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K.-L.G. eye and wing development that Cdh1 is required in vivo to regulate the LATS homolog Warts with CB1954 a conserved mechanism. Cdh1 reduction increased Warts levels, which resulted in reduction of the eye and wing sizes in a Yorkie dependent manner. Therefore, LATS degradation by APC/CCdh1 represents a previously unappreciated and evolutionarily conserved layer of Hippo signaling regulation. Precise growth control in embryonic development and adult tissue regeneration requires tightly regulated cell division and cell loss in response to various changes (1, 2). The Hippo signaling pathway is evolutionarily conserved from nematodes to human and plays essential roles in regulating tissue growth during development and regeneration (1, 3C5). Disruption in Hippo signaling leads to cancer and other devastating diseases (1, 6, 7). Originally identified in as one required to maintain precise organ sizes of the eye and wing by controlling both cell proliferation and survival, the Hippo signaling pathway contains Hippo, Salvador, Warts, and Yorkie as core components (8C12) and receives inputs from extracellular environment as well as intracellular pathways to regulate a number of biological processes (13C18). Central to the Hippo signaling cascade is the regulation of the transcription factor Yorkie by Warts-mediated phosphorylation. Increased Yorkie protein levels and nuclear localization due to CB1954 Warts inactivation result in dramatic tissue overgrowth by activating downstream gene expression to promote cell survival and proliferation. The mammalian Hippo pathway consists of Hippo homologs Ste20-like kinase MST1 and MST2, the scaffolding protein Salvador (SAV, also known as WW45), Warts homologs large tumor suppressor kinase 1/2 CB1954 (LATS1/2), Yorkie homologs transcription coactivators Yes-associated protein (YAP), and TAZ (also known as WWTR1). Activation of MST1/2 kinase inhibits YAP/TAZ by activating LATS1/2 kinases (3, 19). Phosphorylated YAP/TAZ are sequestered in the cytoplasm via interaction with 14-3-3 and subsequently degraded through -TrCPCdependent ubiquitination (20, 21). As cell proliferation is regulated by proper cell cycle progression and Hippo-YAP/TAZ signaling is key to ensure CB1954 precise growth control, apart from promoting cell proliferation, Hippo-YAP/TAZ signaling may also sense changes in cell proliferation and tissue growth and constantly modify cell cycle progression accordingly. YAP/TAZ are likely critical factors that bridge intrinsic and extrinsic changes with cell cycle progression, because YAP/TAZ can be controlled by both intrinsic and extrinsic stimuli that interact with MST1/2 and/or LATS1/2 kinases (1, 19, 22, 23). Cell cycle progression is tightly controlled by periodic expression of key components of cell cycle machinery (24). The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 ubiquitin ligase complex that governs cell cycle progression by regulating cyclic degradation of key cell cycle regulators via two adaptor proteins, CDH1 or CDC20 (24C26). Recent reports have suggested critical roles for APC/C in various cellular processes, including genome stability and tumorigenesis (27). CDH1 degrades a number of proteins in cell cycle-dependent manner, many of which COL12A1 are known to mediate its role in negatively regulating cell proliferation and DNA replication. However, mouse embryonic fibroblast (MEF) cells exhibit premature senescence and slow proliferation (28, 29), suggesting that some of Cdh1s targets may positively regulate cell proliferation. Here, we introduce a cell-intrinsic regulatory mechanism in Hippo signaling by identifying LATS kinases as direct substrates of APC/CCdh1. This evolutionarily conserved mechanism links cell cycle progression directly with Hippo signaling in growth control. Results APC/CCdh1 Is Required for YAP/TAZ Activities. The potent activity of YAP/TAZ in promoting cell proliferation led us to test whether YAP/TAZ activities are intrinsically regulated during cell cycle progression. We therefore examined YAP/TAZ and Hippo signaling activities in different phases of the cell cycle in the double thymidine block (DTB) assay (Fig. 1and and = CB1954 3 independent experiments. Error bars represent SD. (WT or KO MEFs lysates were subjected to Western blotting analysis with indicated antibodies. (and = 3 independent experiments. (< 0.01 (two-tailed Students test). ns, not significant. To further test whether CDH1 or CDC20 of APC/C plays a role in regulating YAP/TAZ protein levels, or was knocked down by two independent siRNAs in various cell lines. Reduction of and and and transcription itself (Fig. 1 and enhanced TAZ-induced reporter activities (is required for YAP/TAZ transcription activities. Because CDH1 is known to have APC/C E3 ligase-dependent.