Tài liệu Báo cáo khoa học: Mammalian Gup1, a homolog of Saccharomyces cerevisiae glycerol uptake/transporter 1, acts as a negative regulator for N-terminal palmitoylation of Sonic hedgehog doc

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Mammalian Gup1, a homolog of Saccharomyces cerevisiaeglycerol uptake/transporter 1, acts as a negative regulatorfor N-terminal palmitoylation of Sonic hedgehogYoichiro Abe1, Yoshiko Kita1and Takako Niikura1,2,*1 Department of Pharmacology, Keio University School of Medicine, Tokyo, Japan2 Department of Neurology, Georgetown University, Washington, DC, USASonic hedgehog (Shh), a member of the vertebrateHedgehog (Hh) family [1–4], is an extracellularsecreted signaling molecule that is involved in embry-onic patterning and organogenesis (for example, in thedorsal–ventral polarity of the spinal cord and in theanterior–posterior polarity in the limb bud) in a con-centration-dependent manner [5].Shh is initially translated as a precursor protein of 45 kDa. After excision of the signal sequence, itundergoes automatic cleavage to release a biologicallyKeywordsGup1; hedgehog acyltransferase;membrane-bound O-acyltransferase;palmitoylation; Sonic hedgehogCorrespondenceY. Abe, Department of Pharmacology,Keio University School of Medicine,35 Shinanomachi, Shinjuku-ku,Tokyo 160-8582, JapanFax: +81 3 3359 8889Tel: +81 3 5363 3750E-mail: yoabe@sc.itc.keio.ac.jp*Present addressDepartment of Neurology, GeorgetownUniversity, Washington, DC, USA(Received 21 August 2007, revised 9November 2007, accepted 20 November2007)doi:10.1111/j.1742-4658.2007.06202.xMammalian glycerol uptake ⁄ transporter 1 (Gup1), a homolog of Saccharo-myces cerevisiae Gup1, is predicted to be a member of the membrane-bound O-acyltransferase family and is highly homologous to mammalianhedgehog acyltransferase, known as Skn, the homolog of the Drosoph-ila skinny hedgehog gene product. Although mammalian Gup1 has asequence conserved among the membrane-bound O-acyltransferase family,the histidine residue in the motif that is indispensable to the acyltransferaseactivity of the family has been replaced with leucine. In this study, wecloned Gup1 cDNA from adult mouse lung and examined whether Gup1is involved in the regulation of N-terminal palmitoylation of Sonic hedge-hog (Shh). Subcellular localization of mouse Gup1 was indistinguishablefrom that of mouse Skn detected using the fluorescence of enhanced greenfluorescent protein that was fused to each C terminus of these proteins.Gup1 and Skn were co-localized with an endoplasmic reticulum marker,78 kDa glucose-regulated protein, suggesting that these two moleculesinteract with overlapped targets, including Shh. In fact, full-length Shhcoprecipitated with FLAG-tagged Gup1 by immunoprecipitation usinganti-FLAG IgG. Ectopic expression of Gup1 with full-length Shh in cellslacking endogenous Skn showed no hedgehog acyltransferase activity asdetermined using the monoclonal antibody 5E1, which was found to recog-nize the palmitoylated N-terminal signaling domain of Shh under denatur-ing conditions. On the other hand, Gup1 interfered with the palmitoylationof Shh catalyzed by endogenous Skn in COS7 and NSC34. These resultssuggest that Gup1 is a negative regulator of N-terminal palmitoylation ofShh and may contribute to the variety of biological actions of Shh.AbbreviationsCHO, Chinese Hamster ovary; CM, conditioned medium; EGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; GRP78,78-kDa glucose-regulated protein; Gup1, glycerol uptake ⁄ transporter 1; HHAT, hedgehog acyltransferase; HRP, horseradish peroxidase;IP, immunoprecipitation; IRES, internal ribosome entry site; MBOAT, membrane-bound O-acyltransferase; Shh, sonic hedgehog; Shh-N,N-terminal signaling domain of Shh without cholesterol modification; Shh-Np, autoprocessed N-terminal signaling domain of Shh;TRITC, tetramethylrhodamine isothiocyanate.318 FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBSactive N-terminal signaling domain of  19 kDa [6–11],which is followed by the addition of cholesterol to itsC-terminal Gly residue, a process catalyzed by theC-terminal catalytic domain [12]. This autoprocessedN-terminal signaling domain of Shh (Shh-Np [9]) is alsopalmitoylated at its N-terminal Cys residue by thehedgehog acyltransferase (HHAT) called Skn [13,14], ahomolog of the Drosophila skinny hedgehog (also calledsightless, central missing,orrasp) [15–18] gene product,in an amide-linked manner [14]. These unique lipidmodifications greatly reduce the diffusibility of Shh-Npand tether it to the cellular membrane. However, theyare necessary to regulate the movement of the proteinto form the proper concentration gradient. The criticalrole of cholesterol modification in the movement ofHh protein has been demonstrated in both vertebratesand invertebrates [9,19–23]. Palmitoylation is alsoinvolved in the regulation of movement of Shh-Np indeveloping mouse embryos. Loss of long-range signal-ing of Shh protein was observed in both Skn null miceand gene-targeted homozygous mice harboring onenucleotide substitution on the Shh locus, from whichpalmitoylation-deficient C25S-Shh is produced [13].One explanation for the loss of long-range signaling ofthe nonpalmitoylated Shh-Np in vivo is its inability toform a diffusible multimeric Hh protein complex[13,24,25].In addition to its role in the movement of Hh pro-tein, palmitoylation is also implicated in the activity ofHh protein in both vertebrates and invertebrates. It isindispensable for the activity of Hh in Drosophila[15–18,26]. Similarly, palmitoylation is also requiredfor the induction of rodent ventral forebrain neurons[27]. Interestingly, in contrast to Drosophila, nonpalmi-toylated Shh-Np is significantly potent in some tissue,for example, in chick embryo neural plate explants andmouse limbs [13,26,28]. Moreover, even in Drosophilatissue, nonpalmitoylated mouse Shh-Np retains somesignaling activity [16]. These findings indicate that bothpalmitoylated and nonpalmitoylated mammalianHh proteins can act as signaling molecules. It is nota-ble that while cholesterylation of Hh protein is anintramolecular event catalyzed by its own C-terminaldomain, palmitoylation is an intermolecular event cat-alyzed by Skn. Therefore, while all Shh-Np certainlypossess cholesterol adduct to their C-terminal regions,palmitoylation of Shh-Np might be controllable. Infact, only 30% of Shh-Np was observed to be palmi-toylated in a mammalian cell line transfected with full-length human Shh [14]. Thus, it is possible that, inaddition to palmitoylated Shh-Np, nonpalmitoylatedShh-Np is also produced in vertebrates in vivo,and that a combination of palmitoylated and non-palmitoylated Shh-Np contributes to cell fate specifica-tion during development.Mammalian glycerol uptake ⁄ transporter 1 (Gup1) isdescribed in the National Center for BiotechnologyInformation gene database as a homolog of Saccharo-myces cerevisiae Gup1 [29] from its sequence homol-ogy. It has also been found to have sequencehomology to Drosophila skinny hedgehog gene productand to mammalian Skn [13,30]. The function of themammalian Gup1 is still unclear. However, it has amotif characteristic of the membrane-bound O-acyl-transferase (MBOAT) superfamily [31], like Drosoph-ila skinny hedgehog gene product and mammalian Skn,as well as yeast Gup1 [32]. One strange thing that hasbeen observed, however, is that in mammalian Gup1,the highly conserved His residue in the motif indis-pensable to the acyltransferase activity of the MBOATsuperfamily has been replaced with a Leu residue.Therefore, it is possible that Gup1 has some functionrelated to the post-translational modification of themammalian hedgehog family, although it may have noacyltransferase activity. In this work we examinedwhether mammalian Gup1 has a role in regulating thepalmitoylation of Shh, by using a novel technique,developed in this study, for detecting the palmitoylatedN-terminal fragment of Shh.ResultsMonoclonal antibody 5E1 recognizes theN-terminal signaling domain of Shh withpalmitoylation under denaturing conditionsTo understand the behavior of N-terminally palmitoy-lated Shh in mammalian systems, we establishedChinese Hamster ovary (CHO) cell clones stablyexpressing full-length mouse Shh either in the presenceor absence of mouse Skn (Y. Abe, Y. Kita & T. Niik-ura, unpublished results). While screening the clones,we found that 5E1, a monoclonal antibody raisedagainst the N-terminal domain of rat Shh expressed ininsect cells [33], recognized Shh-Np in the lysate bywestern blotting only when the clones were transfectedwith both Shh and Skn. 5E1 has been reported not towork well under denaturing conditions such as westernblotting [34], whereas it has been shown to block bind-ing of Shh to its receptor Patched and consequent sig-nal transduction in vivo and in vitro [33]. Therefore,5E1 is believed to recognize a particular conformationof the N-terminal signaling domain of Shh [34]. Ourobservation, however, raises the possibility that Sknhas some function that protects Shh-Np from disrupt-ing the 5E1 epitope, even under denaturing conditions.Y. Abe et al. A negative regulator for palmitoylation of ShhFEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS 319To test this possibility, we first transiently transfectedfull-length mouse Shh cDNA into several cell lines,including CHO, HeLa, COS7 and NSC34 cells(Fig. 1). In our examination so far, the majority of theShh was autoprocessed and 19-kDa Shh-Np was pre-dominantly detected in the lysate of all lines usinganother anti-Shh IgG, H-160, which was raised againstthe N-terminal portion (amino acids 41–200) ofhuman Shh (Fig. 1A–D, lane 2). Consistent with theprevious report, 5E1 failed to recognize Shh-Np in thelysate of CHO cells (Fig. 1A, lane 2), although it rec-ognized full-length Shh (Fig. 1A, lanes 2 and 6). Thiswas also the case with Shh-Np in the lysate of HeLacells (Fig. 1B). Remarkably, 5E1 recognized Shh-Np inthe lysate of COS7 and NSC34 cells, even under dena-turing conditions (Fig. 1C and D, lane 2). The differ-ence between CHO ⁄ HeLa and COS7 ⁄ NSC34 cell linesin the reactivity of 5E1 with Shh-Np was attributed tothe existence of endogenous Skn in the latter lines, asdetermined by RT-PCR analysis (Fig. 2), suggestingthat Skn affects the reactivity of Shh-Np with 5E1,regardless of cell type. To confirm this, we transfectedfull-length Shh together with FLAG-tagged mouse Skninto these lines. As expected, 5E1 efficiently recognizedShh-Np in the lysate of all lines under this experimen-tal condition without affecting the level of Shh-Np(Fig. 1A–D, lane 3). Ectopic expression of Skn led toa reduction in the amount of Shh-Np secreted into theconditioned media (CM) from all lines (Fig. 1A–D,lane 3), suggesting increased hydrophobicity of theprotein, probably as a result of palmitoylationcatalyzed by Skn. Similar results were obtained byusing monoclonal anti-Shh N-terminal fragment,clone 171018 (data not shown). As this antibody alsoacts as a neutralizing antibody, it probably recognizesan epitope overlapping with that of 5E1.The expression of truncated Shh lacking the C-termi-nal domain [Shh (1–198)] results in an N-terminal sig-naling domain of Shh without cholesterol modificationat its C terminus (Shh-N). Using H-160, Shh-N proteinwas detected in the lysate of these cell lines, transientlytransfected with Shh (1–198) cDNA, at a level compara-ble to that of Shh-Np recovered from cells transfectedwith full-length Shh (Fig. 1A–D, lane 4). However, 5E1did not recognize Shh-N in the lysate of all four linesexamined (Fig. 1A–D, lane 4), reflecting the less effi-cient palmitoylation of Shh-N compared with Shh-Np,as previously reported [14]. As seen in cells transfectedwith full-length Shh, the co-expression of FLAG-taggedmouse Skn resulted in greatly reduced secretion of Shh-N into the CM and in the efficient recognition of Shh-Nin lysate by 5E1 under denaturing conditions, withoutaffecting the amount of Shh-N (Fig. 1A–D, lane 5).To examine in greater detail whether the effect ofthe expression of Skn on the 5E1 epitope of Shh-Npunder denaturing conditions is a result of palmitoyla-tion at the N terminus of Shh, we substituted Ser orAla for the Cys25 of full-length Shh, and transientlytransfected these mutants into COS (Fig. 3A) andNSC34 (Fig. 3B) cells. These mutants were expressedat a level comparable to that of wild-type protein, asdetermined using H-160 (Fig. 3A,B, lanes 3–6). Asexpected, neither C25S-Shh-Np nor C25A-Shh-Np wasrecognized by 5E1 (Fig. 3A,B, lanes 3 and 5), whereaswild-type Shh-Np clearly was (Fig. 3A,B, lane 1). Inthe presence of exogenously transfected Skn, C25A-Shh was not recognized by 5E1 (Fig. 3A,B, lane 6).These results indicate a strong correlation between theN-terminal palmitoylation of Shh-N(p) and the reactiv-ity of 5E1 with Shh-N(p). Unexpectedly, C25S-Shh-Npretained the 5E1 epitope when Skn was exogenouslyoverexpressed (Fig. 3A,B, lane 4). Considering thatSkn is a member of the MBOAT superfamily [32], it ispossible that excess Skn transferred an acyl group ontothe hydroxyl group of the N-terminal Ser of C25S-Shh-Np, although the efficiency seems much lowerthan that for wild-type Shh-Np. To confirm this possi-bility, we labeled COS7 cells with [3H]palmitic acidand examined whether the radioactivity is incorporatedinto C25S-Shh-Np, as observed in wild-type Shh(Fig. 3C, lanes 1 and 2). As expected, we detected aband corresponding to C25S-Shh-Np, as well as full-length C25S-Shh, only when Skn was co-expressed(Fig. 3C, lane 4).In COS7 cells, a band migrating more slowly thanShh-N and strongly recognized by 5E1 was observedwhen Shh (1–198) alone was expressed (Fig. 3A,lane 7, asterisk). This species was not prominentlyobserved in lysate from NSC34, CHO, or HeLa cells.Thus, there may be a third post-translational modifica-tion of the N-terminal signaling domain of Shh specificto COS7 cells affecting the 5E1 epitope.Gup1 acts as a negative regulator for N-terminalpalmitoylation of ShhMammalian Gup1 has been described in the gene data-base cited above as a homolog of the S. cerevisiae GUP1gene product, based on its sequence homology. Align-ment of mouse and yeast Gup1 protein sequencesusing the blastp program with BLOSUM62 as amatrix [35] showed that these two proteins are21% identical. However, the same program showedthat mouse Gup1 is more closely related to both mou-se Skn (28%) and Drosophila skinny hedgehog geneproduct (25%). These values were comparable to theA negative regulator for palmitoylation of Shh Y. Abe et al.320 FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBSABCDFig. 1. Expression of Shh protein in transiently transfected mammalian cell lines. CHO (A), HeLa (B), COS7 (C) and NSC34 (D) cells weretransiently transfected with pIRES2-EGFP (IG, lane 1) as a vector control, pCAG-Shh ⁄ CMV-IRES-EGFP (Shh-IG, lane 2), pCAG-Shh ⁄ CMV-Skn-FLAG-IRES-EGFP (Shh-SF-IG, lane 3), pCAG-Shh (1–198) ⁄ CMV-IRES-EGFP (Shh-N-IG, lane 4), pCAG-Shh (1–198) ⁄ CMV-Skn-FLAG-IRES-EGFP(Shh-N-SF-IG, lane 5), pCAG-C199A-Shh ⁄ CMV-IRES-EGFP (C199A-Shh-IG, lane 6), or pCAG-C199A-Shh ⁄ CMV-Skn-FLAG-IRES-EGFP (C199A-Shh-SF-IG, lane 7). Construction of these plasmids is described in detailed in the Experimental procedures. Forty-eight hours after transfec-tion, both conditioned media (indicated as CM) and cell lysates (50 lg) were collected and subjected to western blotting followed by probingwith anti-Shh N-terminal domain H-160, anti-Shh N-terminal domain 5E1, anti-EGFP, or anti-actin IgG. Both full-length Shh and the N-terminalfragment of Shh are indicated by arrows. The C199A mutation blocks autocatalytic cleavage of Shh, resulting in production of only full-lengthShh.Y. Abe et al. A negative regulator for palmitoylation of ShhFEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS 321identity between mouse Skn and Drosophila skinnyhedgehog gene product (29%). Sequence alignmentand calculation of hydrophobicity using severalprograms revealed that both proteins have a similarstructure, with a signal sequence and at least ninetransmembrane domains (Fig. 4). In addition, the openreading frame of both Skn and Gup1 genes consistsof 11 exons; each corresponding exon of the twogenes is similar in size (Fig. 4), suggesting thatthese two genes evolve from the same origin. Thetranscript of Gup1 was detectable in E9.5 mouseembryo (Fig. 2, lane 6), in which Shh transcript is alsodetected [2]. These facts prompted us to examinewhether Gup1 is involved in regulating N-terminalpalmitoylation in the mammalian hedgehog family,including Shh.We cloned Gup1 cDNA by RT-PCR from adultmouse lung poly (A)+RNA and first examined its sub-cellular localization by transiently expressing Gup1,whose C terminus was fused to enhanced green fluores-cent protein (Gup1-EGFP), as well as EGFP-tag-ged Skn (Skn-EGFP) in HeLa cells, which express littleendogenous Skn or Gup1 (Fig. 2, lane 5). Consistentwith a previous report [13], Skn–EGFP (Fig. 5A,C)localized on the endoplasmic reticulum (ER), as deter-mined by immunofluorescent staining of 78-kDaglucose-regulated protein (GRP78) (Fig. 5B,C). Thiswas also the case with Gup1-EGFP (Fig. 5D,F), whichwas co-localized with GRP78 (Fig. 5E,F). These obser-vations imply that these two proteins interact withoverlapped targets. As the intensity of the fluorescenceof these proteins was almost the same, the level ofexpression of these proteins was presumed to be simi-lar. To confirm this, we probed western blots of lysateextracted from COS7 cells, transiently transfected witheach plasmid containing cDNA encoding theseproteins, with anti-GFP IgG. We detected a band, witha molecular mass of  60 kDa, in the lysate of cellstransfected with Gup1-EGFP (Fig. 5G, lane 3). How-ever, we did not detect a band corresponding to that ofGup1-EGFP in the lysate of cells transfected withSkn-EGFP (Fig. 5G, lane 2). Instead, a larger smear,which was also seen in cells transfected withGup1-EGFP, was observed (Fig. 5G, lane 2 and 3). Itis well known that hydrophobic membrane-bound pro-teins are often aggregated in the SDS sample bufferwhen the lysate is boiled. Therefore, we subjected thesamples to western blotting without boiling. Asexpected, we observed double bands, ranging from 60to 70 kDa, and disappearance of the larger smear inlysates of both Skn-EGFP-transfected cells andGup1-EGFP-transfected cells (Fig. 5G, lanes 5 and 6).Under this experimental condition, the level of theexpression of these two proteins was almost the same(Fig. 5G, lanes 5 and 6). Nevertheless, we sometimesobserved a decrease in the intensity of the expectedbands and appearance of a large smear, even in anunboiled sample of cells transfected with Skn-EGFP(data not shown), implying that Skn is more hydro-phobic than Gup1. The expression of FLAG-tag-ged Gup1 in several cell lines, as detected by westernblotting of unboiled samples using anti-FLAG IgG asa probe, revealed two major bands with molecularmasses of  45 and 40 kDa (Fig. 6A–C, lane 3). Theexpression of Skn-FLAG was undetectable in somelines (Fig. 6A,C, lane 2) even when the sampleswere not boiled. However, in COS7 cells transfectedwith Skn-FLAG, a band with a molecular massof  40 kDa was detected when probed with anti-FLAG IgG (Fig. 6B, lane 2). These observationssuggest that Skn without EGFP is more hydrophobicthan Skn with EGFP.As we observed that Gup1 is localized on the ER,we examined whether Gup1 can interact with Shhby immunoprecipitation. We transiently expressedfull-length Shh, together with Gup1-FLAG or Skn-FLAG, in COS7 cells and immunoprecipitated theseproteins using anti-FLAG IgG. As expected, both full-length Shh and the N-terminal fragment of Shh werecoprecipitated with Skn-FLAG (Fig. 7A, upper panel,lane 5), whereas none of the fragment of Shh wasdetected in immunoprecipitate from cells transfectedwith Shh and empty vector (Fig. 7A, upper panel,lane 4). Full-length Shh also coprecipitated with Gup1-FLAG, indicating an interaction between Gup1 andShh (Fig. 7A, upper panel, lane 6).Fig. 2. Expression of Skn and Gup1 transcripts in mammalian celllines. Total RNA extracted from CHO (lane 1), NSC34 (lane 2),COS7 (lane 3), HEK293 (lane 4), HeLa (lane 5) and mouse embry-onic day 9.5 (E9.5) embryo (lane 6) was subjected to RT-PCR analy-sis to detect expression of Skn and Gup1 in these cells. PCRproducts were separated by agarose gel electrophoresis followedby staining with ethidium bromide. A fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internalcontrol.A negative regulator for palmitoylation of Shh Y. Abe et al.322 FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBSWe further assessed whether Gup1 interacts withSkn. We expressed Gup1-EGFP in COS7 cells, togetherwith either Skn-FLAG or empty vector, and subjectedthe cell lysates to immunoprecipitation using anti-FLAG IgG followed by western blotting using anti-GFP IgG (Fig. 7B). We observed a band recognized byanti-GFP IgG only in precipitate from cells cotrans-fected with Gup1-EGFP and Skn-FLAG, suggesting aninteraction between Gup1 and Skn (Fig. 7B, lane 4,arrowhead). In this series of experiments, Skn-FLAGwas barely detectable in both inputs (Fig. 7A, lowerpanel, lane 2, and data not shown) and immunoprecipi-tates (Fig. 7A, lower panel, lane 5, and data notshown) with horseradish peroxidase (HRP)-conjugatedanti-FLAG IgG, probably because of the tendency ofSkn to be aggregated in SDS sample buffer, as demon-strated in Figs 5 and 6. Similarly, the band recognizedwith anti-GFP Ig in precipitate from cells cotransfectedwith Gup1-EGFP and Skn-FLAG was much largerthan expected (Fig. 7B, lane 4, arrowhead). It isprobably aggregated Gup1-EGFP, formed as a resultof boiling to elute proteins from the immunocomplex.Although Gup1 is predicted to be a member of theMBOAT superfamily, the His residue indispensable toMBOAT activity is replaced by Leu (Fig. 4, asterisk).To examine whether Gup1 has HHAT activity, we trans-fected Gup1 cDNA together with full-length Shh cDNAinto CHO cells. As shown in Fig. 6A, Shh-Np in thelysate of CHO cells was not recognized by 5E1 (lane 3),demonstrating that Gup1 has no HHAT activity.Next, we examined whether Gup1 affects the palmi-toylation of Shh-Np in cells expressing endoge-nous Skn, such as COS7 and NSC34 cells, byexpressing full-length Shh in the presence of Gup1-FLAG in these cells. Co-expression of Shh withGup1-FLAG resulted in a reduction of the totalamount of Shh-Np, determined using H-160 in theABCFig. 3. Requirement of Cys25of Shh for Skn-dependent retentionof the 5E1 epitope on the N-terminal fragment of Shh in westernblotting. COS7 (A) and NSC34 (B) cells were transiently transfectedwith pCAG-Shh (lanes 1 and 2), pCAG-C25S-Shh (lanes 3 and 4),pCAG-C25A-Shh (lanes 5 and 6) or pCAG-Shh (1–198) (lanes 7 and8) together with either pFLAG-CMV5a (lanes 1, 3, 5 and 7) as avector control or pCMV-Skn-FLAG (lanes 2, 4, 6 and 8). Cellular pro-teins (50 lg) were subjected to western blotting, followed by prob-ing with anti-Shh N-terminal domain H-160, anti-Shh N-terminaldomain 5E1, or anti-actin IgG. Both full-length Shh and the N-termi-nal fragment of Shh are indicated by arrows. The asterisk indicatesa band in the lysate of COS cells transfected with both pCAG-Shh (1–198) and pFLAG-CMV5a (A, lane 7), migrating more slowlythan Shh-N and strongly recognized with 5E1. (C) COS7 cells weretransiently transfected with pCAG-Shh ⁄ CMV-IRES-EGFP (lane 1),pCAG-Shh ⁄ CMV-Skn-FLAG-IRES-EGFP (lane 2), pCAG-C25S-Shh ⁄ CMV-IRES-EGFP (lane 3), or pCAG-C25S-Shh ⁄ CMV-Skn-FLAG-IRES-EGFP (lane 4). Twenty-four hours after transfection, cellswere labeled with [9,10-3H]palmitic acid for 24 h. Then the cellswere lysed and Shh was immunoprecipitated with 5E1 followed bySDS-PAGE. Dried gel was exposed to an X-ray film to visualizeradiolabeled Shh.Y. Abe et al. A negative regulator for palmitoylation of ShhFEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS 323Fig. 4. Comparison of mouse Gup1 and mouse Skn. Mouse Gup1 (Mo Gup1) and mouse Skn (Mo Skn) were aligned based on amino acidsin their sequences conserved between them, indicated with grey boxes. Amino acids identical among mouse Gup1, mouse Skn and the Dro-sophila skinny hedgehog gene product are indicated in red. The numbers at the right of the alignment indicate the position in the sequence.Arrowheads above the alignment indicate the positions of introns in the encoding genes. The putative signal sequence is shown on a blackbackground. The putative transmembrane domains were estimated on the basis of hydrophobicity calculated using seven programs:TMHMM,TMPRED, HMMTOP, PSORT II, SOSUI, TOPPRED and PREDICTPROTEIN. The range of hydrophobic regions predicted by more than three programs listedabove is indicated with lines on each sequence. Within each range of hydrophobic regions, the overlapped part recognized as a putativetransmembrane domain by all the programs is represented by a thick bar. The position of the His residue in the MBOAT motif indispensableto the activity is indicated with an asterisk.A negative regulator for palmitoylation of Shh Y. Abe et al.324 FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBSlysate of both COS7 and NSC34 cells, to 73.1% and67.1%, respectively, as compared with that in cellscotransfected with full-length Shh and empty vector(Fig. 6B,C, lane 3, and Fig. 6D,E, solid column). Thelevels of modified Shh-Np in COS7 and NSC34 cells,as detected using 5E1, which is expected to recognizepalmitoylated Shh-Np, were further reduced to 6.4%and 10.7%, respectively, as compared with controlcells, suggesting that the expression of Gup1 inhibitspalmitoylation of Shh-Np catalyzed by endoge-nous Skn in these cells (Fig. 6B,C, lane 3, andFig. 6D,E, open column). It seemed that the overex-pression of Skn-FLAG in these cells slightly increasedthe level of palmitoylated Shh-Np, although the differ-ence was not statistically significant (Fig. 6D,E).Taken together, these observations strongly suggestthat mammalian Gup1 acts as a negative regulator ofthe N-terminal palmitoylation of Shh.DiscussionIn this report, we found that mammalian Gup1, a mem-ber of the MBOAT superfamily bearing sequence simi-larity to HHAT, acts as a negative regulator ofN-terminal palmitoylation of Shh. Several reports havedemonstrated the critical role of N-terminal palmitoyla-tion of Hh protein for its activity in Drosophila [15–18,26]. Drosophila Hh protein without palmitoylationnot only loses its activity but also obstructs endogenousHh signaling in vivo [26]. By contrast, mammalian Shhwithout palmitoylation can act in some tissues[13,26,28]. Analysis of both Skn knockout and C25S-Shh knockin mice revealed that the responsiveness tononpalmitoylated Shh-Np varied among tissues [13].Thus, it is possible that while palmitoylated Hh-Np isthe only signaling molecule in Drosophila, both palmi-toylated and nonpalmitoylated Shh-Nps act as signalingmolecules in mammals, and combining these moleculesproduces a variety of effects on developing organs andtissues. If this were the case, the proportion of thesemolecules would have to be controlled precisely. In thepresent study, mammalian Gup1 was found to interactwith full-length Shh, as determined by immunoprecipi-tation (Fig. 7), and to inhibit the N-terminal palmitoy-lation of Shh-Np in multiple mammalian cell lines, asdetermined by western blotting using 5E1 as a probe(Fig. 6). These results, as well as structural similarity toboth mammalian Skn and Drosophila skinny hedgehoggene product (Fig. 4) and subcellular localization ofthese proteins (Fig. 5), strongly suggest that mamma-lian Gup1 may be involved in such a mechanism. It isnot clear how Gup1 decreases the N-terminal palmitoy-lation of Shh. Although the N terminus of the maturesignaling domain of Shh is a Cys residue, N-terminalpalmitoylation is not S-palmitoylation but Na-palmitoy-lation [14]. Other than the hedgehog family, Gasis theonly example that undergoes Na-palmitoylation invertebrates, to our knowledge [36]. How the Na-palmi-toylation of Gasis regulated also remains unclear.However, we assume that mammalian Gup1 competeswith Skn for Shh to prevent palmitoylation ratherthan catalyzing depalmitoylation of Shh becauseother known Na-acylations, namely Na-acetylationand Na-myristylation, are irreversible [37,38]. Furtherin vitro analyses are necessary to determine whetherGup1 can depalmitoylate Shh-Np.GABCDEFFig. 5. Subcellular localization of mouse Skn and Gup1. (A–F) Tovisualize the subcellular localization of mouse Skn (A–C) and Gup1(D–F), EGFP was fused to the C terminus of these proteins andexpressed in HeLa cells. Forty-eight hours after transfection, the cellswere fixed, permeabilized and stained with an ER marker (78-kDaglucose-regulated protein) followed by TRITC-labeled secondary anti-body. The fluorescence of EGFP (A, C, D and F, green) and TRITC(B, C, E and F, red) was observed using a confocal microscope.Scale bar, 25 lm. (G) COS7 cells were transiently transfected withpIRES2-EGFP (lanes 1 and 4) as a vector control, pCMV-Skn-EGFP(lanes 2 and 5) and pCMV-Gup1-EGFP (lanes 3 and 6). Lysates wereextracted from these cells, and boiled (lanes 1–3) or unboiled(lanes 4–6) samples (50 lg) were subjected to western blotting fol-lowed by probing using monoclonal anti-GFP IgG.Y. Abe et al. A negative regulator for palmitoylation of ShhFEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS 325We also demonstrated that 5E1 recognizes the N-ter-minal fragment of Shh under denaturing conditionswhen its N terminus is palmitoylated. The property ofthis antibody is useful in identifying the state of N-ter-minal lipid modification of the protein. Although 5E1is an antibody that recognizes an epitope in the N-ter-minal signaling domain of Shh overlapping with thePatched-binding region and acts as a good neutralizingantibody [33], it was previously reported not to workunder denaturing conditions such as western blotting[34]. However, we found that 5E1 worked in westernblotting when Shh was co-expressed with Skn (Figs 1,3and 5), suggesting that palmitoylation at the N-termi-nal Cys residue of the N-terminal signaling domain ofShh protects the protein from disruption of the 5E1epitope, even under denaturing conditions. One expla-nation for this phenomenon might be that the palmi-tate itself constitutes the epitope when the N-terminalfragment of Shh is denatured. However, our resultsalso showed that both full-length Shh (Fig. 1, lanes 2and 6) and Shh-N with unknown modification inCOS7 cells (Fig. 3A, lane 7, asterisk) were also recog-nized by 5E1 in western blotting, although they werenot expected to undergo palmitoylation under thosetransfection conditions. Therefore, palmitoylation maynot be a component of the 5E1 epitope but may influ-ence the structure of the 5E1 epitope under denaturingconditions. Crystal structure analysis revealed that theresidues Pro42, Lys46, Arg154, Ser157, Ser178and Lys179are located close to each other on the surface of themouse Shh-N protein and are essential for Shh-N tobind both Patched and 5E1 [34,39,40]. Among the resi-dues, Ser178at least is found to be included in the 5E1epitope [39]. In addition, mouse Shh-N lacking theN-terminal 25 amino acids [Shh (50–198)] loses theability to bind not only Patched but also 5E1 inimmunoprecipitation [34]. These observations indicatethe requirement of the N-terminal region of the Shh-N, including Pro42and Lys46, for recognition by 5E1in immunoprecipitation. Therefore, there arise twopossibilities. One is that the N-terminal region, includ-ing Pro42and Lys46, may constitute the epitope for5E1, but that under denaturing conditions it is dissoci-ated from the other parts of the protein, probably theC-terminal region, including Arg154, Ser157, Ser178andLys179. In this case, palmitoylation at the N terminusof Shh-N(p) may support the N-terminal region beinglocated near other amino acids on the C-terminalAB CDEFig. 6. The effect of Gup1 on N-terminalpalmitoylation of Shh-Np. CHO (A), COS7(B) and NSC34 (C) cells were transientlytransfected with pCAG-Shh (lanes 1–3),together with either pFLAG-CMV-5a (lane 1)as a vector control, pCMV-Skn-FLAG(lane 2), or pCMV-Gup1-FLAG (lane 3).Cellular proteins (50 lg) were subjected towestern blotting, using polyclonal anti-Shh N-terminal domain H-160, monoclonalanti-Shh N-terminal domain 5E1, ormonoclonal anti-FLAG IgG. The intensity ofthe signals obtained from the western blotanalysis was quantified usingQUANTITY ONEsoftware (Bio-Rad). The effect of Skn orGup1 on the amount of total Shh-Np,determined with H-160 (solid column), andon the amount of modified Shh-Np, deter-mined with 5E1 (open column), in COS7 (D)and NSC34 (E) cells was expressed as theratio of the intensity of the band of Shh-Npto that from control cells transfected withShh and empty vector in the same blot.Values were the mean ± SD of threeindependent experiments. The differencebetween total Shh-Np and modified Shh-Npwas determined using a paired t-test.*, P < 0.01; NS, not significant. The level ofShh-Np in control cells is shown by thedotted line.A negative regulator for palmitoylation of Shh Y. Abe et al.326 FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBSregion to form the 5E1 epitope, even under denaturingconditions. The other possibility is that the N-terminalportion of Shh-N(p) may not constitute the 5E1epitope but may contribute to stabilization of the 5E1epitope located within the C-terminal portion ofShh-N; when palmitoylated, the N-terminal portionwould retain activity, even under denaturing condi-tions. To understand, in full, the 5E1 epitope underdenaturing conditions, extensive analyses will berequired. One clue may come from identifying thepost-translational modification of Shh-N seen in COS7cells transfected with Shh (1–198) alone (Fig. 3A,lane 7, asterisk).Experimental proceduresPlasmid constructionThe EcoRI–NcoI fragment of mouse Shh cDNA (kindlyprovided by A. P. McMahon) was subcloned between theEcoRI and the SmaI sites of pEGFP-N3 (Clontech, Moun-tain View, CA, USA). Then, it was excised with SpeI,which was blunted with Klenow fragment, and with XhoI,and was inserted between an XhoI and the SwaI sites ofpCALNLw, resulting in pCAG-Shh. pCALNLw vector is a6.6-kbp plasmid derived from a cosmid vector, pA-xCALNLw (Takara, Shiga, Japan), by digestion with SalIfollowed by self-ligation.Mouse Skn was cloned from the total RNA of embry-onic day 9.5 mouse embryo by reverse transcription usingthe avian myeloblastosis virus (AMV) reverse transcriptasefirst-strand synthesis kit (Life Sciences, Inc, St Petersburg,FL, USA) followed by PCR with Taq DNA polymerase(Promega, Madison, WT, USA) using the primers5¢-CACACTACACTGGGAAGCAGAG ACTCCAGC-3¢and 5¢-AGCTGGCCCAGCAGCCATACACAGTTAAAG-3¢. The cDNA was subcloned into the EcoRV site of pBlue-script SK(+) (Stratagene, La Jolla, CA, USA) andsequenced using an automated sequencer (ABI-PRISM310Genetic Analyzer; Perkin-Elmer Applied Biosystems, FosterABFig. 7. Gup1 interacts with both full-length Shh and Skn. (A) COS7 cells were transiently transfected with pCAG-Shh (lanes 1–6), togetherwith pFLAG-CMV-5a (vector) (lanes 1 and 4), pCMV-Skn-FLAG (Skn-F) (lanes 2 and 5), or pCMV-Gup1-FLAG (Gup1-F) (lanes 3 and 6). Cellswere lysed with IP buffer, as described in the Experimental procedures, and subjected to immunoprecipitation using anti-FLAG IgG. Then,the samples were boiled and subjected to western blot analysis using either anti-Shh N-terminal IgG H-160 or HRP-conjugated anti-FLAG IgG(lanes 4–6). Some of the lysate (1 ⁄ 20 volume) was unboiled and also subjected to western blot analysis as input (lanes 1–3). Both full-lengthShh and the N-terminal fragment of Shh are indicated by arrows. (B) COS7 cells were transiently transfected with pCMV-Gup1-EGFP (Gup1-G), together with pFLAG-CMV-5a (vector) (lanes 1 and 3), or pCMV-Skn-FLAG (Skn-F) (lanes 2 and 4). Cells were lysed with IP buffer andsubjected to immunoprecipitation using anti-FLAG IgG. Then, samples were boiled and subjected to western blot analysis using anti-GFP IgGas a probe (lanes 3 and 4). Some of the lysate (1 ⁄ 20 volume) was unboiled and also subjected to western blot analysis as input (lanes 1 and2). Immunoglobulin G heavy and light chains (IgG-H and IgG-L, respectively) are indicated by arrows. Putative Gup1–EGFP is indicated by thearrowhead.Y. Abe et al. A negative regulator for palmitoylation of ShhFEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS 327[...].. .A negative regulator for palmitoylation of Shh Y Abe et al City, CA, USA) A BamHI site was introduced immediately before the termination codon for addition of the FLAG-tag to the C terminus of Skn by PCR using primers 5¢-AA GCTTCCGGAGGCTGCTAGAGAC-3¢ and 5¢-GGATC CAAGAACTGTGTATGTCTG-3¢ The 1.6-kbp fulllength Skn cDNA, whose termination codon was changed to a BamHI site, was inserted between the SalI... 5¢-TCTCCACAGTGACTCCCAGC-3¢; and Gup1, 5¢-GCACAATGGGCCCATGGTACCTGC-3¢ and 5¢-GGATCCCTCCAGCTTCTCTCTGTCCTGC-3¢ These primer sets were designed based on the mouse sequence and were compatible with human species As an internal control, glyceraldehyde-3-phosphate dehydrogenase was amplified using the primers 5¢-TCCACCACCCTGTTGCT GTA-3¢ and 5¢-ACCACAGTCCATGCCATCAC-3¢ (25 cycles at 94 °C for 1 min, 65 °C for 1... Tomohiro Chiba for preparation of embryonic day 9.5 mouse embryos; Dr Dovie Wylie and Ms Takako Hiraki for expert assistance; and all members of the Departments of Pharmacology and Anatomy at Keio University for cooperation The monoclonal antiShh IgG (5E1) developed by Dr Thomas M Jessell was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by... Two hours later, the immunocomplexes were washed four times with the IP buffer Then, the beads were boiled in 20 lL of 2 · SDS-PAGE sample buffer, and the eluted samples were subjected to western blot analysis as described above The authors thank Drs Masato Yasui, Sadakazu Aiso and Masaaki Matsuoka for support; Dr Andrew P McMahon for providing the full-length mouse Shh cDNA; Dr Neil Cashman for providing... Sigma); rabbit anti-actin (1 : 5000; Sigma); HRP-conjugated goat anti-mouse IgG (1 : 5000; BioRad); and HRP-conjugated goat anti-rabbit IgG (1 : 5000; BioRad) A negative regulator for palmitoylation of Shh and EGFP fluorescence was observed using an LSM510 laser-scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) ER was visualized after staining with rabbit anti-GRP-78 Ig (1 : 100; Sigma) followed... insights into the mechanisms of protein palmitoylation Biochemistry 42, 4311–4320 38 Glozak MA, Sengupta N, Zhang X & Seto E (2005) Acetylation and deacetylation of non-histone proteins Gene 363, 15–23 39 Pepinsky RB, Rayhorn P, Day ES, Dergay A, Williams KP, Galdes A, Taylor FR, Boriack-Sjodin PA & Garber EA (2000) Mapping Sonic hedgehog- receptor A negative regulator for palmitoylation of Shh 40 41 42 43... 48 49 interactions by steric interference J Biol Chem 275, 10995–11001 Tanaka Hall TM, Porter JA, Beachy PA & Leahy DJ (1995) A potential catalytic site revealed by the 1.7Angstrom crystal structure of the amino-terminal signalling domain of Sonic hedgehog Nature 378, 212– 216 Cashman NR, Durham HD, Blusztajn JK, Oda K, Tabira T, Shaw IT, Dahrouge S & Antel JP (1992) Neuroblastoma · spinal cord (NSC)... of the signal corresponding to Shh-Np was quantified using quantity one software (BioRad, Hercules, CA, USA) Antibodies used were monoclonal anti-Shh N-terminal fragment (5E1, 1 : 2000; DSHB); rabbit anti-Shh N-terminal (H-160, 1 : 2000; Santa Cruz Biotechnology Inc, Santa Cruz, CA); monoclonal anti-GFP (1E4, 1 : 750; MBL, Nagoya, Japan); HRP-conjugated monoclonal anti-FLAG (M2, 1 : 3000; Sigma); rabbit... RT-PCR analysis Expression of Skn and Gup1 was determined by two-step RT-PCR, as described above, from total RNA extracted using Isogen (Nippon Gene, Tokyo, Japan) For Skn, PCR was performed at 94 °C for 1 min, 65 °C for 1 min and 72 °C for 1 min (35 cycles) For Gup1, PCR was performed at 94 °C for 1 min, 65 °C for 1 min and 72 °C for 3 min (40 cycles) Primers used were Skn, 5¢-CTGCGTGAGCAC CATGTTCA-3¢ and... polyadenylation signals, was inserted into the SalI site of pIRES2-EGFP and pCMVSkn-FLAG-IRES-EGFP, resulting in pCAG-Shh ⁄ CMVIRES-EGFP and pCAG-Shh ⁄ CMV-Skn-FLAG-IRESEGFP, respectively C25S and C19 9A mutations of Shh were introduced by PCR using primers 5¢-CCTGCAGCAGCGGCAGGCA AGGTTATATAG-3¢ and 5¢-GGGCCCAGAGGCCAGG CCGGGGCACACCAG-3¢, and primers 5¢-GGCATGC TGGCTCGCCTGGCTGTGGAAGCA-3¢ and 5¢-GGAT respecCCTGGGAAAGCGCCGCCGGATTTGGC-3¢, . Mammalian Gup1, a homolog of Saccharomyces cerevisiae glycerol uptake/transporter 1, acts as a negative regulator for N-terminal palmitoylation of Sonic. that mammalian Gup1, a mem-ber of the MBOAT superfamily bearing sequence simi-larity to HHAT, acts as a negative regulator of N-terminal palmitoylation
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