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FRMD6 Expression Activates the Hippo Signaling Pathway Kinases in Mammals and Antagonizes Oncogenic YAP

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FRMD6 Expression Activates the Hippo Signaling Pathway Kinases in Mammals and Antagonizes Oncogenic YAP Oncogene (2012) 31, 238–250 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE Willin/FRMD6 expression activates the Hippo signaling pathway kinases in mammals and antagonizes oncogenic YAP L Angus1, S Moleirinho1,2, L Herron1, A Sinha3,4, X Zhang3,4, M Niestrata2, K Dholakia5, MB Prystowsky6, KF Harvey3,4, PA Reynolds2,7 and FJ Gunn-Moore1,7 1School of Biology, University of St Andrews, St Andrews, UK; 2School of Medicine, University of St Andrews, St Andrews, UK; 3Cell Growth and Proliferation Laboratory, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; 4Department of Pathology, University of Melbourne, Parkville, Victoria, Australia; 5School of Physics and Astronomy, University of St Andrews, St Andrews, UK and 6Department of Pathology, Albert Einstein College of Medicine, Bronx, NY, USA The Salvador/Warts/Hippo (Hippo) signaling pathway 2006; Harvey and Tapon, 2007). This pathway limits defines a novel signaling cascade regulating cell contact organ size by inhibiting cell proliferation and promoting inhibition, organ size control, cell growth, proliferation, apoptosis, and is therefore of particular importance apoptosis and cancer development in mammals. The during development and tissue size control (Tapon et al., Drosophila melanogaster protein Expanded acts in 2002; Harvey et al., 2003; Huang et al., 2005; Edgar, the Hippo signaling pathway to control organ size. 2006; Harvey and Tapon, 2007; Pan, 2007). In addition, Previously, willin/FRMD6 has been proposed as the several pieces of evidence suggest that the activities of human orthologue of Expanded. Willin lacks C-terminal multiple Hippo pathway components are deregulated in sequences that are present in Expanded and, to date, little human cancer (Tapon et al., 2002; Harvey and Tapon, is known about the functional role of willin in mammalian 2007; Pan, 2007; Zeng and Hong, 2008). cells. When willin is expressed in D. melanogaster The Hippo pathway consists of a series of kinases and epithelial tissues, it has the same subcellular localization adaptor proteins in which the Hippo kinase, in as Expanded, but cannot rescue growth defects associated association with an adaptor protein Salvador, phos- with expanded deficiency. However, we show that ectopic phorylates and activates Warts kinase, which is asso- willin expression causes an increase in phosphorylation of ciated with an activating subunit, Mats. This core kinase the core Hippo signaling pathway components MST1/2, unit phosphorylates and inactivates the transcriptional LATS1 and YAP, an effect that can be antagonized co-activator Yorkie, thereby suppressing expression of by ezrin. In MCF10A cells, loss of willin expression genes that promote cell survival, growth and prolifera- displays epithelial-to-mesenchymal transition features and tion (Huang et al., 2005). Inactivation of Yorkie in willin overexpression antagonizes YAP activity via the D. melanogaster imaginal disc tissues results in cell cycle N-terminal FERM domain of willin. Therefore, in arrest and apoptosis (Harvey and Tapon, 2007; Pan, mammalian cells willin influences Hippo signaling activity 2007). Ex, Mer and Kibra function upstream of the core by activating the core Hippo pathway kinase cassette. kinase cassette, activating the pathway by an unknown Oncogene (2012) 31, 238–250; doi:10.1038/onc.2011.224; mechanism via Hpo and Wts phosphorylation (Hamar- published online 13 June 2011 atoglu et al., 2006; Genevet et al., 2010; Yu et al., 2010). More recently, Ex has been shown to form a complex Keywords: Willin/FRMD6; Expanded; Hippo pathway; with Yorkie and is proposed to directly regulate its ezrin; merlin activity by the WW domains of YAP and the PPXY motifs of Ex (Badouel et al., 2009). Ex is also thought to function downstream of the Hippo pathway receptor protein, Fat, an atypical cadherin that represses growth Introduction of imaginal discs (Bennett and Harvey, 2006; Silva et al., 2006; Willecke et al., 2006; Tyler and Baker, 2007). Loss Recent studies utilizing Drosophila melanogaster genet- of ex results in the development of hyperplastic imaginal ics have indicated that merlin (Mer) and a second discs and overgrown adult structures such as the wing FERM protein called Expanded (Ex) has an important (Boedigheimer and Laughon, 1993). In addition, over- role in controlling the Salvador/Warts/Hippo (Hippo) expression of ex in the D. melanogaster wing and eye signaling cascade (Edgar 2006; Hamaratoglu et al., leads to a decrease in the number of cells in these tissues (Boedigheimer et al., 1997). Correspondence: Dr FJ Gunn-Moore, Medical and Biological Sciences Less is known about the functioning of the Hippo Building, School of Biology, University of St Andrews, North Haugh, signaling pathway in mammals, particularly the proteins St Andrews, Fife KY16 9TF, UK. that regulate the mammalian Hippo pathway upstream E-mail: [email protected] or [email protected] 7These authors contributed equally to this work. of the core kinase cassette. Components of the Hippo Received 8 March 2011; revised and accepted 6 May 2011; published pathway are conserved in mammals and consist of online 13 June 2011 MST1/2 (Hpo orthologues), WW45/Sav (Sav orthologue), Salvador/Warts/Hippo signaling pathway in mammals L Angus et al 239 LATS1/2 (Wts orthologues), MOB1 (Mats orthologue) (Figure 1b). We noted that YAP phosphorylation could and YAP (Yki orthologue) (Harvey and Tapon, 2007). only be studied over a period of 2 days post-induction, as At least four upstream regulatory branches of the Hippo high cell density caused YAP phosphorylation in unin- pathway exist in Drosophila: Fat/Dachsous, Kibra/Ex/ duced cells as was observed previously (Zhao et al., 2007), Mer, Lethal Giant Larvae/atypical protein kinase C and all experiments were therefore performed at a low cell and Crumbs (Grusche et al., 2010). The mammalian density. No MST1/2, LATS1 and YAP phosphorylation orthologues of Mer (also called NF2, Mer) and Kibra was observed when an empty GFP plasmid was transiently have been shown to function as upstream members of transfected into the stable inducible cell line or when 1 mg/ the Hippo pathway, but the identity of other upstream ml tetracycline was added to cells that did not contain the members of the mammalian Hippo pathway has pcDNA4/TO/myc-his willin-GFP plasmid, but only a remained enigmatic. For example, recent studies suggest plasmid expressing the pcDNA6-TR tetracycline repressor that the Fat/Dachsous pathway does not control organ (data not shown). size and activity of the Yki orthologues, YAP and TAZ, in mice (Saburi et al., 2008; Mao et al., 2011). Ezrin modulates the ability of willin to phosphorylate Interestingly, repression of YAP by AMOT via direct MST1/2 interaction of the WW domains of YAP with the PPXY To investigate the effect of the expression of other motifs of AMOT has been reported (Zhao et al., 2011), a FERM domain-containing proteins on MST1/2 phos- role fulfilled by the C terminus of Ex in D. melanogaster. phorylation, we expressed combinations of Mer and We sought to determine whether the mammalian ezrin in TRex-willin-GFP cells and induced willin-GFP homologue of D. melanogaster Ex, willin/Ex1/FRMD6, expression with 1 mg/ml tetracycline. A hierarchy in the functioned as an upstream member of the mammalian ability to activate MST1/2 phosphorylation was obser- Hippo pathway (Gunn-Moore et al., 2005; Hamaratoglu ved, in which the expression of either Mer or willin et al., 2006). Previously, willin overexpression was shown was sufficient to result in MST1/2 phosphorylation to influence YAP in a luciferase activity, but a mechanistic (Figure 1c). When Mer and willin were co-expressed, a link to the Hippo pathway was not explored (Zhao et al., synergistic effect was observed on MST1/2 phosphor- 2007). We show that increased willin expression causes ylation, in which willin expression enhances Mer’s phosphorylation of MST1/2, LATS1 and YAP. Loss of ability to phosphorylate MST1/2 (Po0.01). Interest- willin expression results in features of an epithelial-to- ingly, ezrin expression was found to have an inhibitory mesenchymal transition (EMT) in MCF10A cells and effect on MST1/2 phosphorylation when co-expressed willin overexpression antagonizes YAP activity by the with Mer, willin or willin and Mer (Po0.01) (Figure 1c). N-terminal FERM domain of willin. The majority of willin localizes to the apical junction of D. melanogaster Willin expression sensitizes HEK-293 cells to apoptotic imaginal disc cells in a manner similar to Ex, although stimuli willin could not rescue growth defects associated with Previous studies have shown that MST1/2 expression ex deficiency. Therefore, willin/Ex1/FRMD6 has both sensitizes cells to death upon addition of tumor necrosis similarities and differences to Ex, but in mammalian cells factor-a (TNFa) and cycloheximide (Lee et al., 2001). willin influences Hippo signaling activity by activating the Therefore, we explored whether willin-GFP expression core Hippo pathway kinase cassette. in HEK-293 cells could also sensitize these cells to cell death signals. Interestingly, MTT (3-(4,5-dimethyl- thiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays Results showed that cell viability dropped by 20% in cells treated with TNFa that expressed willin-GFP as Expression of willin influences phosphorylation of compared with uninduced cells (Figure 2a). Further- MST1/2, LATS1 and YAP more, immunoblot analysis showed that caspase-3 was Mammalian willin and D. melanogaster Ex show 60% cleaved in these cells only upon willin-GFP expression similarity in their N-terminal FERM domain, but the (Figure 2b). A colorimetric assay supported these C-terminal domains are highly divergent with Ex being observations as active caspase-3 was detected in cells much longer and containing additional functional motifs expressing willin-GFP treated with TNFa, whereas no (Figure 1a). Previously, the expression of both willin and caspase-3 activity was observed in the control (TRex) Mer were reported to inactivate the transcriptional co- cells (Figure 2b).
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