Drosophila Schip1 Links Expanded and Tao-1 to Regulate Hippo Signaling

Article

Drosophila Schip1 Links Expanded and Tao-1 to Regulate Hippo Signaling Highlights

Authors

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Schip1 controls Hippo signaling to suppress organ growth in Drosophila

Hyung-Lok Chung, George J. Augustine, Kwang-Wook Choi

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Schip1 forms a complex with Expanded for Hippo localization

Correspondence

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Schip1 directly promotes Tao-1 activity for Hippo phosphorylation

[email protected]

Schip1 links Expanded and Tao-1 to activate Hippo, hence suppressing Yorkie

The Hippo signaling pathway is a major conserved mechanism that controls organ growth in Drosophila and mammals. Here, Chung et al. show that the Drosophila protein Schip1 acts as a linker between Expanded and Tao-1 to control Hippo activity.

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Chung et al., 2016, Developmental Cell 36, 511–524 March 7, 2016 ª2016 Elsevier Inc. http://dx.doi.org/10.1016/j.devcel.2016.02.004

In Brief

Developmental Cell

Article Drosophila Schip1 Links Expanded and Tao-1 to Regulate Hippo Signaling Hyung-Lok Chung,1,2 George J. Augustine,2,3,4 and Kwang-Wook Choi1,* 1Department

of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, South Korea for Functional Connectomics, Korea Institute of Science and Technology, Seoul 136-791, South Korea 3Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 637553, Singapore 4Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2016.02.004 2Center

SUMMARY

Regulation of organ size is essential in animal development, and Hippo (Hpo) signaling is a major conserved mechanism for controlling organ growth. In Drosophila, Hpo and Warts kinases are core components of this pathway and function as tumor suppressors by inhibiting Yorkie (Yki). Expanded (Ex) is a regulator of the Hpo activity, but how they are linked is unknown. Here, we show that Schip1, a Drosophila homolog of the mammalian Schwannomin interacting protein 1 (SCHIP1), provides a link between Ex and Hpo. Ex is required for apical localization of Schip1 in imaginal discs. Schip1 is necessary for promoting membrane localization and phosphorylation of Hpo by recruiting the Hpo kinase Tao-1. Taking these findings together, we conclude that Schip1 directly links Ex to Hpo signaling by recruiting Tao-1. This study provides insights into the mechanism of Tao-1 regulation and a potential growth control function for SCHIP1 in mammals.

INTRODUCTION Organ size in animals is regulated by cell proliferation and apoptosis during development. The Hippo signaling pathway is one of the major conserved mechanisms that control organ growth in both Drosophila and mammals. This is also known as the HSW pathway because the core of this signaling system consists of an Mst family kinase Hippo (Hpo) (Harvey et al., 2003; Jia et al., 2003; Pantalacci et al., 2003; Udan et al., 2003; Wu et al., 2003), a regulatory protein Salvador (Sav) (Kango-Singh et al., 2002; Tapon et al., 2002), and the nuclear Dbf2-related (NDR) family kinase Warts (Wts) (Justice et al., 1995; Xu et al., 1995). The HSW genes are considered to be tumor suppressors, because tumorous tissue growth is induced when the functions of these core components are lost in Drosophila or mammalian systems (Pan, 2010; Zhao et al., 2010). Accumulating evidence also indicates that altered regulation of the Hpo pathway is associated with diverse types of cancers (Halder and Johnson, 2011).

In Drosophila, the activated Hpo-Sav complex phosphorylates and activates Wts (Harvey et al., 2003; Wu et al., 2003), which then phosphorylates and inhibits the activity of the transcriptional coactivator Yorkie (Yki) (Huang et al., 2005). Activated Yki moves into the nucleus and forms a complex with the TEAD/TEF family transcription factor Scalloped (Wu et al., 2008; Zhang et al., 2008) to induce expression of target genes. Yki target genes include cyclin E and diap1, which promote cell proliferation and inhibit apoptosis, respectively (Huang et al., 2005). Extensive progress has been made toward understanding the regulation of HSW core activity. One pathway involves the plasma membrane proteins Fat (Ft) and its ligand Dachsous (Ds) (Bennett and Harvey, 2006; Cho et al., 2006; Silva et al., 2006; Tyler and Baker, 2007; Willecke et al., 2006). Another transmembrane protein, Crumbs (Crb), is part of a different pathway that is in parallel with the Ft-Ds system through the FERM (4.1-Ezrin-Radixin-Moesin)-domain protein, Expanded (Ex) (Chen et al., 2010; Ling et al., 2010; Robinson et al., 2010). Fat is also required for apical localization of Ex (Hamaratoglu et al., 2006; Silva et al., 2006; Willecke et al., 2006), implying that Ex might act downstream of both Crb and Fat. Ex functions together with Merlin (Mer) to regulate proliferation and differentiation (McCartney et al., 2000). Evidence suggests that Ex and Mer function together upstream of Hpo (Hamaratoglu et al., 2006; McCartney et al., 2000). Ex and Mer also form a complex with a WW-domain protein, Kibra (Baumgartner et al., 2010; Genevet et al., 2010; Yu et al., 2010). Mer and Ex are homologs of the mammalian Merlin (also called Schwannomin or NF2 [neurofibromatosis type 2]) and Willin/FRMD6, respectively. Ex and Mer are co-localized in the subapical junctional region of epithelial cells (McCartney et al., 2000). Crb regulates Hpo signaling by directly recruiting Ex to the subapical membrane (Chen et al., 2010; Ling et al., 2010; Robinson et al., 2010). Genetic studies have indicated that Ex and Mer have redundant functions in regulating proliferation and differentiation (Hamaratoglu et al., 2006; Hariharan, 2006). However, evidence suggests that Ex and Mer make distinct contributions to HWS signaling in different tissues. For example, Mer is essential in the oocyte whereas Ex is not (MacDougall et al., 2001; Meignin et al., 2007; Polesello and Tapon, 2007; Yu et al., 2008). Recent studies have shown that Mer has an additional function to directly recruit Wts to the plasma membrane without activating the Hpo kinase (Yin et al., 2013). In contrast, in imaginal discs loss of ex results in hyperproliferation, while loss of mer alone

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does not. Hence, it has been proposed that Ex plays more critical roles than Mer for growth regulation, at least in imaginal discs (Hamaratoglu et al., 2006; McCartney et al., 2000; Pellock et al., 2007). Moreover, it was found that Ex has a role in linking Hippo to Wts as a scaffold (Sun et al., 2015). An important unanswered question is how Ex leads to the regulation of Hpo activity for growth suppression in imaginal discs. Recently, it has been discovered that another Mst family protein kinase, Tao-1, directly phosphorylates the threonine195 (T195) residue of Hpo to activate its activity (Boggiano et al., 2011; Poon et al., 2011). However, it is not yet known how Tao-1 activity is regulated. Tao-1 can phosphorylate Hpo even when Ex and Mer are depleted, suggesting that Ex and Mer act upstream of Tao-1 or are independent of Tao-1. Despite extensive efforts, no physical association has been detected between Tao-1 and any member of the Ex-Mer-Kibra complex (Boggiano et al., 2011). Hence, the relationship between ExMer and Tao-1 is still an open question. In a search for new genes that interact with Crb, we found that Crb function might be related to an uncharacterized gene CG5375 that has sequence similarity to the mammalian Schwannomin/Mer-interacting protein (SCHIP1) gene (Goutebroze et al., 2000). Schwannomin/Mer is a FERM domain-containing tumor-suppressor protein whose mutations lead to neurofibromatosis (McClatchey and Giovannini, 2005). SCHIP1 co-localizes with Schwannomin/Mer (also called NF2) and interacts specifically with spliced isoforms and naturally occurring mutant NF2 proteins (Goutebroze et al., 2000). However, it is unknown whether SCHIP1 plays any role in organ growth by affecting Hpo signaling. In this study we report that CG5375, the Drosophila SCHIP1 homolog (Schip1), is required for suppressing organ growth as a critical member of the Hpo signaling pathway. Ex regulates the localization of Schip1 by physical interaction in imaginal discs. Schip1 recruits Tao-1 and regulates its kinase activity to promote Hpo signaling. Our results identify Schip1 as an essential link between Ex and Tao-1 kinase in the Hpo signaling pathway. RESULTS Knockdown or Loss of Schip1 Causes Overgrowth in Adult Eyes Mammalian SCHIP1 was initially identified as a binding partner of the FERM domain protein Schwannomin/Merlin (Goutebroze et al., 2000). A related Drosophila protein is encoded by an uncharacterized annotated gene CG5375 named Schip1. The C-terminal region of Schip1 is similar to that of a human homolog, SCHIP1, with approximately 60% protein sequence identity. The conserved FEZ-like region located in the C-terminal part of Schip1 and human SCHIP1 is a coiled-coil domain containing a leucine zipper pattern (Goutebroze et al., 2000). In contrast, the N-terminal domains of these proteins are less conserved (Figure S1). Compared with the human form, Schip1 has an additional sequence of about 130 amino acids (aa) in its N-terminal region (Figure 1A). To examine the function of Schip1 in developing tissues in vivo, we used a mutant strain, Schip1GS12074 carrying a P{GSV6} element inserted in the coding region of the second exon of Schip1 (Figure 1B). The Schip1GS12074 homozygote is lethal dur-

ing embryogenesis or an early larval stage, indicating that Schip1 is essential for development. P{GSV6} is an EP element containing the upstream activating sequence (UAS) sequence designed to induce a downstream gene upon Gal4 binding (Igaki et al., 2002). The EP element in Schip1GS12074 is inserted in the reverse orientation to the Schip1 gene, so that it produces anti-sense RNA complementary to the 50 region of the Schip1 transcript. Hence, Schip1GS12074 was used as an RNAi line to knock down the Schip1 gene (see Experimental Procedures and Figures S2A–S2C). When driven by ey-Gal4, this Schip1 RNAi did not cause any noticeable change in eye growth (Figure S2E0 ). However, co-expression with dicer (Lee et al., 2004) led to strong overgrowth of the eye with bulging (Figure S2G). Induction of tissue overgrowth by Schip1 RNAi suggests that Schip1 is involved in negative regulation of tissue growth. To confirm the RNAi effects, we generated a deletion allele Schip149 by an imprecise excision of the GS12074 element. This Schip149 mutant showed a 794-bp deletion, which removes more than half of the coding sequence (aa 241–506) (Figure 1B). Since both Schip149 homozygotes and Schip149/Df(2L)Exel7046 die during embryonic or early larval stages, Schip149 appears to be a null or strong hypomorphic allele. To examine the role of Schip1 in organ growth, we generated Schip149 mutant clones in the eye. Induction of Schip149 mutant clones resulted in enlarged eyes with irregular bulging on the surface (Figures 1C and 1D), consistent with the overgrown eye phenotype induced by Schip1 RNAi. Furthermore, Schip1 mutant clones in mid-pupal eyes had an increase in the number of interommatidial cells (Figures 1H and 1I). Schip1 Mutant Clones Overproliferate in Developing Imaginal Discs To analyze the overgrowth phenotype during development, we generated eye imaginal discs containing Schip149 mutant clones or Schip1+ control clones identified by the lack of the GFP clone marker. As expected, Schip149 mutant clones were much larger than Schip1+ control clones (Figures 1E– 1G), consistent with the overgrowth of mutant clones seen in adult eyes (Figure 1D). This suggests that Schip149 mutant cells have strong growth advantages over adjacent wild-type cells in the developing eye disc. To further analyze the effects of Schip149 mutation on tissue growth, we asked whether mutant cells are more proliferative than wild-type cells. This was done by checking bromodeoxyuridine (BrdU) incorporation, which marks cells in the S phase of the cell cycle. Normally, wild-type cells in the morphogenetic furrow (MF) of eye disc are arrested at the G1 stage. Just posterior to the MF, a column of cells undergoes a final round of division in the second mitotic wave (SMW). Hence, the cell cycle ceases in the region posterior to the SMW in normal eye discs. As shown in Figures 1J and 1J0 , Schip149 cells in mutant clones showed ectopic BrdU incorporation posterior to the SMW. To test whether Schip149 mutant cells have more mitotic activity than normal cells, we stained eye discs containing Schip149 mutant clones with anti-phosphohistone (PH3) antibody. Similar to the pattern of BrdU incorporation, PH3 staining in wild-type eye discs was mainly observed anterior to the MF and in the SMW. In contrast, Schip149 mutant clones showed ectopic PH3 staining posterior to the SMW (Figures 1K and 1K0 ). These

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Figure 1. Loss of Schip1 Causes Overproliferation in the Eye (A) Schematic alignment of Schip1 and human SCHIP1. (B) Schematic structure of the Schip1 gene. Schip1GS12074 has a P element inserted in the second exon. Schip149 has a 794-bp deletion by imprecise excision of GS12074 P element. (C and D) Scanning electron microscopy images of adult eyes. (C) w1118 control shows the normal eye. (D) An eye with Schip149 mutant clones shows an enlargement with folded surface, indicating overgrowth. (E and F) Eye discs stained with the GFP clone marker. (E) Sizes of Schip1+ clones (GFP) are similar to those of GFP+/GFP+ twin-spot clones (cells with brighter green). (F) Sizes of Schip149 mutant clones (/) are much bigger than GFP+ twin-spot clones (+/+). (+/) indicates heterozygous cells. (G) Quantification of the ratio of GFP area/total area (%) for Schip1+ and Schip149 clones (n = 5; error bars represent ±SEM. ***p < 0.001, t test). (H and I) Schip1 mutant clones in mid-pupal eyes show increased number of interommatidial cells. (H) Schip1+ clones. (I) Schip149 clones. Arrows indicate the regions where interommatidial cells are increased. Scale bars, 20 mm (H and I). (J–K0 ) Effects of Schip1 mutant clones on BrdU and PH3 levels in eye discs. (J and J0 ) Schip1 mutant clones posterior to the SMW show ectopic BrdU staining. (J0 ) shows only red channel. (K and K0 ) Schip1 mutant clones posterior to the SMW show ectopic PH3 staining. (K0 ) shows only red channel. Marked area in (J0 ) and (K0 ) indicates the mutant clones posterior to the SMW. Positions of the SMW and the morphogenetic furrow (MF) are indicated by arrows. Scale bars, 50 mm (E, F, J–K0 ).

results indicate that Schip149 mutant cells fail to exit the cell cycle after the SMW and continue to proliferate. Loss of Schip1 Upregulates Yki Target Genes Overproliferation in Schip1 mutant clones raises the possibility that Schip1 might negatively regulate Yki signaling. Increased Yki activity leads to transcriptional activation of its target genes, such as diap1 and cyclin E, resulting in inhibition of cell death and facilitation of cell cycle, respectively (Huang et al., 2005; Oh and Irvine, 2008). Therefore, we examined whether the loss of Schip1 upregulates these Yki target genes. Indeed, many Schip149 mutant clones in eye discs showed higher levels of Diap1 and Cyclin E (Figures 2A–2B00 ). In addition to Diap1 and cyclin E, expression of Hpo signaling components is upregulated by Yki in a feedback loop (Hamaratoglu et al., 2006; Huang et al., 2005). For instance, Ex and Crb, which act upstream of Hpo, are known to be regulated

transcriptionally by Yki (Genevet et al., 2009; Hamaratoglu et al., 2006). Thus, we examined whether the levels of Ex and Crb are also affected by loss of Schip1. When Schip1 RNAi was induced by en-Gal4 in the posterior region of the wing imaginal disc, the level of Ex also increased in the posterior region (Figures S3A–S3A%). Similarly, the level of Ex was significantly increased in Schip149 mutant clones (Figures 2C–2C00 , marked as dashed lines). We then tested whether Ex is transcriptionally regulated by Schip1 using an ex-lacZ reporter. When Schip1 was depleted in the anterior-posterior boundary region of the wing disc by decapentaplegic (dpp)-Gal4, exlacZ reporter was strongly induced in the dpp region compared with control (Figures 2D–2D00 ). Similarly, elevated levels of Crb protein were detected in larval wing discs of Schip149 mutant clones (Figures 2E–2E00 ). Expression of a crb-lacZ reporter was strongly induced along the dorsoventral boundary of normal wing disc (Figure 2F) (Herranz et al., 2006). When

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Figure 2. Schip1 Regulates Hpo Pathway Target Genes

(A and B) Schip149 mutant clones show higher levels of Diap1 and Cyclin E in eye discs. (A–A00 ) Diap1 staining in Schip149 mutant clones. (A) Merge, (A0 ) GFP, (A00 ) Diap1. (B–B00 ) Cyclin E staining in Schip149 mutant clones. (B) Merge, (B0 ) GFP, (B00 ) Cyclin E. The areas marked by dotted lines show clear increases in the level of Cyclin E (B). (C–C00 ) Ex staining in Schip149 mutant clones of wing disc. (C) Merge, (C0 ) GFP, (C00 ) Ex. Marked areas shows clear increases in the Ex level. (D–D00 ) Knockdown of Schip1 by dpp-Gal4 induces ectopic expression of the ex-lacZ reporter in the anterior-posterior boundary region compared with dpp>GFP control. (D) dpp>GFP; ex-lacZ, (D0 ) GFP, (D00 ) dpp>Schip1 RNAi; ex-lacZ. (E and F) Upregulation of Crb level in Schip1 clones. (E–E00 ) Crb staining in Schip149 mutant clones of wing disc. (E) Merge, (E) GFP, (E00 ) Crb. Marked areas show clear increases in the Crb level. (F–F00 ) crb-lacZ is normally induced along the dorsoventral boundary region (arrows). Schip1 knockdown by en-Gal4 induces ectopic expression of crb-lacZ in the posterior compartment (asterisks). (F) Merge, (F0 ) GFP, (F00 ) crb-lacZ. (G and H) Upregulation of Crb by Schip1 RNAi was suppressed by knockdown of Yki. (G–G00 ) Crb level is increased by Schip1 RNAi. (G) Merge, (G0 ) GFP, (G00 ) Crb. (H–H00 ) yki RNAi suppressed the increase of Crb caused by Schip1 RNAi. (H) Merge, (H0 ) GFP, (H00 ) Crb. The areas marked by dotted lines show the boundary between posterior and anterior part of wing discs. Scale bars, 20 mm.

Schip1 was knocked down by en-Gal4, crb-lacZ was ectopically induced in the posterior compartment (Figures 2F–2F00 ). Furthermore, the upregulation of Crb caused by Schip1 RNAi was fully suppressed by knockdown of Yki (Figures 2G–2H00 ). Altogether, these data suggest that Schip1 negatively regulates the expression of Yki target genes. Schip1 Is Antagonistic to Yki Activation The activity of Yki depends on its subcellular localization rather than its expression level (Oh and Irvine, 2008). Thus, we examined whether loss of Schip1 affects the subcellular distribution of Yki. As shown in Figures 3A–3A00 , the expression pattern of Yki was changed in Schip149 mutant clones, especially within the wing pouch region (Figure 3A00 ). Confocal analysis at a higher resolution showed that Yki staining in wild-type cells was local-

ized mainly in the cytoplasmic region around the nucleus (Figures 3B–3B00 ), consistent with the previous report that Yki is predominantly cytoplasmic (Oh and Irvine, 2008). In contrast, Yki staining in most Schip149 mutant cells was more broadly distributed and often filled the nuclear region (Figure 3B00 ). The observed Yki mislocalization suggests that Schip1 might be involved in the retention of Yki in the cytoplasm, thereby inhibiting Yki signaling. Thus, we tested whether Schip1 can affect the Yki activity in vitro by using a luciferase reporter assay in S2 cells. The addition of a Gal4 DNA binding (GDB) domain-Yki fusion gene increased luciferase reporter activity by 3.8-fold from the basal activity. However, when Ex or Hpo was added, Yki activity decreased to approximately 20% and 15% of control levels, respectively (Figure 3C0 ). Adding a similar amount of Schip1 also decreased the Yki activity to 40%, while the maltose binding

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Figure 3. Schip1 Inhibits Yki Activity by Regulating the Level of Yki Phosphorylation and Subcellular Localization (A–A00 ) The pattern of Yki is changed in Schip149 mutant clones. (A) Merge, (A0 ) GFP, (A00 ) Yki. Dashed lines show the regions where the localization of Yki is clearly altered in wing disc. (B–B00 ) Confocal analysis at higher magnification shows altered Yki localization. (B) Merge, (B0 and B00 ) Yki. Dashed lines in (B–B00 ) indicate the clone boundary between GFP-positive (Schip1+/+ and Schip1+/) and GFP-negative cells (Schip1/). Several cells with brighter GFP staining in (B) are twin-spot cells. In (B0 ), approximate positions of nuclei filled with Yki are indicated by dotted circles within the clone. (C and C0 ) Yki luciferase assays show that Schip1 can suppress the transcriptional activity of Yki. (C) Transcriptional activity of Yki measured by relative luciferase ratios in S2 cells transfected with the Gal4 DNA binding domain with Yki (Gal4DBD-Yki), UAS-luciferase, and plasmids expressing MBP as a negative control. (C0 ) Transcriptional activity of Yki measured by relative luciferase ratios in S2 cells transfected with Gal4DBD-Yki, UAS-luciferase, and plasmids expressing Hpo, Ex and Schip1. The transcriptional Yki activity is suppressed by Schip1. (D) The expression level of phosphorylated Yki at S168 residue is increased by Schip1 overexpression, although the total amount of Yki was not noticeably affected. All error bars represent SD (n = 5). Scale bars, 20 mm (A and B).

protein (MBP) used as a negative control did not affect the Yki activity (Figure 3C). These results demonstrate that Schip1 antagonizes the function of Yki in transcription. Because phosphorylated Yki is excluded from the nucleus (Oh and Irvine, 2008, 2009), we next asked whether Schip1 can increase the level of Yki phosphorylation. Schip1 overexpression in the entire wing pouch using nub-Gal4 resulted in early lethality. When Schip1 was overexpressed in the posterior wing compartment by en-GaL4 (en>Schip1), the amount of phosphorylated Yki was increased, although the total amount of Yki was not noticeably changed (Figure 3D). Taken together, these data suggest that Schip1 inhibits Yki activity by regulating the level of Yki phosphorylation and subcellular localization. Schip1 Genetically Interacts with ex Our data thus far indicate that Schip1 negatively regulates Yki activity by enhancing the level of phosphorylated Yki. Because Wts is a protein kinase that phosphorylates Yki, we examined whether Schip1 genetically interacts with wts by determining

whether Wts overexpression can suppress the Schip1 RNAi phenotype. Knockdown of Schip1 by en-Gal4 clearly increased the number of PH3-positive mitotic cells in the posterior compartment compared with the anterior control region (Figures 4A and 4B). Overexpression of Wts suppressed this overproliferation effect of Schip1 knockdown, reducing PH3 staining to a normal level (Figure 4C). We also confirmed this genetic interaction in adult wings: en>Schip1 RNAi at 18 C induced overproliferation in the posterior region of larval wing discs, and adult wings showed an enlargement compared with en>+ (marked as a dotted line). Wts overexpression led to near-complete suppression of the adult wing phenotype of en>Schip1 RNAi (Figures 4A0 , 4B0 , and 4C0 ). These results suggest that Schip1 acts upstream of, or in parallel to, Wts. Given that Crb interacts with the FERM domain protein Ex (Ling et al., 2010) to inhibit Hpo signaling, we tested whether Schip1 genetically interacts with crb. Overexpression of Crbintra by glass multiple reporter (GMR)-Gal4 in the developing retina caused roughening of the eye surface and size reduction. Overexpression of Ex suppressed the Crbintra eye phenotype (Figures 4D and 4E). In contrast, UAS-GFP did not affect the Crb phenotype, indicating that the UAS-Ex effect was not due to additional UAS dosage. Similarly, overexpression of Schip1 also strongly suppressed the Crbintra eye phenotype (Figure 4F), while Schip1

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Figure 4. Genetic Interaction of Schip1 with Wts and Ex (A–C) Overexpression of Wts rescues increased cell proliferation by knockdown of Schip1 in the posterior wing disc (en>Schip1 RNAi). (A) en Gal4/+, (B) en>Schip1 RNAi/+, (C) en>Schip1 RNAi, wts. Dotted lines indicate the anterior-posterior boundaries based on en>GFP (not shown). Anterior is to the left. Scale bars, 50 mm. (A0 –C0 ) Increased adult wing of en>Schip1 RNAi is rescued by Wts overexpression. (A0 ) en Gal4/+, (B0 ) en>Schip1 RNAi/+, (C0 ) en>Schip1 RNAi, wts. Red dashed lines indicate the wild-type wing size. Scale bars, 100 mm. (D–F) Overexpression of Ex or Schip1 suppresses the defects of GMR>crbintra. (D) GMR>crbintra/+, (E) GMR>crbintra>ex, (F) GMR>crbintra>Schip1. (G–I) Knockdown of Schip1 strongly rescues the small eye phenotype of Ex overexpression. (G) GMR Gal4/+, (H) GMR>ex/+, (I) GMR>ex>Schip1 RNAi. (J–L) Overexpression of Schip1 partially rescues the small eye phenotype of Ex knockdown. (J) ey Gal4/+, (K) ey>ex RNAi, (L) ey>ex RNAi>Schip1.

overexpression in the wild-type background showed no detectable effects in the eye. Crb is required for apical localization of Ex but also promotes downregulation of apical Ex (Grzeschik et al., 2010; Robinson et al., 2010) by ubiquitin-dependent degradation (Ribeiro et al., 2014). Indeed, Crbintra overexpression in eye disc resulted in a reduction of apical Ex, as reported earlier (Grzeschik et al., 2010). Schip1 overexpression restored the normal level of apical Ex caused by Crbintra overexpression (Figure S5). These results indicate that Ex and Schip1 might be functionally related. Thus, we examined the genetic interaction between Ex and Schip1. Knockdown of Schip1 almost completely rescued the small eye phenotype of Ex overexpression (Figures 4H and 4I) while UAS-GFP control had no effect (Figure S4D). We then asked whether ectopic Schip1 can recover the ex RNAi defects. Similar to Ex overexpression, knockdown of Ex also results in eye reduction. It has been shown that reduced Ex causes overgrown eye discs but smaller adult eye due to its effect on retinal differentiation (Pellock et al., 2007). Overexpression of Schip1 consistently rescued the eye-size reduction caused by ex RNAi (Figures 4K and 4L).

Schip1 Co-localizes and Physically Interacts with Ex We have shown above that Schip1 genetically interacts with ex. This raises the possibility that Schip1 might co-localize with Ex to function together. To examine the subcellular localization of Schip1 protein, we generated an antibody against the N-terminal part of Schip1. Immunostaining of third instar eye discs showed that Schip1 normally is expressed in all cells of the eye disc and was significantly reduced in Schip149 mutant clones (Figures 5A and 5A0 ). Western blot analysis of adult tissue extracts also showed a strong reduction of Schip1 protein in Schip149/+ heterozygotes compared with the wild-type level (Figure 5A00 ). Next, we checked the subcellular distribution of Schip1 expression. As in eye discs, Schip1 was expressed in all wing disc cells with enriched localization to the cell membrane. Schip1 showed overlapping localization with Ex (Figures 5B and 5C–5E0 ). Ex is known to be localized to apical junctions (Grzeschik et al., 2010; Ribeiro et al., 2014; Robinson et al., 2010). In z sections, significant co-localization of Schip1 with Ex in the apical regions of wing imaginal disc was apparent (Figures 5C–E), although some Ex staining was detected more apically than Schip1. Schip1 showed strong co-localization with the apical marker

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Figure 5. Schip1 Co-localizes and Physically Interacts with Ex (A and A0 ) Schip1 antibody staining is greatly reduced in Schip149 mutant clones (GFP-negative region marked by dashed line). (A) Merge, (A0 ) Schip1. (A00 ) Western blot analysis of adult tissue extracts of w1118 and Schip149/+ heterozygote. The Schip1 level is reduced in heterozygotes. (B–E) Overlapping localization of Ex and Schip1 in wing imaginal discs of w1118. (B) Merge of Ex (green) and Schip1 (red) at a low magnification. (C–E) Z-section view at a high magnification of the box region in (B). (C) Ex, (D) Schip1, (E) merge. Note that some Ex staining is located more apical than Schip1. (C0 –E0 ) Horizontal sections. (C0 ) Ex, (D0 ) Schip1, (E0 ) Merge. (F–H) Schip1 directly interacts with FERM domain of Mer. (F) GST pull-down between MBP-Mer and GST-Schip1. (G) GST pull-down between GSTSchip1, MBP-Mer (1–314), and MBP-Mer (314– 635). (H) CoIP between Schip1 and Mer. (I–K) Schip1 directly binds to the FERM domain of Ex. (I) GST pull-down between Schip1 and Ex. (J) GST pull-down between Ex (N) (N-terminal FERM domain) and Schip1. (K) CoIP between Ex and Schip1. (L–O) Localization of Schip1 is altered by knockdown or overexpression of Ex in wing disc. Green staining indicates GFP expression by ptc-Gal4 or en-Gal4. (L) The apical section of ptc>ex RNAi>GFP. The arrow shows a reduced Schip1 level in the ptc domain where ex RNAi is targeted. (M) Basal section of ptc>ex RNAi>GFP. The arrow shows Schip1 accumulation in the ptc domain. (N) Some Schip1 staining was mislocalized to the basal region in the posterior part of en>ex RNAi>GFP wing disc. Top panels: Z-section images. Bottom panels: Basal section images show a higher level of Schip1 in the posterior region. Apical sections show no significant difference in the posterior region (not shown). (O) Overexpression of Ex increases Schip1 in the apical region. Z sections are shown on the top. The arrow indicates the apical region where ectopic Schip1 is recruited by Ex overexpression. Red dashed lines indicate the approximate position of the apical basal boundary, and white dashed lines show the boundary between posterior and anterior part of wing disc (N and O). Scale bars, 20 mm.

Patj, but some Patj was also detected more apically than Schip1 (Figures S6A0 –S6A%). Schip1 staining showed considerable overlap with the adherens junction marker Arm (Figures S6B0 – S6B%), although Schip1 staining extended more basally than that of Arm. These results indicate that Schip1 is localized to adherens junctions as well as to the region of Ex and Patj. Next, we determined whether Schip1 and Ex also physically interact. Since human SCHIP1 is known to interact with the

FERM domain of Schwannomin/Merlin, we first examined whether Schip1 can bind to Mer. By using bacterially purified proteins, we confirmed direct binding between Schip1 and the FERM domain of Mer (Figures 5F and 5G). Furthermore, the localization of these two proteins partially overlapped (Figures S6C–S6C%). Coimmunoprecipitation (coIP) assays indicated that Schip1 and Mer form a complex in cultured S2 cells (Figure 5H). We then determined whether Schip1 physically interacts with Ex. Glutathione S-transferase (GST) pull-down assays revealed direct binding between Schip1 and Ex in vitro (Figure 5I). Like Mer, Ex has a FERM domain in the N-terminal region. Schip1

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specifically bound to the N-terminal region of Ex, which contains the FERM domain (Figure 5J). Furthermore, coIP assays showed that Ex co-immunoprecipitates with Schip1 (Figure 5K). Taken together, although hSCHIP1 was initially identified as a Merbinding partner, our data provide strong evidence for genetic interaction, co-localization, and physical interaction between Schip1 and Ex. The interactions between Schip1 and Ex suggest that Ex might be involved in the localization of Schip1. Thus, we examined whether the pattern of Schip1 localization in the wing is affected by ex RNAi. Expressing ex RNAi using ptc-Gal4 reduced the amount of apical Schip1 (Figure 5L), and caused Schip1 to mislocalize and accumulate in the basal region (Figure 5M). Reducing Ex in the posterior compartment, by using en-Gal4, also caused Schip1 to mislocalize to the basal region (Figure 5N). We also asked whether overexpressing Ex, by en-Gal4, could recruit Schip1 protein to the apical region. Examination of z sections indicated that the level of Schip in the posterior basal region did not change significantly, while its level in apical sections increased in comparison with the anterior control region. This suggests that Ex overexpression increases the total level of Schip1, with the upregulated Schip1 mostly recruited to the apical region (Figure 5O). Therefore, Ex seems to be both necessary and sufficient for proper apical localization of Schip1. Schip1 Is Required for Promoting Hpo Phosphorylation Our data thus far suggest that Schip1 localization is regulated by Ex, while loss of Schip1 can be suppressed by Wts overexpression. Thus, we reasoned that Schip1 might be involved in the regulation of Hpo function at a step between Ex and Wts. To test this idea, we examined whether Schip1 can regulate the activity of Hpo. Hpo activity depends on its state of phosphorylation (Boggiano et al., 2011). To determine the effects of Schip1 mutation on the phosphorylation level of Hpo in vivo, we analyzed protein extracts from wing discs containing Schip149 mutant clones. We used a heat-shock condition in which about 80% of wing disc samples showed large mutant clones that covered 50% or more of the disc area. For comparison, protein extracts from wing discs containing wild-type GFP-negative clones were used as controls. Protein extracts from these experimental and control groups of wing discs were transferred onto Western blots for immunostaining. As shown in Figures 6A and 6A0 , the level of phosphorylated Hpo was significantly reduced in wing discs containing Schip149 mutant clones. In contrast, wing discs with Schip149 clones showed a higher level of unphosphorylated Hpo protein (Figures 6A and 6A00 ). Because wing discs with Schip149 clones also contain wild-type cells, the actual difference in the phosphorylation level between wildtype and Schip149 mutant cells is likely to be even larger than that shown in Figures 6A–6A00 . We also tested whether Schip1 overexpression can affect Hpo phosphorylation. Overexpression of Schip1 in the posterior compartment of wing disc by en-Gal4 clearly increased the level of phosphorylated Hpo while decreasing the amount of unphosphorylated Hpo (Figures 6B–6B00 ). It has been shown that Hpo must translocate from the cytoplasm to the apical membrane region to be activated by phosphorylation (Deng et al., 2013; Ho et al., 2010). Because Schip1 promotes Hpo phosphorylation, we expect that gain or loss of

Schip1 will alter the subcellular distribution of Hpo. To address this possibility, we performed a c100p100 centrifugation assay (Yin et al., 2013) to measure the relative distribution of Hpo in the membrane and cytosolic fractions. In normal S2 cells, the majority of Hpo was located in the cytosolic fraction. This was true even in the presence of overexpressed Sav, a scaffold protein involved in anchoring of Hpo to the cell membrane (Figure 6C). Overexpression of Schip1 resulted in translocation of most cytosolic Hpo to the membrane fraction, which is consistent with enhanced Hpo phosphorylation. Conversely, knockdown of Schip1 increased the level of cytosolic Hpo without significantly changing the total level of Hpo. To obtain additional evidence for the translocation of Hpo by Schip1 in vivo, we examined the subcellular localization of Hpo in imaginal discs. Because the monoclonal Hpo/MST antibody utilized for the Western blot analysis above (Figure 6B) did not work well for immunocytochemistry, we used an Hpo polyclonal antibody. With this antibody, we observed higher levels of cytoplasmic Hpo protein in Schip149 mutant clones than in wild-type cells within the eyes (Figures 6D–D00 ) and wing discs (Figures 6E– 6E00 ). In most Schip1 mutant cells, Hpo immunostaining was strongly enhanced in the entire intracellular region, whereas wild-type cells showed relatively weaker staining near the cell membrane (Figures 6F and 6F0 ). To define the apical basal pattern of Hpo localization, we examined z-stack images of immunostained wing discs. As Figures 6G and 6G0 show, Hpo in wild-type cells was mainly localized to the apical region. However, Schip149 mutant cells showed strong Hpo staining in both apical and basal regions. Taken together, these results support the conclusion that Schip1 is required for Hpo activity by promoting phosphorylation and proper localization of Hpo. Schip1 Directly Promotes Tao-1 Kinase Activity Our data indicate that Schip1 is required to enhance Hpo phosphorylation. Because Schip1 protein does not have a kinase domain, it could affect Hpo phosphorylation by interacting with another kinase that then regulates Hpo. We hypothesized that Tao-1 could serve as such a kinase, because Tao-1 is a protein kinase that is known to directly phosphorylate Hpo (Boggiano et al., 2011; Poon et al., 2011). To our knowledge, phosphoHpo has been detected only in Western blots and not in tissue staining. Therefore, we first used the polyclonal anti-Hpo antibody to examine the effects of Tao-1 on Hpo phosphorylation in vivo. Interestingly, overexpression of Tao-1 kinase, by enGal4, strongly reduced Hpo staining in the posterior compartment of the wing disc (Figure 7A). Because the total Hpo level in S2 cells did not change significantly in response to Tao-1 overexpression (Myc-Hpo levels in Figure 7H), reduced Hpo staining by Tao-1 overexpression might be due to a higher specificity of the anti-Hpo antibody to unphosphorylated Hpo. However, an alternative possibility, which might be more likely, is that phospho-Hpo is less stable, thus reducing the total level of Hpo in vivo when Hpo is phosphorylated in response to Tao-1 overexpression. We also found that Schip1 overexpression decreased Hpo staining (Figure 7B). Because Schip1 overexpression strongly increases the level of phospho-Hpo on Western blot (Figure 6B), the decreased Hpo staining in wing disc caused by Schip1 seems to be due to a reduction in total Hpo levels. On the contrary, knockdown of Schip1 significantly

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Figure 6. Schip1 Is Required for Hpo Activity by Promoting Phosphorylation and Proper Localization of Hpo (A–A00 ) Loss of Schip1 decreases the phosphorylated Hippo (P-Hpo) level but increases the level of Hpo. (A) Anti-Hpo staining of Western blot of protein extracts from wing discs containing Schip149 mutant clones. (A0 ) Quantification of relative level of P-Hpo band intensity (n = 5). (A00 ) Quantification of relative Hpo band intensity for Schip1+ clones versus Schip149 mutant clones (n = 5). Error bars represent ±SEM. ***p < 0.001; 0.001 < **p < 0.01. (B–B00 ) Overexpression of Schip1 increases the P-Hpo level but decreases the level of Hpo. (B0 ) Quantification of relative level of P-Hpo band intensity (n = 5). (B00 ) Quantification of relative Hpo band intensity for Schip1+ clones versus Schip149 mutant clones (n = 5). Error bars represent ±SEM. ***p < 0.001; 0.001 < **p < 0.01. (C) c100p100 centrifugation assay. Schip1 overexpression increases the Hpo level in the membrane fraction. Schip1 RNAi shows the opposite effects. (D–G0 ) Immunostaining of Hpo in Schip149 mutant clones of developing eye and wing imaginal disc. (D–D00 ) Hpo staining in Schip149 mutant clones in eye disc. (D) Merge, (D0 ) GFP, (D00 ) Hpo. (E–E00 ) Hpo staining in Schip149 mutant clones in wing disc. (E) Merge, (E0 ) GFP, (E00 ) Hpo. (F and F0 ) High magnification of Schip149 mutant clones (the boxed area in E). (F) Merge, (F0 ) Hpo. (G and G0 ) Z-stack image of Hpo staining in the Schip149 mutant clone in eye disc (G) and wing disc (G0 ). Scale bars, 20 mm.

elevated the level of Hpo staining, which is consistent with the Western blot results shown in Figure 6A as well as the increased cytoplasmic staining in Schip1 mutant clones (Figures 6D–6G). We then addressed whether Tao-1 kinase is essential for Schip1 function. Remarkably, the upregulation of Hpo staining by Schip1 RNAi mostly reverted to the normal level by Tao-1 overexpression (Figure 7D). Furthermore, overexpression of Tao-1 was sufficient to rescue wing structure back to normal

(Figures S7A and S7D). Likewise, upregulation of Sav and Crb caused by Schip1 RNAi was suppressed by Tao-1 overexpression (Figures S7A, S7B, S7D, and S7E). In contrast, overexpression of a kinase-dead mutant form of Tao-1 (Tao-1KD) failed to suppress the defects (Figures S7E, S8C, and S8F). We also tested whether Schip1 overexpression can suppress the effects of tao-1 RNAi. Because Tao-1 knockdown by en-Gal4 caused pupal lethality, as reported earlier (Poon et al., 2011), we

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Figure 7. Tao-1 Kinase Functions Downstream of Schip1 through Physical Association (A–E) Effects of Tao-1 and Schip1 on Hpo expression in wing disc. GFP staining indicates the posterior compartment by en>Gal4. (A) Reduced Hpo staining by en>Tao-1 Flag. (B) Reduced Hpo staining by en>Schip1. (C) Enhanced Hpo staining by en>Schip1 RNAi. (D) Tao-1 overexpression suppresses the effects of Schip1 RNAi in en>Schip1 RNAi>Tao-1 Flag (E) CoIP between Schip1 and Tao-1. (F) GST pull-down between MBP-Tao-1 and GST-Schip1. (G) c100p100 centrifugation assay. Schip1 increases Tao-1 level in membrane fraction. (H) Schip1 promotes the kinase activity of Tao-1 in S2 cell. Scale bars, 20 mm (A–D).

examined Ex levels in larval wing discs as readout. tao-1 RNAi by en-Gal4 significantly increased the Ex level in the posterior wing compartment as expected (Figure S7F). However, Schip1 overexpression did not suppress this tao-1 RNAi effect (Figure S7G). Therefore, Tao-1 overexpression can rescue Schip1 defects but not vice versa. Furthermore, coIP and GST pull-down experiments showed that Schip1 physically associates with Tao-1 in a protein complex (Figures 7E and 7F). Because knockdown of Schip1 increased the level of membrane-associated Hpo, we carried out a similar c100p100 assay to examine the localization of Tao-1. In normal control S2 cells, Tao-1 was mostly detected in the cytosol fraction (Figure 7G). Consistent with Hpo, overexpression of Schip1 led to a strong enrichment of Tao-1 in the membrane fraction, while Tao-1 stayed in the cytosolic fraction in Schip1-depleted cells. To verify that Schip1 regulates the activity of Tao-1, we examined the effects of Schip1 on the level of phosphorylation of Hpo residue T195, which is known to be a target for Tao-1 kinase (Boggiano et al., 2011). Overexpression of Tao-1 alone weakly increased the phosphorylated Hpo level compared with the control level without Tao-1. Similarly, Schip1 overexpression slightly increased the amount of phospho-Hpo. However, when both Schip1 and Tao-1 were co-expressed, the level of phospho-Hpo was greatly enhanced (Figure 7H). These data suggest that Schip1 directly interacts with Tao-1 kinase to promote Hpo phosphorylation.

DISCUSSION Schip1 Is a Member of the Hpo Signaling Pathway Hpo, Sav, and Wts form a core complex in the Hpo signaling pathway to suppress the activity of Yki. Hpo activity is regulated by cell membrane-associated upstream factors, including Crb, Ex, and Mer. However, a direct linkage between these upstream factors and Hpo had not been made previously. In this study, we have identified Schip1, a FERM-interacting protein, as a linker between Ex and Tao-1 kinase in the Hpo signaling pathway. A multitude of evidence indicates that Schip1, as a new component of Hpo signaling, is required to suppress organ growth. Reduction or loss of Schip1 results in tissue overgrowth along with increased DNA synthesis and cell proliferation. Furthermore, loss of Schip1 induces upregulation of Yki target genes such as cycE and diap1. Hpo signaling factors, such as Ex and Crb, are transcriptionally induced by a positive-feedback effect of Yki activity (Genevet et al., 2009; Hamaratoglu et al., 2006). Our data demonstrate clear upregulation of Ex and Crb levels in response to reducing Schip1. We also showed that the Crb upregulation caused by Schip1 RNAi is restored by reducing Yki, indicating that the increased Crb level was due to Yki activation by reduced Schip1. Yki activity normally depends on the subcellular localization of Yki protein, rather than the level of Yki expression (Oh and Irvine, 2008, 2009). Our data indicate that loss of Schip1 alters the localization of Yki from the

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wing disc epithelium. The reduced eye phenotype caused by ex RNAi can be strongly suppressed by Schip1 overexpression. Hence, the function of Ex seems to be largely mediated by Schip1.

Figure 8. Proposed Roles of Schip1 in Hippo Signaling Several Hpo signaling components relevant to this study are shown schematically. Hpo together with Sav activates Wts. Activated Wts inhibits Yki activity by excluding Yki from the nucleus. Ex is associated with apical cell membrane. For active Hpo signaling, Ex directly interacts with Schip1 and recruits it to the apical membrane. Schip1 recruits Tao-1, thus leading to Hpo phosphorylation. Ex can also interact with Wts (Sun et al., 2015). It is unknown whether Schip1 regulates Ex-Wts interaction (question mark). Mer genetically acts upstream to Hpo but it can also function in a parallel pathway by directly recruiting Wts to the plasma membrane (Yin et al., 2013). Schip1 can bind Mer, and may also function in this parallel pathway (dotted arrow; see Discussion for details).

cytoplasm to a broader region that includes the nucleus. We also demonstrated that Schip1 overexpression can increase the level of phosphorylated Yki without altering the total amount. These results suggest that Schip1 inhibits Yki signaling by the phosphorylation-dependent retention of Yki in the cytoplasm. More direct evidence for the role of Schip1 in Hpo signaling was provided by the inhibition of Yki transcriptional activity by Schip1 in a luciferase assay. Interaction of Ex and Schip1 The inhibitory effects of Schip1 on Yki activity are similar to the function of Hpo and Wts, suggesting that these factors participate in the same (or related) pathways. Strong suppression of the Schip1 RNAi phenotype by Wts overexpression implies that Schip1 is likely to function upstream of Wts, although these two proteins could also function in a parallel pathway. Interestingly, our data show that Schip1 is also required for proper localization of Hpo (Figure 6C) and its phosphorylation (Figures 6A and 6B). Hence, Schip1 seems to positively regulate the activity of the Hpo-Wts cascade. Our finding that reduced Ex causes basal mislocalization of Schip1 provides evidence that this function of Schip1 depends on Ex, at least in part. Furthermore, overexpression of Ex leads to increased Schip1 in the apical region of

Schip1 Links Ex and Tao-1 Kinase Overexpression of Wts is sufficient to rescue the increased organ growth induced by knockdown of Schip1 (Figures 4B0 and 4C0 ). Our data also indicate that Schip1 is necessary for activation of Hpo kinase. The reduced Hpo phosphorylation evident in Schip1 mutant clones is also consistent with altered subcellular localization of Hpo. These data, together with the genetic and physical interaction between Schip1 and Ex, imply that Schip1 provides a link between Ex and Hpo. Recent findings that Hpo is a direct substrate for Tao-1 protein kinase raised the possibility that Schip1 might regulate Tao-1. Indeed, the rescue of the Schip1 RNAi phenotype by Tao-1 (Figures 7C and 7D) and the physical interaction between Tao-1 and Schip1 (Figures 7E and 7F) strongly suggest that Schip1 directly affects Tao-1 function. Furthermore, we showed that Schip1 can induce the localization of Tao-1 to the membrane, as it does for Hpo. Thus, Schip1dependent translocation of Tao-1 appears to be an important event in Hpo activation. In support of this, our data demonstrate that Tao-1 enhances the level of Hpo phosphorylation in the presence of Schip1. Hpo is a kinase, within the Mst/Ste20 family, that regulates the HSW pathway. Interestingly, Tao-1 is another Ste20 family kinase that also phosphorylates the critical residue (T195) of Hpo. It has been found that Tao-1 fails to form a complex with Ex-Mer, and that depletion of Ex-Mer has no effect on Wts activation by Tao-1. Nonetheless, Wts phosphorylation induced by Ex and Mer expression can be substantially inhibited by Tao-1 knockdown. Based on these findings, it has been proposed that Ex-Mer and Tao-1 cooperate to activate Hpo phosphorylation, perhaps through an unknown transmembrane receptor whose localization or activity is regulated by Ex-Mer (Boggiano et al., 2011). However, our findings lead us to propose that Ex is linked to Tao-1 through a direct mutual interaction with Schip1. In this model (Figure 8), we suggest that Ex is involved in localizing Schip1 to the plasma membrane. Schip1 not only binds to Ex but also physically interacts with Tao-1, resulting in translocation to the membrane and activation of its kinase activity toward Hpo. Furthermore, strong suppression of the Schip1 knockdown phenotype by Tao-1 (Figures 7D and S7) and Hpo (not shown) also supports the conclusion that Schip1 functions through Tao-1 and its target Hpo. A recent study has shown that Hpo pathway activation causes Wts to relocate from adherens junctions to more apical regions to co-localize with Ex. Furthermore, the Wts-Ex co-localization is due to a physical interaction between these two proteins, leading to the proposal that Ex is a scaffold linking Hpo to Wts (Sun et al., 2015). Our study suggests that Schip1 acts as a similar linker to recruit Tao-1 and Hpo to Ex, thereby activating Hpo. It is unknown whether Schip1 might also participate in relocalization of Wts to form a complex with Ex. Only double mutations of both Ex and Mer genes result in significant tissue overgrowth, suggesting that Ex and Mer have redundant tumor-suppressor functions in Hpo signaling (Hamaratoglu et al., 2006; McCartney et al., 2000). Mer and Ex also

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cooperatively modulate endocytic trafficking of cell surface receptors involved in multiple growth signaling pathways (Maitra et al., 2006). Although Ex, Mer, and Kibra form a protein complex, genetic studies suggest that they have different levels of functional contributions in different developmental contexts (Meignin et al., 2007; Pellock et al., 2007; Polesello and Tapon, 2007; Yu et al., 2008, 2010). For instance, reduction of Ex in eye or wing leads to significant growth defects, whereas similar reduction of Mer has very subtle effects in those organs (Milton et al., 2010; Pellock et al., 2007). In contrast, Mer is essential in follicle cells to maintain cell polarity and limit proliferation in the oocyte (MacDougall et al., 2001; Pellock et al., 2007; Yu et al., 2008, 2010). In imaginal discs, loss of Ex is sufficient to induce Yki target gene expression, while mer or kibra mutations are not. These studies, as well as our data, suggest that Ex plays a more critical role than Mer in growth control of imaginal discs. We also noted that unlike ex RNAi, mer RNAi does not significantly affect the size of eyes or wings. However, mer RNAi causes an expansion of head tissues (Figure S8G). This head phenotype of mer RNAi was partially suppressed by Schip1 overexpression (Figure S8G), suggesting that Schip1 genetically interacts with mer in head tissues. Mer can function independent of Hpo by directly regulating the localization of Wts (Yin et al., 2013). Therefore, it is possible that Mer and Schip1 might function in head development in a parallel Mer-Wts pathway. Due to the function of Mer in dual pathways and its context dependence, detailed understanding of the mechanisms for the role of the Mer-Schip1 interaction in other tissues and organs will require future study. Schip1 Is a SCHIP1-Related Protein with Distinct Properties The Schip1 protein sequence is related to a family of mammalian SCHIP1-related proteins. Drosophila Schip1 and SCHIP1 family proteins share conserved domains, such as a serine-rich region and the FEZ-like domain found in mammalian FEZ proteins and Caenorhabditis elegans Unc76 (Goutebroze et al., 2000). However, there are also regions that do not show significant sequence similarity. For example, Schip1 has an N-terminal region of about 130 aa that is absent in human SCHIP1. Some Ser-rich regions, Asp/Glu regions, and the PEST motif shared among vertebrate species are not conserved in Schip1. The FEZ-like coiled-coil domain in the C-terminal part of Schip1 and SCHIP1 has a conserved leucine zipper pattern (Goutebroze et al., 2000). FEZ proteins and Unc76 are known to be involved in axonal outgrowth and fasciculation (Bloom and Horvitz, 1997), but it is unknown whether they play any role in tissue growth. An intriguing property of human SCHIP1 is that it co-immunoprecipitates with specific isoforms or mutant forms of NF2/ Merlin, but not with the wild-type protein. Hence, it has been suggested that SCHIP1 interacts only with specific forms of NF2 altered by posttranslational modification, splicing, or mutations (Goutebroze et al., 2000). In contrast, our data show that Schip1 can form a complex with wild-type Mer protein. We have also shown that Schip1 can bind not only to Mer but also to Ex, and that its interaction with Ex is crucial for its regulation of Hpo. In mammalian systems, Willin/Ex1/FRMD6 is known to be the homolog of Drosophila Ex (Gunn-Moore et al., 2005). Willin expression can increase the phosphorylation of MST1/2, LATS2, and

YAP (Angus et al., 2012), implying that Willin can regulate the Hpo pathway by activating its core components. Furthermore, TAOK1/3 can directly phosphorylate MST1/2 (Boggiano et al., 2011; Poon et al., 2011). It remains to be determined whether SCHIP1 is involved in linking Willin to TAOK1/3 in regulating Hpo signaling in mammals. EXPERIMENTAL PROCEDURES In Vitro GST Pull-Down Assays For GST pull-down, isopropyl b-D-1-thiogalactopyranoside-inducible R2 cells (BL21 derivative) were transformed with plasmids for MBP-Ex, MBP-Mer, and GST-Schip1. Pull-down buffer contained 20 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 10% glycerol, 0.1% Triton X-100, 1 mM DTT, and protease inhibitor cocktail. For Western blotting, a-MBP antibody (NEB) was used as primary antibody. Western Blot Analysis of Imaginal Disc Extracts Wing imaginal discs containing Schip149 or Schip1+ control clones were dissected and homogenized in radioimmunoprecipitation assay buffer containing the complete protease inhibitor cocktail (Roche). Samples containing 20 mg of protein were electrophoresed on a 4%–12% gradient SDS-PAGE gel and transferred to Immobilon-FL polyvinylidene difluoride membranes. Quantification of Western Blots The intensity of each band was measured and normalized to a loading control using ImageJ software. SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures and eight figures and can be found with this article online at http://dx.doi. org/10.1016/j.devcel.2016.02.004. AUTHOR CONTRIBUTIONS H.-L.C. and K.-W.C designed the study, and H.-L.C performed experiments. H.-L.C., G.J.A., and K.-W.C discussed and interpreted the results. H.-L.C. and K.-W.C wrote and G.J.A. edited the manuscript. ACKNOWLEDGMENTS We are grateful to George Halder, Richard Fehon, Nic Tapon, Soon-ji Yoo, the Bloomington Drosophila Stock Center, the Vienna Drosophila RNAi Center, the Drosophila Genomics Resource Center, and the Developmental Studies Hybridoma Bank for reagents and fly stocks. We thank Kyung-Ok Cho for constructive comments on the manuscript. This research was supported by a National Research Laboratory grant (NRF-2011-0028326) and a Global Research Laboratory grant (2014K1A1A2042982) through the National Research Foundation of Korea funded by the Korean Ministry of Education Science & Technology and by the World Class Institute (WCI) Program of the National Research Foundation of Korea (WCI 2009-003). Received: July 22, 2015 Revised: January 26, 2016 Accepted: February 4, 2016 Published: March 7, 2016 REFERENCES Angus, L., Moleirinho, S., Herron, L., Sinha, A., Zhang, X., Niestrata, M., Dholakia, K., Prystowsky, M.B., Harvey, K.F., Reynolds, P.A., et al. (2012). Willin/FRMD6 expression activates the Hippo signaling pathway kinases in mammals and antagonizes oncogenic YAP. Oncogene 31, 238–250.

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