcription coactivators Yes-associated pro- [email protected]. tein (YAP), and TAZ (also known as WWTR1). Activation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. MST1/2 kinase inhibits YAP/TAZ by activating LATS1/2 kinases 1073/pnas.1821370116/-/DCSupplemental. (3, 19). Phosphorylated YAP/TAZ are sequestered in the cyto- www.pnas.org/cgi/doi/10.1073/pnas.1821370116 PNAS Latest Articles | 1of10 Downloaded by guest on September 24, 2021 roles for APC/C in various cellular processes, including genome during cell cycle (24, 32), but only CDH1 oscillated similarly to stability and tumorigenesis (27). CDH1 degrades a number of phosphorylated YAP. We next examined YAP/TAZ nuclear lo- proteins in cell cycle-dependent manner, many of which are known calization, which is regulated by phosphorylation in the DTB assay to mediate its role in negatively regulating cell proliferation and (SI Appendix, Fig. S1 A–C) and found that YAP/TAZ nuclear − − DNA replication. However, Cdh1 / mouse embryonic fibroblast localization was most pronounced in G1 phase (0 and 14 h), but (MEF) cells exhibit premature senescence and slow proliferation much reduced in G2/M phase (6 and 8 h). To further confirm that (28, 29), suggesting that some of Cdh1’stargetsmaypositively YAP/TAZ nuclear localization changes in different phases of cell regulate cell proliferation. Here, weintroduceacell-intrinsicreg- cycle under natural and asynchronous conditions, we used the ulatory mechanism in Hippo signaling by identifying LATS kinases fluorescent ubiquitination-based cell cycle indicator (FUCCI) as direct substrates of APC/CCdh1. This evolutionarily conserved system, a powerful tool for visualizing cell cycle progression in mechanism links cell cycle progression directly with Hippo signaling asynchronous cycling cells (Fig. 1B) (33). FUCCI utilizes the in growth control. phase-dependent proteolysis of the oscillators Cdt1 and Geminin. Fusion protein of Cdt1 or Geminin with the fluorescent mono- Results meric RFP (Cdt-RFP) or GFP (Geminin-GFP) serves as an in- APC/CCdh1 Is Required for YAP/TAZ Activities. The potent activity of dicator of G1 or S and G2 phase, respectively. We found that YAP/TAZ in promoting cell proliferation led us to test whether while YAP/TAZ were clearly localized in the nucleus of cells in YAP/TAZ activities are intrinsically regulated during cell cycle the G1 or G1/S phase, their nuclear localization was much re- progression. We therefore examined YAP/TAZ and Hippo sig- duced in G2 or M phase cells (Fig. 1B). These results indicate that naling activities in different phases of the cell cycle in the double YAP/TAZ activities oscillate during cell cycle and APC/C may thymidine block (DTB) assay (Fig. 1A and SI Appendix,Fig.S1A). regulate YAP/TAZ in a phosphorylation-dependent manner. We found that TAZ and phosphorylated YAP levels (pS127 by To further test whether CDH1 or CDC20 of APC/C plays a LATS kinases) (19, 20, 21, 30) oscillated during cell cycle and, in role in regulating YAP/TAZ protein levels, CDH1 or CDC20 was particular, YAP phosphorylation was reduced, while TAZ protein knocked down by two independent siRNAs in various cell lines.