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Molecular Cell

Article Combined Functional Genomic and Proteomic Approaches Identify a PP2A Complex as a Negative Regulator of Hippo Signaling Paulo S. Ribeiro,1,7 Filipe Josue´,1,5,7 Alexander Wepf,3,4,7 Michael C. Wehr,1 Oliver Rinner,3,6 Gavin Kelly,2 Nicolas Tapon,1,8,* and Matthias Gstaiger3,4,8,* 1Apoptosis

and Proliferation Control Laboratory and Biostatistics Service Cancer Research UK, London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK 3ETH, Institute for Molecular Systems Biology, Wolfgang-Pauli-Street 16, CH-8093 Zurich, Switzerland 4Competence Center for Systems Physiology and Metabolic Diseases, ETH Zurich, 8093 Zurich, Switzerland 5PhD Programme in Biomedicine and Experimental Biology, Center for Neuroscience and Cell Biology, University of Coimbra, 3004-517 Coimbra, Portugal 6Present address: Biognosys AG, Wolfgang-Pauli-Strasse 16, 8093 Zurich, Switzerland 7These authors contributed equally to this work 8These authors contributed equally to this work *Correspondence: [email protected] (N.T.), [email protected] (M.G.) DOI 10.1016/j.molcel.2010.08.002 2Bioinformatics

SUMMARY

The Hippo (Hpo) pathway is a central determinant of tissue size in both Drosophila and higher organisms. The core of the pathway is a kinase cascade composed of an upstream kinase Hpo (MST1/2 in mammals) and a downstream kinase Warts (Wts, Lats1/2 in mammals), as well as several scaffold proteins, Sav, dRASSF, and Mats. Activation of the core kinase cassette results in phosphorylation and inactivation of the progrowth transcriptional coactivator Yki, leading to increased apoptosis and reduced tissue growth. The mechanisms that prevent inappropriate Hpo activation remain unclear, and in particular, the identity of the phosphatase that antagonizes Hpo is unknown. Using combined proteomic and RNAi screening approaches, we identify the dSTRIPAK PP2A complex as a major regulator of Hpo signaling. dSTRIPAK depletion leads to increased Hpo activatory phosphorylation and repression of Yki target genes in vivo, suggesting this phosphatase complex prevents Hpo activation during development.

INTRODUCTION Metazoan organ development requires a strict control of cell proliferation, growth, and death. Studies in Drosophila melanogaster have identified the Hippo (Hpo) pathway as one of the major signaling pathways required for tissue size control (Reddy and Irvine, 2008). The Hpo pathway controls the final tissue and organ size by both inhibiting cell proliferation and promoting apoptosis (Harvey and Tapon, 2007). At the core of the pathway lies a kinase cascade comprised of the Ste20-related kinase

Hpo and the Dbf2-related kinase Warts (Wts) (Harvey et al., 2003; Jia et al., 2003; Justice et al., 1995; Pantalacci et al., 2003; Udan et al., 2003; Wu et al., 2003; Xu et al., 1995). Hpo and Wts, together with their respective scaffold proteins Salvador (Sav) and Mob as tumor suppressor (Mats), phosphorylate and inhibit the transcriptional coactivator Yorkie (Yki) (Huang et al., 2005; Kango-Singh et al., 2002; Lai et al., 2005; Tapon et al., 2002). In addition, the atypical cadherin Fat (Ft), the FERM proteins Expanded (Ex) and Merlin (Mer), and the recently identified Kibra are upstream members of the Hpo pathway that, by an unknown mechanism, relay the information of largely unidentified developmental signals to the core kinase cassette (Baumgartner et al., 2010; Bennett and Harvey, 2006; Cho et al., 2006; Genevet et al., 2010; Hamaratoglu et al., 2006; Pellock et al., 2007; Silva et al., 2006; Willecke et al., 2006; Yu et al., 2010). Activation of the Hpo pathway kinase cassette leads to Wts-dependent phosphorylation of Yki at several evolutionarily conserved Ser residues, leading to 14-3-3-mediated sequestration of Yki in the cytoplasm (Dong et al., 2007; Hao et al., 2008; Huang et al., 2005; Oh and Irvine, 2008, 2009; Oka et al., 2008; Zhang et al., 2008a; Zhao et al., 2007). Although Yki has several Wts phosphorylation target sites, Ser168 appears to be the main regulatory phosphorylation site in Yki controlling nuclear translocation, as mutation of this residue renders Yki largely refractory to Hpo pathway activity (Dong et al., 2007; Oh and Irvine, 2008, 2009). Inhibition of Yki nuclear translocation prevents Yki from associating with Scalloped (Sd), a transcription factor belonging to the TEAD/TEF family that has been shown to mediate Yki function in the Hpo pathway (Goulev et al., 2008; Wu et al., 2008; Zhang et al., 2008b). Yki, together with Sd and potentially other transcription factors, promotes the expression of progrowth and antiapoptotic genes, such as the cell-cycle regulator cycE (cyclin E), the inhibitor of apoptosis diap1, and the microRNA bantam (Huang et al., 2005; Nolo et al., 2006; Tapon et al., 2002; Thompson and Cohen, 2006). In addition, Yki promotes expression of ex, mer, kibra, and dachsous (ds), Molecular Cell 39, 521–534, August 27, 2010 ª2010 Elsevier Inc. 521

Molecular Cell Regulation of Hpo Signaling by the PP2A Complex

suggesting the existence of a regulatory feedback mechanism that tightly regulates the activation level of the Hpo pathway (Cho et al., 2006; Hamaratoglu et al., 2006; Willecke et al., 2006; Genevet et al., 2010). While genetic studies have been extremely fruitful in the identification of Hpo pathway activators, few inhibitors have been identified and, thus, relatively little is known about the mechanisms that negatively regulate Hpo signaling (Harvey and Tapon, 2007). dRASSF inhibits the Hpo pathway signaling by competing with Sav for Hpo binding, thereby blocking the kinase activity of Hpo (Polesello et al., 2006). In turn, the unconventional myosin Dachs inhibits Hpo signaling at a downstream step by directly associating with Wts and, through an unknown mechanism, diminishing Wts protein levels (Cho et al., 2006; Mao et al., 2006). Given that Hpo signaling relies on a kinase cascade core, it is plausible that the pathway is negatively regulated by the action of one or more of the 28 Ser/Thr protein phosphatases encoded by the fly genome (Morrison et al., 2000). However, to date, no phosphatase affecting Hpo signaling has been described. Substrate specificity, as well as temporal and spatial control of phosphatase activity, is thought to be controlled by combinatorial assembly of catalytic phosphatase subunits with multiple regulatory subunits. In the case of the human PP2A subfamily, where complex formation has been studied extensively, it is known that combinatorial assembly of each of the two PP2A catalytic subunits with one of the 15 regulatory B subunits and two scaffolding subunits results in a multitude of phosphatase complexes (Glatter et al., 2009; Goudreault et al., 2009; Janssens et al., 2005). Here we combine mass spectrometry-based analysis of the Hpo interactome with genome-wide RNAi screening to identify dSTRIPAK (Drosophila Striatin-interacting phosphatase and kinase), a Drosophila PP2A phosphatase complex, as a negative regulator of Hpo signaling. Inhibition of the phosphatase complex resulted in enhanced Hpo activatory phosphorylation and hyperactivation of the Hpo pathway in vivo. Finally, dSTRIPAK is specifically associated with Hpo/dRASSF complexes but absent in Hpo/Sav complexes, suggesting that the interplay between dRASSF and Sav determines Hpo dephosphorylation. RESULTS Identification of a Phosphatase Complex as Part of the Hpo Interactome Most known members of the Drosophila Hpo pathway have been identified through genetic screens (Huang et al., 2005; Pantalacci et al., 2003; Polesello et al., 2006; Silva et al., 2006; Tapon et al., 2002). Affinity purification coupled with mass spectrometry (AP-MS) represents an alternative method to identify pathway members but, so far, this technique has not been applied to characterize Hpo signaling in Drosophila cells. To identify Hpo complexes from Drosophila cells using reciprocal AP-MS experiments, we generated a set of Kc167 cells expressing hemagglutinin (HA) epitope-tagged versions of Hpo and its known binding partners Sav and dRASSF. In addition, we included Mob4 and Cka, two proteins that we have identified as Hpo-binding proteins in the course of this study. Protein complexes were recovered by anti HA-immunoaffinity purifica522 Molecular Cell 39, 521–534, August 27, 2010 ª2010 Elsevier Inc.

tion and identified by tandem mass spectrometry from two independent replicate experiments (Figure 1A and see the Experimental Procedures). To generate a high-quality protein interaction network model of the Hpo interactome from AP-MS raw data, we included only those proteins as nodes that have been identified in both replicate purifications and which were absent from an AP-MS control data set obtained with HA-GFP-expressing Kc167 cells (for details of data filtering, see the Experimental Procedures). In Figure 1B, all proteins of the network that are connected by at least two arrows are shown to highlight protein complexes formation. The interaction network recapitulates the known interactions between Hpo, Sav, and dRASSF, validating the experimental approach. Binding of Sav and dRASSF to Hpo seemed to be mutually exclusive, as no Sav peptides were recovered in the dRASSF AP-MS and vice versa, suggesting that, as previously proposed, Sav/Hpo- and dRASSF/Hpo-containing complexes are separate physical entities (Polesello et al., 2006). Besides known Hpo-binding proteins, we also identified a set of additional proteins including the Connector of kinase to AP-1 (Cka) and Mps one binder kinase activator-like 4 (Mob4) in Hpo and dRASSF purifications, but not in Sav purifications. When we included Cka and Mob4 as baits for AP-MS analysis, we discovered a strongly interlinked protein interaction network, which indicates formation of larger complexes by the highly connected proteins Hpo, dRASSF, CG10915, CG11526, CG10158 (the Drosophila ortholog of FGFR1OP2, which we will refer as FGOP2 in the text), Mob4, and Cka (Figure 1B). Remarkably, these proteins represent fly homologs of a set of human proteins found in STRIPAK, a recently discovered PP2A complex, which has been found to associate with a specific set of Ste20-like kinases (Glatter et al., 2009; Goudreault et al., 2009). Similarly, we found that besides Hpo two additional Ste20-like kinases, Misshapen (Msn) and germinal center kinase III (GckIII), are bound to the dSTRIPAK components Cka and Mob4, indicating that dSTRIPAK interacts with a set of Ste20like family members in Drosophila. The finding that the Hpo AP-MS did not reveal the presence of these other kinases suggests that they form mutually exclusive STRIPAK complexes. Not all homologous Drosophila proteins expected from the human STRIPAK complex could be observed in the filtered AP-MS data set that we used to construct our network model (e.g., Pp2A-29B, Mts, CG5073, and CG17494). We have analyzed the unfiltered raw data set for the presence of additional Drosophila STRIPAK homologs in Hpo, dRASSF, Mob4, and Cka purifications, to assess whether these proteins were absent from the final list because they did not pass our stringent filtering criteria. Indeed, Figure 1C shows that, except for CG17494, which corresponds to human SLMAP, all expected homologous components of the human STRIPAK complex could be identified in our AP-MS experiments. Taken together, these results suggest the existence of a homologous dSTRIPAK phosphatase complex associated with Hpo and dRASSF. Identification of Mts, the dSTRIPAK Catalytic Subunit, as a Hpo Pathway Regulator In parallel, we performed a high-throughput RNAi screen in Drosophila tissue culture cells to identify modulators of Hpo


Figure 1. AP-MS Analysis of the Hpo Interactome Reveals Association with dSTRIPAK, a Protein Complex that Is Related to the Human PP2A Complex STRIPAK (A) A reciprocal affinity purification mass spectrometry approach was used to identify interactions of the Drosophila Hpo interactome. AP-MS data from two independent biological replicates were processed for protein identification, and identified proteins were filtered against a contaminant data set obtained from multiple control purifications using GFP as bait (see also the Experimental Procedures). (B) Hpo-related interaction map. From the filtered data set a protein interaction network model was constructed that contains only proteins for which a total of at least five unique peptides have been identified in the two replicate experiments and which are connected to other network components by at least two edges. Red nodes indicate the bait proteins used in this study, whereas orange nodes refer to the identified prey proteins. The total number of identified peptides is displayed by the thickness of the edges between the nodes. (C) Proteins identified in this study contain homologs of the human STRIPAK complex. Drosophila proteins identified in Hpo, RASSF, Cka, and Mob4 AP-MS experiments that are homologous to human STRIPAK components are listed in the table. The table also displays the corresponding human STRIPAK homologs and the degree of homology to the identified corresponding Drosophila proteins. The number of unique peptides identified in indicated AP-MS experiments is depicted in the right part of the table. Asterisks indicate proteins for which peptides have been found also in control AP-MS experiments, which were enriched in Cka and Mob4 purifications.

signaling. We used a luciferase-based assay in which we transfected S2R+ cells with plasmids encoding a GAL4 DNA-binding domain-Yki (G4-DBD-Yki) fusion protein (Huang et al., 2005), firefly luciferase under the control of upstream activating sequences (UAS-FLuc), and Renilla luciferase under the control of the baculoviral OpIE2 promoter (pIZ-Ren) as a control for cell number and transfection efficiency. Modulation of Hpo pathway activity leads to changes in G4-DBD-Yki activity, which then drives the expression of the UAS-FLuc reporter (Huang et al., 2005). Using RNAi to deplete Hpo pathway components, we observed that knockdown of the Hpo signaling activators Wts, Sav, and Hpo induced a robust enhancement of this luciferase readout (Figure 2A). Conversely, dsRNA against Yki was able to strongly inhibit the readout. We further investigated whether this readout could be inhibited by cotransfection of individual Hpo pathway activators. Overexpression of Wts, Hpo, Sav, Ex, and Mer caused robust reduction of UAS-FLuc expression (Figure 2B). Interest-

ingly, the strength of this inhibition was in agreement with the corresponding in vivo phenotype, with Hpo and Wts causing stronger reductions than Sav, Ex, and Mer. Furthermore, the combination of Ex and Mer enhanced the reduction caused by either protein alone. Together, these data show that the assay is sensitive to modulation of Hpo signaling activity. We used this G4-DBD-Yki/UAS-FLuc assay in S2R+ cells in combination with the Ambion Silencer Drosophila RNAi Library, which contains 13,059 unique dsRNAs targeting !92% of the Drosophila genome (Figure 2C). From this genome-wide screen, we recovered microtubule star (mts), a gene that encodes the PP2A catalytic subunit present in the dSTRIPAK complex, as one of the most potent inhibitors of the G4-DBD-Yki/UAS-FLuc signal (Figure 2D and Figure S1). Together with our AP-MS experiments showing the association between dSTRIPAK and Hpo, this strongly suggests a function for dSTRIPAK in antagonizing Hpo function. Molecular Cell 39, 521–534, August 27, 2010 ª2010 Elsevier Inc. 523


Figure 2. A Genome-wide RNAi Screen Identifies mts as a Negative Regulator of Hpo Signaling (A–C) RNAi-mediated depletion (A) or overexpression (B) of known Hpo signaling pathway components is able to modulate G4-DBD-Yki induction of UAS-FLuc. Results are shown as average of relative luciferase units (RLUs). Error bars denote SD. (C) Schematic overview of the genome-wide screen in Drosophila S2R+ cells using G4-DBD-Yki/UAS-FLuc as a readout of Hpo signaling. (D) The PP2A/dSTRIPAK catalytic subunit microtubule star (mts) appears as the top inhibitor of Hpo signaling in a representative plate of our genome-wide screen. yki is also shown as a negative control.

The genome-wide screening data (see the Experimental Procedures) were validated by a secondary RNAi screen performed using alternative nonoverlapping dsRNAs designed against all primary hits (Figure S1C). Most of the primary hits were validated in the secondary screen, and these included five genes involved in nucleic acid synthesis and degradation, five cell-cycle-related genes, two zinc-finger-containing genes, one gene containing a protein-protein interaction domain, and another containing a domain associated with cell polarity. Validation of the Interactions of dSTRIPAK Components with Hpo and dRASSF Both our proteomic and high-throughput functional genomic approaches suggest an involvement of the dSTRIPAK complex in the regulation of Hpo signaling (Figures 1 and 2). To validate the AP-MS analysis, we performed coimmunoprecipitation (coIP) experiments from S2 cell lysates expressing tagged versions of the identified components of the dSTRIPAK complex and members of the Hpo signaling pathway. We initially tested whether FGOP2 and the putative PP2A regulatory protein Cka interact with Hpo, as suggested by the AP-MS results. FLAGtagged Hpo could coIP both Myc-tagged Cka and FGOP2, while no association was observed with the control protein NTAN (Figure 3A). In addition, we have detected a weak but specific interaction between Hpo and Mob4, which may occur via Cka (Figures S2A and S2B). Next, we tested whether dRASSF 524 Molecular Cell 39, 521–534, August 27, 2010 ª2010 Elsevier Inc.

associates with Cka and FGOP2. HA-tagged RASSF was coexpressed in S2 cells with FLAG-tagged NTAN, Cka, FGOP2, or Hpo. As expected, dRASSF associates with Hpo (Figure 3B). Furthermore, dRASSF specifically coIPs with FLAG-tagged Cka and FGOP2, albeit to a lesser extent when compared to Hpo. Together, our coIP results validate the interactions identified by AP-MS and suggest that both Cka and FGOP2 associate with Hpo and RASSF. The dSTRIPAK complex identified in Drosophila tissue culture cells includes Mts, the catalytic subunit of the PP2A complex, which is known to associate with different regulatory subunits that confer specificity to the phosphatase (Eichhorn et al., 2009). According to sequence similarity data, Cka is the Drosophila ortholog of the mammalian Striatin family of B000 PP2A regulatory subunits (Chen et al., 2002). While in mammals Striatin family members have been shown to interact with the PP2A core heterodimer, Cka has so far only been associated with regulation of JNK signaling, and no demonstration of its interaction with Drosophila PP2A has been reported (Bond and Foley, 2009; Chen et al., 2002; Moreno et al., 2000). By performing coIP experiments using either HA- or Myc-tagged Mts, we have found that, in agreement with the mammalian data, Cka efficiently copurified Mts (Figure S2). In addition, Mob4 was also found to specifically interact with Mts. In contrast, FGOP2 failed to interact with Mts under the conditions tested. Moreover, Hpo was found to weakly interact with Myc-tagged Mts but not


Figure 3. The Drosophila dSTRIPAK Complex Interacts with Members of the Hpo Pathway (A and B) Cka and FGOP2 interact with Hpo (A) and RASSF (B). (C and D) Hpo, Cka, and FGOP2 form a trimeric complex in Drosophila S2 cells. (C) Outline of the serial coIP procedure performed. See the Supplemental Experimental Procedures for details. (D) Cka, Hpo, and FGOP2 associate as a trimeric complex. S2 cells transfected with Myc-tagged Hpo, HA-tagged FGOP2, and either FLAG-tagged NTAN or Cka were lysed as detailed in the Experimental Procedures, and lysates were treated as shown in (C). Lysates, post-FLAG IP lysates, FLAG eluates, and Myc-purified immunoprecipitates were analyzed by immunoblotting using the indicated antibodies. (E) Cka associates with Hpo in a RASSF-dependent manner. Cells were lysed as detailed in the Experimental Procedures. CoIP assays were performed in Drosophila S2 cells transfected with the indicated constructs. Lysates and FLAG-purified immunoprecipitates were analyzed by immunoblotting with the indicated antibodies. RNAi treatment of Drosophila S2 cells was performed 72 hr before cell lysis. Tubulin was used as loading control. (F) Quantification of the relative binding between Hpo and endogenous Cka. Shown are the means of the ratio between the anti-Cka and respective anti-FLAG signal intensities relative to control (dsRed RNAi) levels for three independent experiments performed as in (E). Error bars denote SEM.

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HA-tagged Mts. As the interaction correlated with the relative levels of Hpo, these data suggest that the Hpo:Mts interaction is either labile or dependent on other factors, such as Cka. Moreover, the interaction between Hpo and Cka seems to depend on a stable PP2A core complex, as RNAi-mediated depletion of Mts reduced the affinity of Hpo toward Cka (Figure S2E). Next, we tested whether a complex between the dSTRIPAK complex and the core kinase cassette of the Hpo pathway exists. Serial coIP experiments performed in S2 cells as schematized in Figure 3C revealed that Hpo, Cka, and FGOP2 can form a trimeric complex (Figure 3D). Hpo is known to interact with Sav and RASSF in a mutually exclusive manner (Polesello et al., 2006). Interestingly, our AP-MS data suggest that the dSTRIPAK complex associates with dRASSF but not Sav. This observation raises the hypothesis that dRASSF antagonizes Hpo signaling by recruiting the dSTRIPAK complex to act on its Hpo pathway targets. To test this notion, we monitored the interaction between FLAG-tagged Hpo and endogenous Cka in the absence of the Hpo adaptors Sav and RASSF. We chose to focus on the regulatory B000 type subunit Cka, since it is thought that regulatory B subunits confer substrate specificity to the PP2A complex (Janssens and Goris, 2001). Hpo readily interacted with endogenous Cka when S2 cells were treated with control dsRNA targeting DsRed (Figure 3E). Consistent with our hypothesis, RASSF dsRNA markedly reduced the amount of endogenous Cka copurified by Hpo, while treating cells with dsRNA against Sav caused a mild increase in the interaction (Figures 3E and 3F). Furthermore, Cka interacted with RASSF in a SARAH domain and Hpo-independent manner, suggesting that the dSTRIPAK complex functions, at least in part, with RASSF to target Hpo (Figure S2F and data not shown). Together with our proteomics data, these results suggest that dRASSF promotes association of the dSTRIPAK complex with Hpo. dSTRIPAK-Mediated Regulation of Hpo Signaling As Mts was identified as a negative regulator of the Hpo pathway in our genome-wide RNAi screen, we sought to further explore the mechanism by which Mts and, by extension, the dSTRIPAK complex regulate this signaling cascade. To validate the inhibitory effect of the dSTRIPAK complex on Hpo signaling, we used the G4-DBD-Yki reporter used in the genome-wide screen to test the effect of Cka overexpression on Hpo pathway activity (Figure 4A). In the absence of any overexpressed protein, G4-DBD-Yki promoted a robust induction of the luciferase reporter. As expected, overexpression of either Hpo or Wts caused a marked reduction in reporter activity. In contrast, Cka overexpression enhanced Yki-induced luciferase activity, which is consistent with the dSTRIPAK complex acting as a negative regulator of Hpo signaling (Figure 4A). In addition, we assessed whether expression of Cka could revert the Hpo-mediated inhibition of G4-DBD-Yki. Indeed, overexpression of Cka was sufficient to block the action of Hpo on the transcriptional activity of G4-DBD-Yki in a dose-dependent manner (Figure 4B). Next, we examined the effect of PP2A inhibition on Hpo pathway activity by monitoring Yki migration on SDS-PAGE, which has been used as an indirect measure of its phosphoryla526 Molecular Cell 39, 521–534, August 27, 2010 ª2010 Elsevier Inc.

tion state (Figure 4C) (Huang et al., 2005; Oh and Irvine, 2008). In basal conditions, Yki migrates with an approximate molecular weight of 50 kDa. Upon Mts depletion by RNAi, we observed an upward mobility shift in Yki, indicative of an increase in Yki phosphorylation levels. This suggests that inhibiting Mts function leads to enhanced Yki phosphorylation. Accordingly, Mts RNAi caused an increase in the levels of Ser168-phosphorylated Yki. Importantly, we observed a similar effect on Yki migration and phosphorylation levels when Cka was depleted (Figure 4D). Indeed, chemical inhibition of PP2A using okadaic acid caused a similar Yki bandshift (Figure S3A). Interestingly, the effects of both Mts and Cka RNAi on Yki migration and phosphorylation levels were abrogated when cells were additionally treated with dsRNA targeting hpo (Figures 4C and 4D). This indicates that the dSTRIPAK complex acts upstream or at the level of Hpo to induce Yki phosphorylation. To test whether the dSTRIPAK complex directly affects Hpo activity, we performed RNAi-mediated depletion of dSTRIPAK components and monitored Hpo activity using an anti-phospho-PAK antibody as readout. The phospho-PAK antibody cross-reacts with Thr195 of Hpo, a residue in the activation loop of Hpo whose phosphorylation correlates with Hpo activity (Colombani et al., 2006). As expected, RNAi-mediated depletion of the Hpo negative regulator dRASSF resulted in an increase in the phospho-Hpo signal. Depletion of Mts or Cka caused a marked increase in the levels of Thr195-phosphorylated Hpo, indicating that levels of active Hpo were elevated in these cells (Figure 4C). Together with the observation that the dSTRIPAK complex associates with Hpo, this suggests that the dSTRIPAK complex controls Hpo pathway activity by reverting the activating phosphorylation of Hpo. This regulation could be due to a direct effect on Hpo or through regulation of an upstream component that, in turn, regulates Hpo phosphorylation. The dSTRIPAK Complex Interacts Genetically with Hpo To further test the notion that the dSTRIPAK complex regulates Hpo signaling in vivo, we performed genetic interaction experiments in adult flies (Figures 5A–5F). Overexpression of hpo in the developing eye under the control of the Glass Multimer Reporter (GMR) promoter induces extensive apoptosis, leading to a marked reduction of adult eye size and a rough eye phenotype (Harvey et al., 2003; Pantalacci et al., 2003; Udan et al., 2003; Wu et al., 2003; and Figure 5B). Removing one copy of FGOP2 or cka enhanced the GMR-hpo phenotype, while it had no effect on the GMR-GAL4 phenotype (Figures 5C–5F and Figures S4A–S4E). GMR-hpo flies carrying one mutant allele of either FGOP2 or cka displayed small patches of necrotic tissue in the anterior region of the eye, which was not observed in GMR-hpo flies. Moreover, contrary to GMR-hpo alone, which had no effect on viability, quantification of progeny numbers from FGOP2 and cka heterozygotes revealed a significant reduction in expected progeny percentages of the genotypic classes bearing one mutant copy of wild-type FGOP2 or cka (Figure 5G). Thus, the Hpo gain-of-function phenotype is sensitive to the gene dosage of dSTRIPAK components. Next, we used a hpo loss-of-function phenotype in the Drosophila developing wing to further probe the genetic relationship between hpo and the dSTRIPAK complex. Hpo depletion in


Figure 4. PP2A Regulates Hpo and Yki Phosphorylation in a Hpo-Dependent Manner (A) Cka overexpression enhances G4-DBD-Yki transcriptional activity. Luciferase assays were performed as described in the Experimental Procedures. (B) Cka blocks the Hpo-mediated inhibition of G4-DBD-Yki transcriptional activity. Luciferase assays were performed as in (A), but Hpo was cotransfected with G4-DBD-Yki. Differentially shaded bars indicate different Hpo concentrations (dark gray, 20 ng; mild gray, 4 ng; and light gray, 2 ng). Empty vector or Cka (20 or 100 ng) was cotransfected with G4-DBD-Yki and Hpo. Results are shown as average ± SD of RLUs. Inset depicts the data from the highest Hpo concentration alone to highlight the differences between Hpo alone and Hpo plus Cka. (C and D) Mts (C) and Cka (D) inhibit Yki phosphorylation by modulating Hpo activity. RNAi treatment of Drosophila S2 cells was performed 72 hr before cell lysis. Endogenous Yki and phospho-Yki protein levels were detected by western blot analysis using anti-Yki and anti-phospho-Yki antibodies, respectively. Tubulin was used as loading control. (E) Phosphorylation of the activation loop of Hpo is increased when PP2A activity is decreased. Drosophila S2R+ cells were treated with RNAi targeting the indicated genes for 72 hr prior to lysis. Total Hpo and phospho-Hpo protein levels were analyzed by immunoblotting using anti-Hpo and anti-phospho-PAK antibodies, respectively. Asterisk indicates cross-reactive band.

the posterior region of the wing by expressing an RNAi targeting hpo under the control of the engrailed (en) promoter induced posterior compartment overgrowth (Figure 5I). Combining hpo and FGOP2 depletion suppressed the overgrowth phenotype caused by reduction of Hpo levels, as seen by the pronounced

size reduction of the wing posterior compartment (Figure 5J). Indeed, wings of en>>hpo-IR/FGOP2-IR flies had a posterior compartment of approximately wild-type size (Figure S4). Similarly, codepletion of hpo and cka suppressed the hpo RNAi phenotype (Figure 5K). Interestingly, the posterior compartment Molecular Cell 39, 521–534, August 27, 2010 ª2010 Elsevier Inc. 527


Figure 5. cka and FGOP2 Genetically Interact with hpo (A–F) Heterozigosity for FGOP2 or cka enhances the hpo overexpression phenotype in the Drosophila eye, as shown by scanning electron micrographs of Drosophila heads. Arrowhead denotes necrotic tissue. (G) Quantification of the expected progeny from GMR-hpo genetic interaction experiments. Results are shown as average ± SD of progeny cohorts. Total number of flies scored >250. (H–K) RNAi-mediated depletion of FGOP2 and cka in the posterior compartment of the Drosophila wing suppresses the overgrowth phenotype caused by depletion of hpo. The posterior compartment of the wing is falsely colored in blue for clarity purposes. See text for details.

of wings from en>>hpo-IR/cka-IR flies was significantly smaller than that of wild-type flies, which suggests that cka depletion may affect other pathways besides the Hpo signaling cascade (Figure S4). One obvious candidate is the JNK pathway, which has previously been shown to be regulated by Cka (Bond and Foley, 2009; Chen et al., 2002). In addition to cka and FGOP2, depletion of other members of the dSTRIPAK complex, such as mob4, CG5073, CG11526, and CG17494, also suppressed the hpo overgrowth phenotype (Figures S4F–S4M). Together, our 528 Molecular Cell 39, 521–534, August 27, 2010 ª2010 Elsevier Inc.

genetic interaction data suggest that hpo genetically interacts with members of the dSTRIPAK complex, supporting our hypothesis that the dSTRIPAK complex antagonizes Hpo signaling by dephosphorylating Hpo kinase. In Vivo Regulation of Hpo Signaling by dSTRIPAK Complex Members In addition to acting upstream of the core kinase cassette of the Hpo pathway, ex is also a target of Sd/Yki-mediated activity


(Hamaratoglu et al., 2006). Therefore, ex transcript and protein levels can be used as in vivo readouts of pathway activation. We used a patched-GAL4 (ptc) driver combined with UAS-RNAi constructs to specifically deplete the expression of dSTRIPAK complex genes in the anteroposterior (A/P) boundary of the wing imaginal disc (Figure 6). In control discs, Ex was primarily localized in the subapical region of cells, as seen in transverse sections of third-instar wing imaginal discs (Figure 6A). RNAimediated depletion of FGOP2 or Cka caused a marked decrease in the apical level of Ex, which was restricted to the ptc-expressing domain marked by GFP expression (Figures 6B and 6C). This was not due to a general disruption of polarity, since the levels of the basal determinant Discs large (Dlg) and the adherens junction protein E-cadherin (E-cad) were unaltered in flies expressing FGOP2 or cka RNAi (Figures 6D–6F and Figures S5A–S5C). In all cases, similar results were obtained with at least one additional RNAi line (data not shown). Next, we assessed whether the effect of the dSTRIPAK complex on Ex protein levels occurred at the level of ex transcription using an ex-lacZ enhancer trap fly line, which expresses b-galactosidase under the control of the endogenous ex promoter (Figures 6G–6J). As expected, RNAi targeting wts caused an increase in ex-lacZ expression in the ptc domain due to increased Yki activity (Figure 6H). Consistent with an inhibitory effect of the dSTRIPAK complex on Hpo signaling, RNAi-mediated depletion of FGOP2 or Cka markedly decreased ex-lacZ expression at the A/P boundary of wing imaginal discs (Figures 6I and 6J). This effect was specific to members of the dSTRIPAK complex and was not seen in flies expressing RNAi targeting alternative phosphatases and PP2A regulatory subunits (Figures S5D–S5P). Taken together, these data suggest that, in the absence of FGOP2 and Cka, Hpo signaling is hyperactive, leading to a repression of Yki and consequent reduction in ex expression and Ex protein levels. Thus, the dSTRIPAK complex can negatively regulate Hpo signaling in vivo. The dSTRIPAK Complex Acts Upstream of Wts and Yki and Downstream of Sav To further examine the in vivo effect of the dSTRIPAK complex on the regulation of Hpo signaling, we performed genetic epistasis experiments making use of the MARCM system to combine loss of function of dSTRIPAK components with genetic manipulation of Hpo pathway components (Lee and Luo, 1999). Mitotic clones expressing cka RNAi displayed a growth defect when compared with wild-type clones (Figure 7B). This is

Figure 6. The dSTRIPAK Complex Negatively Regulates Hpo Signaling Targets (A–F) Depletion of Cka or FGOP2 reduces the protein levels of Ex at the apical membrane. Transverse sections depicting the protein expression levels of Ex

(A–C) or Dlg (D–F) in third-instar wing discs of ptc-GAL4 >> GFP (A and D), ptcGAL4 >> FGOP2-IR (B and E), or ptc-GAL4 >> cka-IR flies (C and F). The apical surface is localized on the top. The ptc domain is marked by GFP (green), and Ex or Dlg protein levels are represented in gray or red. (G–J) Depletion of the dSTRIPAK complex affects Sd/Yki-dependent ex transcription. The transcriptional activation status of Hpo signaling was monitored in wing discs using an ex-lacZ enhancer trap expressed under the control of ptc-GAL4. XY sections of third-instar wing discs of ptc-GAL4 >> GFP (G), ptc-GAL4 >> wts-IR (H), ptc-GAL4 >> dFGOP2-IR (I), or ptc-GAL4 >> cka-IR (J) flies. Gray or red depicts lacZ staining, whereas DAPI staining is shown as gray or blue. GFP is shown as green in merge images. Ventral is localized on the top.



Figure 7. The dSTRIPAK Complex Regulates Hpo Signaling Upstream of Wts and Yki (A–F) XY sections of third-instar wing discs containing clones induced with the MARCM system. MARCM FRT82B blank clones (A), cka-IR MARCM clones (B), sav3 MARCM clones (C), cka-IR; sav3 MARCM clones (D), wtsX1 MARCM clones (E), and cka-IR; wtsX1 MARCM clones (F) are marked by GFP (green). DAPI staining is shown as blue. Ventral is to the top. (G) Quantification of the percentage of MARCM clone area relative to total area. Results are shown as mean ± SEM of individual wing imaginal discs. (A) n = 28, (B) n = 13, (C) n = 14, (D) n = 13, (E) n = 14, (F) n = 6. x and n.s. represent p < 0.01 and p > 0.01 significance in Student’s t test, respectively. (H–L) XY sections of third-instar wing discs containing clones induced with the MARCM system. MARCM FRT40A blank clones (H), cka1 MARCM clones (I), cka1; UAS-yki MARCM clones (J), FGOP2KG MARCM clones (K), and FGOP2KG; UAS-yki MARCM clones (L) are shown in green and marked by expression of GFP. DAPI staining is shown as blue. Ventral is to the top. (M) Quantification of the percentage of MARCM clone area relative to total area. Results are shown as mean ± SEM of individual wing imaginal discs. (H) n = 11, (I) n = 9, (J) n = 8, (K) n = 12, (L) n = 6. x denotes p < 0.01 significance in Student’s t test.

consistent with Cka negatively regulating Hpo signaling. In contrast, loss-of-function clones for sav and wts displayed a growth advantage when compared to wild-type clones (Figures 7C and 7E). Interestingly, loss of wts suppressed the undergrowth phenotype of cka RNAi clones, while loss of sav had no effect (Figures 7D, 7F, and 7G). This suggests that Cka acts genetically upstream of Wts and downstream of Sav in the Hpo pathway. We also analyzed loss-of-function clones for cka and FGOP2, which, like cka RNAi clones, exhibited a growth defect when compared to wild-type clones (Figures 7H, 7I, and 7K). Impor530 Molecular Cell 39, 521–534, August 27, 2010 ª2010 Elsevier Inc.

tantly, the defect of cka and FGOP2 mitotic clones was rescued by overexpression of yki (Figures 7J, 7L, and 7M). Together, these results suggest that the dSTRIPAK complex acts upstream of Wts and Yki and downstream of Sav, which supports our hypothesis of a direct effect on Hpo activity. DISCUSSION Negative Regulation of the Hpo Pathway Metazoan development is the overall result of the activity of wellorchestrated signaling pathways that instruct cells to grow,


proliferate, polarize, migrate, differentiate, or die. To avoid developmental defects, it is paramount that these evolutionarily conserved pathways are tightly regulated. The Hpo tumor suppressor pathway plays a central role in development and disease, as it controls tissue size by promoting apoptosis and blocking cell proliferation (Harvey and Tapon, 2007; KangoSingh and Singh, 2009; Zhao et al., 2008). Despite the identification of the negative regulators dRASSF and Dachs, little is known about the molecular mechanisms that restrict Hpo signaling (Harvey and Tapon, 2007). Indeed, a clear mammalian homolog of the Drosophila unconventional myosin Dachs is yet to be discovered (Reddy and Irvine, 2008). Moreover, the situation is further complicated by redundancy in the mammalian RASSF family proteins and the fact that some of its members have tumor suppressor properties (Richter et al., 2009; Sherwood et al., 2010). dSTRIPAK, a Hpo Phosphatase Complex Since the core of the Hpo pathway is composed of a kinase cascade, one central question in the field has been the identity of the phosphatase complex that antagonizes Hpo signaling. Our results suggest that the dSTRIPAK PP2A complex is a major influence on Hpo signaling in vitro and in vivo. First, dSTRIPAK components associate with Hpo and dRASSF, as shown by AP-MS and coIP analysis (Figures 1 and 3). Second, we recovered Mts, the dSTRIPAK catalytic subunit as an inhibitor in an unbiased RNAi screen for Hpo pathway regulators (Figure 2). Third, loss of dSTRIPAK components results in elevated phosphorylation of both Hpo and Yki in cell culture (Figure 4). Fourth, the Yki target ex is repressed upon depletion of dSTRIPAK in vivo. Finally, genetic epistasis experiments place the dSTRIPAK complex upstream of wts and yki and downstream of sav in the Hpo pathway, which is consistent with an effect at the level of Hpo. Although dSTRIPAK has a major influence on Hpo pathway activity, it is of course possible that other phosphatase complexes participate in Hpo regulation. PP2A is considered to be a promiscuous phosphatase. However, its activity is specifically targeted to its physiological substrates by the association of the core enzyme with its regulatory B type subunits (Virshup and Shenolikar, 2009). In the case of the recently described human STRIPAK complex, the PP2A core dimer forms a higher-order complex with the B000 regulatory subunits of the Striatin family and a specific set of cellular proteins which include three Ste20-related kinases (Glatter et al., 2009; Goudreault et al., 2009). Cka is the sole Drosophila ortholog of the mammalian Striatin family, which comprises Striatin, Striatin3, or S/G2 nuclear autoantigen (SG2NA) and Striatin4 or Zinedin (Castets et al., 2000; Chen et al., 2002). Our AP-MS experiments suggest the existence of a homologous Drosophila complex, which we refer to as dSTRIPAK. Besides Hpo we found two other Ste20-related kinases, GckIII and Msn associated with the dSTRIPAK components Cka and Mob4, indicating that dSTRIPAK can bind to a specific set of structurally related kinases. Interestingly, both Cka and Msn have been found in genetic experiments as well as large-scale RNAi screens to play a critical role in JNK signaling indicating an additional role for dSTRIPAK beyond Hpo signaling (Bond and Foley, 2009; Chen et al., 2002; Stronach, 2005).

Regulation of Hpo Dephosphorylation How to achieve precisely the correct amount of developmental Hpo signaling is a key issue in growth control. Is it therefore possible that modulating Hpo dephosphorylation provides a regulatory mechanism? The association of Hpo with Cka is, at least in part, dependent on dRASSF (Figure 3E). This suggests that dRASSF may block Hpo signaling via a mechanism that not only relies on its ability to prevent the Hpo:Sav interaction but also helps to recruit dSTRIPAK, thus actively promoting Hpo inactivation by dephosphorylation of Thr195 in Hpo’s activation loop (Polesello et al., 2006). This dual mechanism of action would allow rapid termination of Hpo signaling in response to developmental cues. In contrast, Sav, which acts positively on Hpo signaling, does not associate with dSTRIPAK in our AP-MS data and might therefore protect Hpo from dephosphorylation by displacing dRASSF, with which it competes for Hpo binding (Polesello et al., 2006). Our data therefore provide a model for how the interplay between the activator Sav and the inhibitor dRASSF might control pathway activity through Hpo phosphorylation. Mammalian Hpo Pathway Regulation Is the regulation of Hpo by dSTRIPAK conserved in mammals? The fact that PP2A can control MST1/2 phosphorylation levels in mammalian tissue culture cells supports this notion (Gerd Pfeifer, personal communication). However, the relationship between RASSF proteins and STRIPAK may be more complex in mammals. Unlike dRASSF, RASSF1A, the best-studied mammalian RASSF protein, has been shown to promote activation of MST kinases (Guo et al., 2007). In contrast, RASSF6 has recently been reported to function in an analogous manner to dRASSF (Ikeda et al., 2009). It will therefore be interesting to test whether different mammalian RASSFs have distinct effects on MST1/2 dephosphorylation by PP2A complexes. As the link between Hpo signaling and cancer becomes increasingly apparent (Harvey and Tapon, 2007), targeting the STRIPAK complex may provide an exciting new avenue for cancer therapy. EXPERIMENTAL PROCEDURES Expression Constructs and Cell Line Generation Drosophila ORFs of interest were PCR amplified from Drosophila cDNA pools using Phusion Polymerase (Finnzymes) and ligated into a Gateway (Invitrogen) compatible entry vector (pENTR-D-TOPO). The following primer pairs have been used for cDNA amplification: Hpo, 50 -CACCATGTCTGAGCCAGA GG-30 and 50 - TATCTTAATCAGATTATTATTGAT-30 ; Sav, 50 -CACCATGAACTA TCTAACGATCC-30 and 50 -CTGGTTTTGGTTCTGGTT-30 ; Cka, 50 -TTCCGCGG CCGCCACCATGGGCACCAATTCGGGAG-30 and 50 -AGTCGGCGCCCGACA AAGACCTTGGCGAGGC-30 ; Mob4, 50 -TTCCGCGGCCGCCACCATGAAGATG GCTGACGGCTCGAC-30 and 50 -AGTCGGCGCGCCCAGCCTCGCTTTCGCCA GGG-30 ; and dRASSF, 50 -CACCATGTGGAAGTGCCACAAGTG-30 and 50 -CA AATGCACTTTCAGAGATTC-30 . By LR recombination the sequenced ORFs were transferred from the entry vector into an in-house-designed expression vector (pMHW-Blast) allowing for inducible metallothionein promoter-regulated expression of the bait protein fused to a triple HA affinity tag at its N terminus. Drosophila Kc167 cells were transfected with Effectene transfection reagent (QIAGEN) and grown in Drosophila Schneider medium (Invitrogen) containing 10% FBS, 50 mg/ml penicillin, and 50 mg/ml streptomycin. Blasticidin (10 mg/ml; Invitrogen) was used for selection during 5 weeks. Cell pools



were tested for positive expression by western blotting using anti-HA antibodies (Covance). For bait expression the cells were exposed to 600 mM CuSO4 overnight. Gal4-DBD-Yki Luciferase Assays For the transcriptional G4-DBD-Yki experiments, the long isoform of Yki was recombined into a Gal4-DBD-pDEST-Vector (pAG4W), which is based on the pAGW vector from the DRGC Gateway Vector collection. pAG4W was generated by replacing the EGFP tag with the DNA-binding domain of the yeast GAL4 protein (Gal4-DBD, amino acids 1–147) using AgeI and StuI restriction enzymes. Expression vectors for Wts, Hpo, and Cka were generated using the DRGC Gateway Vector collection. Luciferase assays were performed in S2R+ cells transfected with FugeneHD and pActin5C-hRL as internal Renilla luciferase control. Equal amounts of DNA were transfected in each sample (20 ng for overexpressing plasmid unless otherwise stated, 30 ng for UAS-luciferase, 20 ng for G4-DBD-Yki, and 3 ng for pActin5C-Renilla). Immunoprecipitation and Immunoblot Analysis For FLAG immunoprecipitation assays, cells were lysed in lysis buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% Triton X-100, 10% glycerol, and 1 mM EDTA) supplemented with 0.1 M NaF, phosphatase inhibitor cocktails 1 and 2 (Sigma), and protease inhibitor cocktail (Roche). Cell extracts were spun at 14,000 rpm for 10 min at 4" C. FLAG-tagged proteins were purified using anti-FLAG M2 affinity agarose gel (SIGMA). After 1 hr incubation at 4" C, FLAG immunoprecipitates were washed three to four times with lysis buffer or wash buffer (10 mM Tris [pH 7.5], 150 mM NaCl, 0.1% Triton X-100, and 5% glycerol). Detection of purified proteins and associated complexes was performed by immunoblot analysis using chemiluminescence (GE Healthcare). Western blots were probed with anti-FLAG (mouse M2, SIGMA), anti-Myc (mouse 9E10, Santa Cruz Biotechnology), anti-HA (rat 3F10, Roche Applied Science), anti-Cka (a kind gift from S. Hou), and anti-tubulin (mouse E7, Developmental Studies Hybridoma Bank, DSHB). Anti-Yki, anti-phospho-Yki, anti-Hpo, and anti-phospho-PAK antibodies have been previously described (Colombani et al., 2006; Genevet et al., 2010; Polesello et al., 2006). The NTAN-FLAG plasmid was a kind gift from M. Ditzel. Quantification of immunoblots in Figure 3F was performed by densitometry using ImageJ. Scanning Electron Microscopy Analysis Adult flies were processed for scanning electron microscopy as previously described (Kango-Singh et al., 2002). Immunostaining For loss-of-function analysis, mosaic tissues were obtained with the MARCM system using hsFLP drivers. Mosaics were induced by a 40 min heat shock at 37" C of 48 hr AEL larvae. Larval tissues were processed as in Polesello et al. (Polesello et al., 2006). Primary antibodies were incubated overnight at 4" C. b-galactosidase (Promega) and Dlg (mouse 4F3, DSHB) antibodies were used at 1/500, Ex antibody (a kind gift from Allen Laughon) was used at 1/200, and E-cad antibody (rat DCAD2, DSHB) was used at 1/100. Anti-rabbit, anti-mouse, and anti-rat Rhodamine Red-X-conjugated secondary antibodies (Jackson ImmunoResearch) were used at 1/500 and incubated for at least 2 hr at room temperature. After washes, tissues were mounted in DAPI-containing Vectashield (Vector Labs). Fluorescence images were acquired on a Zeiss LSM510 Meta confocal laser scanning microscope (253 and 403 objective lenses). Analysis of Genetic Interactions in the Drosophila Wing For analysis of genetic interactions in the Drosophila wing, flies with genotype of interest were collected and preserved in 70% EtOH for at least 24 hr. Wings were removed in 100% isopropanol and mounted in microscope slides using Euparal (Agar Scientific) as mounting medium. Euparal was used according to the manufacturer’s specifications. Wings were visualized using a Zeiss Axioplan 2 imaging system, using Optovar optics (1.6x amplification) and a 2.53 objective (Plan-NEOFLUAR, Zeiss). Wings were photographed using a Leica DFC420C camera. Wing areas were measured using the measure module of ImageJ. Images were processed using Adobe Photoshop.

532 Molecular Cell 39, 521–534, August 27, 2010 ª2010 Elsevier Inc.

Genotypes w; FRT40A cka1 was a kind gift from S. Hou. The MARCM makers yw tubGAL4 hsFLP 122 UAS-nucGFPmyc;; FRT82B CD21 y+ tubG80.LL3/TM6 and yw tubGAL4 hsFLP 122 UAS-nucGFPmyc; FRT40A CD21 tubG80.LL3/CyO were gifts from G. Struhl. Genotypes were as follows: Figure 5A, w; Figure 5B, GMR-hpo; Figure 5C, GMR-hpo/ P{SUPor-P}CG10158KG05348; Figure 5D, GMR-hpo/ Df(1)w67c23, y1, P{lacW}Ckak12603; Figure 5E, GMR-hpo/ FRT40A cka1; Figure 5F, GMR-hpo/ Df(1)w67c23, y1, P{lacW}Ckak12606; Figure 5H, en-GAL4/+; Figure 5I, en-GAL4, UAS-hpo-IR/ +; Figure 5J, en-GAL4, UAS-hpo-IR/ UASFGOP2-IR; Figure 5K, en-GAL4, UAS-hpo-IR/ UAS-cka-IR; Figures 6A, 6D, and 6G, ptc-GAL4, UAS-GFP/ +; Figures 6B, 6E, and 6I, ptc-GAL4, UASGFP/ UAS-FGOP2-IR; Figures 6C, 6F, and 6J, ptc-GAL4, UAS-GFP/ UAScka-IR; Figure 6H, ptc-GAL4, UAS-GFP/ UAS-wts-IR; Figure 7A, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc;; FRT82B CD21 y+ tubG80.LL3/ +; Figure 7B, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc; +/ UAS-cka-IR; FRT82B CD21 y+ tubG80.LL3/+; Figure 7C, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc;; FRT82B CD21 y+ tubG80.LL3/ FRT82B sav3; Figure 7D, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc; +/ UAS-cka-IR; FRT82B CD21 y+ tubG80.LL3/ FRT82B sav3; Figure 7E, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc;; FRT82B CD21 y+ tubG80.LL3/ FRT82B wtsX1; Figure 7F, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc; +/ UAS-cka-IR; FRT82B CD21 y+ tubG80.LL3/ FRT82B wtsX1; Figure 7H, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc; FRT40A CD21 tubG80.LL3/+; Figure 7I, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc; FRT40A CD21 tubG80.LL3/ FRT40A cka1; Figure 7J, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc; FRT40A CD21 tubG80.LL3/ FRT40A cka1; + / UAS-yki; Figure 7K, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc; FRT40A CD21 tubG80.LL3/ FRT40A CG10158KG05348; and Figure 7L, yw tubGAL4 hsFLP 122 UAS-nucGFPmyc; FRT40A CD21 tubG80.LL3/ FRT40A CG10158KG05348; +/ UAS-yki. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, five figures, two tables, and Supplemental References and can be found with this article online at doi:10.1016/j.molcel.2010.08.002. ACKNOWLEDGMENTS We would like to thank S. Hou for cka alleles and anti-Cka antibody; D. Pan, M. Boutros, T. Maile, and M. Ditzel for plasmids; G. Struhl, the Vienna Drosophila RNAi Centre, and the Bloomington Drosophila Stock Center for fly stocks; P. Meier for discussions; and G. Pfeifer for sharing unpublished results. We are grateful to J. Dobbelaere, L. Muthusamy, and G. Clark for help with plating the RNAi library, and B. Thompson, T. Maile, A. Genevet, B. Aerne, Y. Zhou, and E. Chan for comments on the manuscript. We thank R. Instrell, B. Saunders, M. Howell, T. Liu, and B. Baum for help and advice with the RNAi screen, and K. Blight for SEM. Research in the Tapon lab is supported by Cancer Research UK. F.J. was the recipient of a predoctoral fellowship from the Portuguese Foundation for Science and Technology (FCT - POCI2010/FSE). M.C.W. was supported by an EMBO Long-Term Fellowship. Received: February 18, 2010 Revised: June 23, 2010 Accepted: July 16, 2010 Published: August 26, 2010 REFERENCES Baumgartner, R., Poernbacher, I., Buser, N., Hafen, E., and Stocker, H. (2010). The WW domain protein Kibra acts upstream of Hippo in Drosophila. Dev. Cell 18, 309–316. Bennett, F.C., and Harvey, K.F. (2006). Fat cadherin modulates organ size in Drosophila via the Salvador/Warts/Hippo signaling pathway. Curr. Biol. 16, 2101–2110.


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