From Cell Structure to Transcription: Hippo Forges a New Path
2006; Cell Press; Volume: 124; Issue: 2 Linguagem: Inglês
10.1016/j.cell.2006.01.005
ISSN1097-4172
Autores Tópico(s)Hereditary Neurological Disorders
ResumoThe control of cell number during animal development is a longstanding puzzle. Recent studies in the fruit fly Drosophila melanogaster have defined a new signaling pathway that restricts cell proliferation in differentiating epithelia. The cytoskeletal proteins Merlin and Expanded, which play a role in cell adhesion and structure, control the activation of the Hippo/Salvador kinase complex, which in turn activates the Warts/Mats kinase complex. Warts/Mats kinase phosphorylates and inhibits Yorkie, a transcriptional coactivator that positively regulates cell growth, survival, and proliferation. This conserved signaling pathway contains several tumor-suppressor genes and regulates the contact inhibition of proliferation in cultured cells. The control of cell number during animal development is a longstanding puzzle. Recent studies in the fruit fly Drosophila melanogaster have defined a new signaling pathway that restricts cell proliferation in differentiating epithelia. The cytoskeletal proteins Merlin and Expanded, which play a role in cell adhesion and structure, control the activation of the Hippo/Salvador kinase complex, which in turn activates the Warts/Mats kinase complex. Warts/Mats kinase phosphorylates and inhibits Yorkie, a transcriptional coactivator that positively regulates cell growth, survival, and proliferation. This conserved signaling pathway contains several tumor-suppressor genes and regulates the contact inhibition of proliferation in cultured cells. Developmental and cancer biologists have long pondered how cell proliferation is restricted, first during embryogenesis to allow morphogenesis and later in adults to maintain homeostasis. I use the term "restricted," rather than "promoted" because, although it is often assumed that cells must be stimulated to proliferate, the basal state of the single-celled eukaryotes that gave rise to metazoa was almost certainly one of proliferation, limited only by nutrients. With the arrival of multicellularity came the requirement that cells acquire characteristics akin to cooperation and altruism, such as the ability to restrain their growth and division in response to signals from other cells and to die when no longer needed. These properties are essential for proper development and for maintaining homeostasis in adults. Acquiring them necessitated the establishment of new mechanisms to allow cells to sense their density or numbers and to apply this information to regulate intrinsic cellular processes including growth, division, and survival. Although it is clear that genetic systems controlling cell numbers are vulnerable (consider cancer as an example), we are still far from understanding how these systems operate during normal development. In this review, I highlight some recent advances relevant to this puzzle. The advent of modern methods for generating genetic mosaics in the fruit fly Drosophila melanogaster (Xu and Rubin, 1993Xu T. Rubin G.M. Analysis of genetic mosaics in developing and adult Drosophila tissues.Development. 1993; 117: 1223-1237Crossref PubMed Google Scholar) prompted screens in which clones of cells with mutations in random genes were produced, and the adults were scored for abnormal tumor-like growths. These screens identified many genes that, when mutated, caused the inappropriate proliferation of cells. Many of these genes turned out to be orthologs of known or suspected human tumor-suppressor genes, such as PTEN or TSC2. Studies of these genes in Drosophila have proven useful for assigning functions to the proteins they encode and mapping their signaling pathways. Some new growth suppressors were also discovered. The first of these was warts/large tumor suppressor (wts/lats), a kinase of the nuclear Dbf-2-related (NDR) family (Justice et al., 1995Justice R.W. Zilian O. Woods D.F. Noll M. Bryant P.J. The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation.Genes Dev. 1995; 9: 534-546Crossref PubMed Scopus (659) Google Scholar, Xu et al., 1995Xu T.A. Wang W.Y. Zhang S. Stewart R.A. Yu W. Identifying tumor suppressors in genetic mosaics—the Drosophila lats gene encodes a putative protein-kinase.Development. 1995; 121: 1053-1063Crossref PubMed Google Scholar). Cells with mutations in the wts gene exhibit dramatic outgrowths in a variety of fly epithelial tissues but do not display overt changes in cell identity or the ability to differentiate. Further screens isolated three other genes—hippo (hpo), salvador (sav), and mob as tumor suppressor (mats)—that gave strikingly similar tumorous phenotypes when clonally deleted in developing eyes, wings, or legs of the fly (Harvey et al., 2003Harvey K.F. Pfleger C.M. Hariharan I.K. The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis.Cell. 2003; 114: 457-467Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, Kango-Singh et al., 2002Kango-Singh M. Nolo R. Tao C. Verstreken P. Hiesinger P.R. Bellen H.J. Halder G. Shar-pei mediates cell proliferation arrest during imaginal disc growth in Drosophila.Development. 2002; 129: 5719-5730Crossref PubMed Scopus (253) Google Scholar, Pantalacci et al., 2003Pantalacci S. Tapon N. Leopold P. The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila.Nat. Cell Biol. 2003; 5: 921-927Crossref PubMed Scopus (413) Google Scholar, Tapon et al., 2002Tapon N. Harvey K.F. Bell D.W. Wahrer D.C. Schiripo T.A. Haber D.A. Hariharan I.K. salvador promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines.Cell. 2002; 110: 467-478Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, Udan et al., 2003Udan R.S. Kango-Singh M. Nolo R. Tao C. Halder G. Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway.Nat. Cell Biol. 2003; 5: 914-920Crossref PubMed Scopus (526) Google Scholar, Wu et al., 2003Wu S. Huang J. Dong J. Pan D. hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts.Cell. 2003; 114: 445-456Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar, Lai et al., 2005Lai Z.C. Wei X. Shimizu T. Ramos E. Rohrbaugh M. Nikolaidis N. Ho L.L. Li Y. Control of cell proliferation and apoptosis by mob as tumor suppressor, mats.Cell. 2005; 120: 675-685Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). The similarities among the phenotypic effects caused by mutations in these four genes suggested that they might function in a common pathway. When deleted in wing epithelial cells, each of them causes a cell shape change termed apical hypertrophy, in which the apical cell surface expands away from the nucleus, taking on a domed shape, and areas of apical-lateral adhesion are reduced (Justice et al., 1995Justice R.W. Zilian O. Woods D.F. Noll M. Bryant P.J. The Drosophila tumor suppressor gene warts encodes a homolog of human myotonic dystrophy kinase and is required for the control of cell shape and proliferation.Genes Dev. 1995; 9: 534-546Crossref PubMed Scopus (659) Google Scholar). These apical-lateral regions are where adherens junctions reside in insect epithelia, suggesting that this set of genes affects the function of adherens junctions (Bilder, 2004Bilder D. Epithelial polarity and proliferation control: links from the Drosophila neoplastic tumor suppressors.Genes Dev. 2004; 18: 1909-1925Crossref PubMed Scopus (442) Google Scholar, Perez-Moreno et al., 2003Perez-Moreno M. Jamora C. Fuchs E. Sticky business: Orchestrating cellular signals at adherens junctions.Cell. 2003; 112: 535-548Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar). Clones of mutant cells produced in the wing or eye primordia (called "discs") also exhibit smooth borders and a rounded shape, indicative of altered cell adhesion, although cell polarity and organization of the epithelial monolayer are maintained. Prior to differentiation, the mutant cells were also observed to grow and divide more rapidly than normal cells, without any change in cell size. A distinct effect was noted in the eye, where mutations in hpo, sav, or wts each cause inappropriate proliferation and survival of nonneural interommatidial cells just after differentiation. This suggested a common function for these genes during eye development (see Figure 1) . Normally, interommatidial cells are produced in excess, and most of them are culled in a wave of apoptosis during eye differentiation. In hpo, sav, or wts mutants, these interommatidial cells fail to die but instead divide several extra times, causing expansion and some disorganization of the eye. Mutations in the mats gene were reported to cause a somewhat more severe phenotype, in which differentiation was abrogated and, perhaps as a result, substantial apoptosis occurred at late stages (Lai et al., 2005Lai Z.C. Wei X. Shimizu T. Ramos E. Rohrbaugh M. Nikolaidis N. Ho L.L. Li Y. Control of cell proliferation and apoptosis by mob as tumor suppressor, mats.Cell. 2005; 120: 675-685Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). When very large cell clones with mutations in wts or hpo were produced, however, they too showed pronounced defects in cell differentiation (Wu et al., 2003Wu S. Huang J. Dong J. Pan D. hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts.Cell. 2003; 114: 445-456Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar, Xu et al., 1995Xu T.A. Wang W.Y. Zhang S. Stewart R.A. Yu W. Identifying tumor suppressors in genetic mosaics—the Drosophila lats gene encodes a putative protein-kinase.Development. 1995; 121: 1053-1063Crossref PubMed Google Scholar), suggesting that the severe phenotype conferred by mats mutations may not actually reflect a distinct function. The cause of these differentiation defects is unclear, but given that they vary according to clone size, they could be due to the failure of the mutant cells to effectively transduce long-range differentiation signals. Given these similarities in mutant phenotypes, perhaps it is not surprising that the Hippo and Sav proteins were found to physically interact with each other, as were the Warts and Mats proteins (see Figure 2) . Biochemical assays showed that Hippo, a Ste-20/MST2 type kinase, phosphorylates and activates Warts and that Sav, a WW-repeat protein, facilitates this reaction potentially by acting as a scaffold. Furthermore, Mats was found to bind to Warts, but not to Hippo, and to stimulate Warts kinase activity. Genetic tests performed in vivo supported these interactions. Mats has also been shown to physically and genetically interact with another NDR-family kinase, tricornered (trc), which is required for the morphogenesis of epidermal hairs, bristles, and dendrites (He et al., 2005He Y. Emoto K. Fang X. Ren N. Tian X. Jan Y.N. Adler P.N. Drosophila mob family proteins interact with the related tricornered (trc) and warts (wts) kinases.Mol. Biol. Cell. 2005; 16: 4139-4152Crossref PubMed Scopus (65) Google Scholar). This might explain the relative severity of the mats mutant phenotype. Targeted searches for downstream effectors revealed that the loss of hpo, sav, wts, or mats caused a cell-autonomous increase in the amounts of the Drosophila inhibitor of apoptosis gene product, DIAP-1, and the cell cycle regulator Cyclin E (Harvey et al., 2003Harvey K.F. Pfleger C.M. Hariharan I.K. The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis.Cell. 2003; 114: 457-467Abstract Full Text Full Text PDF PubMed Scopus (661) Google Scholar, Pantalacci et al., 2003Pantalacci S. Tapon N. Leopold P. The Salvador partner Hippo promotes apoptosis and cell-cycle exit in Drosophila.Nat. Cell Biol. 2003; 5: 921-927Crossref PubMed Scopus (413) Google Scholar, Tapon et al., 2002Tapon N. Harvey K.F. Bell D.W. Wahrer D.C. Schiripo T.A. Haber D.A. Hariharan I.K. salvador promotes both cell cycle exit and apoptosis in Drosophila and is mutated in human cancer cell lines.Cell. 2002; 110: 467-478Abstract Full Text Full Text PDF PubMed Scopus (622) Google Scholar, Udan et al., 2003Udan R.S. Kango-Singh M. Nolo R. Tao C. Halder G. Hippo promotes proliferation arrest and apoptosis in the Salvador/Warts pathway.Nat. Cell Biol. 2003; 5: 914-920Crossref PubMed Scopus (526) Google Scholar, Wu et al., 2003Wu S. Huang J. Dong J. Pan D. hippo encodes a Ste-20 family protein kinase that restricts cell proliferation and promotes apoptosis in conjunction with salvador and warts.Cell. 2003; 114: 445-456Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar). DIAP-1 and Cyclin E are important regulators of cell survival and cell cycle progression, respectively, and thus they probably account, at least in part, for the survival and continued proliferation of cells with mutations in these genes. Other potential clues to function have come from studies of the mammalian Warts orthologs, LATS1 and LATS2/Kpm, which implicate both proteins in the regulation of mitosis. LATS1 and LATS2/Kpm are phosphorylated and activated in mitosis, and when overexpressed they can bind to and inhibit the mitotic kinase Cdk1 (Tao et al., 1999Tao W. Zhang S. Turenchalk G.S. Stewart R.A. St. John M.A. Chen W. Xu T. Human homologue of the Drosophila melanogaster lats tumour suppressor modulates CDC2 activity.Nat. Genet. 1999; 21: 177-181Crossref PubMed Scopus (224) Google Scholar, Yang et al., 2001Yang X. Li D.M. Chen W. Xu T. Human homologue of Drosophila lats, LATS1, negatively regulates growth by inducing G(2)/M arrest or apoptosis.Oncogene. 2001; 20: 6516-6523Crossref PubMed Scopus (104) Google Scholar, Xia et al., 2002Xia H. Qi H. Li Y. Pei J. Barton J. Blackstad M. Xu T. Tao W. LATS1 tumor suppressor regulates G2/M transition and apoptosis.Oncogene. 2002; 21: 1233-1241Crossref PubMed Scopus (129) Google Scholar, Kamikubo et al., 2003Kamikubo Y. Takaori-Kondo A. Uchiyama T. Hori T. Inhibition of cell growth by conditional expression of kpm, a human homologue of Drosophila warts/lats tumor suppressor.J. Biol. Chem. 2003; 278: 17609-17614Crossref PubMed Scopus (60) Google Scholar, Iida et al., 2004Iida S. Hirota T. Morisaki T. Marumoto T. Hara T. Kuninaka S. Honda S. Kosai K. Kawasuji M. Pallas D.C. Saya H. Tumor suppressor WARTS ensures genomic integrity by regulating both mitotic progression and G1 tetraploidy checkpoint function.Oncogene. 2004; 23: 5266-5274Crossref PubMed Scopus (74) Google Scholar). This suggests a possible explanation for LATS-dependent growth suppression. Perhaps more importantly, loss-of-function studies revealed that cells lacking LATS1 or LATS2 exhibit defects in exiting mitosis (Bothos et al., 2005Bothos J. Tuttle R.L. Ottey M. Luca F.C. Halazonetis T.D. Human LATS1 is a mitotic exit network kinase.Cancer Res. 2005; 65: 6568-6575Crossref PubMed Scopus (108) Google Scholar) and in cytokinesis (Yang et al., 2004Yang X. Yu K. Hao Y. Li D.M. Stewart R. Insogna K.L. Xu T. LATS1 tumour suppressor affects cytokinesis by inhibiting LIMK1.Nat. Cell Biol. 2004; 6: 609-617Crossref PubMed Scopus (149) Google Scholar, McPherson et al., 2004McPherson J.P. Tamblyn L. Elia A. Migon E. Shehabeldin A. Matysiak-Zablocki E. Lemmers B. Salmena L. Hakem A. Fish J. et al.Lats2/Kpm is required for embryonic development, proliferation control and genomic integrity.EMBO J. 2004; 23: 3677-3688Crossref PubMed Scopus (164) Google Scholar) that lead to multinucleate cells, centrosome amplification, and genomic instability. Functions in mitosis or cytokinesis might contribute to the tumor-suppressor activity of the LATS genes in mammals (Takahashi et al., 2005Takahashi Y. Miyoshi Y. Takahata C. Irahara N. Taguchi T. Tamaki Y. Noguchi S. Down-regulation of LATS1 and LATS2 mRNA expression by promoter hypermethylation and its association with biologically aggressive phenotype in human breast cancers.Clin. Cancer Res. 2005; 11: 1380-1385Crossref PubMed Scopus (241) Google Scholar, St. John et al., 1999St. John M.A. Tao W. Fei X. Fukumoto R. Carcangiu M.L. Brownstein D.G. Parlow A.F. McGrath J. Xu T. Mice deficient of Lats1 develop soft-tissue sarcomas, ovarian tumours and pituitary dysfunction.Nat. Genet. 1999; 21: 182-186Crossref PubMed Scopus (349) Google Scholar), but such defects do not provide a ready explanation for the increased cell growth and survival seen in wts mutant cells in Drosophila. Moreover, as mitotic defects have not been observed in fly cells with mutations in wts, hpo, sav, or mats, these functions may be specific to the mammalian LATS1 and LATS2 genes. Thus, despite the intriguing overgrowth phenotypes conferred by loss of genes encoding Hippo complex components, Hippo has until recently remained an orphan signaling entity, lacking known upstream inputs and, for the most part, critical downstream effectors. Given that the Hippo and Warts complexes could not easily be slotted into any known signaling pathway, it was assumed that they must define a new signaling pathway important for containing proliferation, at least in epithelial cells. Although the increase in Cyclin E and DIAP-1 may explain the boost in cell numbers seen in mutant tissues, these two target molecules cannot explain the stimulation of cell growth (increased cell mass) or the intriguing cell shape and adhesion defects displayed by these cells. Moreover, how the Warts kinase complex controls even its known targets (cyclin E, diap-1) is not obvious, with some researchers suggesting transcriptional control and others favoring regulation at the protein level. Two recent papers have redrawn this lonely scene, adding a downstream effector (Huang et al., 2005Huang J. Wu S. Barrera J. Matthews K. Pan D. The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila homolog of YAP.Cell. 2005; 122: 421-434Abstract Full Text Full Text PDF PubMed Scopus (1181) Google Scholar), some upstream inputs (Hamaratoglu et al., 2005Hamaratoglu F. Kango-Singh M. Nolo R. Hyun E. Tao C. Jafar-Nejad H. Halder G. The tumor suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis.Nat. Cell Biol. 2005; 8 (Published online December 11, 2005): 27-36https://doi.org/10.1038/ncb1339Crossref PubMed Scopus (545) Google Scholar), and an interesting feedback loop. The downstream effector, Yorkie (Yki), was discovered by Duojia "DJ" Pan's group not by phenotypic screens in flies but by virtue of its ability to bind to Warts in a yeast two-hybrid screen (Huang et al., 2005Huang J. Wu S. Barrera J. Matthews K. Pan D. The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila homolog of YAP.Cell. 2005; 122: 421-434Abstract Full Text Full Text PDF PubMed Scopus (1181) Google Scholar). Yorkie is the Drosophila ortholog of YAP (Yes-associated protein), which has been characterized as a non-DNA binding transcriptional coactivator in mammalian cells (Yagi et al., 1999Yagi R. Chen L.F. Shigesada K. Murakami Y. Ito Y. A WW domain-containing yes-associated protein (YAP) is a novel transcriptional co-activator.EMBO J. 1999; 18: 2551-2562Crossref PubMed Scopus (404) Google Scholar). Yorkie fulfills all of the criteria required of a Warts effector: it not only binds to Warts but appears to recapitulate all of its tumor-suppressor functions when overexpressed in cell clones in developing wings and eyes of the fly. Moreover, loss-of-function mutations in yki arrest cell growth and are epistatic for hpo and wts, meaning that hpo, yki, or wts, yki double-mutant cells grow just as poorly as yki single-mutant cells. This suggested that the Hippo/Sav and Warts/Mats complexes might act upstream of Yorkie as negative regulators, and indeed, they were shown experimentally to suppress transcriptional activation by Yorkie. Biochemical analysis of cultured Drosophila cells showed that Yorkie is phosphorylated by Warts in a Hippo/Sav-dependent fashion, suggesting that Warts normally suppresses Yorkie activity by phosphorylating it. The specific role, if any, that phosphorylation plays in regulating the activity of Yorkie remains to be determined. The discovery of Yorkie is a substantial advance because it will allow the identification of the binding partners that bring it to the promoters of target genes. This will facilitate the comprehensive cataloging of Hippo/Warts targets that are regulated transcriptionally. Given the near-complete phenocopy of wts, hpo, and sav mutant phenotypes by overproduced Yorkie, this may cover most of the relevant targets and thus explain the cell adhesion, cell shape, and growth effects of the pathway, which are presently mysterious. The study from Huang et al., 2005Huang J. Wu S. Barrera J. Matthews K. Pan D. The Hippo signaling pathway coordinately regulates cell proliferation and apoptosis by inactivating Yorkie, the Drosophila homolog of YAP.Cell. 2005; 122: 421-434Abstract Full Text Full Text PDF PubMed Scopus (1181) Google Scholar may provide the means to answer other pressing questions about the Hippo pathway. For instance, it has been suspected that Hippo/Warts activity may increase as cells differentiate, thereby suppressing proliferation and promoting apoptosis, but so far there has been little evidence that the pathway is subject to spatiotemporal regulation at the onset of differentiation. Despite the observation that it has more profound effects in differentiating cells than proliferating ones, and in some cell types (interommatidial) than others (photoreceptors), all Hippo pathway components appear to be uniformly expressed during the stages of eye development when the mutant phenotypes unfold. Assays of Yorkie activity, most likely using phosphospecific antibodies but potentially based on Yorkie nuclear localization or yki reporter gene expression, may reveal how the Hippo pathway is regulated in space and time during development. Such studies are likely to uncover considerable complexity, as evidenced by a striking recent report showing that Warts, Hippo, and Sav play a second role as essential regulators of photoreceptor cell type choice in the postmitotic Drosophila eye (Mikeladze-Dvali et al., 2005Mikeladze-Dvali T. Wernet M.F. Pistillo D. Mazzoni E.O. Teleman A.A. Chen Y.W. Cohen S. Desplan C. The growth regulators warts/lats and melted interact in a bistable loop to specify opposite fates in Drosophila R8 photoreceptors.Cell. 2005; 122: 775-787Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In this case, the transcriptional targets, which might again be regulated by Yorkie, are rhodopsin genes essential for color vision and have no apparent relevance to growth suppression. This suggests that Yorkie probably activates distinct sets of genes using different DNA binding partners and that its spectrum of targets will vary greatly according to developmental stage and cellular context. Interestingly, Mikeladze-Dvali et al., 2005Mikeladze-Dvali T. Wernet M.F. Pistillo D. Mazzoni E.O. Teleman A.A. Chen Y.W. Cohen S. Desplan C. The growth regulators warts/lats and melted interact in a bistable loop to specify opposite fates in Drosophila R8 photoreceptors.Cell. 2005; 122: 775-787Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar also found that wts transcript expression correlated with photoreceptor cell type and was regulated by a conserved pleckstrin-homology (PH) domain-containing protein, Melted, in a bistable loop in which Melted and Warts can mutually repress each other at the transcriptional level. This loop ensures that photoreceptors express either Melted and one rhodopsin isoform (rh5, used for shorter wavelength colors) or Warts and a different rhodopsin isoform (rh6, used for longer wavelength colors), to guarantee that no cells express both rhodopsins simultaneously. Interactions between melted and wts, or cell type-specific transcription of wts, have not yet been noted during the growth phase in the eye or other organs. This leads us to another mystery that has dogged researchers in this field for several years: what are the upstream inputs that regulate the Hippo/Warts pathway, if it is regulated? Again, to the chagrin of the fly geneticists, forward genetic screens gave few clues. A candidate gene approach taken by Georg Halder's group, however, recently provided a lead that should help to focus the search (Hamaratoglu et al., 2005Hamaratoglu F. Kango-Singh M. Nolo R. Hyun E. Tao C. Jafar-Nejad H. Halder G. The tumor suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis.Nat. Cell Biol. 2005; 8 (Published online December 11, 2005): 27-36https://doi.org/10.1038/ncb1339Crossref PubMed Scopus (545) Google Scholar). This study followed up on earlier work (McCartney et al., 2000McCartney B.M. Kulikauskas R.M. LaJeunesse D.R. Fehon R.G. The neurofibromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation.Development. 2000; 127: 1315-1324PubMed Google Scholar) that showed that cells doubly mutant for two genes that encode band 4.1 superfamily proteins, merlin and expanded, exhibited epidermal outgrowths similar to those caused by loss of hpo, wts, or sav. merlin and expanded encode members of the ezrin/radixin/moesin (ERM) family of FERM domain-containing proteins. They are partially redundant for function and can form heterodimeric complexes. Both proteins localize adjacent to adherens junctions and are thought to link transmembrane proteins to the actin cytoskeleton (Bretscher et al., 2002Bretscher A. Edwards K. Fehon R.G. ERM proteins and merlin: integrators at the cell cortex.Nat. Rev. Mol. Cell Biol. 2002; 3: 586-599Crossref PubMed Scopus (1098) Google Scholar, Sun et al., 2002Sun C.X. Robb V.A. Gutmann D.H. Protein 4.1 tumor suppressors: getting a FERM grip on growth regulation.J. Cell Sci. 2002; 115: 3991-4000Crossref PubMed Scopus (153) Google Scholar, McClatchey and Giovanni, 2005McClatchey A.I. Giovanni M. Membrane organization and tumorigenesis—the NF2 tumor suppressor, Merlin.Genes Dev. 2005; 19: 2265-2277Crossref PubMed Scopus (198) Google Scholar). expanded mutants exhibit mild overgrowth phenotypes in flies (Boedigheimer et al., 1997Boedigheimer M.J. Nguyen K.P. Bryant P.J. Expanded functions in the apical cell domain to regulate the growth rate of imaginal discs.Dev. Genet. 1997; 20: 103-110Crossref PubMed Scopus (48) Google Scholar, Boedigheimer and Laughon, 1993Boedigheimer M. Laughon A. Expanded: a gene involved in the control of cell proliferation in imaginal discs.Development. 1993; 118: 1291-1301PubMed Google Scholar), and mammalian fibroblasts and endothelial cells lacking Merlin, encoded by the neurofibromatosis 2 (NF2) tumor-suppressor gene, are resistant to contact inhibition of proliferation (Lallemand et al., 2003Lallemand D. Curto M. Saotome I. Giovannini M. McClatchey A.I. NF2 deficiency promotes tumorigenesis and metastasis by destabilizing adherens junctions.Genes Dev. 2003; 17: 1090-1100Crossref PubMed Scopus (236) Google Scholar, Okada et al., 2005Okada T. Lopez-Lago M. Giancotti F.G. Merlin/NF-2 mediates contact inhibition of growth by suppressing recruitment of Rac to the plasma membrane.J. Cell Biol. 2005; 171: 361-371Crossref PubMed Scopus (137) Google Scholar). Interestingly, Lats2 mutant mouse embryonic fibroblasts also exhibit this defect (McPherson et al., 2004McPherson J.P. Tamblyn L. Elia A. Migon E. Shehabeldin A. Matysiak-Zablocki E. Lemmers B. Salmena L. Hakem A. Fish J. et al.Lats2/Kpm is required for embryonic development, proliferation control and genomic integrity.EMBO J. 2004; 23: 3677-3688Crossref PubMed Scopus (164) Google Scholar), suggesting that LATS2 may act downstream of Merlin in mammals as its ortholog, Warts, does in Drosophila (see below). In flies, the overgrowth phenotype of merlin, expanded double-mutant cells is much more severe than that caused by either single mutant. Notably, the double-mutant phenotype is strikingly similar to that conferred by loss of wts, hpo, or sav (see Figure 1). When generated in the Drosophila eye, merlin, expanded double-mutant cells grow into smooth-bordered clones that proliferate inappropriately during the differentiation phase and then evade the normal wave of apoptosis that culls excess interommatidial cells. As with hpo and wts complex mutants, merlin, expanded double-mutant cells also sustain increased transcription of cyclin E and diap-1. Hamaratoglu et al., 2005Hamaratoglu F. Kango-Singh M. Nolo R. Hyun E. Tao C. Jafar-Nejad H. Halder G. The tumor suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis.Nat. Cell Biol. 2005; 8 (Published online December 11, 2005): 27-36https://doi.org/10.1038/ncb1339Crossref PubMed Scopus (545) Google Scholar went on to use a clever battery of epistasis tests to show that hpo and wts are required for and act genetically downstream of merlin and expanded in containing growth and culling excess interommatidial cells at differentiation. For instance, overexpressed expanded promoted apoptosis but was incapable of doing so in hpo mutant tissue. Biochemical tests indicated that Merlin and Expanded do not bind directly to Hippo, Sav, or Warts; however, they do stimulate the phosphorylation of Warts by Hippo and thereby probably activate Warts to signal downstream. Consistent with this notion, transcriptional activation by Yorkie was shown to be suppressed by high levels of Merlin and Expanded. How these ERM proteins stimulate the interaction between Hippo and Warts is still
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