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

Mammalian Gup1, a homolog of Saccharomyces cerevisiae glycerol uptake/transporter 1, acts as a negative regulator for N-terminal palmitoylation of Sonic hedgehog Yoichiro Abe1, Yoshiko Kita1 and Takako Niikura1,2,* Department of Pharmacology, Keio University School of Medicine, Tokyo, Japan Department of Neurology, Georgetown University, Washington, DC, USA Keywords Gup1; hedgehog acyltransferase; membrane-bound O-acyltransferase; palmitoylation; Sonic hedgehog Correspondence Y Abe, Department of Pharmacology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Fax: +81 3359 8889 Tel: +81 5363 3750 E-mail: yoabe@sc.itc.keio.ac.jp *Present address Department of Neurology, Georgetown University, Washington, DC, USA (Received 21 August 2007, revised November 2007, accepted 20 November 2007) doi:10.1111/j.1742-4658.2007.06202.x Mammalian glycerol uptake ⁄ transporter (Gup1), a homolog of Saccharomyces cerevisiae Gup1, is predicted to be a member of the membranebound O-acyltransferase family and is highly homologous to mammalian hedgehog acyltransferase, known as Skn, the homolog of the Drosophila skinny hedgehog gene product Although mammalian Gup1 has a sequence conserved among the membrane-bound O-acyltransferase family, the histidine residue in the motif that is indispensable to the acyltransferase activity of the family has been replaced with leucine In this study, we cloned Gup1 cDNA from adult mouse lung and examined whether Gup1 is involved in the regulation of N-terminal palmitoylation of Sonic hedgehog (Shh) Subcellular localization of mouse Gup1 was indistinguishable from that of mouse Skn detected using the fluorescence of enhanced green fluorescent 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 molecules interact with overlapped targets, including Shh In fact, full-length Shh coprecipitated with FLAG-tagged Gup1 by immunoprecipitation using anti-FLAG IgG Ectopic expression of Gup1 with full-length Shh in cells lacking endogenous Skn showed no hedgehog acyltransferase activity as determined using the monoclonal antibody 5E1, which was found to recognize the palmitoylated N-terminal signaling domain of Shh under denaturing conditions On the other hand, Gup1 interfered with the palmitoylation of Shh catalyzed by endogenous Skn in COS7 and NSC34 These results suggest that Gup1 is a negative regulator of N-terminal palmitoylation of Shh and may contribute to the variety of biological actions of Shh Sonic hedgehog (Shh), a member of the vertebrate Hedgehog (Hh) family [1–4], is an extracellular secreted signaling molecule that is involved in embryonic patterning and organogenesis (for example, in the dorsal–ventral polarity of the spinal cord and in the anterior–posterior polarity in the limb bud) in a concentration-dependent manner [5] Shh is initially translated as a precursor protein of  45 kDa After excision of the signal sequence, it undergoes automatic cleavage to release a biologically Abbreviations CHO, 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 FEBS Y Abe et al active N-terminal signaling domain of  19 kDa [6–11], which is followed by the addition of cholesterol to its C-terminal Gly residue, a process catalyzed by the C-terminal catalytic domain [12] This autoprocessed N-terminal signaling domain of Shh (Shh-Np [9]) is also palmitoylated at its N-terminal Cys residue by the hedgehog acyltransferase (HHAT) called Skn [13,14], a homolog of the Drosophila skinny hedgehog (also called sightless, central missing, or rasp) [15–18] gene product, in an amide-linked manner [14] These unique lipid modifications greatly reduce the diffusibility of Shh-Np and tether it to the cellular membrane However, they are necessary to regulate the movement of the protein to form the proper concentration gradient The critical role of cholesterol modification in the movement of Hh protein has been demonstrated in both vertebrates and invertebrates [9,19–23] Palmitoylation is also involved in the regulation of movement of Shh-Np in developing mouse embryos Loss of long-range signaling of Shh protein was observed in both Skn null mice and gene-targeted homozygous mice harboring one nucleotide substitution on the Shh locus, from which palmitoylation-deficient C25S-Shh is produced [13] One explanation for the loss of long-range signaling of the nonpalmitoylated Shh-Np in vivo is its inability to form a diffusible multimeric Hh protein complex [13,24,25] In addition to its role in the movement of Hh protein, palmitoylation is also implicated in the activity of Hh protein in both vertebrates and invertebrates It is indispensable for the activity of Hh in Drosophila [15–18,26] Similarly, palmitoylation is also required for the induction of rodent ventral forebrain neurons [27] Interestingly, in contrast to Drosophila, nonpalmitoylated Shh-Np is significantly potent in some tissue, for example, in chick embryo neural plate explants and mouse limbs [13,26,28] Moreover, even in Drosophila tissue, nonpalmitoylated mouse Shh-Np retains some signaling activity [16] These findings indicate that both palmitoylated and nonpalmitoylated mammalian Hh proteins can act as signaling molecules It is notable that while cholesterylation of Hh protein is an intramolecular event catalyzed by its own C-terminal domain, palmitoylation is an intermolecular event catalyzed by Skn Therefore, while all Shh-Np certainly possess cholesterol adduct to their C-terminal regions, palmitoylation of Shh-Np might be controllable In fact, only 30% of Shh-Np was observed to be palmitoylated in a mammalian cell line transfected with fulllength human Shh [14] Thus, it is possible that, in addition to palmitoylated Shh-Np, nonpalmitoylated Shh-Np is also produced in vertebrates in vivo, and that a combination of palmitoylated and non- A negative regulator for palmitoylation of Shh palmitoylated Shh-Np contributes to cell fate specification during development Mammalian glycerol uptake ⁄ transporter (Gup1) is described in the National Center for Biotechnology Information gene database as a homolog of Saccharomyces cerevisiae Gup1 [29] from its sequence homology It has also been found to have sequence homology to Drosophila skinny hedgehog gene product and to mammalian Skn [13,30] The function of the mammalian Gup1 is still unclear However, it has a motif characteristic of the membrane-bound O-acyltransferase (MBOAT) superfamily [31], like Drosophila skinny hedgehog gene product and mammalian Skn, as well as yeast Gup1 [32] One strange thing that has been observed, however, is that in mammalian Gup1, the highly conserved His residue in the motif indispensable to the acyltransferase activity of the MBOAT superfamily has been replaced with a Leu residue Therefore, it is possible that Gup1 has some function related to the post-translational modification of the mammalian hedgehog family, although it may have no acyltransferase activity In this work we examined whether mammalian Gup1 has a role in regulating the palmitoylation of Shh, by using a novel technique, developed in this study, for detecting the palmitoylated N-terminal fragment of Shh Results Monoclonal antibody 5E1 recognizes the N-terminal signaling domain of Shh with palmitoylation under denaturing conditions To understand the behavior of N-terminally palmitoylated Shh in mammalian systems, we established Chinese Hamster ovary (CHO) cell clones stably expressing full-length mouse Shh either in the presence or absence of mouse Skn (Y Abe, Y Kita & T Niikura, unpublished results) While screening the clones, we found that 5E1, a monoclonal antibody raised against the N-terminal domain of rat Shh expressed in insect cells [33], recognized Shh-Np in the lysate by western blotting only when the clones were transfected with both Shh and Skn 5E1 has been reported not to work well under denaturing conditions such as western blotting [34], whereas it has been shown to block binding of Shh to its receptor Patched and consequent signal transduction in vivo and in vitro [33] Therefore, 5E1 is believed to recognize a particular conformation of the N-terminal signaling domain of Shh [34] Our observation, however, raises the possibility that Skn has some function that protects Shh-Np from disrupting the 5E1 epitope, even under denaturing conditions FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS 319 A negative regulator for palmitoylation of Shh Y Abe et al To test this possibility, we first transiently transfected full-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 the Shh was autoprocessed and 19-kDa Shh-Np was predominantly detected in the lysate of all lines using another anti-Shh IgG, H-160, which was raised against the N-terminal portion (amino acids 41–200) of human Shh (Fig 1A–D, lane 2) Consistent with the previous report, 5E1 failed to recognize Shh-Np in the lysate of CHO cells (Fig 1A, lane 2), although it recognized full-length Shh (Fig 1A, lanes and 6) This was also the case with Shh-Np in the lysate of HeLa cells (Fig 1B) Remarkably, 5E1 recognized Shh-Np in the lysate of COS7 and NSC34 cells, even under denaturing conditions (Fig 1C and D, lane 2) The difference between CHO ⁄ HeLa and COS7 ⁄ NSC34 cell lines in the reactivity of 5E1 with Shh-Np was attributed to the existence of endogenous Skn in the latter lines, as determined by RT-PCR analysis (Fig 2), suggesting that Skn affects the reactivity of Shh-Np with 5E1, regardless of cell type To confirm this, we transfected full-length Shh together with FLAG-tagged mouse Skn into these lines As expected, 5E1 efficiently recognized Shh-Np in the lysate of all lines under this experimental condition without affecting the level of Shh-Np (Fig 1A–D, lane 3) Ectopic expression of Skn led to a reduction in the amount of Shh-Np secreted into the conditioned media (CM) from all lines (Fig 1A–D, lane 3), suggesting increased hydrophobicity of the protein, probably as a result of palmitoylation catalyzed by Skn Similar results were obtained by using monoclonal anti-Shh N-terminal fragment, clone 171018 (data not shown) As this antibody also acts as a neutralizing antibody, it probably recognizes an epitope overlapping with that of 5E1 The expression of truncated Shh lacking the C-terminal domain [Shh (1–198)] results in an N-terminal signaling domain of Shh without cholesterol modification at its C terminus (Shh-N) Using H-160, Shh-N protein was detected in the lysate of these cell lines, transiently transfected with Shh (1–198) cDNA, at a level comparable to that of Shh-Np recovered from cells transfected with full-length Shh (Fig 1A–D, lane 4) However, 5E1 did not recognize Shh-N in the lysate of all four lines examined (Fig 1A–D, lane 4), reflecting the less efficient palmitoylation of Shh-N compared with Shh-Np, as previously reported [14] As seen in cells transfected with full-length Shh, the co-expression of FLAG-tagged mouse Skn resulted in greatly reduced secretion of ShhN into the CM and in the efficient recognition of Shh-N in lysate by 5E1 under denaturing conditions, without affecting the amount of Shh-N (Fig 1A–D, lane 5) 320 To examine in greater detail whether the effect of the expression of Skn on the 5E1 epitope of Shh-Np under denaturing conditions is a result of palmitoylation at the N terminus of Shh, we substituted Ser or Ala for the Cys25 of full-length Shh, and transiently transfected these mutants into COS (Fig 3A) and NSC34 (Fig 3B) cells These mutants were expressed at a level comparable to that of wild-type protein, as determined using H-160 (Fig 3A,B, lanes 3–6) As expected, neither C25S-Shh-Np nor C25A-Shh-Np was recognized by 5E1 (Fig 3A,B, lanes and 5), whereas wild-type Shh-Np clearly was (Fig 3A,B, lane 1) In the presence of exogenously transfected Skn, C25AShh was not recognized by 5E1 (Fig 3A,B, lane 6) These results indicate a strong correlation between the N-terminal palmitoylation of Shh-N(p) and the reactivity of 5E1 with Shh-N(p) Unexpectedly, C25S-Shh-Np retained the 5E1 epitope when Skn was exogenously overexpressed (Fig 3A,B, lane 4) Considering that Skn is a member of the MBOAT superfamily [32], it is possible that excess Skn transferred an acyl group onto the hydroxyl group of the N-terminal Ser of C25SShh-Np, although the efficiency seems much lower than that for wild-type Shh-Np To confirm this possibility, we labeled COS7 cells with [3H]palmitic acid and examined whether the radioactivity is incorporated into C25S-Shh-Np, as observed in wild-type Shh (Fig 3C, lanes and 2) As expected, we detected a band corresponding to C25S-Shh-Np, as well as fulllength C25S-Shh, only when Skn was co-expressed (Fig 3C, lane 4) In COS7 cells, a band migrating more slowly than Shh-N and strongly recognized by 5E1 was observed when Shh (1–198) alone was expressed (Fig 3A, lane 7, asterisk) This species was not prominently observed in lysate from NSC34, CHO, or HeLa cells Thus, there may be a third post-translational modification of the N-terminal signaling domain of Shh specific to COS7 cells affecting the 5E1 epitope Gup1 acts as a negative regulator for N-terminal palmitoylation of Shh Mammalian Gup1 has been described in the gene database cited above as a homolog of the S cerevisiae GUP1 gene product, based on its sequence homology Alignment of mouse and yeast Gup1 protein sequences using the blastp program with BLOSUM62 as a matrix [35] showed that these two proteins are 21% identical However, the same program showed that mouse Gup1 is more closely related to both mouse Skn (28%) and Drosophila skinny hedgehog gene product (25%) These values were comparable to the FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Abe et al A negative regulator for palmitoylation of Shh A B C D Fig Expression of Shh protein in transiently transfected mammalian cell lines CHO (A), HeLa (B), COS7 (C) and NSC34 (D) cells were transiently transfected with pIRES2-EGFP (IG, lane 1) as a vector control, pCAG-Shh ⁄ CMV-IRES-EGFP (Shh-IG, lane 2), pCAG-Shh ⁄ CMV-SknFLAG-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 (C199AShh-SF-IG, lane 7) Construction of these plasmids is described in detailed in the Experimental procedures Forty-eight hours after transfection, both conditioned media (indicated as CM) and cell lysates (50 lg) were collected and subjected to western blotting followed by probing with 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-terminal fragment of Shh are indicated by arrows The C199A mutation blocks autocatalytic cleavage of Shh, resulting in production of only full-length Shh FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS 321 A negative regulator for palmitoylation of Shh Y Abe et al Fig Expression of Skn and Gup1 transcripts in mammalian cell lines Total RNA extracted from CHO (lane 1), NSC34 (lane 2), COS7 (lane 3), HEK293 (lane 4), HeLa (lane 5) and mouse embryonic day 9.5 (E9.5) embryo (lane 6) was subjected to RT-PCR analysis to detect expression of Skn and Gup1 in these cells PCR products were separated by agarose gel electrophoresis followed by staining with ethidium bromide A fragment of glyceraldehyde-3phosphate dehydrogenase (GAPDH) was amplified as an internal control identity between mouse Skn and Drosophila skinny hedgehog gene product (29%) Sequence alignment and calculation of hydrophobicity using several programs revealed that both proteins have a similar structure, with a signal sequence and at least nine transmembrane domains (Fig 4) In addition, the open reading frame of both Skn and Gup1 genes consists of 11 exons; each corresponding exon of the two genes is similar in size (Fig 4), suggesting that these two genes evolve from the same origin The transcript of Gup1 was detectable in E9.5 mouse embryo (Fig 2, lane 6), in which Shh transcript is also detected [2] These facts prompted us to examine whether Gup1 is involved in regulating N-terminal palmitoylation in the mammalian hedgehog family, including Shh We cloned Gup1 cDNA by RT-PCR from adult mouse lung poly (A)+ RNA and first examined its subcellular localization by transiently expressing Gup1, whose C terminus was fused to enhanced green fluorescent protein (Gup1-EGFP), as well as EGFP-tagged Skn (Skn-EGFP) in HeLa cells, which express little endogenous Skn or Gup1 (Fig 2, lane 5) Consistent with a previous report [13], Skn–EGFP (Fig 5A,C) localized on the endoplasmic reticulum (ER), as determined by immunofluorescent staining of 78-kDa glucose-regulated protein (GRP78) (Fig 5B,C) This was also the case with Gup1-EGFP (Fig 5D,F), which was co-localized with GRP78 (Fig 5E,F) These observations imply that these two proteins interact with overlapped targets As the intensity of the fluorescence of these proteins was almost the same, the level of expression of these proteins was presumed to be simi322 lar To confirm this, we probed western blots of lysate extracted from COS7 cells, transiently transfected with each plasmid containing cDNA encoding these proteins, with anti-GFP IgG We detected a band, with a molecular mass of  60 kDa, in the lysate of cells transfected with Gup1-EGFP (Fig 5G, lane 3) However, we did not detect a band corresponding to that of Gup1-EGFP in the lysate of cells transfected with Skn-EGFP (Fig 5G, lane 2) Instead, a larger smear, which was also seen in cells transfected with Gup1-EGFP, was observed (Fig 5G, lane and 3) It is well known that hydrophobic membrane-bound proteins are often aggregated in the SDS sample buffer when the lysate is boiled Therefore, we subjected the samples to western blotting without boiling As expected, we observed double bands, ranging from 60 to 70 kDa, and disappearance of the larger smear in lysates of both Skn-EGFP-transfected cells and Gup1-EGFP-transfected cells (Fig 5G, lanes and 6) Under this experimental condition, the level of the expression of these two proteins was almost the same (Fig 5G, lanes and 6) Nevertheless, we sometimes observed a decrease in the intensity of the expected bands and appearance of a large smear, even in an unboiled sample of cells transfected with Skn-EGFP (data not shown), implying that Skn is more hydrophobic than Gup1 The expression of FLAG-tagged Gup1 in several cell lines, as detected by western blotting of unboiled samples using anti-FLAG IgG as a probe, revealed two major bands with molecular masses of  45 and 40 kDa (Fig 6A–C, lane 3) The expression of Skn-FLAG was undetectable in some lines (Fig 6A,C, lane 2) even when the samples were not boiled However, in COS7 cells transfected with Skn-FLAG, a band with a molecular mass of  40 kDa was detected when probed with antiFLAG IgG (Fig 6B, lane 2) These observations suggest that Skn without EGFP is more hydrophobic than Skn with EGFP As we observed that Gup1 is localized on the ER, we examined whether Gup1 can interact with Shh by immunoprecipitation We transiently expressed full-length Shh, together with Gup1-FLAG or SknFLAG, in COS7 cells and immunoprecipitated these proteins using anti-FLAG IgG As expected, both fulllength Shh and the N-terminal fragment of Shh were coprecipitated with Skn-FLAG (Fig 7A, upper panel, lane 5), whereas none of the fragment of Shh was detected in immunoprecipitate from cells transfected with Shh and empty vector (Fig 7A, upper panel, lane 4) Full-length Shh also coprecipitated with Gup1FLAG, indicating an interaction between Gup1 and Shh (Fig 7A, upper panel, lane 6) FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Abe et al Fig Requirement of Cys25 of Shh for Skn-dependent retention of the 5E1 epitope on the N-terminal fragment of Shh in western blotting COS7 (A) and NSC34 (B) cells were transiently transfected with pCAG-Shh (lanes and 2), pCAG-C25S-Shh (lanes and 4), pCAG-C25A-Shh (lanes and 6) or pCAG-Shh (1–198) (lanes and 8) together with either pFLAG-CMV5a (lanes 1, 3, and 7) as a vector control or pCMV-Skn-FLAG (lanes 2, 4, and 8) Cellular proteins (50 lg) were subjected to western blotting, followed by probing with anti-Shh N-terminal domain H-160, anti-Shh N-terminal domain 5E1, or anti-actin IgG Both full-length Shh and the N-terminal fragment of Shh are indicated by arrows The asterisk indicates a band in the lysate of COS cells transfected with both pCAGShh (1–198) and pFLAG-CMV5a (A, lane 7), migrating more slowly than Shh-N and strongly recognized with 5E1 (C) COS7 cells were transiently transfected with pCAG-Shh ⁄ CMV-IRES-EGFP (lane 1), pCAG-Shh ⁄ CMV-Skn-FLAG-IRES-EGFP (lane 2), pCAG-C25SShh ⁄ CMV-IRES-EGFP (lane 3), or pCAG-C25S-Shh ⁄ CMV-Skn-FLAGIRES-EGFP (lane 4) Twenty-four hours after transfection, cells were labeled with [9,10-3H]palmitic acid for 24 h Then the cells were lysed and Shh was immunoprecipitated with 5E1 followed by SDS-PAGE Dried gel was exposed to an X-ray film to visualize radiolabeled Shh We further assessed whether Gup1 interacts with Skn We expressed Gup1-EGFP in COS7 cells, together with either Skn-FLAG or empty vector, and subjected the cell lysates to immunoprecipitation using antiFLAG IgG followed by western blotting using antiGFP IgG (Fig 7B) We observed a band recognized by anti-GFP IgG only in precipitate from cells cotransfected with Gup1-EGFP and Skn-FLAG, suggesting an interaction between Gup1 and Skn (Fig 7B, lane 4, arrowhead) In this series of experiments, Skn-FLAG was barely detectable in both inputs (Fig 7A, lower panel, lane 2, and data not shown) and immunoprecipitates (Fig 7A, lower panel, lane 5, and data not shown) with horseradish peroxidase (HRP)-conjugated anti-FLAG IgG, probably because of the tendency of Skn to be aggregated in SDS sample buffer, as demonstrated in Figs and Similarly, the band recognized with anti-GFP Ig in precipitate from cells cotransfected with Gup1-EGFP and Skn-FLAG was much larger than expected (Fig 7B, lane 4, arrowhead) It is probably aggregated Gup1-EGFP, formed as a result of boiling to elute proteins from the immunocomplex Although Gup1 is predicted to be a member of the MBOAT superfamily, the His residue indispensable to MBOAT activity is replaced by Leu (Fig 4, asterisk) To examine whether Gup1 has HHAT activity, we transfected Gup1 cDNA together with full-length Shh cDNA into CHO cells As shown in Fig 6A, Shh-Np in the lysate of CHO cells was not recognized by 5E1 (lane 3), demonstrating that Gup1 has no HHAT activity Next, we examined whether Gup1 affects the palmitoylation of Shh-Np in cells expressing endoge- A negative regulator for palmitoylation of Shh A B C nous Skn, such as COS7 and NSC34 cells, by expressing full-length Shh in the presence of Gup1FLAG in these cells Co-expression of Shh with Gup1-FLAG resulted in a reduction of the total amount of Shh-Np, determined using H-160 in the FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS 323 A negative regulator for palmitoylation of Shh Y Abe et al Fig Comparison of mouse Gup1 and mouse Skn Mouse Gup1 (Mo Gup1) and mouse Skn (Mo Skn) were aligned based on amino acids in their sequences conserved between them, indicated with grey boxes Amino acids identical among mouse Gup1, mouse Skn and the Drosophila 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 black background 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 listed above is indicated with lines on each sequence Within each range of hydrophobic regions, the overlapped part recognized as a putative transmembrane domain by all the programs is represented by a thick bar The position of the His residue in the MBOAT motif indispensable to the activity is indicated with an asterisk 324 FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Abe et al A negative regulator for palmitoylation of Shh A B C D E the level of palmitoylated Shh-Np, although the difference was not statistically significant (Fig 6D,E) Taken together, these observations strongly suggest that mammalian Gup1 acts as a negative regulator of the N-terminal palmitoylation of Shh F Discussion G Fig Subcellular localization of mouse Skn and Gup1 (A–F) To visualize the subcellular localization of mouse Skn (A–C) and Gup1 (D–F), EGFP was fused to the C terminus of these proteins and expressed in HeLa cells Forty-eight hours after transfection, the cells were fixed, permeabilized and stained with an ER marker (78-kDa glucose-regulated protein) followed by TRITC-labeled secondary antibody 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 with pIRES2-EGFP (lanes and 4) as a vector control, pCMV-Skn-EGFP (lanes and 5) and pCMV-Gup1-EGFP (lanes and 6) Lysates were extracted from these cells, and boiled (lanes 1–3) or unboiled (lanes 4–6) samples (50 lg) were subjected to western blotting followed by probing using monoclonal anti-GFP IgG lysate of both COS7 and NSC34 cells, to 73.1% and 67.1%, respectively, as compared with that in cells cotransfected with full-length Shh and empty vector (Fig 6B,C, lane 3, and Fig 6D,E, solid column) The levels of modified Shh-Np in COS7 and NSC34 cells, as detected using 5E1, which is expected to recognize palmitoylated Shh-Np, were further reduced to 6.4% and 10.7%, respectively, as compared with control cells, suggesting that the expression of Gup1 inhibits palmitoylation of Shh-Np catalyzed by endogenous Skn in these cells (Fig 6B,C, lane 3, and Fig 6D,E, open column) It seemed that the overexpression of Skn-FLAG in these cells slightly increased In this report, we found that mammalian Gup1, a member of the MBOAT superfamily bearing sequence similarity to HHAT, acts as a negative regulator of N-terminal palmitoylation of Shh Several reports have demonstrated the critical role of N-terminal palmitoylation of Hh protein for its activity in Drosophila [15– 18,26] Drosophila Hh protein without palmitoylation not only loses its activity but also obstructs endogenous Hh signaling in vivo [26] By contrast, mammalian Shh without palmitoylation can act in some tissues [13,26,28] Analysis of both Skn knockout and C25SShh knockin mice revealed that the responsiveness to nonpalmitoylated Shh-Np varied among tissues [13] Thus, it is possible that while palmitoylated Hh-Np is the only signaling molecule in Drosophila, both palmitoylated and nonpalmitoylated Shh-Nps act as signaling molecules in mammals, and combining these molecules produces a variety of effects on developing organs and tissues If this were the case, the proportion of these molecules would have to be controlled precisely In the present study, mammalian Gup1 was found to interact with full-length Shh, as determined by immunoprecipitation (Fig 7), and to inhibit the N-terminal palmitoylation of Shh-Np in multiple mammalian cell lines, as determined by western blotting using 5E1 as a probe (Fig 6) These results, as well as structural similarity to both mammalian Skn and Drosophila skinny hedgehog gene product (Fig 4) and subcellular localization of these proteins (Fig 5), strongly suggest that mammalian Gup1 may be involved in such a mechanism It is not clear how Gup1 decreases the N-terminal palmitoylation of Shh Although the N terminus of the mature signaling domain of Shh is a Cys residue, N-terminal palmitoylation is not S-palmitoylation but Na-palmitoylation [14] Other than the hedgehog family, Gas is the only example that undergoes Na-palmitoylation in vertebrates, to our knowledge [36] How the Na-palmitoylation of Gas is regulated also remains unclear However, we assume that mammalian Gup1 competes with Skn for Shh to prevent palmitoylation rather than catalyzing depalmitoylation of Shh because other known Na-acylations, namely Na-acetylation and Na-myristylation, are irreversible [37,38] Further in vitro analyses are necessary to determine whether Gup1 can depalmitoylate Shh-Np FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS 325 A negative regulator for palmitoylation of Shh A D B Y Abe et al C Fig The effect of Gup1 on N-terminal palmitoylation of Shh-Np CHO (A), COS7 (B) and NSC34 (C) cells were transiently transfected 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 to western blotting, using polyclonal antiShh N-terminal domain H-160, monoclonal anti-Shh N-terminal domain 5E1, or monoclonal anti-FLAG IgG The intensity of the signals obtained from the western blot analysis was quantified using QUANTITY ONE software (Bio-Rad) The effect of Skn or Gup1 on the amount of total Shh-Np, determined with H-160 (solid column), and on the amount of modified Shh-Np, determined with 5E1 (open column), in COS7 (D) and NSC34 (E) cells was expressed as the ratio of the intensity of the band of Shh-Np to that from control cells transfected with Shh and empty vector in the same blot Values were the mean ± SD of three independent experiments The difference between total Shh-Np and modified Shh-Np was determined using a paired t-test *, P < 0.01; NS, not significant The level of Shh-Np in control cells is shown by the dotted line E We also demonstrated that 5E1 recognizes the N-terminal fragment of Shh under denaturing conditions when its N terminus is palmitoylated The property of this antibody is useful in identifying the state of N-terminal lipid modification of the protein Although 5E1 is an antibody that recognizes an epitope in the N-terminal signaling domain of Shh overlapping with the Patched-binding region and acts as a good neutralizing antibody [33], it was previously reported not to work under denaturing conditions such as western blotting [34] However, we found that 5E1 worked in western blotting when Shh was co-expressed with Skn (Figs 1,3 and 5), suggesting that palmitoylation at the N-terminal Cys residue of the N-terminal signaling domain of Shh protects the protein from disruption of the 5E1 epitope, even under denaturing conditions One explanation for this phenomenon might be that the palmitate itself constitutes the epitope when the N-terminal fragment of Shh is denatured However, our results also showed that both full-length Shh (Fig 1, lanes and 6) and Shh-N with unknown modification in COS7 cells (Fig 3A, lane 7, asterisk) were also recognized by 5E1 in western blotting, although they were not expected to undergo palmitoylation under those 326 transfection conditions Therefore, palmitoylation may not be a component of the 5E1 epitope but may influence the structure of the 5E1 epitope under denaturing conditions Crystal structure analysis revealed that the residues Pro42, Lys46, Arg154, Ser157, Ser178 and Lys179 are located close to each other on the surface of the mouse Shh-N protein and are essential for Shh-N to bind both Patched and 5E1 [34,39,40] Among the residues, Ser178 at least is found to be included in the 5E1 epitope [39] In addition, mouse Shh-N lacking the N-terminal 25 amino acids [Shh (50–198)] loses the ability to bind not only Patched but also 5E1 in immunoprecipitation [34] These observations indicate the requirement of the N-terminal region of the ShhN, including Pro42 and Lys46, for recognition by 5E1 in immunoprecipitation Therefore, there arise two possibilities One is that the N-terminal region, including Pro42 and Lys46, may constitute the epitope for 5E1, but that under denaturing conditions it is dissociated from the other parts of the protein, probably the C-terminal region, including Arg154, Ser157, Ser178 and Lys179 In this case, palmitoylation at the N terminus of Shh-N(p) may support the N-terminal region being located near other amino acids on the C-terminal FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Abe et al A negative regulator for palmitoylation of Shh A B Fig Gup1 interacts with both full-length Shh and Skn (A) COS7 cells were transiently transfected with pCAG-Shh (lanes 1–6), together with pFLAG-CMV-5a (vector) (lanes and 4), pCMV-Skn-FLAG (Skn-F) (lanes and 5), or pCMV-Gup1-FLAG (Gup1-F) (lanes and 6) Cells were 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-length Shh and the N-terminal fragment of Shh are indicated by arrows (B) COS7 cells were transiently transfected with pCMV-Gup1-EGFP (Gup1G), together with pFLAG-CMV-5a (vector) (lanes and 3), or pCMV-Skn-FLAG (Skn-F) (lanes and 4) Cells were lysed with IP buffer and subjected to immunoprecipitation using anti-FLAG IgG Then, samples were boiled and subjected to western blot analysis using anti-GFP IgG as a probe (lanes and 4) Some of the lysate (1 ⁄ 20 volume) was unboiled and also subjected to western blot analysis as input (lanes and 2) Immunoglobulin G heavy and light chains (IgG-H and IgG-L, respectively) are indicated by arrows Putative Gup1–EGFP is indicated by the arrowhead region to form the 5E1 epitope, even under denaturing conditions The other possibility is that the N-terminal portion of Shh-N(p) may not constitute the 5E1 epitope but may contribute to stabilization of the 5E1 epitope located within the C-terminal portion of Shh-N; when palmitoylated, the N-terminal portion would retain activity, even under denaturing conditions To understand, in full, the 5E1 epitope under denaturing conditions, extensive analyses will be required One clue may come from identifying the post-translational modification of Shh-N seen in COS7 cells transfected with Shh (1–198) alone (Fig 3A, lane 7, asterisk) Experimental procedures Plasmid construction The EcoRI–NcoI fragment of mouse Shh cDNA (kindly provided by A P McMahon) was subcloned between the EcoRI and the SmaI sites of pEGFP-N3 (Clontech, Mountain 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 of pCALNLw, resulting in pCAG-Shh pCALNLw vector is a 6.6-kbp plasmid derived from a cosmid vector, pAxCALNLw (Takara, Shiga, Japan), by digestion with SalI followed by self-ligation Mouse Skn was cloned from the total RNA of embryonic day 9.5 mouse embryo by reverse transcription using the avian myeloblastosis virus (AMV) reverse transcriptase first-strand synthesis kit (Life Sciences, Inc, St Petersburg, FL, USA) followed by PCR with Taq DNA polymerase (Promega, Madison, WT, USA) using the primers 5¢-CACACTACACTGGGAAGCAGAGACTCCAGC-3¢ and 5¢-AGCTGGCCCAGCAGCCATACACAGTTAAAG3¢ The cDNA was subcloned into the EcoRV site of pBluescript SK(+) (Stratagene, La Jolla, CA, USA) and sequenced using an automated sequencer (ABI-PRISM310 Genetic Analyzer; Perkin-Elmer Applied Biosystems, Foster FEBS 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 and the BamHI sites of pFLAG-CMV5a (Sigma, St Louis, MO, USA) (pCMV-Skn-FLAG, SF) or between the XhoI and the BamHI sites of pEGFP-N3 (pCMV-Skn-EGFP) The cDNA encoding FLAG-tagged Skn was then excised by digestion with BglII and ScaI and inserted between the BglII and the SmaI sites of the p-internal ribosome entry site (IRES2)-EGFP (IG) vector (Clontech), resulting in pCMV-Skn-FLAG-IRES-EGFP (SF-IG) Mouse Gup1 was cloned from the poly (A)+ RNA of adult mouse lung by RT-PCR using primers 5¢-CTCG AGGCCATGGGCATCAAGACAGC-3¢ and 5¢-GGATC CCTCCAGCTTCTCTCTGTCCTGC-3¢, which remove the termination codon and add a XhoI and a BamHI site to the 5¢ and 3¢ ends, respectively It was then subcloned into pGEM-T vector (Promega) for sequencing Then, the cDNA was excised with XhoI and BamHI and subcloned between the HindIII and the BamHI sites of pFLAG-CMV5a (pCMV-Gup1-FLAG) or between the XhoI and the BamHI sites of pEGFP-N3 (pCMV-Gup1EGFP) The EcoRI site of pCAG-Shh, and the AseI site of both pIRES2-EGFP and pCMV-Skn-FLAG-IRES-EGFP, were changed to a SalI site by linker ligation Subsequently, pCAG-Shh was digested with SalI and the fragment of  5.5 kbp, containing CAG promoter, full-length Shh cDNA and rabbit globin 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 C199A 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¢, tively Shh (1–198) (Shh lacking the C-terminal catalytic domain) was constructed by PCR, which changed the codon TGT corresponding to the Cys199 to TGA (Stop) and added an EcoRV site immediately after the stop codon using a sense primer 5¢-GGCATGCTGGCT CGCCTGGCTGTGGAAGCA-3¢ and an antisense primer 5¢-AAGCTTGATATCTCAGCCGCCGGATTTGGC-3¢ C25A mutation of Shh was introduced by a QuikChange site-directed mutagenesis kit (Stratagene) using primers 5¢-CCGGGCTGGCCGCTGGCCCCGGCAGGGG-3¢ and 5¢-CCCCTGCCGGGGCCAGCGGCCAGCCCGG-3¢ 328 Cell culture and transient transfection CHO cells were cultured in Ham’s F12 supplemented with 10% fetal bovine serum, 50 unitsỈmL)1 of penicillin and 50 lgỈmL)1 of streptomycin COS7, HeLa, HEK293 and NSC34 [41] cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 50 unitsỈmL)1 of penicillin and 50 lgỈmL)1 of streptomycin CHO (5 · 105 cells ⁄ dish), NSC34 (2 · 105 cells ⁄ dish), COS7 (2 · 105 cells ⁄ dish) and HeLa (2 · 105 cells ⁄ dish) cells seeded onto 60-mm dishes were transfected with each plasmid using Lipofectamine reagent (Invitrogen, Carlsbad, CA, USA) The transfected cells were used 48 h after transfection unless otherwise indicated 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 min, 65 °C for and 72 °C for (35 cycles) For Gup1, PCR was performed at 94 °C for min, 65 °C for and 72 °C for (40 cycles) Primers used were Skn, 5¢-CTGCGTGAGCAC CATGTTCA-3¢ and 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 min, 65 °C for and 72 °C for min) Western blot analysis Cells were washed twice with NaCl ⁄ Pi (PBS) and lysed with lysis buffer containing 20 mm Tris-HCl (pH 7.5), mm EDTA, 1% Triton X-100 and CompleteTM protease inhibitor cocktail tablets (Roche Diagnostics, Indianapolis, IN, USA) Each sample was analyzed using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA), and 30–50 lg of cellular protein was subjected to SDS-PAGE, followed by transfer to polyvinylidene difluoride membrane (Pall Life Sciences, East Hills, NY, USA) and blocking with 10% skim milk (Becton-Dickinson, Franklin Lakes, NJ, USA) in NaCl ⁄ Pi containing 0.1% Tween 20 (Wako, Osaka, Japan) Signals were detected with enhanced chemiluminescence reagents (GE Healthcare Bio-Sciences, Piscataway, NJ) As for the analysis of the secreted N-terminal signaling domain of Shh, 20 lL of CM was diluted with an equivalent volume of 2· SDS-PAGE sample buffer and subjected to SDSPAGE, followed by western blotting as described above FEBS Journal 275 (2008) 318–331 ª 2007 The Authors Journal compilation ª 2007 FEBS Y Abe et al To quantify the amount of total and modified 19-kDa Shh-Np, the same blots were probed with both H-160 and 5E1, respectively The intensity 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, : 2000; DSHB); rabbit anti-Shh N-terminal (H-160, : 2000; Santa Cruz Biotechnology Inc, Santa Cruz, CA); monoclonal anti-GFP (1E4, : 750; MBL, Nagoya, Japan); HRP-conjugated monoclonal anti-FLAG (M2, : 3000; 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 by tetramethylrhodamine isothiocyanate (TRITC)-labeled swine anti-rabbit IgG (1 : 100; DAKO, Glostrup, Denmark) Structural analysis The putative signal sequence of mouse Skn and Gup1 was calculated using signalp 3.0 [42] Hydrophobicity of the proteins was calculated using seven programs: tmhmm, tmpred, hmmtop, psort ii, sosui, toppred, and predictprotein [43–49] Immunoprecipitation Acknowledgements Transfected COS cells on 60-mm dishes were washed twice with NaCl ⁄ Pi and lysed with immunoprecipitation (IP) buffer containing 25 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% Nonidet P-40, and CompleteTM protease inhibitor cocktail tablets (Roche Diagnostics) Then, the lysate was incubated with lg of anti-FLAG IgG overnight at °C, followed by the addition of 20 lL of Protein G–Sepharose Fast Flow beads (GE Healthcare Bio-Sciences) Two hours later, the immunocomplexes were washed four times with the IP buffer Then, the beads were boiled in 20 lL of · 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 NSC34 cells; Dr 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 the University of Iowa Department of Biological Sciences (Iowa City, IA 52242, USA) This work was supported, in part, by grants from Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (C) 16590845 (YA), 17590893 (YK), 17590894 (TN), Keio Gijuku Academic Development funds (YA and TN), National Grant-in-Aid for the Establishment of High-Tech Research Center in a Private University (YA), and the Nakabayashi Trust for ALS Research (TN) The authors gratefully dedicate this article to late Professor Ikuo Nishimoto Isotope labeling with [3H]palmitic acid COS7 cells seeded onto 60-mm dishes at a density of · 105 cells ⁄ dish were transiently transfected with pCAGShh ⁄ CMV-IRES-EGFP, pCAG-Shh ⁄ CMV-Skn-FLAGIRES-EGFP, pCAG-C25S-Shh ⁄ CMV-IRES-EGFP, or pCAG-C25S-Shh ⁄ CMV-Skn-FLAG-IRES-EGFP Twentyfour hours after transfection, cells were incubated in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum containing 200 lCiỈmL)1 of [9,10-3H]palmitic acid (Perkin Elmer) for 24 h and lysed with IP buffer Shh was immunoprecipitated from the lysate as described above using 5E1 and subjected to SDS-PAGE The gel was fixed with isopropanol ⁄ water ⁄ acetic acid (25 : 65 : 10, v ⁄ v ⁄ v) for 30 and treated with Amplify Fluorographic Reagent (GE Healthcare Bio-Sciences) for 30 Then, the gel was dried and exposed to an X-ray film at 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Na -palmitoylation of Gas is regulated also remains unclear However, we assume that mammalian Gup1 competes with Skn for Shh to prevent palmitoylation rather than catalyzing depalmitoylation of. .. 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