A longstanding question in biology is how the size of an organ is determined (Conlon and Raff, 1999
). While environmental cues such as nutrient availability play an important role in regulating organ size, developing organs also possess intrinsic information about their final size. Accumulating evidence suggest that a “size checkpoint” operates at the level of the organ’s total mass, rather than the size or the number of the constituent cells. At present, the molecular nature of this organ size checkpoint remains a mystery.
Recent studies in the fruit fly Drosophila
have implicated the Hippo signaling pathway as an intrinsic mechanism that restricts organ size in development (Edgar, 2006
; Pan, 2007
). This pathway is defined by a kinase cascade whereby the Ste20-like kinase Hippo (Hpo), facilitated by the WW-domain-containing adaptor protein Salvador (Sav), phosphorylates and activates the NDR family kinase Warts (Wts). Wts, in turn, phosphorylates and inactivates the transcriptional coactivator Yorkie (Yki), leading to transcriptional downregulation of target genes such as the cell-cycle regulator cyclin E
, the cell death inhibitor diap1
, and the microRNA bantam
. Inactivation of the Hpo, Sav, or Wts tumor suppressors, or overexpression of the Yki oncoprotein, results in massive tissue overgrowth characterized by excessive cell proliferation and diminished apoptosis.
Several lines of evidence suggest that Yki represents the most critical effector of Wts in the Hippo growth-control pathway (Huang et al., 2005
). First, Yki is phosphorylated by Wts in a Hpo-dependent manner. Second, Yki is required for normal diap1
transcription and tissue growth in Drosophila
imaginal discs, and genetic analysis placed Yki downstream of hpo
, and wts
. Most importantly, overexpression of Yki recapitulates the loss-of-function phenotypes of hpo
, and wts
, such as increased diap1
transcription, increased cell proliferation, and diminished apoptosis, as well as tissue overgrowth. While these findings have established Yki as a critical nuclear effector of the Hippo pathway, the molecular mechanism by which Hippo signaling inactivates Yki function remains to be determined.
A longstanding issue in Hippo signaling concerns the composition and physiological role of this pathway in mammals. Despite the presence of mammalian homologs for all the known components of the Drosophila
Hippo pathway (Mst1/2 for Hpo, WW45 for Sav, Lats1/2 for Wts, and YAP for Yki), previous studies in mammals have failed to unite these proteins in a physiologically relevant signaling cascade. Instead, these proteins have been associated with a wide range of biochemical and physiological roles. For example, Mst1/2 was reported to phosphorylate histone H2B (Cheung et al., 2003
) and FOXO transcription factors (Lehtinen et al., 2006
). Lats1/2 was reported to regulate mitosis and cytokinesis by interacting with cdc2 (Tao et al., 1999
), zyxin (Hirota et al., 2000
), and LIMK1 (Yang et al., 2004
). YAP was reported to play diverse functions ranging from protein trafficking to transcription by binding to at least nine different targets (Sudol, 1994
; Mohler et al., 1999
; Yagi et al., 1999
; Espanel and Sudol, 2001
; Vassilev et al., 2001
; Ferrigno et al., 2002
; Komuro et al., 2003
; Basu et al., 2003
; Howell et al., 2004
). Most critical to the establishment of a mammalian Hippo pathway is a physiologically relevant assay that measures pathway output, which unfortunately has not been available.
In this report, we first investigate the mechanism by which Hippo signaling antagonizes Yki in Drosophila. We show that Hippo signaling inactivates Yki by excluding it from the nucleus via phosphorylation of a critical residue (S168). We provide functional evidence that phosphorylation of this residue mediates the growth-suppressive output of the Hippo signaling pathway. Based on the functional conservation of this phosphorylation site in the mammalian YAP protein, we delineate a mammalian Hippo signaling pathway that links Mst1/2, WW45, and Lats1/2 to YAP phosphorylation. Finally, we explore the physiological function of the mammalian Hippo pathway using a conditional YAP transgenic mouse model. We demonstrate that the mammalian Hippo pathway is a potent regulator of organ size, and that its dysregulation leads to tumorigenesis. These results implicate the Hippo pathway as a universal regulator of tissue homeostasis in metazoan animals.