The deubiquitinase UCHL5/UCH37 positively regulates Hedgehog signaling by deubiquitinating Smoothened

The deubiquitinase UCHL5/UCH37 positively regulates Hedgehog signaling by deubiquitinating... Abstract The Hedgehog (Hh) signaling pathway plays important roles in developmental processes including pattern formation and tissue homeostasis. The seven-pass transmembrane receptor Smoothened (Smo) is the pivotal transducer in the pathway; it, and thus the pathway overall, is regulated by ubiquitin-mediated degradation, which occurs in the absence of Hh. In the presence of Hh, the ubiquitination levels of Smo are decreased, but the molecular basis for this outcome is not well understood. Here, we identify the deubiquitinase UCHL5 as a positive regulator of the Hh pathway. We provide both genetic and biochemical evidence that UCHL5 interacts with and deubiquitinates Smo, increasing stability and promoting accumulation at the cell membrane. Strikingly, we find that Hh enhances the interaction between UCHL5 and Smo, thereby stabilizing Smo. We also find that proteasome subunit RPN13, an activator of UCHL5, could enhance the effect of UCHL5 on Smo protein level. More importantly, we find that the mammalian counterpart of UCHL5, UCH37, plays the same role in the regulation of Hh signaling by modulating hSmo ubiquitination and stability. Our findings thus identify UCHL5/UCH37 as a critical regulator of Hh signaling and potential therapeutic target for cancers. Hedgehog, Smoothened, ubiquitination, UCHL5, UCH37 Introduction The Hh pathway plays vital roles in governing animal embryonic development and adult tissue homeostasis (Ingham and McMahon, 2001; Jia and Jiang, 2006; Jiang and Hui, 2008; Briscoe and Therond, 2013). The pathway sits at the cross-road of cell survival and differentiation decisions, and deregulated Hh signaling can lead to hyperproliferation and tumorigenesis (Hooper and Scott, 2005; Ingham et al., 2011). Indeed, mutations of pathway components have been implicated in numerous human disorders, including birth defects and several types of cancers (Berman et al., 2003; Ingham and Placzek, 2006). The key components and regulatory systems of the Hh signaling are conserved from Drosophila to mammals (Jiang and Hui, 2008; Wilson and Chuang, 2010). Therefore, Drosophila is an ideal model to study Hh signaling. In Drosophila wing discs, Hh proteins are produced and processed by posterior (P) compartment cells. Mature Hh proteins secreted by P cells move into the anterior (A) compartment to form a gradient. In A pathway cells near A/P boundary, Hh signaling is initiated at the cell membrane by the inactivation of the 12-span transmembrane receptor Patched (Ptc) upon binding of the Hh ligands (Jeong and McMahon, 2002; Hooper and Scott, 2005). Ptc inactivation relieves its inhibitory effect on the GPCR family protein Smoothened (Smo), leading to the activation of the unique transcription factor Cubitus interruptus (Ci) and thereby the expression of Hh target genes decapentaplegic (dpp), ptc, and engrailed (en) in a Hh-concentration-dependent manner (Lum and Beachy, 2004; Jiang and Hui, 2008). The 7-span transmembrane receptor Smo is essential for transducing Hh signal across the cell plasma membrane. In the absence of Hh, Ptc inhibits Smo activity and its accumulation on the cell membrane. The intracellular Smo protein undergoes ubiquitination, leading to endocytosis-mediated Smo degradation (Li et al., 2012; Xia et al., 2012). Upon different concentrations of Hh, Smo is differentially phosphorylated by protein kinase A (PKA) and casein kinase I (CKI) and phosphorylation antagonizes the ubiquitination of Smo, culminating in Smo accumulation on the plasma membrane (Jia et al., 2004; Apionishev et al., 2005). The activity of Smo is strictly regulated by multiple mechanisms, one of which is the ubiquitin-mediated protein modification. Loss of uba1, which encodes the unique ubiquitin-activating enzyme in Drosophila, results in Smo accumulation in the wing disc, suggesting that Smo undergoes ubiquitin-mediated degradation (Li et al., 2012). Ubiquitin fused Smo protein (Smo-Ub) predominantly localizes in Rab7-labeled late endosomes, indicating that ubiquitination also alters the localization of Smo (Xia et al., 2012). Hh treatment attenuates Smo ubiquitination and promotes its cell surface accumulation (Li et al., 2012; Xia et al., 2012). Ubiquitination is an enzymatic process by which the substrate is covalently modified with the 76 amino acid protein named ubiquitin (Ub) (Hochstrasser, 1995). Ubiquitination has been important not only in the destabilization of proteins, but also in the regulation of protein functions, including protein localization and protein–protein interaction (Hicke, 2001; Weissman, 2001). Like other protein modifications, the process of ubiquitination is also reversible after the identification of many deubiquitinating enzymes (DUBs), which counteract the ubiquitination process by removing Ub conjugates from target proteins (Wilkinson, 2000). DUBs are highly conserved proteases that catalyze the cleavage of the isopeptide bond between ubiquitin and the target protein. Due to distinct catalytic domains, DUBs are classified into five subfamilies: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), otubain proteases (OTUs), JAB1/MPN/Mov34 domain proteases (JAMMs) and Machado-Joseph disease domain proteases (MJDs) (Nijman et al., 2005; Zhang et al., 2012). UCHL5 (also known as UCH37) is a 37 kDa deubiquitinase composed of an N-terminal UCH domain and a C-terminal extension domain (Jiao et al., 2014). UCHL5 was first identified as a component of the proteasome that is thought to be involved in the cleavage of polyubiquitin chains from ubiquitinated substrates (Lam et al., 1997). Increasing substrates of UCHL5 have been identified, including type I TGF-β receptor (TGF-βRI) (Wicks et al., 2005), and E2 promoter binding factor 1 (E2F1) (Mahanic et al., 2015), underscoring that UCHL5 plays versatile roles in multiple cellular processes. The full-length UCHL5 exhibits low deubiquitinating activity through autoinhibition mediated by the C-terminal oligomerization, while the inhibitory effect is alleviated by Rpn13-UCHL5 interaction (Yao et al., 2006, 2008; Jiao et al., 2014). Thus, Rpn13 strongly stimulates UCHL5 deubiquitinating activity (Chen and Walters, 2015; VanderLinden et al., 2015). Given that the Smo undergoes ubiquitination-medated destabilization, it is fruitful to find the deubiquitinase of Smo (Shi et al., 2013; Yang et al., 2013). Although Usp8 is identified as a deubiquitinating enzyme of Smo, its interaction with Smo is not regulated by Hh signaling, suggesting that Hh-mediated Smo stabilization is probably not through Usp8 (Li et al., 2012). Therefore, the underlying mechanism of Hh inhibiting Smo ubiquitination is still unknown. In this study, we identified UCHL5 as a positive modulator of Hh pathway through stabilizing Smo. We also provided evidence that UCHL5 binds to Smo and recruits Rpn13 to form a trimetric complex that decreases Smo ubiquitination and promotes the cell surface accumulation of Smo. In addition, we found that Hh promotes the interaction between UCHL5 and Smo, and that UCHL5 is required for Hh-induced Smo deubiquitination and cell surface accumulation. Moreover, the mammalian counterpart of UCHL5, UCH37, plays a conserved role in the regulation of Hh signaling. Results Knockdown of UCHL5 attenuates Hh signaling in Drosophila The adult Drosophila wing is formed by an epithelial sheet, named wing disc. A large number of genes, including many Hh pathway components, have previously been shown to be required for differentiation of wings (Ingham and McMahon, 2001). Hh stabilizes Ci in A/P boundary of wing disc to control Hh target gene expression, which finally determines the space between vein L3 and L4 (Jia et al., 2010; Mao et al., 2014). In our previous published paper, we carried out a modified genetic screening and identified that knockdown of UCHL5 narrowed the width between vein 3 and vein 4 in wings expressing a dominant negative Smo (Smo–PKA), provides a sensitive background of Hh pathway (Zhou et al., 2015b). We confirmed the screening result and found that knockdown of UCHL5 using UCHL5-RNAi (35433) from Bloomington Stock Center (BSC) indeed resulted in a narrow L3/L4 intervein (Figure 1A and B). Given that the space between vein 3 and vein 4 is a characteristic monitor of Hh pathway activity in adult wings, knockdown of UCHL5 narrowing the space indicates that UCHL5 may positively regulate Hh pathway. Figure 1 View largeDownload slide Knockdown of UCHL5 represses Hh signaling. All wing imaginal discs shown in this study were oriented with anterior to the left and ventral up. (A and B) Comparison of adult wing phenotypes between control (A) and UCHL5 knockdown flies (B). Arrows indicate the space between vein 3 and vein 4. (C and D) The expression pattern of UCHL5 in wing discs was determined by in situ hybridization with DIG-labeled mRNA probe against UCHL5. The sense probe acts as negative control (C). Of note, UCHL5 ubiquitously expresses in wing discs. (E–H”) Knockdown of UCHL5 by MS1096-Gal4 under the background of Smo–PKA attenuated the expression of Ptc (compare F–F” with E–E”) and ptc-lacZ. Arrows indicate the decrease of Ptc and ptc-lacZ. (I–I”) UAS-GFP (green) marks the apG4-mediated gene expression pattern. apG4 drives UAS transgenes to be specifically expressed in the dorsal region of wing discs. (J–J”) Knockdown of UCHL5 by apG4 attenuated the expression of ptc-lacZ (arrow). (K–L”) Knockdown of UCHL5 with apG4 attenuated the expression of En (compare L–L” with K–K”). Figure 1 View largeDownload slide Knockdown of UCHL5 represses Hh signaling. All wing imaginal discs shown in this study were oriented with anterior to the left and ventral up. (A and B) Comparison of adult wing phenotypes between control (A) and UCHL5 knockdown flies (B). Arrows indicate the space between vein 3 and vein 4. (C and D) The expression pattern of UCHL5 in wing discs was determined by in situ hybridization with DIG-labeled mRNA probe against UCHL5. The sense probe acts as negative control (C). Of note, UCHL5 ubiquitously expresses in wing discs. (E–H”) Knockdown of UCHL5 by MS1096-Gal4 under the background of Smo–PKA attenuated the expression of Ptc (compare F–F” with E–E”) and ptc-lacZ. Arrows indicate the decrease of Ptc and ptc-lacZ. (I–I”) UAS-GFP (green) marks the apG4-mediated gene expression pattern. apG4 drives UAS transgenes to be specifically expressed in the dorsal region of wing discs. (J–J”) Knockdown of UCHL5 by apG4 attenuated the expression of ptc-lacZ (arrow). (K–L”) Knockdown of UCHL5 with apG4 attenuated the expression of En (compare L–L” with K–K”). At first, we tested the expression pattern of UCHL5 in wing via in situ hybridization and found that UCHL5 ubiquitiously expressed throughout the wing disc (Figure 1C and D). To confirm whether UCHL5 regulates Hh pathway, we employed immunostaining to test the expression of Hh target genes. Compared with control discs (Figure 1E–E” and G–G”), knockdown of UCHL5 by MS1096-Gal4 apparently decreased Ptc (Figure 1F–F”) and ptc-lacZ (Figure 1H–H”) levels under Smo–PKA background. In addition, we used a stronger gal4 driver ap-Gal4 (apG4) to knockdown UCHL5 in the dorsal region of wing discs and found that knockdown of UCHL5 compromised ptc-lacZ (compare Figure 1J–J” with I–I”) and En (compare Figure 1L–L” with K–K”). The decreased Hh signaling induced by UCHL5 knockdown was unlikely due to an off-target effect because expression of two different RNAi lines (V-34618 from VDRC and 3431R-1 from NIG) targeting nonoverlapping regions of a UCHL5 sequence produced a similar phenotype (Supplementary Figure S1). Taken together, these results suggest that UCHL5 is a positive regulator of Hh pathway. To determine the specificity of UCHL5, we tested whether UCHL5 is involved in modulating other important signaling pathways, such as Hippo, Notch and Wingless pathways. Compared with the control discs (Supplementary Figure S2A–A”, C–C”, and E–E”), knockdown of UCHL5 did not affect the expression of Hippo pathway target gene ex-lacZ (Supplementary Figure S2B–B”), Notch pathway target gene cut (Supplementary Figure S2D–D”), and Wingless pathway target gene vg (Supplementary Figure S2F–F”). UCHL5 binds to and stabilizes Smo Given that the deubiquitinase plays its roles always through recognizing and deubiquitinating substrates (Nijman et al., 2005; Huang and Cochran, 2013), we speculated that interaction with a component of Hh pathway is essential for UCHL5 regulating Hh signaling. To test this possibility, co-IP experiments were carried out in S2 cells. UCHL5 exclusively interacted with Smo (Figure 2A and B), not with other components including Ci (Supplementary Figure S3A), Fu (Supplementary Figure S3B), Cos2 (Supplementary Figure S3C) and Sufu (Supplementary Figure S3D). Figure 2 View largeDownload slide UCHL5 binds to and stabilizes Smo. (A and B) HA-UCHL5 interacted with Myc-Smo (A) and endogenous Smo (B) in S2 cells. (C–D”) Knockdown of UCHL5 with apG4 decreased the protein level of Smo (compare D–D” with C–C”). (E) Relative mRNA levels of UCHL5 from hetero and homo UCHL5j2B8 larval. Of note, UCHL5j2B8 mutant showed residual UCHL5 expression. (F–G”) Wing discs carrying UCHL5j2B8 clones were immunostained to show the expression of GFP (green) and Smo (white) at low (F–F”) and high (G–G”) magnifications. UCHL5j2B8 clones are recognized by the lack of GFP. Of note, Smo is decreased in UCHL5 mutant cells. (H) UCHL5 could hamper exogenous Smo degradation. The results were presented as mean ± SD of values from three independent experiments. Of note, proteins loaded had been adjusted such that the amounts of Smo at T = 0 were equivalent. Figure 2 View largeDownload slide UCHL5 binds to and stabilizes Smo. (A and B) HA-UCHL5 interacted with Myc-Smo (A) and endogenous Smo (B) in S2 cells. (C–D”) Knockdown of UCHL5 with apG4 decreased the protein level of Smo (compare D–D” with C–C”). (E) Relative mRNA levels of UCHL5 from hetero and homo UCHL5j2B8 larval. Of note, UCHL5j2B8 mutant showed residual UCHL5 expression. (F–G”) Wing discs carrying UCHL5j2B8 clones were immunostained to show the expression of GFP (green) and Smo (white) at low (F–F”) and high (G–G”) magnifications. UCHL5j2B8 clones are recognized by the lack of GFP. Of note, Smo is decreased in UCHL5 mutant cells. (H) UCHL5 could hamper exogenous Smo degradation. The results were presented as mean ± SD of values from three independent experiments. Of note, proteins loaded had been adjusted such that the amounts of Smo at T = 0 were equivalent. The binding between UCHL5 and Smo indicates that UCHL5 possibly modulates Hh signaling through Smo. Compared with the control disc (Figure 2C–C”), knockdown of UCHL5 indeed decreased Smo protein level (Figure 2D–D”). To confirm this result, a hypomorphic allele of UCHL5, UCHL5j2B8, was employed to generate mutant clones. The RT-PCR result proved that UCHL5j2B8 was a moderate, not a strong mutant allele (Figure 2E). We found that the Smo level was decreased in UCHL5j2B8 mutant cells, which were marked by the lack of GFP expression (Figure 2F–G”). However, the decrease of Smo in UCHL5j2B8 clones was clear, but not severe, probably because of residual UCHL5 expression. The main function of deubiquitinase is to remove the ubiquitin chains from the substrate, culminating in preventing proteasome-mediated substrate proteolysis. The interaction of UCHL5 and Smo indicates that Smo acts as a potential target of UCHL5. To test this possibility, we measured Smo protein stability in S2 cells treated with Cycloheximide (CHX) to block protein synthesis. The results showed that overexpression of UCHL5 significantly prevented Smo degradation (Figure 2H). Collectively, UCHL5 positively regulates Hh signaling through binding to and stabilizing Smo. UCHL5 binds to the SAID domain of Smo via its N-terminal region UCHL5 is comprised of an N-terminal UCH domain and C-terminal extension region (Figure 3A). To map which domain of UCHL5 is essential for its binding with Smo, we generated Fg-UCHL5-N and Fg-UCHL5-C truncated constructs (Figure 3A). Through co-IP experiments, we found the N-terminally located UCH domain is both necessary and sufficient to mediate Smo binding (Figure 3C). Figure 3 View largeDownload slide UCHL5 binds to the SAID domain of Smo through its N-terminal UCH fragment. (A and B) Schematic drawings show the domains or motifs in UCHL5 and Smo and their truncated fragments used in subsequent co-IP assay. (A) Red and blue bars represent N-terminal UCH domain and C-terminal extension region of UCHL5, respectively. (C) UCHL5 interacts with Smo through its N-terminus in S2 cells. (D and E) UCHL5 binds with the C-terminus of Smo (compare E with D). (F) SAID domain-deleted form of Smo did not bind to UCHL5 in S2 cells. (G) Fg-UCHL5 associated with Myc-SAID in S2 cells. (H and I) Hh-conditioned medium treatment promoted UCHL5 binding with Myc-Smo (H) and Smo (I) in S2 cells (mean ± SD from three independent experiments; ***P≤ 0.005, student’s t test). (J) Hh-conditioned medium treatment promoted UCHL5-N binding with Myc-Smo in S2 cells (mean ± SD from three independent experiments; ***P ≤ 0.005, student’s t test). Figure 3 View largeDownload slide UCHL5 binds to the SAID domain of Smo through its N-terminal UCH fragment. (A and B) Schematic drawings show the domains or motifs in UCHL5 and Smo and their truncated fragments used in subsequent co-IP assay. (A) Red and blue bars represent N-terminal UCH domain and C-terminal extension region of UCHL5, respectively. (C) UCHL5 interacts with Smo through its N-terminus in S2 cells. (D and E) UCHL5 binds with the C-terminus of Smo (compare E with D). (F) SAID domain-deleted form of Smo did not bind to UCHL5 in S2 cells. (G) Fg-UCHL5 associated with Myc-SAID in S2 cells. (H and I) Hh-conditioned medium treatment promoted UCHL5 binding with Myc-Smo (H) and Smo (I) in S2 cells (mean ± SD from three independent experiments; ***P≤ 0.005, student’s t test). (J) Hh-conditioned medium treatment promoted UCHL5-N binding with Myc-Smo in S2 cells (mean ± SD from three independent experiments; ***P ≤ 0.005, student’s t test). To determine which region in Smo is responsible for binding to UCHL5, various Myc-Smo truncated mutants (Figure 3B) were transfected into S2 cells with HA-UCHL5, followed by co-IP assays. As shown in Figure 3D and E, Smo interacted with UCHL5 using its C-terminal fragment. Previous studies have demonstrated that the C-terminally located Smo auto-inhibitory domain (SAID) plays an important role in regulating Smo activity and ubiquitination (Zhao et al., 2007; Li et al., 2012). To test whether the SAID domain mediates Smo binding to UCHL5, we generated SAID-deleted Smo construct (Myc-Δ SAID). The co-IP results showed that Myc-ΔSAID failed to bind to UCHL5 (Figure 3F), while the SAID domain could interact with UCHL5 (Figure 3G). Taken together, UCHL5 interacts with the SAID domain of Smo using its N-terminal UCH fragment. The ubiquitination status of Smo is tightly controlled by the Hh pathway (Zhao et al., 2007; Li et al., 2012; Xia et al., 2012). In the presence of Hh, the ubiquitination of Smo is apparently suppressed (Li et al., 2012). Hh might prevent Smo ubiquitination by regulating UCHL5. However, Hh did not affect the expression of UCHL5 because in situ hybridization showed that UCHL5 expressed evenly in the wing disc (Figure 1C and D). The finding that UCHL5 interacted with Smo led to the hypothesis that Hh might govern the affinity between UCHL5 and Smo. To test this, we treated the S2 cells with conditional Hh medium, followed by co-IP experiment. The result revealed that Hh promoted UCHL5–Smo and UCHL5–N–Smo interactions (Figure 3H–J). UCHL5 deubiquitinates Smo UCHL5 belongs to the ubiquitin carboxy-terminal hydrolase family and contains a characteristic catalytic cysteine (Cys) at the core enzymatic domain (UCH domain) (Johnston et al., 1997; Nishio et al., 2009; Maiti et al., 2011). Mutation of the Cys leads to an inactive form of the enzyme. To test whether UCHL5 regulates Hh pathway through its deubiquitinase activity, we generated a point mutant of UCHL5 with its catalytic Cys replaced by Ala (Fg-UCHL5CA). Analogous to UCHL5-RNAi, overexpression of UCHL5CA decreased the space between vein 3 and vein 4 (Figure 4A and B), suggesting that UCHL5CA plays a dominant-negative role. In addition, through adult rescue assays, we found only wild-type UCHL5 restored the phenotype induced by UCHL5-RNAi, while UCHL5CA deteriorated the wing defect (Figure 4C–E). Consistently, overexpression of UCHL5CA apparently downregulated Ptc and Smo levels (Figure 4F–G’) in wing discs and significantly promoted Smo degradation in S2 cells (Figure 4H). In sum, these observations indicate that the deubiquitinase activity is essential for UCHL5 regulating Smo. Figure 4 View largeDownload slide UCHL5 deubiquitinates Smo. (A–D) Comparison of adult wing phenotypes under the background of Smo–PKA from flies of UCHL5 knockdown (A), Fg-UCHL5CA expression (B), Fg-UCHL5 expression in UCHL5 knockdown background (C), and Fg-UCHL5CA expression in UCHL5 knockdown background (D). Arrows mark the space between vein 3 and vein 4. (E) Quantification of the adult wing phenotypes. (F–F”) A wing disc expressing UCHL5CA with MS1096 under the background of Smo–PKA was stained for Ptc (white). UCHL5CA overexpression decreased Ptc expression. (G–G’) A wing disc expressing UCHL5CA by apG4 was stained for Smo (white). UCHL5CA overexpression decreased Smo protein level (arrow). (H) UCHL5CA promoted Smo degradation. The results were presented as mean ± SD of values from three independent experiments. Proteins loaded had been adjusted such that the amounts of Smo at T = 0 were equivalent. (I) S2 cells transfected with the indicated plasmids were analyzed by western blotting. Of note, Fg-UCHL5 decreased, but Fg-UCHL5CA promoted Smo ubiquitination. (J) Knockdown of UCHL5 using UCHL5-dsRNA promoted Smo ubiquitination in S2 cells. UCHL5-dsRNA could effectively knock down UCHL5 mRNA level in S2 cells (bottom two panels). (K) Knockdown of UCHL5 blocked Hh-mediated Myc-Smo deubiquitination (compare lanes 4, 5 with lane 2). (L) UCHL5 attenuated the interaction between Smo and Hrs. (M) Fg-UCHL5 decreased Smo ubiquitination mediated by Hrs. (N–O’) Wing discs expressing Fg-Hrs alone (N–N’) or Fg-Hrs/HA-UCHL5 together (O–O’) by MS1096 were immunostained with Smo (white). UCHL5 could inhibit Hrs-mediated Smo destabilization (arrows). Figure 4 View largeDownload slide UCHL5 deubiquitinates Smo. (A–D) Comparison of adult wing phenotypes under the background of Smo–PKA from flies of UCHL5 knockdown (A), Fg-UCHL5CA expression (B), Fg-UCHL5 expression in UCHL5 knockdown background (C), and Fg-UCHL5CA expression in UCHL5 knockdown background (D). Arrows mark the space between vein 3 and vein 4. (E) Quantification of the adult wing phenotypes. (F–F”) A wing disc expressing UCHL5CA with MS1096 under the background of Smo–PKA was stained for Ptc (white). UCHL5CA overexpression decreased Ptc expression. (G–G’) A wing disc expressing UCHL5CA by apG4 was stained for Smo (white). UCHL5CA overexpression decreased Smo protein level (arrow). (H) UCHL5CA promoted Smo degradation. The results were presented as mean ± SD of values from three independent experiments. Proteins loaded had been adjusted such that the amounts of Smo at T = 0 were equivalent. (I) S2 cells transfected with the indicated plasmids were analyzed by western blotting. Of note, Fg-UCHL5 decreased, but Fg-UCHL5CA promoted Smo ubiquitination. (J) Knockdown of UCHL5 using UCHL5-dsRNA promoted Smo ubiquitination in S2 cells. UCHL5-dsRNA could effectively knock down UCHL5 mRNA level in S2 cells (bottom two panels). (K) Knockdown of UCHL5 blocked Hh-mediated Myc-Smo deubiquitination (compare lanes 4, 5 with lane 2). (L) UCHL5 attenuated the interaction between Smo and Hrs. (M) Fg-UCHL5 decreased Smo ubiquitination mediated by Hrs. (N–O’) Wing discs expressing Fg-Hrs alone (N–N’) or Fg-Hrs/HA-UCHL5 together (O–O’) by MS1096 were immunostained with Smo (white). UCHL5 could inhibit Hrs-mediated Smo destabilization (arrows). Since UCHL5 regulates Smo through deubiquitinase activity, it is necessary to test whether UCHL5 deubiquitinates Smo directly. Via cell-based ubiquitination assay (Zhou et al., 2015a, b), we found that UCHL5 decreased whereas UCHL5CA increased Smo ubiquitination (Figure 4I). Furthermore, knockdown of the endogenous UCHL5 using dsRNA promoted Smo ubiquitination (Figure 4J, top). The efficiency of UCHL5-dsRNA was confirmed by semi-quantitative PCR analysis (Figure 4J, bottom). Previous studies have demonstrated that Hh hampers Smo ubiquitination through an unclear mechanism (Li et al., 2012; Jiang and Jia, 2015). Hh enhancing UCHL5–Smo affinity provided a possibility that Hh regulates Smo through UCHL5. Knockdown of UCHL5 indeed blocked Hh-mediated Smo deubiquitination (Figure 4K), suggesting that Hh stabilizes Smo, at least in part, through UCHL5. Albeit Smo is regulated by ubiquitination, its corresponding E3 ligase remains unknown. Hrs has been reported to promote Smo ubiquitination and destabilization through binding to SAID domain of Smo (Fan et al., 2013). We found that UCHL5 inhibited Hrs-Smo interaction (Figure 4L) and Hrs-mediated Smo ubiquitination (Figure 4M). Compared with the control disc (Figure 4N–N’), UCHL5 hampered Hrs-mediated Smo degradation (Figure 4O–O’). UCHL5 regulates Smo through UCHL5−Rpn13 complex Proteasomal receptors that recognize ubiquitin chains attached to substrates are key mediators of selective protein degradation in eukaryotes. As a ubiquitin receptor, Rpn13 associates with UCHL5 to enhance its deubiquitinating activity in vivo (Qiu et al., 2006; Yao et al., 2006). Given that UCHL5 deubiquitinates Smo, it is interesting to test whether Rpn13 cooperates with UCHL5 to regulate Smo. We first knocked down Rpn13 in the wing disc and found that knockdown of Rpn13 decreased Smo level (Figure 5A–A”). In addition, immunostaining results showed that Rpn13 and UCHL5 co-localized in the cytoplasm of S2 cells (Figure 5B–B’”). We performed a two-step immunoprecipitation experiment and found that Rpn13, UCHL5 and Smo formed a trimeric complex when co-expressed in S2 cells (Figure 5C). Since UCHL5 binds to Smo through its N-terminal UCH domain, we speculated that UCHL5 recruits Rpn13 through its C-terminal fragment. Consistently, co-IP experiments revealed that only UCHL5-C, but not UCHL5-N interacted with Rpn13 (Figure 5D), suggesting that UCHL5 bridges Rpn13 and Smo via distinct regions. Figure 5 View largeDownload slide UCHL5 regulates Smo through UCHL5−Rpn13 complex. (A–A”) A wing disc expressing Rpn13-RNAi by apG4 was immunostained to show the expression of Smo (white). Of note, Rpn13 knockdown resulted in a decrease of Smo (arrow). (B–B’”) Fg-UCHL5 (green) and HA-Rpn13 (red) co-localized in the cytoplasm of S2 cells. (C) The two-step immunoprecipitation indicated UCHL5, Rpn13, and Smo in the same complex. (D) UCHL5 interacted with Rpn13 through its C-terminal region in S2 cells. (E–G) Wing discs expressing UCHL5-RNAi alone (E), Rpn13-RNAi alone (F), or UCHL5-RNAi plus Rpn13-RNAi (G) were immunostained to show Smo expression (white). Expression of UCHL5-RNAi plus Rpn13-RNAi resulted in a more dramatic decrease in Smo expression (arrows). (H) Quantification of the Smo intensity in wing discs. White bars represent the wild-type Smo fluorescence; black bars represent Smo fluorescence in the gene knockdown region (mean ± SD from three independent experiments; *P < 0.05, **P ≤ 0.01, student’s t test). Figure 5 View largeDownload slide UCHL5 regulates Smo through UCHL5−Rpn13 complex. (A–A”) A wing disc expressing Rpn13-RNAi by apG4 was immunostained to show the expression of Smo (white). Of note, Rpn13 knockdown resulted in a decrease of Smo (arrow). (B–B’”) Fg-UCHL5 (green) and HA-Rpn13 (red) co-localized in the cytoplasm of S2 cells. (C) The two-step immunoprecipitation indicated UCHL5, Rpn13, and Smo in the same complex. (D) UCHL5 interacted with Rpn13 through its C-terminal region in S2 cells. (E–G) Wing discs expressing UCHL5-RNAi alone (E), Rpn13-RNAi alone (F), or UCHL5-RNAi plus Rpn13-RNAi (G) were immunostained to show Smo expression (white). Expression of UCHL5-RNAi plus Rpn13-RNAi resulted in a more dramatic decrease in Smo expression (arrows). (H) Quantification of the Smo intensity in wing discs. White bars represent the wild-type Smo fluorescence; black bars represent Smo fluorescence in the gene knockdown region (mean ± SD from three independent experiments; *P < 0.05, **P ≤ 0.01, student’s t test). To further test whether UCHL5 synergizes with Rpn13 to stabilize Smo, we knocked down single or both UCHL5 and Rpn13 in wing discs. Knockdown of UCHL5 (Figure 5E) or Rpn13 (Figure 5F) clearly attenuated Smo. However, simultaneous knockdown of both UCHL5 and Rpn13 resulted in a more dramatic decrease of Smo protein (Figure 5G and H). Taken together, these results suggest that UCHL5 most likely forms a complex with Rpn13 to deubiquitinate Smo. UCHL5 promotes the cell surface accumulation of Smo The activity of Smo is tightly linked with its localization. It has been revealed that forced localization of Smo to the cell surface enhances Smo activity, whereas endoplasmic retention of an activated form of Smo inhibits its activity (Zhu et al., 2003). It also has been reported that the distribution of Smo is governed by ubiquitination (Li et al., 2012; Xia et al., 2012). We next applied a cell-based immunostaining assay to assess whether UCHL5 regulates the cell surface expression of Smo (Jia et al., 2004). All S2 cells for experiments were transfected with N-terminally tagged Myc-Smo. Cell surface and total Myc-Smo were visualized via immunostaining with anti-Myc antibody before and after cell membrane permeabilization, respectively. Compared with the control cells (Figure 6A–B’), UCHL5 transfection apparently promoted the cell surface accumulation of Smo (Figure 6C–D’), whereas the dominant negative form UCHL5CA decreased the cell surface expression of Smo (Figure 6E–F’). In addition, we treated the transfected S2 cells with control dsRNA or UCHL5-dsRNA to knock down the endogenous UCHL5. Compared with GFP-dsRNA treatment (Figure 6G–H’), UCHL5-dsRNA attenuated the cell surface expression of Smo (Figure 6I–J’). Figure 6 View largeDownload slide UCHL5 positively regulates the cell surface expression of Smo. (A–F’) S2 cells transfected with the indicated constructs were stained by Myc antibody and DAPI with or without cell membrane permeabilization. Regular staining showed the total Myc-Smo protein, while cell surface staining indicated the cell membrane Myc-Smo protein. Of note, UCHL5 promoted the cell membrane accumulation of Smo (compare D–D’ with B–B’), but UCHL5CA inhibited the cell surface localization of Smo (compare F–F’ with B–B’). The nuclei were shown by DAPI staining. (G–J’) S2 cells were treated with GFP-dsRNA or UCHL5-dsRNA for 24 h, and then transfected with Myc-Smo plasmid. After 48 h, cells were collected for immunostaining. Compared with GFP-dsRNA (H–H’), UCHL5-dsRNA treatment apparently attenuated the cell surface accumulation of Myc-Smo (J–J’). The nuclei were marked by DAPI staining. (K–N’) S2 cells were treated with GFP-dsRNA or UCHL5-dsRNA for 24 h, and then transfected with Myc-Smo plasmid. After 24 h, cells were stimulated by Hh conditional medium for additional 24 h, followed by cell harvesting and staining. Hh medium treatment indeed promoted the cell surface accumulation of Smo (compare L–L’ with H–H’). Knockdown of UCHL5 compromised Hh-mediated the cell membrane accumulation of Smo (compare N–N’ with L–L’). The nuclei were indicated by DAPI staining. (O) Quantification of the Myc-Smo intensity on S2 cell membrane. (P–Q’’’) Myc-Smo protein (green) expressed by MS1096 alone or plus UCHL5 RNAi under the background of Hh overexpression in wing discs. UCHL5 RNAi apparently decreased the cell surface localization of Myc-Smo. The nuclei were marked by DAPI (blue). (R) Quantification of the Myc-Smo intensity in wing discs. Figure 6 View largeDownload slide UCHL5 positively regulates the cell surface expression of Smo. (A–F’) S2 cells transfected with the indicated constructs were stained by Myc antibody and DAPI with or without cell membrane permeabilization. Regular staining showed the total Myc-Smo protein, while cell surface staining indicated the cell membrane Myc-Smo protein. Of note, UCHL5 promoted the cell membrane accumulation of Smo (compare D–D’ with B–B’), but UCHL5CA inhibited the cell surface localization of Smo (compare F–F’ with B–B’). The nuclei were shown by DAPI staining. (G–J’) S2 cells were treated with GFP-dsRNA or UCHL5-dsRNA for 24 h, and then transfected with Myc-Smo plasmid. After 48 h, cells were collected for immunostaining. Compared with GFP-dsRNA (H–H’), UCHL5-dsRNA treatment apparently attenuated the cell surface accumulation of Myc-Smo (J–J’). The nuclei were marked by DAPI staining. (K–N’) S2 cells were treated with GFP-dsRNA or UCHL5-dsRNA for 24 h, and then transfected with Myc-Smo plasmid. After 24 h, cells were stimulated by Hh conditional medium for additional 24 h, followed by cell harvesting and staining. Hh medium treatment indeed promoted the cell surface accumulation of Smo (compare L–L’ with H–H’). Knockdown of UCHL5 compromised Hh-mediated the cell membrane accumulation of Smo (compare N–N’ with L–L’). The nuclei were indicated by DAPI staining. (O) Quantification of the Myc-Smo intensity on S2 cell membrane. (P–Q’’’) Myc-Smo protein (green) expressed by MS1096 alone or plus UCHL5 RNAi under the background of Hh overexpression in wing discs. UCHL5 RNAi apparently decreased the cell surface localization of Myc-Smo. The nuclei were marked by DAPI (blue). (R) Quantification of the Myc-Smo intensity in wing discs. The localization of Smo is controlled by Hh pathway activity. An antibody uptake experiment using cultured S2 cells suggested that Hh regulates the cell surface expression of Smo through promoting the recycling of Smo (Jia et al., 2004). In addition, Smo is preferentially localized in the cytoplasm of anterior compartment cells in wing discs where Hh is not present, whereas enriched on the cell membrane of posterior compartment cells where Hh stimulation occurs (Nakano et al., 2004). To test whether the accumulation of Smo induced by Hh stimulation is due to the effect of UCHL5, S2 cells transfected with Myc-Smo were treated with GFP-dsRNA (control) or UCHL5-dsRNA and then treated with Hh-conditioned medium or control medium. Consistent with the previous observations (Liu et al., 2007; Zhao et al., 2007), Hh stimulation indeed promote the cell surface accumulation of Smo (compare Figure 6K–L’ with 6G–H’). Intriguingly, the cell surface accumulation of Smo was attenuated by UCHL5-dsRNA treatment in the presence of Hh stimulation both in S2 cells (Figure 6M–N’) and in wing discs (compare Figure 6Q–Q’” with 6P–P”’), indicating that Hh promotes the cell surface expression of Smo, at least in part, through UCHL5. In addition, we confirmed that the localization of endogenous Smo protein was also regulated by Hh signals (Supplementary Figure S4A–L’’’). Taken together, these data suggest that UCHL5 promotes the cell surface accumulation of Smo (Figure 6O and R). UCHL5 cooperates with Usp8 to regulate Smo The previous study has demonstrated that Usp8 acts as a deubiquitinase of Smo protein (Li et al., 2012; Xia et al., 2012). It is necessary to assess whether Usp8 and UCHL5 redundantly deubiquitinate Smo. Knockdown of UCHL5 (Figure 7A) or Usp8 (Figure 7B) alone decreased Smo protein levels, suggesting that both deubiquitinases could stabilize Smo independently. However combined knockdown of UCHL5 and Usp8 resulted in a dramatic decrease of Smo (Figure 7C and D). Through cell-based ubiquitination assays, we found that knockdown of UCHL5 and Usp8 caused a higher ubiquitination Smo than knockdown of UCHL5 or Usp8 alone (Figure 7E and F). These results show that UCHL5 cooperates with Usp8 to deubiquitinate and stabilize Smo. Figure 7 View largeDownload slide UCHL5 coordinates with Usp8 to regulate Smo. (A–C) Wing discs expressing UCHL5-RNAi alone (A), Usp8-RNAi alone (B), or UCHL5-RNAi plus Usp8-RNAi (C) were immunostained to show Smo expression (white). Expression of UCHL5-RNAi plus Usp8-RNAi resulted in a more dramatic decrease in Smo expression (arrows). (D) Quantification of the Smo intensity in wing discs. White bars represent the wild-type Smo fluorescence; black bars represent Smo fluorescence in the gene knockdown region (mean ± SD from three independent experiments; *P < 0.05, **P ≤ 0.01, student’s t test). (E and F) S2 cells were treated with UCHL5-dsRNA alone, Usp8-dsRNA alone, or UCHL5-dsRNA plus Usp8-dsRNA. Combined knockdown of UCHL5 and Usp8 apparently promoted the ubiquitination of Smo in S2 cells (compare lane 4 with lanes 2, 3). Figure 7 View largeDownload slide UCHL5 coordinates with Usp8 to regulate Smo. (A–C) Wing discs expressing UCHL5-RNAi alone (A), Usp8-RNAi alone (B), or UCHL5-RNAi plus Usp8-RNAi (C) were immunostained to show Smo expression (white). Expression of UCHL5-RNAi plus Usp8-RNAi resulted in a more dramatic decrease in Smo expression (arrows). (D) Quantification of the Smo intensity in wing discs. White bars represent the wild-type Smo fluorescence; black bars represent Smo fluorescence in the gene knockdown region (mean ± SD from three independent experiments; *P < 0.05, **P ≤ 0.01, student’s t test). (E and F) S2 cells were treated with UCHL5-dsRNA alone, Usp8-dsRNA alone, or UCHL5-dsRNA plus Usp8-dsRNA. Combined knockdown of UCHL5 and Usp8 apparently promoted the ubiquitination of Smo in S2 cells (compare lane 4 with lanes 2, 3). UCHL5 plays a conserved role in mammalian cells In mammal, UCHL5 has a homolog UCH37, which encodes a 37 kDa protein (Yao et al., 2008; Nishio et al., 2009). To assess whether the regulation of UCHL5 on Smo is conserved from Drosophila to mammals, we generated a HA-UCH37 transgenic fly (HA-UCH37). Overexpression of HA-UCH37 apparently restored the wing phenotypes induced by UCHL5 knockdown in Drosophila (Figure 8A and B). In addition, we found that the decrease of Ptc expression caused by UCHL5 knockdown (Figure 8C–C”) was rescued via overexpressing HA-UCHL5 (Figure 8D–D”) or HA-UCH37 (Figure 8E–E”), suggesting that UCH37 could functionally replace UCHL5 in the regulation of Hh pathway. Figure 8 View largeDownload slide UCH37 deubiquitinates hSmo and promotes Hh Signaling activity in mammalian cells. (A and B) Adult wings express UCHL5-RNAi alone or UCHL5-RNAi plus HA-tagged UCH37 under the background of Smo–PKA through MS1096. (C–E”) Wing discs expressing UCHL5-RNAi alone, UCHL5-RNAi plus HA-UCHL5, or UCHL5-RNAi plus HA-UCH37 by MS1096 under the background of Smo–PKA were stained for Ptc (white). The decrease of Ptc protein caused by UCHL5 knockdown (C–C”) was restored by overexpressing HA-UCHL5 (D–D”) or HA-UCH37 (E–E”) (arrows). (F) UCH37 could bind to hSmo in NIH-3T3 cells. (G) ShhN treatment promoted the binding between UCH37 and hSmo in NIH-3T3 cells (mean ± SD from three independent experiments; ***P ≤ 0.005, student’s t test). (H) UCH37 inhibited the ubiquitination of hSmo protein in NIH-3T3 cells. (I) Knockdown of UCH37 decreased hSmo levels and Hh pathway activity in NIH-3T3 cells (mean ± SD; ***P ≤ 0.005, student’s t test). (J) Relative mRNA levels of gli1, ptc1, and hhip were revealed by real-time PCR in UCH37 knockdown NIH-3T3 cells (mean ± SD from three independent experiments; ***P≤ 0.005, student’s t test). (K) Gli-luciferase (Gli-luc) reporter assay in NIH-3T3 cells transfected with the indicated constructs. Gli luciferase activities were normalized to Renilla luciferase activities. UCH37 promoted, but UCH37CA attenuated Shh pathway activity (mean ± SD from three independent experiments; **P ≤ 0.01, student’s t test). Figure 8 View largeDownload slide UCH37 deubiquitinates hSmo and promotes Hh Signaling activity in mammalian cells. (A and B) Adult wings express UCHL5-RNAi alone or UCHL5-RNAi plus HA-tagged UCH37 under the background of Smo–PKA through MS1096. (C–E”) Wing discs expressing UCHL5-RNAi alone, UCHL5-RNAi plus HA-UCHL5, or UCHL5-RNAi plus HA-UCH37 by MS1096 under the background of Smo–PKA were stained for Ptc (white). The decrease of Ptc protein caused by UCHL5 knockdown (C–C”) was restored by overexpressing HA-UCHL5 (D–D”) or HA-UCH37 (E–E”) (arrows). (F) UCH37 could bind to hSmo in NIH-3T3 cells. (G) ShhN treatment promoted the binding between UCH37 and hSmo in NIH-3T3 cells (mean ± SD from three independent experiments; ***P ≤ 0.005, student’s t test). (H) UCH37 inhibited the ubiquitination of hSmo protein in NIH-3T3 cells. (I) Knockdown of UCH37 decreased hSmo levels and Hh pathway activity in NIH-3T3 cells (mean ± SD; ***P ≤ 0.005, student’s t test). (J) Relative mRNA levels of gli1, ptc1, and hhip were revealed by real-time PCR in UCH37 knockdown NIH-3T3 cells (mean ± SD from three independent experiments; ***P≤ 0.005, student’s t test). (K) Gli-luciferase (Gli-luc) reporter assay in NIH-3T3 cells transfected with the indicated constructs. Gli luciferase activities were normalized to Renilla luciferase activities. UCH37 promoted, but UCH37CA attenuated Shh pathway activity (mean ± SD from three independent experiments; **P ≤ 0.01, student’s t test). We next determined whether UCH37 is involved in the regulation of hSmo ubiquitination and Shh pathway activity in mammalian cells. First, the co-IP experiments revealed that UCH37 interacted with hSmo (Figure 8F) and Hh promoted their interaction (Figure 8G). Furthermore, we found that UCH37 indeed decreased the ubiquitination level of hSmo (Figure 8H), indicating that UCH37 can deubiquitinate hSmo. The ability of UCH37 to suppress hSmo ubiquitination implies that UCH37 possibly regulates Shh pathway activity. To test this possibility, we performed Gli-luciferase (Gli-Luc) reporter assay in NIH-3T3 cells, which express Shh pathway components and target genes (Zhou et al., 2015b). Knockdown of UCH37 attenuated hSmo levels, suppressed Shh pathway activity (Figure 8I) and the expression of Shh target genes gli1, ptch1 and hhip (Figure 8J). Consistently, overexpression of UCH37 enhanced, whereas UCH37CA attenuated Shh pathway activity (Figure 8K). In mammals, Hh signal transduction occurs at the primary cilium. In the presence of Hh, Smo protein is accumulated to the cilium where it converts Gli proteins from transcriptional repressors to activators (Huangfu and Anderson, 2006; Ma et al., 2016). To determine whether UCH37 regulates hSmo ciliary accumulation, NIH-3T3 cells were transfected with Myc-hSmo plus Sstr3-GFP (for labeling primiary cilia) or with Fg-UCH37CA with or without ShhN treatment. According to our experimental statistics, only ~22% of the cells contained ciliary Myc-hSmo signals (Supplementary Figure S5A and E). While ~85% of the cells exhibited ciliary Myc-hSmo signals after treated with ShhN (Supplementary Figure S5B and E). Importantly, expression of Fg-UCH37CA caused less and weaker Shh-induced hSmo ciliary localization (Supplementary Figure S5C–E). Taken together, these results suggest that UCH37 plays a conserved role in regulating Shh pathway. Discussion As one of the essential components of Hh pathway, Smo contributes to transducing the signal across the cell plasma membrane (Ingham and McMahon, 2001). Accumulation of Smo on the membrane and its ubiquitination are finely governed by the Hh signal. In the absence of Hh, Smo undergoes ubiquitination in the cytoplasm and fails to accumulate on the cell membrane, culminating in turning off Hh pathway and target gene expression. In the presence of Hh, Ptc relieves its inhibitory effect on Smo activity by binding to Hh, resulting in the cell membrane accumulation of Smo and Hh pathway activation (Jia et al., 2004; Li et al., 2012; Jiang and Jia, 2015). The ubiquitination of Smo and its localization are tightly linked. However, it remains largely unknown how Smo ubiquitination is modulated during Hh signaling transduction. In this study, we identified the deubiquitinase UCHL5 as a positive regulator of Hh signaling through genetic screening. In addition, we provided both genetic and biochemical evidences that UCHL5 binds to and deubiquitinates Smo, culminating in stabilizing Smo. We also figured out that the recognition of Smo by UCHL5 is dependent on the SAID domain of Smo C-tail. Moreover, UCHL5 promoted the cell membrane localization of Smo protein. Finally, we found that the mammalian homolog UCH37 plays a conserved role in regulating Smo and Hh pathway. Our findings thus unveil that a conserved deubiquitinase UCHL5/UCH37 regulates the ubiquitination and localization of Smo to achieve optimal Hh pathway activity. UCHL5 is a member of the UCH-containing deubiquitinases in Drosophila. UCHL5 binds to Smo through its N-terminally located UCH domain (Figure 3C). In addition to UCHL5, Drosophila also has two other UCH-containing proteins: UCH-D (CG4265) and UCHL5R (CG1950). Intriguingly, we revealed that only UCHL5 and UCHL5R form a complex with Smo, while UCH-D did not show any interaction with Smo (Supplementary Figure S3E and F). These observations suggest that the amino acid composition in UCH domain may contribute to the substrate specificity of different deubiquitinases, albeit their space structure of UCH domainsis similar. Moreover, we found that knockdown of UCHL5R decreased Smo and Hh pathway activity (data not shown), indicating UCHL5R also plays as a potential deubiquitinase of Smo. Taken together, it is possible that the different deubiquitinases may play additive roles to achieve most efficient deubiquitination of Smo. The SAID domain, which is important for the maintenance of Smo ‘closed’ conformation by arginine-rich motifs, has been found to be critical for the cell membrane accumulation of Smo (Zhao et al., 2007). In addition, Hrs binds to the SAID domain to promote Smo ubiquitination and degradation (Fan et al., 2013). These data strongly suggest that the SAID domain plays a negative role in regulating Smo activity. Our work uncovered that the deubiquitinase UCHL5 stabilizes Smo via binding to the SAID domain on Smo (Figure 3D–G), indicating that the SAID domain also has positive effect on Smo activity. These two view points are not paradoxical because the SAID domain we studied in this work covers 158 amino acids. We next want to find which amino acids on Smo are responsible for Smo-UCHL5 interaction. It is likely that the arginine-rich motifs in the SAID domain are not involved in mediating its interaction with UCHL5. In addition, since both UCHL5 and Hrs bind to the SAID domain of Smo and play opposite roles on the Smo stability, it’s interesting to further investigate whether they occupy the SAID domain of Smo in a competitive way. Hh blocks Smo ubiquitination, which is vital for its activity. What is the underlying mechanism? Although the previous data have revealed that Usp8 decreases Smo ubiquitination, this event is possible out of Hh control (Li et al., 2012). Usp8 attenuates Smo ubiquitination regardless of the Hh signaling status and the interaction between Usp8 and Smo is not apparently modulated by Hh stimulation (Li et al., 2012), suggesting that Smo deubiquitination by Usp8 is unlikely to be a mechanism by which Hh blocks Smo ubiquitination. In this study, we found that Hh stimulation boosted UCHL5–Smo association (Figure 3H–J), indicating that the effect of UCHL5 on Smo is amplified by Hh signaling. In addition, we found that knockdown of UCHL5 inhibited Hh-mediated Smo deubiquitination (Figure 4J), suggesting that Hh blocks Smo ubiquitination, at least partly through promoting UCHL5–Smo interaction. The deubiquitinating function of UCH37 on hSmo we unveiled here suggests that UCH37 may play a role in regulating the Hh pathway in mammalian systems. Deletion of UCH37 resulted in prenatal lethality in mice associated with severe defect in embryonic brain development (Al-Shami et al., 2010). It is well known that the Hh pathway plays an indispensible role during the development of brain (Rowitch et al., 1999; Ruiz i Altaba et al., 2002; Farmer et al., 2016). Therefore, it would be interesting to investigate whether UCH37 deficiency embryos show any defects in the Hh pathway. Increasing observations indicate that UCH37 is also involved in many pathological processes, including tumorigenesis. The expressions of UCH37 are upregulated in most epithelial ovarian cancer patients (Wang et al., 2014). UCH37 is increased in many patients of esophageal squamous cell carcinoma (Chen et al., 2012). In addition, UCH37 upregulation has been tightly linked to human hepatocellular carcinoma (Fang et al., 2012). These data suggest that UCH37 possibly plays an oncogenic role in tumorigenesis. Since Hh pathway hyperactivation induces many tumors, it should be fruitful to determine whether Hh-Smo axis contributes to UCH37-induced tumors. Multiple studies have already revealed that UCH37 acts as a potential target for cancer therapy (Chen et al., 2013). Our finding that UCH37 positively regulates the Hh pathway through deubiquitinating Smo provides UCH37 as a potential drug target for Hh-related tumors. Materials and methods Immunostaining and in situ hybridization of wing discs, cell culture, transfection, immunoprecipitation, western blot, and real-time PCR were performed according to standard protocols. For detailed procedures, fly strains, and constructs used in this study, see the Supplementary Experimental Procedures. Generating mutant clones Clones of mutant cells were generated by the FLP-FRT system as described (Banning et al., 2011). The genotype for generating UCHL5 clone is hs-flp; UCHL5j2B8FRT80B/ubi-GFP FRT80B. The mutant clones were marked by loss of GFP expression. RNA interference To knock down genes in S2 cells, the double stranded RNA (dsRNA) was generated by MEGAscript High Yield Transcription Kit (Ambion) according to the manufacturer’s instructions. DNA templates targeting UCHL5 (aa1-324), UCHL5 (aa1-221), UCHL5 (aa153-324) and Usp8 (aa274-557) were generated by PCR and used for generating dsRNA. dsRNA targeting the GFP full-length coding sequence was used as a control. The dsRNA-mediated gene knockdown was performed as previously described (Lawrence Lum et al., 2003; Li et al., 2012). To silence UCH37 expression in mammalian cells, a mixture of two different siRNAs was used. The siRNA sequences were UCH37-siRNA-1 (5′-CCGAGCUCAUUAAAGGAUUdTd-3′), UCH37-siRNA-2 (5′-CCGAUUGAUUUGGUGCAUdTdT-3′), and mock-siRNA (5′-UUCUCCGAACGUGUCACGUdTdT-3′). All siRNA duplexes were transfectedat a final concentration of 100 nM using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Protein stability assays and ubiquitination assays S2 cells were plated in 10-cm dishes and transfected with plasmids after 18–24 h. After another 24 h, the cells were split and transferred into 6-well cell culture plates at equivalent densities. Cells were treated with 20 μg/ml CHX (Calbiochem) for the indicated times before harvesting. After western blots, the band intensity was measured by Image J. To examine the levels of ubiquitinated Smo, S2 cells were transfected with Smo and then lysed with denaturing buffer (1% SDS, 50 mM Tris, pH 7.5, 0.5 mM EDTA, and 1 mM DTT) and ultrasonic. The lysates were then diluted 10-fold with regular lysis buffer and subjected to immunoprecipitation and western blot analysis. Statistical analysis Relative intensity of Smo in wing discs was the Smo fluorescence intensity in RNAi expression region divided by the Smo fluorescence intensity in wild-type region. Similarly, relative intensity of cilia-located Smo was the cilia-located Smo fluorescence intensity divided by the whole-cell Smo fluorescence intensity. relative intensity of Smo on the membrane with various treatment was obtained, with the wild-type Smo on the membrane normalized to 1. Imaging data were analyzed in the program Image J. The data shown in the figures were representative of three or more independent experiments and were analyzed by Student’s t-test, with P < 0.05 considered significant. Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. Acknowledgements We thank Dr Yun Zhao (Shanghai Institute of Biochemistry and Cell Biology, CAS), Dr Steven Y. Cheng (Nanjing Medical University), and Dr Yongbin Chen (Kunming Institute of Zoology, CAS) for providing plasmids and reagents. We also appreciate National Institute of Genetics of Japan (NIG), Vienna Drosophila RNAi Center (VDRC), and the Bloomington Stock Center (BSC) for providing fly stocks. We apologize to colleagues whose works were not cited owing to space limitation. Funding This work was supported by grants from the National Basic Research Program of China (2011CB943902), the National Natural Science Foundation of China (30971679, 31071264, and 31271531), and the Fundamental Research Funds for the Central Universities (090314380019). Conflict of interest: none declared. Author contributions: Q.Z. and Z.Z. designed the experiments. Z.Z., X.Y., S.P., P.C., W.J., and Z.S performed the experiments. Z.Z., X.Y., and Q.Z. wrote the manuscript. References Al-Shami, A., Jhaver, K.G., Vogel, P., et al.  . ( 2010). Regulators of the proteasome pathway, Uch37 and Rpn13, play distinct roles in mouse development. PLoS One  5, e13654. 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( 2003). Altered localization of Drosophila Smoothened protein activates Hedgehog signal transduction. Genes Dev.  17, 1240– 1252. Google Scholar CrossRef Search ADS PubMed  © The Author (2017). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Molecular Cell Biology Oxford University Press

The deubiquitinase UCHL5/UCH37 positively regulates Hedgehog signaling by deubiquitinating Smoothened

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Oxford University Press
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© The Author (2017). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.
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1674-2788
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10.1093/jmcb/mjx036
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Abstract

Abstract The Hedgehog (Hh) signaling pathway plays important roles in developmental processes including pattern formation and tissue homeostasis. The seven-pass transmembrane receptor Smoothened (Smo) is the pivotal transducer in the pathway; it, and thus the pathway overall, is regulated by ubiquitin-mediated degradation, which occurs in the absence of Hh. In the presence of Hh, the ubiquitination levels of Smo are decreased, but the molecular basis for this outcome is not well understood. Here, we identify the deubiquitinase UCHL5 as a positive regulator of the Hh pathway. We provide both genetic and biochemical evidence that UCHL5 interacts with and deubiquitinates Smo, increasing stability and promoting accumulation at the cell membrane. Strikingly, we find that Hh enhances the interaction between UCHL5 and Smo, thereby stabilizing Smo. We also find that proteasome subunit RPN13, an activator of UCHL5, could enhance the effect of UCHL5 on Smo protein level. More importantly, we find that the mammalian counterpart of UCHL5, UCH37, plays the same role in the regulation of Hh signaling by modulating hSmo ubiquitination and stability. Our findings thus identify UCHL5/UCH37 as a critical regulator of Hh signaling and potential therapeutic target for cancers. Hedgehog, Smoothened, ubiquitination, UCHL5, UCH37 Introduction The Hh pathway plays vital roles in governing animal embryonic development and adult tissue homeostasis (Ingham and McMahon, 2001; Jia and Jiang, 2006; Jiang and Hui, 2008; Briscoe and Therond, 2013). The pathway sits at the cross-road of cell survival and differentiation decisions, and deregulated Hh signaling can lead to hyperproliferation and tumorigenesis (Hooper and Scott, 2005; Ingham et al., 2011). Indeed, mutations of pathway components have been implicated in numerous human disorders, including birth defects and several types of cancers (Berman et al., 2003; Ingham and Placzek, 2006). The key components and regulatory systems of the Hh signaling are conserved from Drosophila to mammals (Jiang and Hui, 2008; Wilson and Chuang, 2010). Therefore, Drosophila is an ideal model to study Hh signaling. In Drosophila wing discs, Hh proteins are produced and processed by posterior (P) compartment cells. Mature Hh proteins secreted by P cells move into the anterior (A) compartment to form a gradient. In A pathway cells near A/P boundary, Hh signaling is initiated at the cell membrane by the inactivation of the 12-span transmembrane receptor Patched (Ptc) upon binding of the Hh ligands (Jeong and McMahon, 2002; Hooper and Scott, 2005). Ptc inactivation relieves its inhibitory effect on the GPCR family protein Smoothened (Smo), leading to the activation of the unique transcription factor Cubitus interruptus (Ci) and thereby the expression of Hh target genes decapentaplegic (dpp), ptc, and engrailed (en) in a Hh-concentration-dependent manner (Lum and Beachy, 2004; Jiang and Hui, 2008). The 7-span transmembrane receptor Smo is essential for transducing Hh signal across the cell plasma membrane. In the absence of Hh, Ptc inhibits Smo activity and its accumulation on the cell membrane. The intracellular Smo protein undergoes ubiquitination, leading to endocytosis-mediated Smo degradation (Li et al., 2012; Xia et al., 2012). Upon different concentrations of Hh, Smo is differentially phosphorylated by protein kinase A (PKA) and casein kinase I (CKI) and phosphorylation antagonizes the ubiquitination of Smo, culminating in Smo accumulation on the plasma membrane (Jia et al., 2004; Apionishev et al., 2005). The activity of Smo is strictly regulated by multiple mechanisms, one of which is the ubiquitin-mediated protein modification. Loss of uba1, which encodes the unique ubiquitin-activating enzyme in Drosophila, results in Smo accumulation in the wing disc, suggesting that Smo undergoes ubiquitin-mediated degradation (Li et al., 2012). Ubiquitin fused Smo protein (Smo-Ub) predominantly localizes in Rab7-labeled late endosomes, indicating that ubiquitination also alters the localization of Smo (Xia et al., 2012). Hh treatment attenuates Smo ubiquitination and promotes its cell surface accumulation (Li et al., 2012; Xia et al., 2012). Ubiquitination is an enzymatic process by which the substrate is covalently modified with the 76 amino acid protein named ubiquitin (Ub) (Hochstrasser, 1995). Ubiquitination has been important not only in the destabilization of proteins, but also in the regulation of protein functions, including protein localization and protein–protein interaction (Hicke, 2001; Weissman, 2001). Like other protein modifications, the process of ubiquitination is also reversible after the identification of many deubiquitinating enzymes (DUBs), which counteract the ubiquitination process by removing Ub conjugates from target proteins (Wilkinson, 2000). DUBs are highly conserved proteases that catalyze the cleavage of the isopeptide bond between ubiquitin and the target protein. Due to distinct catalytic domains, DUBs are classified into five subfamilies: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), otubain proteases (OTUs), JAB1/MPN/Mov34 domain proteases (JAMMs) and Machado-Joseph disease domain proteases (MJDs) (Nijman et al., 2005; Zhang et al., 2012). UCHL5 (also known as UCH37) is a 37 kDa deubiquitinase composed of an N-terminal UCH domain and a C-terminal extension domain (Jiao et al., 2014). UCHL5 was first identified as a component of the proteasome that is thought to be involved in the cleavage of polyubiquitin chains from ubiquitinated substrates (Lam et al., 1997). Increasing substrates of UCHL5 have been identified, including type I TGF-β receptor (TGF-βRI) (Wicks et al., 2005), and E2 promoter binding factor 1 (E2F1) (Mahanic et al., 2015), underscoring that UCHL5 plays versatile roles in multiple cellular processes. The full-length UCHL5 exhibits low deubiquitinating activity through autoinhibition mediated by the C-terminal oligomerization, while the inhibitory effect is alleviated by Rpn13-UCHL5 interaction (Yao et al., 2006, 2008; Jiao et al., 2014). Thus, Rpn13 strongly stimulates UCHL5 deubiquitinating activity (Chen and Walters, 2015; VanderLinden et al., 2015). Given that the Smo undergoes ubiquitination-medated destabilization, it is fruitful to find the deubiquitinase of Smo (Shi et al., 2013; Yang et al., 2013). Although Usp8 is identified as a deubiquitinating enzyme of Smo, its interaction with Smo is not regulated by Hh signaling, suggesting that Hh-mediated Smo stabilization is probably not through Usp8 (Li et al., 2012). Therefore, the underlying mechanism of Hh inhibiting Smo ubiquitination is still unknown. In this study, we identified UCHL5 as a positive modulator of Hh pathway through stabilizing Smo. We also provided evidence that UCHL5 binds to Smo and recruits Rpn13 to form a trimetric complex that decreases Smo ubiquitination and promotes the cell surface accumulation of Smo. In addition, we found that Hh promotes the interaction between UCHL5 and Smo, and that UCHL5 is required for Hh-induced Smo deubiquitination and cell surface accumulation. Moreover, the mammalian counterpart of UCHL5, UCH37, plays a conserved role in the regulation of Hh signaling. Results Knockdown of UCHL5 attenuates Hh signaling in Drosophila The adult Drosophila wing is formed by an epithelial sheet, named wing disc. A large number of genes, including many Hh pathway components, have previously been shown to be required for differentiation of wings (Ingham and McMahon, 2001). Hh stabilizes Ci in A/P boundary of wing disc to control Hh target gene expression, which finally determines the space between vein L3 and L4 (Jia et al., 2010; Mao et al., 2014). In our previous published paper, we carried out a modified genetic screening and identified that knockdown of UCHL5 narrowed the width between vein 3 and vein 4 in wings expressing a dominant negative Smo (Smo–PKA), provides a sensitive background of Hh pathway (Zhou et al., 2015b). We confirmed the screening result and found that knockdown of UCHL5 using UCHL5-RNAi (35433) from Bloomington Stock Center (BSC) indeed resulted in a narrow L3/L4 intervein (Figure 1A and B). Given that the space between vein 3 and vein 4 is a characteristic monitor of Hh pathway activity in adult wings, knockdown of UCHL5 narrowing the space indicates that UCHL5 may positively regulate Hh pathway. Figure 1 View largeDownload slide Knockdown of UCHL5 represses Hh signaling. All wing imaginal discs shown in this study were oriented with anterior to the left and ventral up. (A and B) Comparison of adult wing phenotypes between control (A) and UCHL5 knockdown flies (B). Arrows indicate the space between vein 3 and vein 4. (C and D) The expression pattern of UCHL5 in wing discs was determined by in situ hybridization with DIG-labeled mRNA probe against UCHL5. The sense probe acts as negative control (C). Of note, UCHL5 ubiquitously expresses in wing discs. (E–H”) Knockdown of UCHL5 by MS1096-Gal4 under the background of Smo–PKA attenuated the expression of Ptc (compare F–F” with E–E”) and ptc-lacZ. Arrows indicate the decrease of Ptc and ptc-lacZ. (I–I”) UAS-GFP (green) marks the apG4-mediated gene expression pattern. apG4 drives UAS transgenes to be specifically expressed in the dorsal region of wing discs. (J–J”) Knockdown of UCHL5 by apG4 attenuated the expression of ptc-lacZ (arrow). (K–L”) Knockdown of UCHL5 with apG4 attenuated the expression of En (compare L–L” with K–K”). Figure 1 View largeDownload slide Knockdown of UCHL5 represses Hh signaling. All wing imaginal discs shown in this study were oriented with anterior to the left and ventral up. (A and B) Comparison of adult wing phenotypes between control (A) and UCHL5 knockdown flies (B). Arrows indicate the space between vein 3 and vein 4. (C and D) The expression pattern of UCHL5 in wing discs was determined by in situ hybridization with DIG-labeled mRNA probe against UCHL5. The sense probe acts as negative control (C). Of note, UCHL5 ubiquitously expresses in wing discs. (E–H”) Knockdown of UCHL5 by MS1096-Gal4 under the background of Smo–PKA attenuated the expression of Ptc (compare F–F” with E–E”) and ptc-lacZ. Arrows indicate the decrease of Ptc and ptc-lacZ. (I–I”) UAS-GFP (green) marks the apG4-mediated gene expression pattern. apG4 drives UAS transgenes to be specifically expressed in the dorsal region of wing discs. (J–J”) Knockdown of UCHL5 by apG4 attenuated the expression of ptc-lacZ (arrow). (K–L”) Knockdown of UCHL5 with apG4 attenuated the expression of En (compare L–L” with K–K”). At first, we tested the expression pattern of UCHL5 in wing via in situ hybridization and found that UCHL5 ubiquitiously expressed throughout the wing disc (Figure 1C and D). To confirm whether UCHL5 regulates Hh pathway, we employed immunostaining to test the expression of Hh target genes. Compared with control discs (Figure 1E–E” and G–G”), knockdown of UCHL5 by MS1096-Gal4 apparently decreased Ptc (Figure 1F–F”) and ptc-lacZ (Figure 1H–H”) levels under Smo–PKA background. In addition, we used a stronger gal4 driver ap-Gal4 (apG4) to knockdown UCHL5 in the dorsal region of wing discs and found that knockdown of UCHL5 compromised ptc-lacZ (compare Figure 1J–J” with I–I”) and En (compare Figure 1L–L” with K–K”). The decreased Hh signaling induced by UCHL5 knockdown was unlikely due to an off-target effect because expression of two different RNAi lines (V-34618 from VDRC and 3431R-1 from NIG) targeting nonoverlapping regions of a UCHL5 sequence produced a similar phenotype (Supplementary Figure S1). Taken together, these results suggest that UCHL5 is a positive regulator of Hh pathway. To determine the specificity of UCHL5, we tested whether UCHL5 is involved in modulating other important signaling pathways, such as Hippo, Notch and Wingless pathways. Compared with the control discs (Supplementary Figure S2A–A”, C–C”, and E–E”), knockdown of UCHL5 did not affect the expression of Hippo pathway target gene ex-lacZ (Supplementary Figure S2B–B”), Notch pathway target gene cut (Supplementary Figure S2D–D”), and Wingless pathway target gene vg (Supplementary Figure S2F–F”). UCHL5 binds to and stabilizes Smo Given that the deubiquitinase plays its roles always through recognizing and deubiquitinating substrates (Nijman et al., 2005; Huang and Cochran, 2013), we speculated that interaction with a component of Hh pathway is essential for UCHL5 regulating Hh signaling. To test this possibility, co-IP experiments were carried out in S2 cells. UCHL5 exclusively interacted with Smo (Figure 2A and B), not with other components including Ci (Supplementary Figure S3A), Fu (Supplementary Figure S3B), Cos2 (Supplementary Figure S3C) and Sufu (Supplementary Figure S3D). Figure 2 View largeDownload slide UCHL5 binds to and stabilizes Smo. (A and B) HA-UCHL5 interacted with Myc-Smo (A) and endogenous Smo (B) in S2 cells. (C–D”) Knockdown of UCHL5 with apG4 decreased the protein level of Smo (compare D–D” with C–C”). (E) Relative mRNA levels of UCHL5 from hetero and homo UCHL5j2B8 larval. Of note, UCHL5j2B8 mutant showed residual UCHL5 expression. (F–G”) Wing discs carrying UCHL5j2B8 clones were immunostained to show the expression of GFP (green) and Smo (white) at low (F–F”) and high (G–G”) magnifications. UCHL5j2B8 clones are recognized by the lack of GFP. Of note, Smo is decreased in UCHL5 mutant cells. (H) UCHL5 could hamper exogenous Smo degradation. The results were presented as mean ± SD of values from three independent experiments. Of note, proteins loaded had been adjusted such that the amounts of Smo at T = 0 were equivalent. Figure 2 View largeDownload slide UCHL5 binds to and stabilizes Smo. (A and B) HA-UCHL5 interacted with Myc-Smo (A) and endogenous Smo (B) in S2 cells. (C–D”) Knockdown of UCHL5 with apG4 decreased the protein level of Smo (compare D–D” with C–C”). (E) Relative mRNA levels of UCHL5 from hetero and homo UCHL5j2B8 larval. Of note, UCHL5j2B8 mutant showed residual UCHL5 expression. (F–G”) Wing discs carrying UCHL5j2B8 clones were immunostained to show the expression of GFP (green) and Smo (white) at low (F–F”) and high (G–G”) magnifications. UCHL5j2B8 clones are recognized by the lack of GFP. Of note, Smo is decreased in UCHL5 mutant cells. (H) UCHL5 could hamper exogenous Smo degradation. The results were presented as mean ± SD of values from three independent experiments. Of note, proteins loaded had been adjusted such that the amounts of Smo at T = 0 were equivalent. The binding between UCHL5 and Smo indicates that UCHL5 possibly modulates Hh signaling through Smo. Compared with the control disc (Figure 2C–C”), knockdown of UCHL5 indeed decreased Smo protein level (Figure 2D–D”). To confirm this result, a hypomorphic allele of UCHL5, UCHL5j2B8, was employed to generate mutant clones. The RT-PCR result proved that UCHL5j2B8 was a moderate, not a strong mutant allele (Figure 2E). We found that the Smo level was decreased in UCHL5j2B8 mutant cells, which were marked by the lack of GFP expression (Figure 2F–G”). However, the decrease of Smo in UCHL5j2B8 clones was clear, but not severe, probably because of residual UCHL5 expression. The main function of deubiquitinase is to remove the ubiquitin chains from the substrate, culminating in preventing proteasome-mediated substrate proteolysis. The interaction of UCHL5 and Smo indicates that Smo acts as a potential target of UCHL5. To test this possibility, we measured Smo protein stability in S2 cells treated with Cycloheximide (CHX) to block protein synthesis. The results showed that overexpression of UCHL5 significantly prevented Smo degradation (Figure 2H). Collectively, UCHL5 positively regulates Hh signaling through binding to and stabilizing Smo. UCHL5 binds to the SAID domain of Smo via its N-terminal region UCHL5 is comprised of an N-terminal UCH domain and C-terminal extension region (Figure 3A). To map which domain of UCHL5 is essential for its binding with Smo, we generated Fg-UCHL5-N and Fg-UCHL5-C truncated constructs (Figure 3A). Through co-IP experiments, we found the N-terminally located UCH domain is both necessary and sufficient to mediate Smo binding (Figure 3C). Figure 3 View largeDownload slide UCHL5 binds to the SAID domain of Smo through its N-terminal UCH fragment. (A and B) Schematic drawings show the domains or motifs in UCHL5 and Smo and their truncated fragments used in subsequent co-IP assay. (A) Red and blue bars represent N-terminal UCH domain and C-terminal extension region of UCHL5, respectively. (C) UCHL5 interacts with Smo through its N-terminus in S2 cells. (D and E) UCHL5 binds with the C-terminus of Smo (compare E with D). (F) SAID domain-deleted form of Smo did not bind to UCHL5 in S2 cells. (G) Fg-UCHL5 associated with Myc-SAID in S2 cells. (H and I) Hh-conditioned medium treatment promoted UCHL5 binding with Myc-Smo (H) and Smo (I) in S2 cells (mean ± SD from three independent experiments; ***P≤ 0.005, student’s t test). (J) Hh-conditioned medium treatment promoted UCHL5-N binding with Myc-Smo in S2 cells (mean ± SD from three independent experiments; ***P ≤ 0.005, student’s t test). Figure 3 View largeDownload slide UCHL5 binds to the SAID domain of Smo through its N-terminal UCH fragment. (A and B) Schematic drawings show the domains or motifs in UCHL5 and Smo and their truncated fragments used in subsequent co-IP assay. (A) Red and blue bars represent N-terminal UCH domain and C-terminal extension region of UCHL5, respectively. (C) UCHL5 interacts with Smo through its N-terminus in S2 cells. (D and E) UCHL5 binds with the C-terminus of Smo (compare E with D). (F) SAID domain-deleted form of Smo did not bind to UCHL5 in S2 cells. (G) Fg-UCHL5 associated with Myc-SAID in S2 cells. (H and I) Hh-conditioned medium treatment promoted UCHL5 binding with Myc-Smo (H) and Smo (I) in S2 cells (mean ± SD from three independent experiments; ***P≤ 0.005, student’s t test). (J) Hh-conditioned medium treatment promoted UCHL5-N binding with Myc-Smo in S2 cells (mean ± SD from three independent experiments; ***P ≤ 0.005, student’s t test). To determine which region in Smo is responsible for binding to UCHL5, various Myc-Smo truncated mutants (Figure 3B) were transfected into S2 cells with HA-UCHL5, followed by co-IP assays. As shown in Figure 3D and E, Smo interacted with UCHL5 using its C-terminal fragment. Previous studies have demonstrated that the C-terminally located Smo auto-inhibitory domain (SAID) plays an important role in regulating Smo activity and ubiquitination (Zhao et al., 2007; Li et al., 2012). To test whether the SAID domain mediates Smo binding to UCHL5, we generated SAID-deleted Smo construct (Myc-Δ SAID). The co-IP results showed that Myc-ΔSAID failed to bind to UCHL5 (Figure 3F), while the SAID domain could interact with UCHL5 (Figure 3G). Taken together, UCHL5 interacts with the SAID domain of Smo using its N-terminal UCH fragment. The ubiquitination status of Smo is tightly controlled by the Hh pathway (Zhao et al., 2007; Li et al., 2012; Xia et al., 2012). In the presence of Hh, the ubiquitination of Smo is apparently suppressed (Li et al., 2012). Hh might prevent Smo ubiquitination by regulating UCHL5. However, Hh did not affect the expression of UCHL5 because in situ hybridization showed that UCHL5 expressed evenly in the wing disc (Figure 1C and D). The finding that UCHL5 interacted with Smo led to the hypothesis that Hh might govern the affinity between UCHL5 and Smo. To test this, we treated the S2 cells with conditional Hh medium, followed by co-IP experiment. The result revealed that Hh promoted UCHL5–Smo and UCHL5–N–Smo interactions (Figure 3H–J). UCHL5 deubiquitinates Smo UCHL5 belongs to the ubiquitin carboxy-terminal hydrolase family and contains a characteristic catalytic cysteine (Cys) at the core enzymatic domain (UCH domain) (Johnston et al., 1997; Nishio et al., 2009; Maiti et al., 2011). Mutation of the Cys leads to an inactive form of the enzyme. To test whether UCHL5 regulates Hh pathway through its deubiquitinase activity, we generated a point mutant of UCHL5 with its catalytic Cys replaced by Ala (Fg-UCHL5CA). Analogous to UCHL5-RNAi, overexpression of UCHL5CA decreased the space between vein 3 and vein 4 (Figure 4A and B), suggesting that UCHL5CA plays a dominant-negative role. In addition, through adult rescue assays, we found only wild-type UCHL5 restored the phenotype induced by UCHL5-RNAi, while UCHL5CA deteriorated the wing defect (Figure 4C–E). Consistently, overexpression of UCHL5CA apparently downregulated Ptc and Smo levels (Figure 4F–G’) in wing discs and significantly promoted Smo degradation in S2 cells (Figure 4H). In sum, these observations indicate that the deubiquitinase activity is essential for UCHL5 regulating Smo. Figure 4 View largeDownload slide UCHL5 deubiquitinates Smo. (A–D) Comparison of adult wing phenotypes under the background of Smo–PKA from flies of UCHL5 knockdown (A), Fg-UCHL5CA expression (B), Fg-UCHL5 expression in UCHL5 knockdown background (C), and Fg-UCHL5CA expression in UCHL5 knockdown background (D). Arrows mark the space between vein 3 and vein 4. (E) Quantification of the adult wing phenotypes. (F–F”) A wing disc expressing UCHL5CA with MS1096 under the background of Smo–PKA was stained for Ptc (white). UCHL5CA overexpression decreased Ptc expression. (G–G’) A wing disc expressing UCHL5CA by apG4 was stained for Smo (white). UCHL5CA overexpression decreased Smo protein level (arrow). (H) UCHL5CA promoted Smo degradation. The results were presented as mean ± SD of values from three independent experiments. Proteins loaded had been adjusted such that the amounts of Smo at T = 0 were equivalent. (I) S2 cells transfected with the indicated plasmids were analyzed by western blotting. Of note, Fg-UCHL5 decreased, but Fg-UCHL5CA promoted Smo ubiquitination. (J) Knockdown of UCHL5 using UCHL5-dsRNA promoted Smo ubiquitination in S2 cells. UCHL5-dsRNA could effectively knock down UCHL5 mRNA level in S2 cells (bottom two panels). (K) Knockdown of UCHL5 blocked Hh-mediated Myc-Smo deubiquitination (compare lanes 4, 5 with lane 2). (L) UCHL5 attenuated the interaction between Smo and Hrs. (M) Fg-UCHL5 decreased Smo ubiquitination mediated by Hrs. (N–O’) Wing discs expressing Fg-Hrs alone (N–N’) or Fg-Hrs/HA-UCHL5 together (O–O’) by MS1096 were immunostained with Smo (white). UCHL5 could inhibit Hrs-mediated Smo destabilization (arrows). Figure 4 View largeDownload slide UCHL5 deubiquitinates Smo. (A–D) Comparison of adult wing phenotypes under the background of Smo–PKA from flies of UCHL5 knockdown (A), Fg-UCHL5CA expression (B), Fg-UCHL5 expression in UCHL5 knockdown background (C), and Fg-UCHL5CA expression in UCHL5 knockdown background (D). Arrows mark the space between vein 3 and vein 4. (E) Quantification of the adult wing phenotypes. (F–F”) A wing disc expressing UCHL5CA with MS1096 under the background of Smo–PKA was stained for Ptc (white). UCHL5CA overexpression decreased Ptc expression. (G–G’) A wing disc expressing UCHL5CA by apG4 was stained for Smo (white). UCHL5CA overexpression decreased Smo protein level (arrow). (H) UCHL5CA promoted Smo degradation. The results were presented as mean ± SD of values from three independent experiments. Proteins loaded had been adjusted such that the amounts of Smo at T = 0 were equivalent. (I) S2 cells transfected with the indicated plasmids were analyzed by western blotting. Of note, Fg-UCHL5 decreased, but Fg-UCHL5CA promoted Smo ubiquitination. (J) Knockdown of UCHL5 using UCHL5-dsRNA promoted Smo ubiquitination in S2 cells. UCHL5-dsRNA could effectively knock down UCHL5 mRNA level in S2 cells (bottom two panels). (K) Knockdown of UCHL5 blocked Hh-mediated Myc-Smo deubiquitination (compare lanes 4, 5 with lane 2). (L) UCHL5 attenuated the interaction between Smo and Hrs. (M) Fg-UCHL5 decreased Smo ubiquitination mediated by Hrs. (N–O’) Wing discs expressing Fg-Hrs alone (N–N’) or Fg-Hrs/HA-UCHL5 together (O–O’) by MS1096 were immunostained with Smo (white). UCHL5 could inhibit Hrs-mediated Smo destabilization (arrows). Since UCHL5 regulates Smo through deubiquitinase activity, it is necessary to test whether UCHL5 deubiquitinates Smo directly. Via cell-based ubiquitination assay (Zhou et al., 2015a, b), we found that UCHL5 decreased whereas UCHL5CA increased Smo ubiquitination (Figure 4I). Furthermore, knockdown of the endogenous UCHL5 using dsRNA promoted Smo ubiquitination (Figure 4J, top). The efficiency of UCHL5-dsRNA was confirmed by semi-quantitative PCR analysis (Figure 4J, bottom). Previous studies have demonstrated that Hh hampers Smo ubiquitination through an unclear mechanism (Li et al., 2012; Jiang and Jia, 2015). Hh enhancing UCHL5–Smo affinity provided a possibility that Hh regulates Smo through UCHL5. Knockdown of UCHL5 indeed blocked Hh-mediated Smo deubiquitination (Figure 4K), suggesting that Hh stabilizes Smo, at least in part, through UCHL5. Albeit Smo is regulated by ubiquitination, its corresponding E3 ligase remains unknown. Hrs has been reported to promote Smo ubiquitination and destabilization through binding to SAID domain of Smo (Fan et al., 2013). We found that UCHL5 inhibited Hrs-Smo interaction (Figure 4L) and Hrs-mediated Smo ubiquitination (Figure 4M). Compared with the control disc (Figure 4N–N’), UCHL5 hampered Hrs-mediated Smo degradation (Figure 4O–O’). UCHL5 regulates Smo through UCHL5−Rpn13 complex Proteasomal receptors that recognize ubiquitin chains attached to substrates are key mediators of selective protein degradation in eukaryotes. As a ubiquitin receptor, Rpn13 associates with UCHL5 to enhance its deubiquitinating activity in vivo (Qiu et al., 2006; Yao et al., 2006). Given that UCHL5 deubiquitinates Smo, it is interesting to test whether Rpn13 cooperates with UCHL5 to regulate Smo. We first knocked down Rpn13 in the wing disc and found that knockdown of Rpn13 decreased Smo level (Figure 5A–A”). In addition, immunostaining results showed that Rpn13 and UCHL5 co-localized in the cytoplasm of S2 cells (Figure 5B–B’”). We performed a two-step immunoprecipitation experiment and found that Rpn13, UCHL5 and Smo formed a trimeric complex when co-expressed in S2 cells (Figure 5C). Since UCHL5 binds to Smo through its N-terminal UCH domain, we speculated that UCHL5 recruits Rpn13 through its C-terminal fragment. Consistently, co-IP experiments revealed that only UCHL5-C, but not UCHL5-N interacted with Rpn13 (Figure 5D), suggesting that UCHL5 bridges Rpn13 and Smo via distinct regions. Figure 5 View largeDownload slide UCHL5 regulates Smo through UCHL5−Rpn13 complex. (A–A”) A wing disc expressing Rpn13-RNAi by apG4 was immunostained to show the expression of Smo (white). Of note, Rpn13 knockdown resulted in a decrease of Smo (arrow). (B–B’”) Fg-UCHL5 (green) and HA-Rpn13 (red) co-localized in the cytoplasm of S2 cells. (C) The two-step immunoprecipitation indicated UCHL5, Rpn13, and Smo in the same complex. (D) UCHL5 interacted with Rpn13 through its C-terminal region in S2 cells. (E–G) Wing discs expressing UCHL5-RNAi alone (E), Rpn13-RNAi alone (F), or UCHL5-RNAi plus Rpn13-RNAi (G) were immunostained to show Smo expression (white). Expression of UCHL5-RNAi plus Rpn13-RNAi resulted in a more dramatic decrease in Smo expression (arrows). (H) Quantification of the Smo intensity in wing discs. White bars represent the wild-type Smo fluorescence; black bars represent Smo fluorescence in the gene knockdown region (mean ± SD from three independent experiments; *P < 0.05, **P ≤ 0.01, student’s t test). Figure 5 View largeDownload slide UCHL5 regulates Smo through UCHL5−Rpn13 complex. (A–A”) A wing disc expressing Rpn13-RNAi by apG4 was immunostained to show the expression of Smo (white). Of note, Rpn13 knockdown resulted in a decrease of Smo (arrow). (B–B’”) Fg-UCHL5 (green) and HA-Rpn13 (red) co-localized in the cytoplasm of S2 cells. (C) The two-step immunoprecipitation indicated UCHL5, Rpn13, and Smo in the same complex. (D) UCHL5 interacted with Rpn13 through its C-terminal region in S2 cells. (E–G) Wing discs expressing UCHL5-RNAi alone (E), Rpn13-RNAi alone (F), or UCHL5-RNAi plus Rpn13-RNAi (G) were immunostained to show Smo expression (white). Expression of UCHL5-RNAi plus Rpn13-RNAi resulted in a more dramatic decrease in Smo expression (arrows). (H) Quantification of the Smo intensity in wing discs. White bars represent the wild-type Smo fluorescence; black bars represent Smo fluorescence in the gene knockdown region (mean ± SD from three independent experiments; *P < 0.05, **P ≤ 0.01, student’s t test). To further test whether UCHL5 synergizes with Rpn13 to stabilize Smo, we knocked down single or both UCHL5 and Rpn13 in wing discs. Knockdown of UCHL5 (Figure 5E) or Rpn13 (Figure 5F) clearly attenuated Smo. However, simultaneous knockdown of both UCHL5 and Rpn13 resulted in a more dramatic decrease of Smo protein (Figure 5G and H). Taken together, these results suggest that UCHL5 most likely forms a complex with Rpn13 to deubiquitinate Smo. UCHL5 promotes the cell surface accumulation of Smo The activity of Smo is tightly linked with its localization. It has been revealed that forced localization of Smo to the cell surface enhances Smo activity, whereas endoplasmic retention of an activated form of Smo inhibits its activity (Zhu et al., 2003). It also has been reported that the distribution of Smo is governed by ubiquitination (Li et al., 2012; Xia et al., 2012). We next applied a cell-based immunostaining assay to assess whether UCHL5 regulates the cell surface expression of Smo (Jia et al., 2004). All S2 cells for experiments were transfected with N-terminally tagged Myc-Smo. Cell surface and total Myc-Smo were visualized via immunostaining with anti-Myc antibody before and after cell membrane permeabilization, respectively. Compared with the control cells (Figure 6A–B’), UCHL5 transfection apparently promoted the cell surface accumulation of Smo (Figure 6C–D’), whereas the dominant negative form UCHL5CA decreased the cell surface expression of Smo (Figure 6E–F’). In addition, we treated the transfected S2 cells with control dsRNA or UCHL5-dsRNA to knock down the endogenous UCHL5. Compared with GFP-dsRNA treatment (Figure 6G–H’), UCHL5-dsRNA attenuated the cell surface expression of Smo (Figure 6I–J’). Figure 6 View largeDownload slide UCHL5 positively regulates the cell surface expression of Smo. (A–F’) S2 cells transfected with the indicated constructs were stained by Myc antibody and DAPI with or without cell membrane permeabilization. Regular staining showed the total Myc-Smo protein, while cell surface staining indicated the cell membrane Myc-Smo protein. Of note, UCHL5 promoted the cell membrane accumulation of Smo (compare D–D’ with B–B’), but UCHL5CA inhibited the cell surface localization of Smo (compare F–F’ with B–B’). The nuclei were shown by DAPI staining. (G–J’) S2 cells were treated with GFP-dsRNA or UCHL5-dsRNA for 24 h, and then transfected with Myc-Smo plasmid. After 48 h, cells were collected for immunostaining. Compared with GFP-dsRNA (H–H’), UCHL5-dsRNA treatment apparently attenuated the cell surface accumulation of Myc-Smo (J–J’). The nuclei were marked by DAPI staining. (K–N’) S2 cells were treated with GFP-dsRNA or UCHL5-dsRNA for 24 h, and then transfected with Myc-Smo plasmid. After 24 h, cells were stimulated by Hh conditional medium for additional 24 h, followed by cell harvesting and staining. Hh medium treatment indeed promoted the cell surface accumulation of Smo (compare L–L’ with H–H’). Knockdown of UCHL5 compromised Hh-mediated the cell membrane accumulation of Smo (compare N–N’ with L–L’). The nuclei were indicated by DAPI staining. (O) Quantification of the Myc-Smo intensity on S2 cell membrane. (P–Q’’’) Myc-Smo protein (green) expressed by MS1096 alone or plus UCHL5 RNAi under the background of Hh overexpression in wing discs. UCHL5 RNAi apparently decreased the cell surface localization of Myc-Smo. The nuclei were marked by DAPI (blue). (R) Quantification of the Myc-Smo intensity in wing discs. Figure 6 View largeDownload slide UCHL5 positively regulates the cell surface expression of Smo. (A–F’) S2 cells transfected with the indicated constructs were stained by Myc antibody and DAPI with or without cell membrane permeabilization. Regular staining showed the total Myc-Smo protein, while cell surface staining indicated the cell membrane Myc-Smo protein. Of note, UCHL5 promoted the cell membrane accumulation of Smo (compare D–D’ with B–B’), but UCHL5CA inhibited the cell surface localization of Smo (compare F–F’ with B–B’). The nuclei were shown by DAPI staining. (G–J’) S2 cells were treated with GFP-dsRNA or UCHL5-dsRNA for 24 h, and then transfected with Myc-Smo plasmid. After 48 h, cells were collected for immunostaining. Compared with GFP-dsRNA (H–H’), UCHL5-dsRNA treatment apparently attenuated the cell surface accumulation of Myc-Smo (J–J’). The nuclei were marked by DAPI staining. (K–N’) S2 cells were treated with GFP-dsRNA or UCHL5-dsRNA for 24 h, and then transfected with Myc-Smo plasmid. After 24 h, cells were stimulated by Hh conditional medium for additional 24 h, followed by cell harvesting and staining. Hh medium treatment indeed promoted the cell surface accumulation of Smo (compare L–L’ with H–H’). Knockdown of UCHL5 compromised Hh-mediated the cell membrane accumulation of Smo (compare N–N’ with L–L’). The nuclei were indicated by DAPI staining. (O) Quantification of the Myc-Smo intensity on S2 cell membrane. (P–Q’’’) Myc-Smo protein (green) expressed by MS1096 alone or plus UCHL5 RNAi under the background of Hh overexpression in wing discs. UCHL5 RNAi apparently decreased the cell surface localization of Myc-Smo. The nuclei were marked by DAPI (blue). (R) Quantification of the Myc-Smo intensity in wing discs. The localization of Smo is controlled by Hh pathway activity. An antibody uptake experiment using cultured S2 cells suggested that Hh regulates the cell surface expression of Smo through promoting the recycling of Smo (Jia et al., 2004). In addition, Smo is preferentially localized in the cytoplasm of anterior compartment cells in wing discs where Hh is not present, whereas enriched on the cell membrane of posterior compartment cells where Hh stimulation occurs (Nakano et al., 2004). To test whether the accumulation of Smo induced by Hh stimulation is due to the effect of UCHL5, S2 cells transfected with Myc-Smo were treated with GFP-dsRNA (control) or UCHL5-dsRNA and then treated with Hh-conditioned medium or control medium. Consistent with the previous observations (Liu et al., 2007; Zhao et al., 2007), Hh stimulation indeed promote the cell surface accumulation of Smo (compare Figure 6K–L’ with 6G–H’). Intriguingly, the cell surface accumulation of Smo was attenuated by UCHL5-dsRNA treatment in the presence of Hh stimulation both in S2 cells (Figure 6M–N’) and in wing discs (compare Figure 6Q–Q’” with 6P–P”’), indicating that Hh promotes the cell surface expression of Smo, at least in part, through UCHL5. In addition, we confirmed that the localization of endogenous Smo protein was also regulated by Hh signals (Supplementary Figure S4A–L’’’). Taken together, these data suggest that UCHL5 promotes the cell surface accumulation of Smo (Figure 6O and R). UCHL5 cooperates with Usp8 to regulate Smo The previous study has demonstrated that Usp8 acts as a deubiquitinase of Smo protein (Li et al., 2012; Xia et al., 2012). It is necessary to assess whether Usp8 and UCHL5 redundantly deubiquitinate Smo. Knockdown of UCHL5 (Figure 7A) or Usp8 (Figure 7B) alone decreased Smo protein levels, suggesting that both deubiquitinases could stabilize Smo independently. However combined knockdown of UCHL5 and Usp8 resulted in a dramatic decrease of Smo (Figure 7C and D). Through cell-based ubiquitination assays, we found that knockdown of UCHL5 and Usp8 caused a higher ubiquitination Smo than knockdown of UCHL5 or Usp8 alone (Figure 7E and F). These results show that UCHL5 cooperates with Usp8 to deubiquitinate and stabilize Smo. Figure 7 View largeDownload slide UCHL5 coordinates with Usp8 to regulate Smo. (A–C) Wing discs expressing UCHL5-RNAi alone (A), Usp8-RNAi alone (B), or UCHL5-RNAi plus Usp8-RNAi (C) were immunostained to show Smo expression (white). Expression of UCHL5-RNAi plus Usp8-RNAi resulted in a more dramatic decrease in Smo expression (arrows). (D) Quantification of the Smo intensity in wing discs. White bars represent the wild-type Smo fluorescence; black bars represent Smo fluorescence in the gene knockdown region (mean ± SD from three independent experiments; *P < 0.05, **P ≤ 0.01, student’s t test). (E and F) S2 cells were treated with UCHL5-dsRNA alone, Usp8-dsRNA alone, or UCHL5-dsRNA plus Usp8-dsRNA. Combined knockdown of UCHL5 and Usp8 apparently promoted the ubiquitination of Smo in S2 cells (compare lane 4 with lanes 2, 3). Figure 7 View largeDownload slide UCHL5 coordinates with Usp8 to regulate Smo. (A–C) Wing discs expressing UCHL5-RNAi alone (A), Usp8-RNAi alone (B), or UCHL5-RNAi plus Usp8-RNAi (C) were immunostained to show Smo expression (white). Expression of UCHL5-RNAi plus Usp8-RNAi resulted in a more dramatic decrease in Smo expression (arrows). (D) Quantification of the Smo intensity in wing discs. White bars represent the wild-type Smo fluorescence; black bars represent Smo fluorescence in the gene knockdown region (mean ± SD from three independent experiments; *P < 0.05, **P ≤ 0.01, student’s t test). (E and F) S2 cells were treated with UCHL5-dsRNA alone, Usp8-dsRNA alone, or UCHL5-dsRNA plus Usp8-dsRNA. Combined knockdown of UCHL5 and Usp8 apparently promoted the ubiquitination of Smo in S2 cells (compare lane 4 with lanes 2, 3). UCHL5 plays a conserved role in mammalian cells In mammal, UCHL5 has a homolog UCH37, which encodes a 37 kDa protein (Yao et al., 2008; Nishio et al., 2009). To assess whether the regulation of UCHL5 on Smo is conserved from Drosophila to mammals, we generated a HA-UCH37 transgenic fly (HA-UCH37). Overexpression of HA-UCH37 apparently restored the wing phenotypes induced by UCHL5 knockdown in Drosophila (Figure 8A and B). In addition, we found that the decrease of Ptc expression caused by UCHL5 knockdown (Figure 8C–C”) was rescued via overexpressing HA-UCHL5 (Figure 8D–D”) or HA-UCH37 (Figure 8E–E”), suggesting that UCH37 could functionally replace UCHL5 in the regulation of Hh pathway. Figure 8 View largeDownload slide UCH37 deubiquitinates hSmo and promotes Hh Signaling activity in mammalian cells. (A and B) Adult wings express UCHL5-RNAi alone or UCHL5-RNAi plus HA-tagged UCH37 under the background of Smo–PKA through MS1096. (C–E”) Wing discs expressing UCHL5-RNAi alone, UCHL5-RNAi plus HA-UCHL5, or UCHL5-RNAi plus HA-UCH37 by MS1096 under the background of Smo–PKA were stained for Ptc (white). The decrease of Ptc protein caused by UCHL5 knockdown (C–C”) was restored by overexpressing HA-UCHL5 (D–D”) or HA-UCH37 (E–E”) (arrows). (F) UCH37 could bind to hSmo in NIH-3T3 cells. (G) ShhN treatment promoted the binding between UCH37 and hSmo in NIH-3T3 cells (mean ± SD from three independent experiments; ***P ≤ 0.005, student’s t test). (H) UCH37 inhibited the ubiquitination of hSmo protein in NIH-3T3 cells. (I) Knockdown of UCH37 decreased hSmo levels and Hh pathway activity in NIH-3T3 cells (mean ± SD; ***P ≤ 0.005, student’s t test). (J) Relative mRNA levels of gli1, ptc1, and hhip were revealed by real-time PCR in UCH37 knockdown NIH-3T3 cells (mean ± SD from three independent experiments; ***P≤ 0.005, student’s t test). (K) Gli-luciferase (Gli-luc) reporter assay in NIH-3T3 cells transfected with the indicated constructs. Gli luciferase activities were normalized to Renilla luciferase activities. UCH37 promoted, but UCH37CA attenuated Shh pathway activity (mean ± SD from three independent experiments; **P ≤ 0.01, student’s t test). Figure 8 View largeDownload slide UCH37 deubiquitinates hSmo and promotes Hh Signaling activity in mammalian cells. (A and B) Adult wings express UCHL5-RNAi alone or UCHL5-RNAi plus HA-tagged UCH37 under the background of Smo–PKA through MS1096. (C–E”) Wing discs expressing UCHL5-RNAi alone, UCHL5-RNAi plus HA-UCHL5, or UCHL5-RNAi plus HA-UCH37 by MS1096 under the background of Smo–PKA were stained for Ptc (white). The decrease of Ptc protein caused by UCHL5 knockdown (C–C”) was restored by overexpressing HA-UCHL5 (D–D”) or HA-UCH37 (E–E”) (arrows). (F) UCH37 could bind to hSmo in NIH-3T3 cells. (G) ShhN treatment promoted the binding between UCH37 and hSmo in NIH-3T3 cells (mean ± SD from three independent experiments; ***P ≤ 0.005, student’s t test). (H) UCH37 inhibited the ubiquitination of hSmo protein in NIH-3T3 cells. (I) Knockdown of UCH37 decreased hSmo levels and Hh pathway activity in NIH-3T3 cells (mean ± SD; ***P ≤ 0.005, student’s t test). (J) Relative mRNA levels of gli1, ptc1, and hhip were revealed by real-time PCR in UCH37 knockdown NIH-3T3 cells (mean ± SD from three independent experiments; ***P≤ 0.005, student’s t test). (K) Gli-luciferase (Gli-luc) reporter assay in NIH-3T3 cells transfected with the indicated constructs. Gli luciferase activities were normalized to Renilla luciferase activities. UCH37 promoted, but UCH37CA attenuated Shh pathway activity (mean ± SD from three independent experiments; **P ≤ 0.01, student’s t test). We next determined whether UCH37 is involved in the regulation of hSmo ubiquitination and Shh pathway activity in mammalian cells. First, the co-IP experiments revealed that UCH37 interacted with hSmo (Figure 8F) and Hh promoted their interaction (Figure 8G). Furthermore, we found that UCH37 indeed decreased the ubiquitination level of hSmo (Figure 8H), indicating that UCH37 can deubiquitinate hSmo. The ability of UCH37 to suppress hSmo ubiquitination implies that UCH37 possibly regulates Shh pathway activity. To test this possibility, we performed Gli-luciferase (Gli-Luc) reporter assay in NIH-3T3 cells, which express Shh pathway components and target genes (Zhou et al., 2015b). Knockdown of UCH37 attenuated hSmo levels, suppressed Shh pathway activity (Figure 8I) and the expression of Shh target genes gli1, ptch1 and hhip (Figure 8J). Consistently, overexpression of UCH37 enhanced, whereas UCH37CA attenuated Shh pathway activity (Figure 8K). In mammals, Hh signal transduction occurs at the primary cilium. In the presence of Hh, Smo protein is accumulated to the cilium where it converts Gli proteins from transcriptional repressors to activators (Huangfu and Anderson, 2006; Ma et al., 2016). To determine whether UCH37 regulates hSmo ciliary accumulation, NIH-3T3 cells were transfected with Myc-hSmo plus Sstr3-GFP (for labeling primiary cilia) or with Fg-UCH37CA with or without ShhN treatment. According to our experimental statistics, only ~22% of the cells contained ciliary Myc-hSmo signals (Supplementary Figure S5A and E). While ~85% of the cells exhibited ciliary Myc-hSmo signals after treated with ShhN (Supplementary Figure S5B and E). Importantly, expression of Fg-UCH37CA caused less and weaker Shh-induced hSmo ciliary localization (Supplementary Figure S5C–E). Taken together, these results suggest that UCH37 plays a conserved role in regulating Shh pathway. Discussion As one of the essential components of Hh pathway, Smo contributes to transducing the signal across the cell plasma membrane (Ingham and McMahon, 2001). Accumulation of Smo on the membrane and its ubiquitination are finely governed by the Hh signal. In the absence of Hh, Smo undergoes ubiquitination in the cytoplasm and fails to accumulate on the cell membrane, culminating in turning off Hh pathway and target gene expression. In the presence of Hh, Ptc relieves its inhibitory effect on Smo activity by binding to Hh, resulting in the cell membrane accumulation of Smo and Hh pathway activation (Jia et al., 2004; Li et al., 2012; Jiang and Jia, 2015). The ubiquitination of Smo and its localization are tightly linked. However, it remains largely unknown how Smo ubiquitination is modulated during Hh signaling transduction. In this study, we identified the deubiquitinase UCHL5 as a positive regulator of Hh signaling through genetic screening. In addition, we provided both genetic and biochemical evidences that UCHL5 binds to and deubiquitinates Smo, culminating in stabilizing Smo. We also figured out that the recognition of Smo by UCHL5 is dependent on the SAID domain of Smo C-tail. Moreover, UCHL5 promoted the cell membrane localization of Smo protein. Finally, we found that the mammalian homolog UCH37 plays a conserved role in regulating Smo and Hh pathway. Our findings thus unveil that a conserved deubiquitinase UCHL5/UCH37 regulates the ubiquitination and localization of Smo to achieve optimal Hh pathway activity. UCHL5 is a member of the UCH-containing deubiquitinases in Drosophila. UCHL5 binds to Smo through its N-terminally located UCH domain (Figure 3C). In addition to UCHL5, Drosophila also has two other UCH-containing proteins: UCH-D (CG4265) and UCHL5R (CG1950). Intriguingly, we revealed that only UCHL5 and UCHL5R form a complex with Smo, while UCH-D did not show any interaction with Smo (Supplementary Figure S3E and F). These observations suggest that the amino acid composition in UCH domain may contribute to the substrate specificity of different deubiquitinases, albeit their space structure of UCH domainsis similar. Moreover, we found that knockdown of UCHL5R decreased Smo and Hh pathway activity (data not shown), indicating UCHL5R also plays as a potential deubiquitinase of Smo. Taken together, it is possible that the different deubiquitinases may play additive roles to achieve most efficient deubiquitination of Smo. The SAID domain, which is important for the maintenance of Smo ‘closed’ conformation by arginine-rich motifs, has been found to be critical for the cell membrane accumulation of Smo (Zhao et al., 2007). In addition, Hrs binds to the SAID domain to promote Smo ubiquitination and degradation (Fan et al., 2013). These data strongly suggest that the SAID domain plays a negative role in regulating Smo activity. Our work uncovered that the deubiquitinase UCHL5 stabilizes Smo via binding to the SAID domain on Smo (Figure 3D–G), indicating that the SAID domain also has positive effect on Smo activity. These two view points are not paradoxical because the SAID domain we studied in this work covers 158 amino acids. We next want to find which amino acids on Smo are responsible for Smo-UCHL5 interaction. It is likely that the arginine-rich motifs in the SAID domain are not involved in mediating its interaction with UCHL5. In addition, since both UCHL5 and Hrs bind to the SAID domain of Smo and play opposite roles on the Smo stability, it’s interesting to further investigate whether they occupy the SAID domain of Smo in a competitive way. Hh blocks Smo ubiquitination, which is vital for its activity. What is the underlying mechanism? Although the previous data have revealed that Usp8 decreases Smo ubiquitination, this event is possible out of Hh control (Li et al., 2012). Usp8 attenuates Smo ubiquitination regardless of the Hh signaling status and the interaction between Usp8 and Smo is not apparently modulated by Hh stimulation (Li et al., 2012), suggesting that Smo deubiquitination by Usp8 is unlikely to be a mechanism by which Hh blocks Smo ubiquitination. In this study, we found that Hh stimulation boosted UCHL5–Smo association (Figure 3H–J), indicating that the effect of UCHL5 on Smo is amplified by Hh signaling. In addition, we found that knockdown of UCHL5 inhibited Hh-mediated Smo deubiquitination (Figure 4J), suggesting that Hh blocks Smo ubiquitination, at least partly through promoting UCHL5–Smo interaction. The deubiquitinating function of UCH37 on hSmo we unveiled here suggests that UCH37 may play a role in regulating the Hh pathway in mammalian systems. Deletion of UCH37 resulted in prenatal lethality in mice associated with severe defect in embryonic brain development (Al-Shami et al., 2010). It is well known that the Hh pathway plays an indispensible role during the development of brain (Rowitch et al., 1999; Ruiz i Altaba et al., 2002; Farmer et al., 2016). Therefore, it would be interesting to investigate whether UCH37 deficiency embryos show any defects in the Hh pathway. Increasing observations indicate that UCH37 is also involved in many pathological processes, including tumorigenesis. The expressions of UCH37 are upregulated in most epithelial ovarian cancer patients (Wang et al., 2014). UCH37 is increased in many patients of esophageal squamous cell carcinoma (Chen et al., 2012). In addition, UCH37 upregulation has been tightly linked to human hepatocellular carcinoma (Fang et al., 2012). These data suggest that UCH37 possibly plays an oncogenic role in tumorigenesis. Since Hh pathway hyperactivation induces many tumors, it should be fruitful to determine whether Hh-Smo axis contributes to UCH37-induced tumors. Multiple studies have already revealed that UCH37 acts as a potential target for cancer therapy (Chen et al., 2013). Our finding that UCH37 positively regulates the Hh pathway through deubiquitinating Smo provides UCH37 as a potential drug target for Hh-related tumors. Materials and methods Immunostaining and in situ hybridization of wing discs, cell culture, transfection, immunoprecipitation, western blot, and real-time PCR were performed according to standard protocols. For detailed procedures, fly strains, and constructs used in this study, see the Supplementary Experimental Procedures. Generating mutant clones Clones of mutant cells were generated by the FLP-FRT system as described (Banning et al., 2011). The genotype for generating UCHL5 clone is hs-flp; UCHL5j2B8FRT80B/ubi-GFP FRT80B. The mutant clones were marked by loss of GFP expression. RNA interference To knock down genes in S2 cells, the double stranded RNA (dsRNA) was generated by MEGAscript High Yield Transcription Kit (Ambion) according to the manufacturer’s instructions. DNA templates targeting UCHL5 (aa1-324), UCHL5 (aa1-221), UCHL5 (aa153-324) and Usp8 (aa274-557) were generated by PCR and used for generating dsRNA. dsRNA targeting the GFP full-length coding sequence was used as a control. The dsRNA-mediated gene knockdown was performed as previously described (Lawrence Lum et al., 2003; Li et al., 2012). To silence UCH37 expression in mammalian cells, a mixture of two different siRNAs was used. The siRNA sequences were UCH37-siRNA-1 (5′-CCGAGCUCAUUAAAGGAUUdTd-3′), UCH37-siRNA-2 (5′-CCGAUUGAUUUGGUGCAUdTdT-3′), and mock-siRNA (5′-UUCUCCGAACGUGUCACGUdTdT-3′). All siRNA duplexes were transfectedat a final concentration of 100 nM using lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Protein stability assays and ubiquitination assays S2 cells were plated in 10-cm dishes and transfected with plasmids after 18–24 h. After another 24 h, the cells were split and transferred into 6-well cell culture plates at equivalent densities. Cells were treated with 20 μg/ml CHX (Calbiochem) for the indicated times before harvesting. After western blots, the band intensity was measured by Image J. To examine the levels of ubiquitinated Smo, S2 cells were transfected with Smo and then lysed with denaturing buffer (1% SDS, 50 mM Tris, pH 7.5, 0.5 mM EDTA, and 1 mM DTT) and ultrasonic. The lysates were then diluted 10-fold with regular lysis buffer and subjected to immunoprecipitation and western blot analysis. Statistical analysis Relative intensity of Smo in wing discs was the Smo fluorescence intensity in RNAi expression region divided by the Smo fluorescence intensity in wild-type region. Similarly, relative intensity of cilia-located Smo was the cilia-located Smo fluorescence intensity divided by the whole-cell Smo fluorescence intensity. relative intensity of Smo on the membrane with various treatment was obtained, with the wild-type Smo on the membrane normalized to 1. Imaging data were analyzed in the program Image J. The data shown in the figures were representative of three or more independent experiments and were analyzed by Student’s t-test, with P < 0.05 considered significant. Supplementary material Supplementary material is available at Journal of Molecular Cell Biology online. Acknowledgements We thank Dr Yun Zhao (Shanghai Institute of Biochemistry and Cell Biology, CAS), Dr Steven Y. Cheng (Nanjing Medical University), and Dr Yongbin Chen (Kunming Institute of Zoology, CAS) for providing plasmids and reagents. We also appreciate National Institute of Genetics of Japan (NIG), Vienna Drosophila RNAi Center (VDRC), and the Bloomington Stock Center (BSC) for providing fly stocks. We apologize to colleagues whose works were not cited owing to space limitation. Funding This work was supported by grants from the National Basic Research Program of China (2011CB943902), the National Natural Science Foundation of China (30971679, 31071264, and 31271531), and the Fundamental Research Funds for the Central Universities (090314380019). Conflict of interest: none declared. Author contributions: Q.Z. and Z.Z. designed the experiments. Z.Z., X.Y., S.P., P.C., W.J., and Z.S performed the experiments. Z.Z., X.Y., and Q.Z. wrote the manuscript. References Al-Shami, A., Jhaver, K.G., Vogel, P., et al.  . ( 2010). Regulators of the proteasome pathway, Uch37 and Rpn13, play distinct roles in mouse development. PLoS One  5, e13654. 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Published: Oct 13, 2017

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