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• W2079559905 abstract "The neutral sphingomyelinases (nSMases) are considered major candidates for mediating the stress-induced production of ceramide. nSMase2, which has two hydrophobic segments near the NH2-terminal region, has been reported to be located at the plasma membrane and play important roles in ceramide-mediated signaling. In this study, we found that nSMase2 is palmitoylated on multiple cysteine residues via thioester bonds. Site-directed mutagenesis of cysteine residues to alanine indicated that two cysteine clusters of the enzyme are multiply palmitoylated; one cluster is located between the two hydrophobic segments, and the second one is located in the middle of the catalytic region of the protein. When overexpressed in the confluent phase of MCF-7 cells, wild-type nSMase2 was strictly localized in the plasma membranes, and the cysteine mutants of each palmitoylated cysteine cluster were seen not only at the plasma membrane but also in some punctate structures. Furthermore, mutation of all potential palmitoylation sites resulted in a dramatic reduction in the plasma membrane distribution and an increase in the punctate structures. The palmitoylation-deficient mutant was directed to lysosomes and rapidly degraded. Palmitoylation had no effect on enzyme activity but affected membrane-association properties of the protein. Finally, the catalytic region of nSMase2 where palmitoylation occurs was found to be localized at the inner leaflet of the plasma membrane. In summary, the results from this study reveal for the first time the palmitoylation of nSMase2 via thioester bonds and its importance in the subcellular localization and stability of this protein. The neutral sphingomyelinases (nSMases) are considered major candidates for mediating the stress-induced production of ceramide. nSMase2, which has two hydrophobic segments near the NH2-terminal region, has been reported to be located at the plasma membrane and play important roles in ceramide-mediated signaling. In this study, we found that nSMase2 is palmitoylated on multiple cysteine residues via thioester bonds. Site-directed mutagenesis of cysteine residues to alanine indicated that two cysteine clusters of the enzyme are multiply palmitoylated; one cluster is located between the two hydrophobic segments, and the second one is located in the middle of the catalytic region of the protein. When overexpressed in the confluent phase of MCF-7 cells, wild-type nSMase2 was strictly localized in the plasma membranes, and the cysteine mutants of each palmitoylated cysteine cluster were seen not only at the plasma membrane but also in some punctate structures. Furthermore, mutation of all potential palmitoylation sites resulted in a dramatic reduction in the plasma membrane distribution and an increase in the punctate structures. The palmitoylation-deficient mutant was directed to lysosomes and rapidly degraded. Palmitoylation had no effect on enzyme activity but affected membrane-association properties of the protein. Finally, the catalytic region of nSMase2 where palmitoylation occurs was found to be localized at the inner leaflet of the plasma membrane. In summary, the results from this study reveal for the first time the palmitoylation of nSMase2 via thioester bonds and its importance in the subcellular localization and stability of this protein. Ceramide, a bioactive sphingolipid, is involved in the regulation of diverse cellular functions, including cell growth, apoptosis, and differentiation (1Hannun Y.A. Luberto C. Trends Cell Biol. 2000; 10: 73-80Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar). Sphingomyelinase (EC 3.1.4.12, SMase), 2The abbreviations used are: SMase, sphingomyelinase; nSMase, neutral SMase; CHX, cycloheximide; FBS, fetal bovine serum; GFP, green fluorescent protein; PBS, phosphate-buffed saline; SNARE, soluble NSF attachment protein receptors. an enzyme that catalyzes the hydrolysis of the phosphodiester bond of sphingomyelin to produce ceramide and phosphocholine, has emerged as a major pathway of stress-induced ceramide production (2Levade T. Jaffrezou J.P. Biochim. Biophys. Acta. 1999; 1438: 1-17Crossref PubMed Scopus (283) Google Scholar). To date, three classes of SMase, acid, neutral, and alkaline, have been identified that are clearly distinguished by catalytic pH optimum, cation dependence, primary structure, and subcellular localization (3Tani M. Ito M. Igarashi Y. Cell. Signal. 2007; 19: 229-237Crossref PubMed Scopus (122) Google Scholar, 4Marchesini N. Hannun Y.A. Biochem. Cell Biol. 2004; 82 (2004): 27-44Crossref PubMed Scopus (273) Google Scholar). The Mg2+-dependent neutral SMases (nSMases) have emerged as major candidates for mediating ceramide-induced signal transduction (5Clarke C.J. Snook C.F. Tani M. Matmati N. Marchesini N. Hannun Y.A. Biochemistry. 2006; 45: 11247-11256Crossref PubMed Scopus (141) Google Scholar). Recent advances have resulted in molecular identification of at least three distinct nSMases in mammals, nSMases1, -2, and -3 (6Tomiuk S. Hofmann K. Nix M. Zumbansen M. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1999; 95: 3638-3643Crossref Scopus (259) Google Scholar, 7Hofmann K. Tomiuk S. Wolff G. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5895-5900Crossref PubMed Scopus (260) Google Scholar, 8Krut O. Wiegmann K. Kashkar H. Yazdanpanah B. Kronke M. J. Biol. Chem. 2006; 281: 13784-13793Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). nSMase1 was the first identified mammalian nSMase, which was cloned by remote sequence similarity with bacterial SMase (6Tomiuk S. Hofmann K. Nix M. Zumbansen M. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1999; 95: 3638-3643Crossref Scopus (259) Google Scholar). Although this enzyme exhibits in vitro SMase activity (6Tomiuk S. Hofmann K. Nix M. Zumbansen M. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 1999; 95: 3638-3643Crossref Scopus (259) Google Scholar), cells overexpressing it did not show changes in sphingomyelin or ceramide metabolism (9Sawai H. Domae N. Nagan N. Hannun Y.A. J. Biol. Chem. 1999; 274: 38131-38139Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), and the nSMase1 knock-out mouse appeared to exhibit a normal phenotype (10Zumbansen M. Stoffel W. Mol. Cell. Biol. 2002; 22: 3633-3638Crossref PubMed Scopus (53) Google Scholar). nSMase2 has been also cloned by data base search using bacterial SMase genes (7Hofmann K. Tomiuk S. Wolff G. Stoffel W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5895-5900Crossref PubMed Scopus (260) Google Scholar). The enzyme is a membrane-bound protein and has two highly hydrophobic segments near the NH2-terminal region, both of which are thought to function as transmembrane domains. In contrast to nSMase1, nSMase2 possesses in vivo SMase activity when overexpressed in mammalian cells (11Marchesini N. Luberto C. Hannun Y.A. J. Biol. Chem. 2003; 278: 13775-13783Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). A number of studies using culture cell lines have focused on potential signaling roles of nSMase2. Studies in MCF-7 cells have shown that nSMase2 is up-regulated during cell growth and is required for cells to undergo confluence-induced cell cycle arrest (12Marchesini N. Osta W. Bielawski J. Luberto C. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2004; 279: 25101-25111Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Interestingly, nSMase2 had been previously isolated as a confluence-induced gene in rat 3Y1 fibroblasts (13Hayashi Y. Kiyono T. Fujita M. Ishibashi M. J. Biol. Chem. 1997; 272: 18082-18086Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). nSMase2 has also been implicated in signal transduction events in response to cytokines (14Karakashian A.A. Giltiay N.V. Smith G.M. Nikolova-Karakashian M.N. FASEB J. 2004; 18: 968-970Crossref PubMed Scopus (74) Google Scholar, 15Clarke C.J. Truong T.G. Hannun Y.A. J. Biol. Chem. 2007; 282: 1384-1396Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 16De Palma C. Meacci E. Perrotta C. Bruni P. Clementi E. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 99-105Crossref PubMed Scopus (130) Google Scholar), oxidative stress (17Levy M. Castillo S.S. Goldkorn T. Biochem. Biophys. Res. Commun. 2006; 344: 900-905Crossref PubMed Scopus (96) Google Scholar), or amyloid β-peptide (18Jana A. Pahan K. J. Biol. Chem. 2004; 279: 51451-51459Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). In addition, the nSMase2 knock-out mice developed growth retardation that remained throughout development (19Stoffel W. Jenke B. Block B. Zumbansen M. Koebke J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 4554-4559Crossref PubMed Scopus (136) Google Scholar). In an independent study, Aubin et al. (20Aubin I. Adams C.P. Opsahl S. Septier D. Bishop C.E. Auge N. Salvayre R. Negre-Salvayre A. Goldberg M. Guenet J.-L. Poirier C. Nat. Genet. 2005; 37: 803-805Crossref PubMed Scopus (141) Google Scholar) identified a deletion in the gene encoding nSMase2 in the fragilitas ossium or “fro” mouse, a model of osteogenesis imperfecta. Bacterial SMase, which has remote sequence similarity with eukaryotic nSMases, has provided important information on the catalytic mechanism of the enzyme with preservation of key catalytic amino acid residues between the bacterial and eukaryotic enzymes (21Tamura H. Tameishi K. Yamada A. Tomita M. Matsuo Y. Nishikawa K. Ikezawa H. Biochem. J. 1995; 309: 757-764Crossref PubMed Scopus (22) Google Scholar, 22Ago H. Oda M. Takahashi M. Tsuge H. Ochi S. Katunuma N. Miyano M. Sakurai J. J. Biol. Chem. 2006; 281: 16157-16167Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 23Openshaw A.E. Race P.R. Monzo H.J. Vazquez-Boland J.A. Banfield M.J. J. Biol. Chem. 2005; 280: 35011-35017Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Site-directed mutagenesis analyses of mammalian nSMase1 and the yeast nSMase, Isc1p, revealed that these key amino acid residues are essential for catalytic activity of the enzyme as well as bacterial SMases (24Rodrigues-Lima F. Fensome A.C. Josephs M. Evans J. Veldman R.J. Katan M. J. Biol. Chem. 2000; 275: 28316-28325Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 25Okamoto Y. Vaena de Avalos S. Hannun Y.A. Biochemistry. 2003; 42: 7855-7862Crossref PubMed Scopus (23) Google Scholar). In contrast to the accumulated evidence for the catalytic mechanism of nSMases from molecular aspects, there is little information about other primary features, post-translational modifications, or subcellular localization and topology of the protein. nSMase2 strictly localized at the plasma membrane in the confluence phase of MCF-7 cells and in primary hepatocytes when overexpressed (12Marchesini N. Osta W. Bielawski J. Luberto C. Obeid L.M. Hannun Y.A. J. Biol. Chem. 2004; 279: 25101-25111Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 14Karakashian A.A. Giltiay N.V. Smith G.M. Nikolova-Karakashian M.N. FASEB J. 2004; 18: 968-970Crossref PubMed Scopus (74) Google Scholar); however, there is no information on the mechanisms that determine this localization, except for the presence of putative transmembrane regions at the NH2 terminus. In this study, we found that nSMase2 is palmitoylated via thioester bonds, and this post-translational modification has crucial roles for determination of subcellular localization and life span of the protein. This is the first report of posttranslational analysis of nSMase2. The significance of this modification is discussed. Materials—[choline-methyl-14C]Sphingomyelin was provided by Dr. Alicia Bielawska (Medical University of South Carolina, Charleston, SC). [9,10-3H]Palmitic acid (50 Ci/mmol) was from American Radiolabeled Chemicals, Inc. The scintillation mixture Safety Solve was from Research Products International. Other chemicals were from Sigma. Culture media were obtained from Invitrogen. Cell Culture and cDNA Transfection—MCF-7 cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified 5% CO2 incubator. For cDNA transfection, MCF-7 cells were plated in 6-well plates or 35-mm glass bottom microwell dishes at a density of 2.3 × 105 cells/well and cultured for 24 h. Transfections were done using Effectene® transfection reagent (Qiagen) as recommended by the manufacturer. After transfection, cells were cultured for an additional 24 h and used for activity assays, immunoblotting, and immunofluorescence analyses. To obtain tetracycline-inducible stable cell lines, MCF-7 cells were first transfected with pcDNA6/TR vector (Invitrogen) and selected in RPMI 1640 medium supplemented with 10% FBS and 7 μg/ml blasticidin (Invitrogen). The blasticidin-resistant cells were then transfected with the expression construct pcDNA4-NSM2 or pcDNA4-Cys3A5A and selected in RPMI 1640 medium supplemented with 10% FBS, 100 μg/ml Zeocin (Invitrogen), and 7 μg/ml blasticidin. The antibiotic-resistant clones were screened for expression of nSMase2 by immunofluorescence analysis using anti-Myc antibody. A representative clone, termed NSM2-Tet-On or CYS3A5A-Tet-On, was chosen and maintained in RPMI 1640 medium supplemented with 10% Tet System Approved FBS (Clontech) and 7 μg/ml blasticidin. nSMase Activity—MCF-7 cells transfected with plasmids were washed twice with PBS and lysed by syringe passage in buffer containing 25 mm Tris-HCl (pH 7.4), 1 mm EDTA, 0.2% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 1× protease inhibitor mixture (Roche Diagnostics). Post-nuclear supernatants were prepared by centrifugation at 600 × g for 10 min and used for enzyme assays. One hundred μm [cholinemethyl-14C]sphingomyelin (10 cpm/pmol) was incubated at 37 °C for 30 min with an appropriate amount of cell lysate in 100 μl of reaction mixture (100 mm Tris-HCl (pH 7.4), 5 mm MgCl2, 2.5 mm dithiothreitol, 0.2% Triton X-100, and 50 μm phosphatidylserine). The reaction was stopped by adding 500 μl of chloroform/methanol (2:1, v/v), and the resulting 180-μl upper phase was mixed with 4 ml of Safety Solve (Research Products International) for liquid scintillation counting. Preparation of Rabbit Polyclonal Antibody against nSMase2—The antibody was prepared by the Medical University of South Carolina antibody facility. Briefly, a synthetic oligopeptide corresponding to the 337-353 amino acids (RRRHPDEAFDHEVSAFF) of the human nSMase2 (this sequence is identical to the 335-351 amino acids of the mouse enzyme) was used for immunization. Antiserum was affinity-purified over a cyanogen bromide-activated agarose column bound with the same oligopeptide. Protein Determination, SDS-PAGE, and Western Blotting—Protein content was determined by the bicinchoninic acid protein assay (Pierce) with bovine serum albumin as a standard. Samples for gel electrophoresis were combined with 5× SDS sample buffer containing 5% 2-mercaptoethanol and separated by SDS-PAGE. For Western blotting, following separation by SDS-PAGE, proteins were electrotransferred to a nitrocellulose membrane. The membrane was blocked with PBS, 0.1% Tween 20 (PBS-T) containing 3% dried milk. Proteins were identified by incubating with anti-V5 (0.2 μg/ml; Invitrogen) or anti-glyceraldehyde-3-phosphate dehydrogenase (1 μg/ml; Ambion) in 3% dried milk/PBS-T at 4 °C for overnight. Secondary antibodies, horseradish peroxidase-conjugated anti-mouse IgG (Jackson ImmunoResearch) diluted 1:10,000, were incubated in 3% dried milk/PBS-T at room temperature for 1 h. Finally, proteins were visualized using enhanced chemiluminescence (Pierce) with exposure to CL-X Posure™ film (Pierce). Immunoprecipitation—The cells were lysed with 100 μl of 20 mm Tris-HCl (pH 7.4) containing 1% SDS and 1% 2-mercaptoethanol and were boiled for 5 min. Samples were centrifuged at 10,000 × g for 5 min, and the supernatant was mixed with 1 ml of 50 mm Tris-HCl (pH 7.4) containing 150 mm NaCl, 2 mm EDTA, and 1% Triton X-100. The mixture was incubated with 1 μg of anti-V5 antibody at 4 °C for 12 h, and then 10 μl of protein A/G-agarose (Santa Cruz Biotechnology) was added, and the mixture was incubated at 4 °C for 3 h. The precipitate was spun down by centrifugation, washed with PBS five times, and then suspended in 20 μl of SDS sample buffer. After boiling for 5 min, the sample was subjected to SDS-PAGE, followed by Western blotting analysis as described above. In Vivo [3H]Palmitic Acid Labeling—MCF-7 cells grown on a 6-well plate were transfected with plasmids and incubated for 24 h at 37 °C. Culture medium was then changed to 1.5 ml of serum-free RPMI 1640 medium. After a 1-h incubation at 37 °C, cells were labeled with 100 μCi of [3H]palmitic acid (50 Ci/mmol; American Radiolabeled Chemicals, Inc.) at 37 °C for 3 h. Cells were washed twice with PBS, and V5-tagged nSMase2 was immunoprecipitated using anti-V5 antibody and protein A/G-agarose as described above. Immunoprecipitates, suspended in SDS sample buffer containing 1% 2-mercaptoethanol, were boiled for 3 min, separated by SDS-PAGE, and visualized by autoradiography using the fluorographic reagent EN3HANCE™ (PerkinElmer Life Sciences). 1% of respective cell lysates was analyzed by Western blotting with anti-V5 antibody as described above. Preparation of Soluble and Membrane Fractions—MCF-7 cells transfected with plasmids were washed twice with PBS, suspended in buffer A (20 mm Tris-HCl (pH 7.4), 0.25 m sucrose, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, and 1× protease inhibitor mixture (Roche Diagnostics)), and sonicated. After removal of cell debris by centrifugation at 500 × g for 5 min at 4 °C, cell lysates were centrifuged at 100,000 × g for 90 min at 4 °C. Occasionally, the cell lysates were treated with an equal volume of buffer A containing 1% Triton X-100, 1% Tween 20, or 0.5% deoxycholate for 1 h on ice before centrifugation. The resulting supernatant and pellet were used as soluble and membrane fractions, respectively. Immunofluorescence and Confocal Microscopy—Transfected cells were cultured on cover glass and then fixed with 3% paraformaldehyde in PBS for 15 min. After being rinsed with PBS and 50 mm NH4Cl in PBS, cells were permeabilized by 0.1% Triton X-100 in PBS for 5 min at room temperature. After treatment with blocking buffer (2% FBS in PBS) for 15 min, the samples were incubated with anti-V5 (3 μg/ml; Invitrogen), anti-Myc (2.4 μg/ml; Invitrogen), anti-Giantin (1:300 dilution; Abcam), or anti-Lamp1 antibody (2 μg/ml; Santa Cruz Biotechnology) with blocking buffer at room temperature for 2 h followed by secondary antibody at room temperature for 1 h. All confocal images were taken with a laser-scanning confocal microscope (LSM 510 Meta; Carl Zeiss, Thornwood, NY). Each microscopic image is representative of 20 fields over at least three experiments, and all images were taken at the equatorial plane of the cell. Raw data images were cropped in Adobe Photoshop® 7.0 for publication. Plasmid Construction—Wild-type mouse nSMase2 tagged with V5 at the COOH terminus (pEF6NSM2) was constructed by PCR using a 5′ primer with an XhoI restriction site (5′-TCCGCTCGAGAATGGTTTTGTACACGAC-3′) and a 3′ primer disrupted stop codon (5′-CGCCTCCTCTTCCCCTGCAGACA-3′) and subcloned into pEF6/V5-His-TOPO vector (Invitrogen). To generate a construct expressing the GFP-fused nSMase2 (NSM2GFP), pEF6NSM2 was treated with XhoI and EcoRV and subcloned into pEGFP-N2 (Clontech), which was treated with XhoI and SmaI. Tetracycline-inducible wild-type nSMase2 (pcDNA4-NSM2) or Cys3A5A mutant (pcDNA4-Cys3A5A) was constructed by PCR using a 5′ primer with a KpnI restriction site (5′-GGGGTACCATGGTTTTGTACACGACCCCCTTTCCT-3′) and a 3′ primer with an XhoI restriction site disrupted stop codon (5′-TTTCCGCTCGAGTGCCTCCTCTTCCCCTGCAGACAC-3′) and subcloned into pcDNA4/TO/myc-HisA (Invitrogen). Point mutants of nSMase2 were constructed by fusing the NH2- and COOH-terminal fragment of the enzyme. The NH2-terminal fragments were amplified by PCR using a 5′ primer containing the sequence of pEF6/V5-His TOPO vector (5′-TAATACGACTCACTATAGGG-3′), 3′ primers (see supplemental Table 1), and pEF6NSM2 as a template. The COOH-terminal fragments were amplified by PCR using 5′ primers (see supplemental Table 1) and a 3′ primer (5′-CGCCTCCTCTTCCCCTGCAGACA-3′). These fragments were extended with Pfx50™ DNA polymerase (Invitrogen), digested with SpeI and EcoRI, and subcloned into pEF6NSM2. The sequences of all constructs were verified with an ABI377 DNA sequencer. nSMase2 Is Palmitoylated via Thioester Bonds—Several membrane proteins have lipid modifications, and palmitoylation is one of these important lipid modifications. To determine whether nSMase2 is palmitoylated, MCF-7 cells overexpressing nSMase2 with a V5 tag at the COOH terminus were labeled with [3H]palmitic acid, and the protein was immunoprecipitated using anti-V5 antibody and subjected to SDS-PAGE analysis and fluorography. As shown in Fig. 1A,[3H]palmitic acid was incorporated into a 76-kDa protein that co-migrated with nSMase2 on Western blotting, whereas no labeled proteins were detected in mock-transfected cells. In many cases, covalent attachment of palmitic acid into proteins occurs through a thioester bonds to Cys residues (26Smotrys J.E. Linder M.E. Annu. Rev. Biochem. 2004; 73: 559-587Crossref PubMed Scopus (486) Google Scholar). The thioester is cleaved by treatment with weak nucleophiles such as neutral hydroxylamine. Hydroxylamine treatment removed the entire incorporated label from nSMase2, demonstrating that the acyl moiety on the enzyme was attached via a thioester bond (Fig. 1B). Identification of Two Palmitoylated Cys Clusters in nSMase2—There is no well defined consensus sequence for palmitoylation other than a requirement for Cys. Palmitoylation frequently occurs at Cys residues arranged in clusters (26Smotrys J.E. Linder M.E. Annu. Rev. Biochem. 2004; 73: 559-587Crossref PubMed Scopus (486) Google Scholar). We found three Cys clusters in the mouse nSMase2 amino acid sequence as follows: a cluster containing three Cys residues, Cys53, Cys54, and Cys59 (Cys3 cluster), which are located between the two hydrophobic segments; two Cys residues, Cys125 and Cys132 (Cys4 cluster); and four Cys residues, Cys392, Cys395, Cys396, and Cys400 (Cys5 cluster), which are located in the middle of the putative catalytic region of the protein (Fig. 2A). Many proteins are also palmitoylated at Cys residues near the NH2 terminus (26Smotrys J.E. Linder M.E. Annu. Rev. Biochem. 2004; 73: 559-587Crossref PubMed Scopus (486) Google Scholar). In nSMase2 there are two Cys residues, Cys12 (Cys1 position) and Cys27 (Cys2 position) in the NH2-terminal region (Fig. 2A). To determine whether these Cys residues are involved in palmitoylation, we created Cys to Ala mutants of each Cys cluster as well as in combinations (Table 1, all constructs used in this study are shown in Table 1). All mutants were tagged with V5 at their COOH terminus. These mutants were transfected into MCF-7 cells, labeled with [3H]palmitic acid, and analyzed by SDS-PAGE and fluorography or Western blotting using anti-V5 antibody. As shown in Fig. 2B, although all mutant proteins were expressed in the cells, the incorporation of [3H]palmitic acid into each protein was quite different. The mutation of Cys1, -2, or -4 positions to Ala (Cys1A, Cys2A, or Cys4A mutant) did not have significant effects on the incorporation of [3H]palmitate. On the contrary, 3H-palmitoylated Cys3A and Cys5A mutants (mutated Cys3 or Cys5 positions to Ala, respectively) were observed but far less than the wild-type protein. Furthermore, the Cys3A5A combination mutant almost completely lost the incorporation of 3H palmitoylation (Fig. 2B). These results clearly indicated that nSMase2 is palmitoylated in Cys residues of two Cys clusters of the protein.TABLE 1Identification of the different nSMase2 mutants used in this studyNamePosition of mutation (Cys to Ala)Fusion tag (COOH terminus)Wild typeNoneV5NSM2GFPNoneGFPCys1A10NSALS14V5Cys2A25FPAYW29V5Cys3A52PAALQLFAT60V5Cys4A124FAFATANVAL133V5Cys5A391GAHGAANFKALN402V5Cys3A5A52PAALQLFAT60391GAHGAANFKALN402V5GFP-tagged Cys3A5A52PAALQLFAT60391GAHGAANFKALN402GFPCys5A-C53A52PACLQLFCT60391GAHGAANFKALN402V5Cys5A-C54A52PCALQLFCT60391GAHGAANFKALN402V5Cys5A-C59A52PCCLQLFAT60391GAHGAANFKALN402V5Cys5A-C53A,C54A52PAALQLFCT60391GAHGAANFKALN402V5Cys5A-C54A,C59A52PCALQLFAT60391GAHGAANFKALN402V5Cys5A-C53A,C59A52PACLQLFAT60391GAHGAANFKALN402V5Cys3A-C392A52PAALQLFAT60391GAHGCCNFKCLN402V5Cys3A-C395A52PAALQLFAT60391GCHGACNFKCLN402V5Cys3A-C396A52PAALQLFAT60391GCHGCANFKCLN402V5Cys3A-C400A52PAALQLFAT60391GCHGCCNFKALN402V5Cys3A-C395A,C396A52PAALQLFAT60391GCHGAANFKCLN402V5 Open table in a new tab Effect of Palmitoylation on the Localization of nSMase2—Generally, protein palmitoylation appears to play an important role in subcellular trafficking of proteins, in modulating protein activity, and/or in protein-protein interactions (26Smotrys J.E. Linder M.E. Annu. Rev. Biochem. 2004; 73: 559-587Crossref PubMed Scopus (486) Google Scholar). We initially examined whether palmitoylation affects the subcellular localization of the enzyme in the confluence phase of MCF-7 cells. As reported previously, wild-type nSMase2 was localized at the plasma membrane when overexpressed in the confluent culture of MCF-7 cells (Fig. 3A, panel a). Cys1A, Cys2A, and Cys4A mutants showed similar expression patterns to the wildtype protein (Fig. 3A, panels b, c, and e). However, the Cys3A and Cys5A mutants were seen not only in the plasma membrane but also in some punctate structures (Fig. 3A, panels d and f). The Cys3 and -5 double mutant (Cys3A5A) showed a very different distribution, displaying a dramatic reduction in the plasma membrane distribution and an increase in the punctate structures (Fig. 3A, panel g). To eliminate the possibility that this altered localization was somehow caused by the presence of the Ala residues, we also constructed Cys to Ser mutants of each or both Cys clusters. The Ser mutation also caused a dramatic reduction in the [3H]palmitic acid labeling of the proteins and resulted in identical localization to the Ala mutants (data not shown). To avert the possibility that extreme and transient overproduction of a membrane protein would affect its localization, we also isolated stable transfectant clones using a tetracycline-inducible expression system. As shown in Fig. 3B, tetracycline-induced wild-type protein was strictly expressed in plasma membranes, whereas the Cys3A5A protein hardly expressed in the plasma membranes. To confirm the role of palmitoylation in the subcellular localization, 2-bromopalmitate, an inhibitor of protein palmitoylation (27Webb Y. Hermida-Matsumoto L. Resh M.D. J. Biol. Chem. 2000; 275: 261-270Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar), was used. As shown in Fig. 3C, treatment with 2-bromopalmitate resulted in a dramatic reduction of the plasma membrane distribution of tetracycline-induced wild-type protein. Taken together, these results indicate that the lack of palmitoylation in nSMase2 greatly affects its subcellular localization. Identification of Palmitoylated Cys Residues in nSMase2—Next, we investigated which Cys residue is palmitoylated in each Cys cluster of nSMase2. To identify the specific palmitoylated amino acid in the Cys3 position, Cys to Ala mutations of Cys in the Cys3 cluster (Cys54 and Cys59, Cys53 and Cys59, or Cys53 and Cys54) were conducted using the Cys5A quadruple mutant as the receiving strain (Fig. 4A and Table 1). These mutants were overexpressed in MCF-7 cells and labeled with [3H]palmitic acid. It was found that [3H]palmitic acid incorporated into all three mutant proteins but at significantly reduced levels (Fig. 4A, compare 3rd to 5th lanes with 2nd lane). These results indicate that the three Cys residues in the Cys3 cluster are palmitoylated (Fig. 4A), and thus contribute to the overall palmitoylation. Likewise, Cys to Ala mutations in the Cys5 cluster were introduced in the background of the Cys3A triple mutant (Fig. 4B and Table 1). Point mutation of Cys395 or Cys396 in the Cys3A mutant showed that these Cys residues were still palmitoylated, whereas mutation of both Cys395 and Cys396 caused almost total loss of palmitoylation (Fig. 4B), indicating that these contiguous Cys residues serve as palmitoylation sites in the Cys5 cluster. In summary, these results suggest that each Cys cluster of nSMase2 is multiply palmitoylated and contributes to the overall palmitoylation. To investigate which Cys residue in the Cys3 and Cys5 clusters is important for the plasma membrane localization of nSMase2 in MCF-7 cells, each Cys residue in the clusters was changed individually to Ala. In this experiment, point mutation analysis was conducted in the Cys3 position using the Cys5A mutant or the Cys5 position using the Cys3A mutant (Fig. 5 and Table 1). In the point mutation analysis of the Cys3 position, there were no differences in subcellular localization between Cys5A and Cys5A-C59A (Fig. 5, panels b and e), whereas point mutation of Cys53 or Cys54 to Ala resulted in an increase in the distribution of intracellular punctate structures and a decrease in the plasma membrane localization (Fig. 5, panels c and d). The Cys53 and Cys54 double mutation resulted in a predominant punctate distribution, similar to that seen with Cys3A5A mutant (Fig. 5, panels f and m), indicating that Cys53 and Cys54, but not Cys59, in the Cys3 cluster are important for plasma membrane localization of nSMase2. For the Cys5 cluster, although point mutation of Cys392 or Cys400 of Cys3A did not exert significant effects on the plasma membrane localization (Fig. 5, panels g, h, and k), the distribution of punctate structures of the Cys3A-C395A or -C396A was increased compared with that of Cys3A" @default.
• W2079559905 created "2016-06-24" @default.
• W2079559905 creator A5069434243 @default.
• W2079559905 creator A5090422329 @default.
• W2079559905 date "2007-03-01" @default.
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