Balanced ubiquitylation and deubiquitylation of Frizzled regulate cellular responsiveness to Wg/Wnt
2010; Springer Nature; Volume: 29; Issue: 13 Linguagem: Inglês
10.1038/emboj.2010.100
ISSN1460-2075
AutoresAkiko Mukai, Miki Yamamoto‐Hino, Wakae Awano, Wakako Watanabe, Masayuki Komada, Satoshi Goto,
Tópico(s)Developmental Biology and Gene Regulation
ResumoArticle21 May 2010free access Balanced ubiquitylation and deubiquitylation of Frizzled regulate cellular responsiveness to Wg/Wnt Akiko Mukai Akiko Mukai Research Group of Glycobiology and Glycotechnology, Mitsubishi-Kagaku Institute of Life Sciences, Machida, Japan Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan Search for more papers by this author Miki Yamamoto-Hino Miki Yamamoto-Hino Research Group of Glycobiology and Glycotechnology, Mitsubishi-Kagaku Institute of Life Sciences, Machida, JapanPresent address: Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Search for more papers by this author Wakae Awano Wakae Awano Mutant Flies Laboratory, Mitsubishi-Kagaku Institute of Life Sciences, Machida, Japan Search for more papers by this author Wakako Watanabe Wakako Watanabe Research Division, Advanced Medical Research Laboratory, Mitsubishi Tanabe Pharma, Yokohama, Japan Search for more papers by this author Masayuki Komada Corresponding Author Masayuki Komada Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan Search for more papers by this author Satoshi Goto Corresponding Author Satoshi Goto Research Group of Glycobiology and Glycotechnology, Mitsubishi-Kagaku Institute of Life Sciences, Machida, Japan Mutant Flies Laboratory, Mitsubishi-Kagaku Institute of Life Sciences, Machida, JapanPresent address: Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Search for more papers by this author Akiko Mukai Akiko Mukai Research Group of Glycobiology and Glycotechnology, Mitsubishi-Kagaku Institute of Life Sciences, Machida, Japan Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan Search for more papers by this author Miki Yamamoto-Hino Miki Yamamoto-Hino Research Group of Glycobiology and Glycotechnology, Mitsubishi-Kagaku Institute of Life Sciences, Machida, JapanPresent address: Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Search for more papers by this author Wakae Awano Wakae Awano Mutant Flies Laboratory, Mitsubishi-Kagaku Institute of Life Sciences, Machida, Japan Search for more papers by this author Wakako Watanabe Wakako Watanabe Research Division, Advanced Medical Research Laboratory, Mitsubishi Tanabe Pharma, Yokohama, Japan Search for more papers by this author Masayuki Komada Corresponding Author Masayuki Komada Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan Search for more papers by this author Satoshi Goto Corresponding Author Satoshi Goto Research Group of Glycobiology and Glycotechnology, Mitsubishi-Kagaku Institute of Life Sciences, Machida, Japan Mutant Flies Laboratory, Mitsubishi-Kagaku Institute of Life Sciences, Machida, JapanPresent address: Department of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Search for more papers by this author Author Information Akiko Mukai1,2, Miki Yamamoto-Hino1, Wakae Awano3, Wakako Watanabe4, Masayuki Komada 2 and Satoshi Goto 1,3 1Research Group of Glycobiology and Glycotechnology, Mitsubishi-Kagaku Institute of Life Sciences, Machida, Japan 2Department of Biological Sciences, Tokyo Institute of Technology, Yokohama, Japan 3Mutant Flies Laboratory, Mitsubishi-Kagaku Institute of Life Sciences, Machida, Japan 4Research Division, Advanced Medical Research Laboratory, Mitsubishi Tanabe Pharma, Yokohama, Japan *Corresponding authors: Department of Biological Sciences, Tokyo Institute of Technology, 4259-B16 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Tel.: +81 45 924 5703; Fax: +81 45 924 5771; E-mail: [email protected] of Physiology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. Tel.: +81 3 3353 1211; Fax: +81 3 3357 5445; E-mail: [email protected] The EMBO Journal (2010)29:2114-2125https://doi.org/10.1038/emboj.2010.100 There is a Have you seen ...? (July 2010) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Wingless (Wg)/Wnt has been proposed to exert various functions as a morphogen depending on the levels of its signalling. Therefore, not just the concentration of Wg/Wnt, but also the responsiveness of Wg/Wnt-target cells to the ligand, must have a crucial function in controlling cellular outputs. Here, we show that a balance of ubiquitylation and deubiquitylation of the Wg/Wnt receptor Frizzled determines the cellular responsiveness to Wg/Wnt both in mammalian cells and in Drosophila, and that the cell surface level of Frizzled is regulated by deubiquitylating enzyme UBPY/ubiquitin-specific protease 8 (USP8). Although ubiquitylated Frizzled underwent lysosomal trafficking and degradation, UBPY/USP8-dependent deubiquitylation led to recycling of Frizzled to the plasma membrane, thereby elevating its surface level. Importantly, a gain and loss of UBPY/USP8 function led to up- and down-regulation, respectively, of canonical Wg/Wnt signalling. These results unveil a novel mechanism that regulates the cellular responsiveness to Wg/Wnt by controlling the cell surface level of Frizzled. Introduction Wingless (Wg)/Wnt proteins are secreted lipoglycoprotein ligands that control various aspects of development, including cell proliferation, migration, fate specification, and polarity formation (Veeman et al, 2003; Logan and Nusse, 2004; Klein and Mlodzik, 2005; Clevers, 2006). Wg/Wnt signals are transmitted through three major pathways in target cells: the canonical, planar cell polarity, and Ca2+ pathways (Schulte and Bryja, 2007). As a morphogen, Wg/Wnt regulates tissue patterning depending on the strength of the canonical pathway signalling generated in target cells. Morphogens are secreted by restricted groups of cells and transported through tissues to form a graded distribution (Kornberg and Guha, 2007). In the Drosophila wing disc, Wg is expressed in the dorso-ventral border and diffuses gradually in the wing pouch. In regions proximal to the border, sensory organ precursor (SOP) cells are induced by strong Wg signals to differentiate into mechanosensory and chemosensory bristles at the adult wing margin (Couso et al, 1994). On the other hand, the threshold of Wg signals required for the expression of Distal-less (Dll) and vestigial (vg) [QE] is lower. Thus, their expression expands to regions more distal to the border, and induces proliferation of the distal cells (Zecca et al, 1996; Neumann and Cohen, 1997). Forced expression of DFrizzled-2 (DFz2), the high-affinity Wg receptor in Drosophila, in the wing disc results in excess Wg signalling and misregulated extracellular Wg distribution, and induces ectopic sensory bristles in regions distal to the border (Cadigan et al, 1998), suggesting that Wg signalling is controlled by the cell surface Fz level of target cells. Therefore, regulation of the Fz level is crucial for proper response to Wg. Endocytosis is one of the central mechanisms that control the amount of plasma membrane proteins. Fz endocytosis and subsequent lysosomal trafficking have been reported in Drosophila and mammalian cells (Chen et al, 2003; Piddini et al, 2005; Blitzer and Nusse, 2006; Rives et al, 2006; Seto and Bellen, 2006; Yamamoto et al, 2006). However, it is unclear whether Fz endocytosis affects Wg signalling negatively or positively (Kikuchi and Yamamoto, 2007; Gagliardi et al, 2008). In addition, although the involvement of various proteins including β-arrestin 2, Dishevelled-2 (Dvl-2), protein kinase C, Arrow (Arr)/LRP 5 and 6, and AP2 in clathrin- and caveolin-mediated Fz endocytosis has been shown (Chen et al, 2003; Yamamoto et al, 2006; Yu et al, 2007), regulatory mechanisms underlying lysosomal trafficking of endocytosed Fz are not clear. Accordingly, regulation of the cell surface Fz level by endocytosis and lysosomal degradation is largely unknown. Mechanisms of lysosomal trafficking of receptor tyrosine kinases (RTKs) have been extensively studied (Gruenberg and Stenmark, 2004; Saksena et al, 2007). On ligand binding, activated RTKs undergo rapid endocytosis and are transported to the early endosome. RTKs are subsequently incorporated into lumenal vesicles that invaginate from the endosomal limiting membrane. Finally, fusion of such late endosomes, referred to as multivesicular bodies (MVBs), with the lysosome delivers RTKs into the lysosomal lumen. In this trafficking process, ubiquitylation of RTKs serves as a sorting signal that targets the receptors from the early endosome to the lysosome through the MVB. On the endosomal membrane, ubiquitylated RTKs are sorted by four protein complexes termed as endosomal sorting complex required for transport (ESCRT)-0, I, II, and III, which recognize the ubiquitin (Ub) moieties of RTKs and direct the cargo proteins into the invaginating MVB vesicle (Gruenberg and Stenmark, 2004; Saksena et al, 2007). UBPY/Ub-specific protease 8 (USP8), a deubiquitylating enzyme of the USP family, participates in the endosomal sorting of ubiquitylated RTKs through interaction with ESCRT-0 and ESCRT-III (Mizuno et al, 2005; Row et al, 2007). UBPY deubiquitylates ligand-activated epidermal growth factor receptor (EGFR) on the endosome and regulates its lysosomal traffic, although it is under debate whether deubiquitylation promotes or suppresses EGFR degradation (Mizuno et al, 2005; Row et al, 2006; Niendorf et al, 2007; Komada, 2008). In this study, we show that Fz undergoes ubiquitylation-dependent trafficking to the lysosome and that UBPY facilitates Wg/Wnt signalling by suppressing the trafficking/degradation of Fz and increasing its cell surface level through its recycling, providing a novel mechanism that regulates the cellular responsiveness to Wg/Wnt. Results Drosophila UBPY is required for sensory bristle formation in the Drosophila wing To find a new regulator of Wg signalling at the level of protein localization, transport, or degradation, we screened ∼400 genes implicated in membrane trafficking in the genome-wide Drosophila RNA interference (RNAi) library (http://www.shigen.nig.ac.jp/fly/nigfly/about/aboutRnai.jsp). We induced double-strand RNAs (dsRNAs) corresponding to 500 nucleotides in the open reading frames of such genes in the wing pouch by crossing with the scalloped (sd)-Gal4 driver, and examined the formation of sensory bristles at the wing margin. This screen led to the identification of nine genes, knockdown of which resulted in various defects in bristle formation (data not shown). Among them, depletion of an orthologue of the mammalian deubiquitylating enzyme UBPY/USP8 (Figure 1A) (Naviglio et al, 1998; Kato et al, 2000) caused loss of sensory bristles (Figure 1B and C). Drosophila UBPY (dUBPY, CG5798) exhibits an overall amino-acid sequence identity of 28% to human UBPY, and harbours the Cys- and His-box motifs that form the catalytic core, as well as most of the hallmark features of mammalian UBPY (Figure 1A) (Komada, 2008). Real-time PCR analysis showed that the dsRNA for dUBPY, when induced in whole fly bodies by actin-Gal4, suppresses the expression of dUBPY mRNA to ∼30% of that in controls, verifying the effectiveness of this in vivo RNAi system (data not shown). Figure 1.dUBPY regulates Wg signalling in the wing disc. (A) Domain structures of human hUBPY and Drosophila dUBPY. Positions of the Cys- and His-boxes, MIT domain, rhodanese homology (RH) domain, and SH3-binding motifs (SBMs), as well as the dsRNA-target sites used in this study, are indicated. (B–E) Top panels show wings from (B) control (sd-Gal; UAS-GFP RNAi), (C) RNAi-1 (sd-Gal; UAS-lacZ/UAS-dUBPY RNAi-1), (D) RNAi-2 (sd-Gal4; UAS-lacZ/UAS-dUBPY RNAi-2), and (E) partially rescued knockout (UAS-dUBPY; UBPY K.O.) flies. Bottom panels show high-magnification images of the wing margins indicated by arrowheads in top panels. (F–I) Activity staining of β-gal derived from neuralized-lacZ (A101) (F, G) and wg-lacZ (H, I) in control (F, H) and dUBPY RNAi (G, I) wing discs. Arrows indicate the positions of the dorso-ventral border (F, G). (J–K″) Anti-Sens (J) and anti-Arm (K) staining of dUBPY knockout clones, which are negative for β-gal (J′, K′, −/−), in the wing disc. J″ and K″ are merged images. Bars, 100 μm (F–I); 10 μm (J″, K″). Download figure Download PowerPoint The possibility that the observed wing defect was due to an off-target effect of the dsRNA was excluded by the following experiments. First, knockdown by a second set of dsRNA (Figure 1A) also caused the loss of sensory bristles (Figure 1D). Second, we generated dUBPY knockout mutant flies (Supplementary Figure S1). As the knockout caused embryonic lethality, partially rescued knockout strains were developed. These adults exhibited the same loss of sensory bristles as seen in the knockdown flies (Figure 1E). Accordingly, we concluded that this phenotype results from the dysfunction of dUBPY. dUBPY facilitates canonical Wg signalling in Drosophila To elucidate the cause of the loss of sensory bristles on dUBPY knockdown, we examined the specification of SOP cells, which can be marked by neuralized-lacZ (A101) (Campuzano and Modolell, 1992), along the dorso-ventral border in the wing disc (Figure 1F, arrow). In the dUBPY knockdown wing disc, expression of neuralized-lacZ was barely detectable in this region (Figure 1G, arrow). As SOP cells are specified by sequential activation of the Notch and Wg signalling pathways (Couso et al, 1994; de Celis and Bray, 1997), expression of the Notch-target genes Cut, vgBE-lacZ, and wg-lacZ (Kim et al, 1996) was examined. These genes were normally expressed in the knockdown disc (Figure 1H and I; Supplementary Figure S2A–D), suggesting that Notch signalling is not compromised by knockdown of dUBPY. In contrast, the number of cells expressing the Wg-target protein Senseless (Sens; Nolo et al, 2000) and the amount of Armadillo (Arm)/β-catenin protein, which accumulates intracellularly in response to Wg signal, were obviously reduced in the dUBPY knockout clones in the wing disc (Figure 1J–K″). These results suggested that canonical Wg signalling is impaired in the absence of dUBPY and thus that dUBPY is a positive regulator of Wg signalling in Drosophila. As the level of wg expression was not affected in the dUBPY knockdown wing disc (Figure 1H and I), and as the amount and distribution of extracellularly secreted Wg were indistinguishable between the control and dUBPY knockdown discs (Supplementary Figure S2E and F), dUBPY was suggested to participate in Wg signalling in Wg-target cells. UBPY facilitates canonical Wnt signalling in mammalian cells To biochemically address how UBPY regulates Wg/Wnt signalling, we used mammalian cells in culture. First, we examined whether UBPY is required for the Wnt canonical pathway in mammals. The activity of the TCF/LEF transcription factors that induces Wnt-target gene expression (MacDonald et al, 2009) was tested using the TOP-FLASH luciferase reporter gene assay in HEK293T cells. The level of Wnt-target expression induced by Wnt3a was slightly up-regulated by overexpression of wild-type UBPY (UBPYWT) (Figure 2A). However, UBPYC748A,a catalytically inactive mutant that acts in a dominant-negative manner (Mizuno et al, 2005), suppressed expression to the same level as seen in unstimulated cells (Figure 2A). In contrast, overexpression of UBPYS680A, a constitutively active mutant that does not undergo 14-3-3-mediated catalytic inhibition (Mizuno et al, 2007), caused a significant increase in Wnt3a-induced target expression (Figure 2A). Figure 2.UBPY facilitates canonical Wnt signalling in mammalian cells. (A) HEK293T cells were transfected with control FOP-FLASH or TOP-FLASH luciferase together with the indicated FLAG-UBPY constructs, and treated with Wnt3a overnight. Relative luciferase activity in the cell lysates is shown as mean±s.d. (n=4, *P<0.02, t-test). (B) NIH3T3 cells were transfected with the indicated FLAG-UBPY constructs, treated with Wnt3a for 1.5 h, and the lysates were immunoblotted with indicated antibodies. Positions of phosphorylated (P-Dvl-2) and non-phosphorylated Dvl-2 are indicated (top). The ratios of P-Dvl-2 to Dvl-2, which are normalized to that in mock-transfected cells stimulated with Wnt3a, are indicated below the top panel. (C) HEK293T cells were transfected with the indicated FLAG-UBPY constructs, treated with Wnt3a overnight, and their lysates were separated into cytoplasmic and membrane fractions. Each fraction was immunoblotted with indicated antibodies. FLAG-UBPY was solely recovered in the membrane fraction as described earlier (Mizuno et al, 2005). (D) HeLa cells were transfected with or without UBPY siRNAs and treated with Wnt3a for 5 h in the presence of cycloheximide. Cell lysates were immunoblotted with indicated antibodies. Relative intensity of the β-catenin bands is indicated (C, D). Download figure Download PowerPoint To examine whether UBPY regulates the Wnt canonical pathway at the level of Fz receptors, phosphorylation of Dvl-2, which is induced by its direct interaction with activated Fz (MacDonald et al, 2009), was examined. Although the level of Wnt3a-induced Dvl-2 phosphorylation was similar between mock- and UBPYWT-transfected NIH3T3 cells, it was lower in UBPYC748A-expressing cells (Figure 2B). Conversely, when UBPYS680A was expressed, Dvl-2 phosphorylation was even detected in unstimulated cells, and its level after Wnt3a stimulation was much higher than in control cells (Figure 2B). As the canonical pathway signalling relies on stabilization of β-catenin acting downstream of Dvl (MacDonald et al, 2009), regulation of the cytoplasmic β-catenin level by UBPY was also examined. HEK293T cells transfected with UBPYWT, UBPYC748A, or UBPYS680A were stimulated with Wnt3a, and their cytoplasmic fractions were immunoblotted with anti-β-catenin antibody. Compared with cells expressing UBPYWT, the level of cytoplasmic β-catenin was decreased in UBPYC748A-expressing cells and increased in UBPYS680A-expressing cells (Figure 2C). Similarly, RNAi-mediated knockdown of endogenous UBPY in HeLa cells also resulted in a reduced level of β-catenin accumulation on Wnt3a stimulation (Figure 2D). Collectively, these results suggested that the UBPY-mediated regulation of canonical Wg/Wnt signalling is conserved in mammalian cells, and that Fz is a target of UBPY. Fz undergoes ubiquitylation and deubiquitylation in mammalian cells Mammalian UBPY deubiquitylates ligand-activated RTKs on the endosome (Mizuno et al, 2005; Row et al, 2006). However, it remains unknown whether Fz undergoes ubiquitylation. To examine this question, Frizzled-4 (Fz4; Figure 3A), a widely expressed member of the mammalian Fz family, was tagged with the FLAG epitope and expressed in HeLa cells. The cells were treated with or without Wnt3a for 15 min and lysed using a hot lysis method to disrupt Fz4 interaction with other ubiquitylated proteins. FLAG-Fz4 was immunoprecipitated from the lysates with anti-FLAG antibody and immunoblotted with anti-Ub antibody. Fz4 was detected as high-molecular-weight band shifts, suggesting that Fz4 undergoes ubiquitylation (Figure 3B). When FLAG-tagged Ub was co-transfected with HA-tagged Fz4, ubiquitylated Fz4 was detected by anti-FLAG immunoblotting of anti-HA immunoprecipitate (Figure 3C, left, top). To elucidate whether Fz is conjugated with poly-Ub or mono-Ub, we used several Ub mutants. As Lys48 and Lys63 are the major sites used for poly-Ub chain formation (Haglund and Dikic, 2005), we co-expressed UbK48R and UbK63R, in which Lys48 and Lys63, respectively, are replaced to Arg, with HA-Fz4. Fz4 was similarly ubiquitylated in cells expressing these mutants (Figure 3C, left, top). We further examined additional Ub mutants: UbK0, UbR48K, and UbR63K. UbK0 harbours Lys-to-Arg replacement at all seven Lys residues. UbR48K and UbR63K are mutants in which all Lys residues except for Lys48 and Lys63, respectively, are replaced with Arg. The pattern of Fz4 ubiquitylation was similar among cells expressing these Ub mutants (Figure 3C, right) and wild-type Ub (Figure 3C, left). These results suggested that none of the Lys residues in Ub (including Lys48 and Lys63) are required for Fz4 ubiquitylation. We thus suggest that Fz4 is monoubiquitylated at multiple sites. Figure 3.Fz4 undergoes ubiquitylation and deubiquitylation in mammalian cells. (A) Schematic structure of mouse Fz4. Positions of the 12 Lys residues facing the cytoplasm are indicated. (B) HeLa cells were transfected with FLAG-Fz4 with or without HA-UBPYC748A, and treated with Wnt3a for 15 min. FLAG-Fz4 was immunoprecipitated and immunoblotted with indicated antibodies. (C) HeLa cells were transfected with HA-Fz4 with the indicated FLAG-Ub constructs. HA-Fz4 was immunoprecipitated and immunoblotted with indicated antibodies. (D) HeLa cells were transfected with FLAG-Fz4 with or without UBPY siRNAs, and treated with Wnt3a for 1 h. FLAG-Fz4 was immunoprecipitated and immunoblotted with indicated antibodies. (E) HeLa cells were transfected with FLAG-tagged Fz4 or Fz4K0 with or without HA-UBPYC748A, and labelled with biotin on the cell surface. Biotinylated FLAG-Fz4 proteins were immunoprecipitated and immunoblotted with indicated antibodies. (F) HeLa cells were transfected with FLAG-Fz4 with HA-tagged UBPYWT or UBPYC748A, and treated with Wnt3a for 1 h. FLAG-Fz4 was immunoprecipitated and immunoblotted with indicated antibodies. Total lysates were also immunoblotted with indicated antibodies (B–F, lower panels). Asterisks indicate the IgG heavy chain used for immunoprecipitation (B, C, E, F). (G) FLAG-UBPY proteins, expressed in COS-7 cells and immunopurified with anti-FLAG beads, were stained with Coomassie Brilliant Blue (CBB) after electrophoresis (left). HA-Fz4 was expressed in HeLa cells, immunoprecipitated with anti-HA antibody, and incubated with the purified UBPY proteins. The reaction mixtures were immunoblotted with indicated antibodies (right). Download figure Download PowerPoint The level of Fz4 ubiquitylation was drastically elevated when dominant-negative UBPYC748A was co-expressed (Figure 3B) or UBPY was depleted using RNAi (Figure 3D). We also constructed a Fz4K0 mutant in which all 12 of the Lys residues facing the cytoplasm (Figure 3A) were replaced by Arg, and expressed it in HeLa cells. Cell surface FLAG-Fz4K0 was labelled with biotin and sequentially precipitated with streptavidin and anti-FLAG antibody. Immunoblotting of precipitated FLAG-Fz4K0 with anti-Ub antibody showed that this mutant is no longer ubiquitylated on the cell surface even when UBPYC748A is expressed (Figure 3E). In addition, interaction between Fz4 and UBPY was observed by co-immunoprecipitation of HA-tagged UBPY proteins with FLAG-Fz4 from cell lysates (Figure 3F). Finally, we examined whether UBPY deubiquitylates Fz4 in vitro. FLAG-tagged UBPY proteins were immunopurified from transfected cells using anti-FLAG antibody (Figure 3G, left) and incubated with ubiquitylated HA-Fz4, which was also immunoprecipitated from transfected cells. As expected, wild-type UBPY, but not catalytically inactive UBPYC748A, deubiquitylated Fz4 (Figure 3G, right). In addition, constitutively active UBPYS680A exhibited higher activity than wild-type UBPY (Figure 3G, right). Collectively, these data showed that Fz4 is a substrate for UBPY. However, ubiquitylation (Figure 3B and D) and UBPY binding (Figure 3F) of Fz4 were not enhanced by Wnt3a, suggesting that unlike RTKs, the level of Fz ubiquitylation is not regulated by ligand stimulation. UBPY regulates degradation and cell surface level of Fz in mammalian cells On the basis of the finding that Fz4 undergoes ubiquitylation, we next investigated the degradation of Fz4. We first compared the rate of Fz4 degradation in unstimulated and Wnt-stimulated cells. HeLa cells were transfected with FLAG-Fz4, and cell surface proteins were pulse labelled with biotin and chased in the presence or absence of Wnt3a. Immunoprecipitation with anti-FLAG antibody followed by blotting with peroxidase-conjugated streptavidin detected cell surface FLAG-Fz4 mainly as a ladder of ∼55-, 63-, and 70-kDa bands (Figure 4A, top, arrow, closed arrowhead, and open arrowhead). These bands probably represent Fz4 proteins conjugated with zero, one, and two Ub molecules, respectively, because the 63- and 70-kDa forms were not detected when non-ubiquitylatable Fz4K0 was expressed (Figure 4G, top). The 63-kDa band was faint in Figure 3 because of a lower amount of anti-Ub antibody bound to mono- than to multiply ubiquitylated proteins. The intensities of these bands were summed, and the ratio of the intensity at 3 and 6 h of chase to that at 0 h was determined (Figure 4A, bottom). This comparison showed that the degradation rate of biotinylated FLAG-Fz4 is similar in unstimulated and Wnt-stimulated cells, suggesting that ligand stimulation does not accelerate Fz degradation. This was consistent with the results showing that Wnt did not enhance Fz ubiquitylation (Figure 3B and D). To determine how Fz is degraded, we examined the effects of a lysosome inhibitor, bafilomycin A1, and a proteasome inhibitor, MG132. Degradation of biotinylated cell surface FLAG-Fz4 was delayed by treating cells with bafilomycin A1, but not with MG132, suggesting that Fz4 is degraded in the lysosome (Supplementary Figure S3). Figure 4.UBPY regulates degradation and cell surface level of Fz4 in mammalian cells. (A) HeLa cells were transfected with FLAG-Fz4, labelled with biotin on the cell surface, and treated with or without Wnt3a for 3 or 6 h. FLAG-Fz4 was immunoprecipitated and blotted with streptavidin and anti-FLAG antibody. Arrows, closed arrowheads, and open arrowheads indicate Fz proteins conjugated with zero, one, and two Ub molecules, respectively. (B) HeLa cells were transfected with FLAG-Fz4 with HA-tagged UBPYC748A or UBPYS680A, labelled with biotin, and chased for 6 h. FLAG-Fz4 was immunoprecipitated and blotted with streptavidin and anti-FLAG antibody. Total lysates were immunoblotted with anti-HA antibody. (C–D″) HEK293T cells were transfected with Fz4, which was FLAG-tagged extracellularly, together with HA-tagged UBPYS680A (C–C″) or UBPYC748A (D–D″). Living cells were stained with anti-FLAG antibody (C, D). After fixation, cells were further stained with anti-HA antibody and TO-PRO-3 (C′, D′). Arrows indicate cells expressing UBPYS680A or UBPYC748A. Arrowheads indicate cells expressing no ectopic UBPY. C″ and D″ are merged images. Bars, 10 μm. (E, F) Anti-FLAG fluorescence intensity of FLAG-Fz4-expressing cells in the experiments in C–C″ and D–D″ was quantified and shown as mean±s.d. (n [field of view]=10–25, *P<0.01, t-test). (G) HeLa cells were transfected with FLAG-tagged Fz4 or Fz4K0, labelled with biotin, and chased for 6 h. FLAG-Fz4 proteins were immunoprecipitated and blotted with streptavidin and anti-FLAG antibody. The intensity of the biotinylated Fz4 bands was quantified, and the ratio of the intensity after 3 or 6 h of chase to that at 0 h is shown as mean±s.d. (n=5, *P<0.01, t-test) (A, B, bottom; G, right). (H) HEK293T cells were transfected with FOP-FLASH or TOP-FLASH luciferase together with the indicated Fz4 or UBPY constructs, and treated with Wnt3a overnight. Relative luciferase activity in the cell lysates is shown as mean±s.d. (n=3, *P<0.02, t-test). (I) HEK293T cells were transfected with TOP-FLASH luciferase together with the indicated Fz4 and UBPY constructs and treated with Wnt3a overnight. Relative luciferase activity in UBPYC748A- and UBPYS680A-expressing cells to that in UBPYWT-expressing cells is shown (mean±s.d.; n=3, *P<0.02, t-test). Download figure Download PowerPoint When dominant-negative UBPYC748A was overexpressed, non-ubiquitylated Fz4 was lost (Figure 4B, top, arrow), but degradation of ubiquitylated Fz4 was delayed (Figure 4B, top, closed, and open arrowheads) during the chase period. This delayed degradation of ubiquitylated Fz will be discussed below (see Discussion). More importantly, Fz4 degradation was also inhibited when constitutively active UBPYS680A was expressed (Figure 4B). In this case, however, non-ubiquitylated Fz4 accumulated (Figure 4B, top, arrow), suggesting that deubiquitylated Fz escapes from degradation. After endocytosis, non-ubiquitylated plasma membrane proteins are recycled back to the cell surface (Gruenberg and Stenmark, 2004). To test whether deubiquitylated Fz4 is returned to the cell surface in UBPYS680A-expressing cells, we examined the cell surface level of Fz4 in two experiments. First, we performed surface immunostaining of living HEK293T cells transfected with N-terminally (extracellularly) FLAG-tagged Fz4 together with HA-tagged UBPY proteins. Cells were incubated with anti-FLAG antibody at 4°C to block endocytosis, and cell surface Fz4 levels were quantified by measuring the anti-FLAG fluorescence intensity of UBPY-transfected and -untransfected cells in the same microscopic fields, typical examples of which are shown in Figure 4C–C″ and D–D″. In UBPYS680A-expressing cells, the level of surface anti-FLAG staining was increased to ∼190% of that in untransfected cells, suggesting that Fz4 accumulates on the cell surface when deubiquitylated by UBPY (Fig
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