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• W2065269093 abstract "Protein N-myristoylation has been recognized as a cotranslational protein modification. Recently, it was demonstrated that protein N-myristoylation could occur posttranslationally, as in the case of the pro-apoptotic protein BID and cytoskeletal actin. Our previous study showed that the N-terminal nine residues of the C-terminal caspase cleavage product of human gelsolin, an actin-regulatory protein, efficiently direct the protein N-myristoylation. In this study, to analyze the posttranslational N-myristoylation of gelsolin during apoptosis, metabolic labeling of gelsolin and its caspase cleavage products expressed in COS-1 cells with [3H]myristic acid was performed. It was found that the C-terminal caspase cleavage product of human gelsolin (tGelsolin) was efficiently N-myristoylated. When COS-1 cells transiently transfected with gelsolin cDNA were treated with etoposide or staurosporine, apoptosis-inducing agents, N-myristoylated tGelsolin was generated, as demonstrated by in vivo metabolic labeling. The generation of posttranslationally N-myristoylated tGelsolin during apoptosis was also observed on endogenous gelsolin expressed in HeLa cells. Immunofluorescence staining and subcellular fractionation experiment revealed that exogenously expressed tGelsolin did not localize to mitochondria but rather was diffusely distributed in the cytoplasm. To study the role of this modification in the anti-apoptotic activity of tGelsolin, we constructed the bicistronic expression plasmid tGelsolin-IRES-EGFP capable of overexpressing tGelsolin concomitantly with EGFP. Overexpression of N-myristoylated tGelsolin in COS-1 cells using this plasmid significantly inhibited etoposide-induced apoptosis, whereas overexpression of the non-myristoylated tGelsolinG2A mutant did not cause resistance to apoptosis. These results indicate that posttranslational N-myristoylation of tGelsolin does not direct mitochondrial targeting, but this modification is involved in the anti-apoptotic activity of tGelsolin. Protein N-myristoylation has been recognized as a cotranslational protein modification. Recently, it was demonstrated that protein N-myristoylation could occur posttranslationally, as in the case of the pro-apoptotic protein BID and cytoskeletal actin. Our previous study showed that the N-terminal nine residues of the C-terminal caspase cleavage product of human gelsolin, an actin-regulatory protein, efficiently direct the protein N-myristoylation. In this study, to analyze the posttranslational N-myristoylation of gelsolin during apoptosis, metabolic labeling of gelsolin and its caspase cleavage products expressed in COS-1 cells with [3H]myristic acid was performed. It was found that the C-terminal caspase cleavage product of human gelsolin (tGelsolin) was efficiently N-myristoylated. When COS-1 cells transiently transfected with gelsolin cDNA were treated with etoposide or staurosporine, apoptosis-inducing agents, N-myristoylated tGelsolin was generated, as demonstrated by in vivo metabolic labeling. The generation of posttranslationally N-myristoylated tGelsolin during apoptosis was also observed on endogenous gelsolin expressed in HeLa cells. Immunofluorescence staining and subcellular fractionation experiment revealed that exogenously expressed tGelsolin did not localize to mitochondria but rather was diffusely distributed in the cytoplasm. To study the role of this modification in the anti-apoptotic activity of tGelsolin, we constructed the bicistronic expression plasmid tGelsolin-IRES-EGFP capable of overexpressing tGelsolin concomitantly with EGFP. Overexpression of N-myristoylated tGelsolin in COS-1 cells using this plasmid significantly inhibited etoposide-induced apoptosis, whereas overexpression of the non-myristoylated tGelsolinG2A mutant did not cause resistance to apoptosis. These results indicate that posttranslational N-myristoylation of tGelsolin does not direct mitochondrial targeting, but this modification is involved in the anti-apoptotic activity of tGelsolin. Protein N-myristoylation is a well recognized form of lipid modification that occurs on eukaryotic and viral proteins (1Towler D.A. Gordon J.I. Adams S.P. Glaser L. Annu. Rev. Biochem. 1988; 57: 69-99Crossref PubMed Google Scholar, 2Spiegel A.M. Backlund P.S. Butrynski J.E. Jones T.L.Z. Simonds W.F. Trends Biochem. Sci. 1991; 16: 338-341Abstract Full Text PDF PubMed Scopus (91) Google Scholar, 3Boutin J.A. Cell. Signal. 1997; 9: 15-35Crossref PubMed Scopus (350) Google Scholar, 4Resh M.D. Biochim. Biophys. Acta. 1999; 1451: 1-16Crossref PubMed Scopus (1074) Google Scholar, 5Farazi T.A. Waksman G. Gordon J.I. J. Biol. Chem. 2001; 276: 39501-39504Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). Many N-myristoylated proteins play critical roles in regulating cellular structure and function. They include proteins involved in a wide variety of cellular signal transduction pathways such as protein kinases, phosphatases, guanine nucleotide-binding proteins, and Ca2+-binding proteins. In many cases, the functions of these N-myristoylated proteins are regulated by reversible protein-membrane and protein-protein interactions mediated by protein N-myristoylation. Generally, protein N-myristoylation is the result of cotranslational attachment of myristic acid, a 14-carbon saturated fatty acid, to a Gly residue at the extreme N terminus after removal of the initiating Met. A stable amide bond links myristic acid irreversibly to proteins. In addition to the cotranslational protein N-myristoylation, it was demonstrated that posttranslational protein N-myristoylation can also occur, as in the case of the pro-apoptotic protein BID. In this case, proteolytic cleavage of BID by caspase-8 caused exposure of an internal N-myristoylation motif (6Zha J. Weiler S. Oh K.-J. Wei M.C. Korsmeyer S.J. Science. 2000; 290: 1761-1765Crossref PubMed Scopus (472) Google Scholar). The exposed internal N-myristoylation motif was recognized by N-myristoyltransferase, the enzyme responsible for cotranslational N-myristoylation, and posttranslational N-myristoylation reaction occurred. It was also revealed that this postproteolytic N-myristoylation of BID plays critical role in the targeting of BID to mitochondria, its insertion into the outer membrane of mitochondria, the release of cytochrome c, and the killing of cells. Thus, posttranslational N-myristoylation plays a crucial role in the biological activity of BID.Until recently, BID was the only protein that had been demonstrated to be posttranslationally N-myristoylated. However, we recently showed that the C-terminal 15-kDa fragment of cytoskeletal actin is posttranslationally N-myristoylated upon caspase-mediated cleavage and specifically targeted to mitochondria (7Utsumi T. Sakurai N. Nakano K. Ishisaka R. FEBS Lett. 2003; 539: 37-44Crossref PubMed Scopus (106) Google Scholar). In this case, tActin localized at mitochondria did not induce cellular apoptosis. The biological roles of the mitochondrial localization of tActin remain to be clarified. During the analysis of posttranslational N-myristoylation of tActin, we also found that the N-terminal nine residues of the newly exposed N terminus of the caspase cleavage product of human gelsolin, an actin-regulatory protein, efficiently direct the protein N-myristoylation. Gelsolin is a member of a large family of actin-severing and -capping proteins (8Yin H.L. Stossel T.P. Nature. 1979; 281: 583-586Crossref PubMed Scopus (542) Google Scholar, 9Cunningham C.C. Stossel T.P. Kwiatkowski D.J. Science. 1998; 251: 1233-1236Crossref Scopus (256) Google Scholar). Human gelsolin has been shown to inhibit apoptosis through its ability to block the loss of mitochondrial membrane potential and to inhibit caspase activity (10Ohtsu M. Sakai N. Fujita H. Kashiwagi M. Gasa S. Shimizu S. Eguchi Y. Tsujimoto Y. Sakiyama Y. Kobayashi K. Kuzumaki N. EMBO J. 1997; 16: 4650-4656Crossref PubMed Scopus (116) Google Scholar, 11Kwiatkowski D.J. Curr. Opin. Cell Biol. 1999; 11: 103-108Crossref PubMed Scopus (326) Google Scholar, 12Koya R.C. Fujita H. Shimizu S. Ohtsu M. Takimoto M. Tsujimoto Y. Kuzumaki N. J. Biol. Chem. 2000; 275: 15343-15349Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 13Kusano H. Shimizu S. Koya R.C. Fujita H. Kamada S. Kuzumaki N. Tsujimoto Y. Oncogene. 2000; 19: 4807-4814Crossref PubMed Scopus (145) Google Scholar, 14Azuma T. Koths K. Flanagan L. Kwiatkowski D.J. J. Biol. Chem. 2000; 275: 3761-3766Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The role of gelsolin in apoptosis is complicated by the fact that gelsolin is also a substrate for caspase-3, caspase-7, and caspase-9, which generate two cleavage products with opposite function, an N-terminal fragment with pro-apoptotic activity and a C-terminal fragment with anti-apoptotic activity (12Koya R.C. Fujita H. Shimizu S. Ohtsu M. Takimoto M. Tsujimoto Y. Kuzumaki N. J. Biol. Chem. 2000; 275: 15343-15349Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 14Azuma T. Koths K. Flanagan L. Kwiatkowski D.J. J. Biol. Chem. 2000; 275: 3761-3766Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 15Kothakota S. Azuma T. Reinhard C. Klippel A. Tang J. Chu K. McGarry T.J. Kirschner M.W. Koths K. Kwiatkowski D.J. Williams L.T. Science. 1997; 278: 294-298Crossref PubMed Scopus (1031) Google Scholar, 16Kamada S. Kusano H. Fujita H. Ohtsu M. Koya R.C. Kuzumaki N. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8532-8537Crossref PubMed Scopus (95) Google Scholar). The precise mechanisms by which the two fragments affect cellular apoptosis remain to be elucidated.In the present study, the posttranslational N-myristoylation of the caspase cleavage products of human gelsolin was studied by metabolic labeling using cells transfected with either full-length gelsolin or its N- or C-terminal cleavage products. The effects of posttranslational N-myristoylation of the C-terminal cleavage product of gelsolin (tGelsolin) 2The abbreviations used are: tGelsolin, truncated gelsolin; tActin, truncated actin; TNF, tumor necrosis factor; DPBS, Dulbecco's phosphate-buffered saline; EGFP, enhanced green fluorescent protein; ECFP, enhanced cyan fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; VDAC, voltage-dependent anion channel; PDI, protein-disulfide isomerase; ER, endoplasmic reticulum. 2The abbreviations used are: tGelsolin, truncated gelsolin; tActin, truncated actin; TNF, tumor necrosis factor; DPBS, Dulbecco's phosphate-buffered saline; EGFP, enhanced green fluorescent protein; ECFP, enhanced cyan fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; VDAC, voltage-dependent anion channel; PDI, protein-disulfide isomerase; ER, endoplasmic reticulum. on its intracellular targeting and on the anti-apoptotic activity were then investigated. The results showed that human tGelsolin was posttranslationally N-myristoylated upon caspase-mediated cleavage. This posttranslationally N-myristoylated tGelsolin was not targeted to mitochondria but rather was diffusely distributed in the cytoplasm. Overexpression of N-myristoylated tGelsolin in COS-1 cells using the plasmid tGelsolin-IRES-EGFP significantly inhibited etoposide-induced apoptosis, whereas overexpression of the non-myristoylated tGelsolinG2A mutant did not cause resistance to apoptosis.These results indicated that posttranslational N-myristoylation of tGelsolin is involved in the anti-apoptotic activity of tGelsolin.EXPERIMENTAL PROCEDURESMaterials—Restriction endonucleases, DNA-modifying enzymes, RNase inhibitor, and Taq DNA polymerase were purchased from Takara Shuzo, Kyoto, Japan. RNase was obtained from Roche Applied Science. [3H]Leucine, [3H]myristic acid, and Amplify were from Amersham Biosciences. The dye terminator cycle sequencing kit was from Applied Biosystems. Anti-FLAG monoclonal antibody, anti-gelsolin C-terminal fragment monoclonal antibody, anti-heat shock protein 70 (Hsp70) monoclonal antibody, and fluorescein isothiocyanate-conjugated anti-mouse IgG antibody were purchased from Sigma. Anti-voltage-dependent anion channel (VDAC) polyclonal antibody and ProteoExtract™ subcellular proteome extraction kit were purchased from Merck KGaA. Anti-EGFP antibody was from Santa Cruz Biotechnology. MitoTracker Red CMXRos, Alexa Fluor 594 goat anti-mouse IgG antibody, and Hoechst 33342 were obtained from Molecular Probes. Protein G-Sepharose was from Pharmacia Biotech. Plasmid pCMV-gelsolin was from OriGene Technologies. Plasmid pECFP-ER and pIRES2-EGFP were obtained from Clontech. Other reagents were purchased from Wako Pure Chemical, Daiichi Pure Chemicals, or Seikagaku Kogyo and were of analytical or DNA grade.Plasmid Construction—Plasmid pBluescript II SK(+) lacking ApaI and HindIII sites was constructed as described previously (17Utsumi T. Akimaru K. Kawabata Z. Levitan A. Tokunaga T. Tang P. Ide A. Hung M.-C. Klostergaard J. Mol. Cell. Biol. 1995; 15: 6389-6405Crossref Scopus (29) Google Scholar) and designated pB. Plasmid pBGi1α-TNF, which contains a cDNA coding for Gi1α-TNF in which N-terminal 10 residues of Gi1α protein were linked to the N terminus of the mature domain of TNF, were constructed as described previously (18Utsumi T. Tou E. Takemura D. Ishisaka R. Yabuki M. Iwata H. Arch. Biochem. Biophys. 1998; 349: 216-224Crossref PubMed Scopus (8) Google Scholar). Plasmid pB-FLAG, which contains the sequence for the FLAG epitope at the C terminus, was constructed as described in Ref. 7Utsumi T. Sakurai N. Nakano K. Ishisaka R. FEBS Lett. 2003; 539: 37-44Crossref PubMed Scopus (106) Google Scholar. Plasmid pBtGelsolin-FLAG, which contains a cDNA coding for FLAG-tagged tGelsolin, was constructed by utilizing PCR. For this procedure, pCMV-gelsolin (OriGene) served as a template and two oligonucleotides (T-GELSO, 5′-ATATGGATCCATGGGCCTGGGCTTGTCC-3′ and GELSO-C, 5′-ATATGAATTCGGCAGCCAGCTCAGC-3′) as primers. After digestion with BamHI and EcoRI, the amplified products were subcloned into pB-FLAG at the BamHI and EcoRI sites. Plasmid pBtGelsolinG2A-FLAG was constructed by a method similar to that used to construct pBtGelsolin-FLAG using two oligonucleotides (T-GELSOG2A, 5′-ATATGGATTCATGGCCCTGGGCTTGTCC-3′ and GELSO-C, 5′-ATATGAATTCGGCAGCCAGCTCAGC-3′) as primers. Plasmid pBgelsolin-FLAG, which contains a cDNA coding for FLAG-tagged full-length gelsolin, was constructed by a method similar to that used to construct pBtGelsolin-FLAG using two oligonuclotides (N-GELSO, 5′-ATATGGATCCATGGCTCCGCACCGC-3′ and GELSO-C, 5′-ATATGAATTCGGCAGCCAGCTCAGC-3′) as primers. Plasmid pBN-gelsolin-FLAG, which contains a cDNA coding for the FLAG-tagged N-terminal caspase cleavage product of gelsolin, was constructed by a method similar to that used to construct pBtGelsolin-FLAG using two oligonucleotides (N-GELSO, 5′-ATATGGATCCATGGCTCCGCACCGC-3′ and N-GELSO-C, 5′-GCGCGAATTCATCTGTCTGGTC-3′) as primers. Plasmid pBtActin-FLAG was constructed as described previously (7Utsumi T. Sakurai N. Nakano K. Ishisaka R. FEBS Lett. 2003; 539: 37-44Crossref PubMed Scopus (106) Google Scholar). Plasmid pB Gi1α-TNF-FLAG, which contains a cDNA coding for FLAG-tagged Gi1α-TNF, was constructed by a method similar to that used to construct pBtGelsolin-FLAG. For this procedure, pBGi1α-TNF served as a template and two oligonucleotides (T3, 5′-AATTAACCCTCACTAAAGGG-3′ and TNFΔC, 5′-GCGCGAATTCCAGGGCAATGATCCC-3′) as primers. After digestion with BamHI and EcoRI, the amplified products were subcloned into pB-FLAG at the BamHI and EcoRI sites. All the cDNAs in pB vector were subcloned into pcDNA3 and used for transfection assays.The bicistronic expression vector pIRES2-EGFP lacking a BamHI site (pIRES2(ΔB)) was constructed by digesting pIRES2-EGFP (Clontech) with BamHI, blunt-ending with mung bean nuclease, and ligating with T4 ligase. Plasmid pIRES2(ΔB)-FLAG, which contains the sequence for the FLAG epitope at the C terminus, was constructed by utilizing PCR. For this procedure, pB-FLAG served as a template and two oligonucleotides (T3, 5′-AATTAACCCTCACTAAAGGG-3′ and C-FLAG-PST, 5′-GCGCCTGCAGCTACTTATGGTC-3′) as primers. After digestion with SacI and PstI, the amplified product was subcloned into pIRES2(ΔB) at the SacI and PstI sites. Plasmid pIRES2(ΔB)-tGelsolin-FLAG was constructed as follows. The BamHI/EcoRI fragment coding for tGelsolin was excised from pB-tGelsolin-FLAG and then subcloned into pIRES2(ΔB)-FLAG at the BamHI and EcoRI sites. Plasmids pIRES2(ΔB)-tGelsolinG2A-FLAG, pIRES2(ΔB)-gelsolin-FLAG, and pIRES2(ΔB)-tActin-FLAG were constructed by a method similar to that used to construct pIRES2(ΔB)-tGelsolin-FLAG.The DNA sequences of these recombinant cDNAs were confirmed by the dideoxy-nucleotide chain termination method (19Sanger F. Nickelen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52359) Google Scholar).Transfection of COS-1 Cells and Determination of N-Myristoylated Proteins—The simian virus 40-transformed African green monkey kidney cell line, COS-1, was maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% fetal calf serum (FCS; Invitrogen). Cells (2 × 105) were plated onto 35-mm diameter dishes 1 day before transfection. pcDNA3 or pIRES2(ΔB) construct (2 μg) containing cDNA coding for mutant gelsolin was used to transfect each plate of COS-1 cells along with 4 μl of Lipofectamine (2 mg/ml; Invitrogen) in 1 ml of serum-free medium. After incubation for 5 h at 37 °C, the cells were refed with serum-containing medium and incubated again at 37 °C for 48 h. The cells were then washed twice with 1 ml of serum-free DMEM and incubated for 4 h at 37 °C in 1 ml of DMEM with 2% FCS containing [3H]myristic acid (100 μCi/ml). For the treatment with etoposide or staurosporine, 48 h after transfection, the cells were incubated with 200 nm etoposide or 2 μm staurosporine at 37 °C for 18 h in 1 ml of DMEM with 5% FCS containing [3H]myristic acid (100 μCi/ml). For the treatment with Z-VAD-fmk, the cells were pretreated with 100 μm Z-VAD-fmk for 3 h before addition of etoposide or staurosporine. Subsequently, the cells were washed three times with Dulbecco's phosphate-buffered saline (DPBS) and collected with a cell scraper and then lysed with 200 μl of RIPA buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and proteinase inhibitors) on ice for 20 min. After immunoprecipitation with anti-FLAG antibody, the samples were analyzed by SDS-PAGE and fluorography.Western Blotting—The cell lysates of each group of transfected cells were prepared 48 h after transfection and resolved by 12.5% SDS-PAGE and then transferred to an Immobilon-P transfer membrane (Millipore). After blocking with nonfat milk, the membrane was probed with a specific anti-FLAG, anti-gelsolin, anti-protein-disulfide isomerase (PDI), anti-VDAC, anti-Hsp70, or anti-EGFP antibody as described previously (20Utsumi T. Sato M. Nakano K. Takemura D. Iwata H. Ishisaka R. J. Biol. Chem. 2001; 276: 10505-10513Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Immunoreactive proteins were specifically detected by incubation with horseradish peroxidase-conjugated protein G (Bio-Rad). The membrane was developed with enhanced chemiluminescence Western blotting reagent (Amersham Biosciences) and exposed to x-ray film (Eastman Kodak Co.).Immunoprecipitation—Samples were immunoprecipitated with a specific anti-FLAG or anti-gelsolin antibody as described in Ref. 17Utsumi T. Akimaru K. Kawabata Z. Levitan A. Tokunaga T. Tang P. Ide A. Hung M.-C. Klostergaard J. Mol. Cell. Biol. 1995; 15: 6389-6405Crossref Scopus (29) Google Scholar.SDS-PAGE and Fluorography—Samples were denatured by boiling for 3 min in SDS-sample buffer followed by analysis by SDS-PAGE on a 12.5% gel. Thereafter, the gel was fixed and soaked in Amplify™ (Amersham Biosciences) for 20 min. The gel was dried under vacuum and exposed to x-ray film (Kodak) for an appropriate period.Immunofluorescence Analysis and Fluorescence Microscopy—Immunofluorescence analysis of transfected cells was performed 48 h after transfection. At this time, the maximum level of protein expression was achieved. For immunocyotochemistry, cells were washed with DPBS, fixed in 4% paraformaldehyde in DPBS for 15 min, and permeabilized with 0.1% Triton X-100 in DPBS for 10 min at room temperature, followed by washing with 0.1% gelatin in DPBS. The permeabilized cells were incubated with anti-FLAG antibody (1:1000) in DPBS for 1 h at room temperature. After washing with 0.1% gelatin in DPBS, the cells were incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG antibody or Alexa Fluor 594 goat anti-mouse IgG antibody for 1 h at room temperature. Mitochondria were identified by incubating the cells with 300 nm MitoTracker Red for 30 min before fixation. After washing with 0.1% gelatin in DPBS, the cells were observed under a Zeiss Axiovert fluorescence microscope.Subcellular Fractionation—Subcellular fractionation of COS-1 cells expressing either tGelsolin-FLAG or tGelsolinG2A-FLAG was performed by using ProteoExtract™ subcellular proteome extraction kit (Merck) according to the manufacturer's instructions. Briefly, COS-1 cells (2 × 105) were transfected with 2 μg of pcDNA3tGelsolin-FLAG or pcDNA3tGelsolinG2A-FLAG as described earlier and incubated at 37 °C for 48 h. After washing twice with ice-cold Wash Buffer, cells were incubated with 0.5 ml of ice-cold Extraction Buffer I at 4 °C for 10 min, and then the supernatant was collected and used as a cytosolic fraction. Subsequently, cells were incubated with 0.5 ml of ice-cold Extraction Buffer II at 4 °C for 30 min, and then the supernatant was collected and used as a membrane/organelle fraction. The cells were then incubated with 0.5 ml ice-cold Extraction Buffer III at 4 °C for 10 min, then the supernatant was collected and used as a nucleic fraction.Induction and Detection of Apoptosis—The transfected COS-1 cells were incubated with 200 nm etoposide for 24 h. The cells were stained with 1 μm Hoechst 33342 and observed under a Zeiss Axiovert fluorescence microscope. The cell viability was assessed by examining the nuclear morphology. The numbers of total EGFP-positive cells and EGFP-positive cells showing apoptotic phenotype (Hoechst-positive cells) were counted, and the percent of apoptosis was calculated. In this case, 4-5 × 102 cells found in eight randomly selected area were counted in each sample and data are expressed as mean ± S.D. of three independent experiments.Detection of Generation of Posttranslationally N-Myristoylated tGelsolin from Endogenous Gelsolin—HeLa cells were grown in DMEM supplemented with 10% FCS. The cells (6 × 105) were incubated with 2 μm staurosporine at 37 °C for 10 h in 1 ml of DMEM with 5% FCS containing 200 μCi [3H] myristic acid. For the treatment with Z-VAD-fmk, the cells were pretreated with 100 μm Z-VAD-fmk for 3 h before addition of staurosporine. After incubation, the cells were harvested and lysed with RIPA buffer as described earlier and gelsolin and tGelsolin were immunoprecipitated with anti-gelsolin C-terminal fragment antibody (GS-2C4, Sigma). The samples were then analyzed by Western blotting using anti-gelsolin antibody or by SDS-PAGE and fluorograpy.RESULTSN Terminus of C-terminal Caspase Cleavage Product of Gelsolin Is N-Myristoylated—Our previous study showed that the N-terminal nine residues of the C-terminal caspase cleavage product of human gelsolin (tGelsolin) efficiently direct the protein N-myristoylation (7Utsumi T. Sakurai N. Nakano K. Ishisaka R. FEBS Lett. 2003; 539: 37-44Crossref PubMed Scopus (106) Google Scholar). To confirm that the full-length tGelsolin is N-myristoylated, cDNAs coding for epitope-tagged gelsolin and tGelsolin were generated and their susceptibility to protein N-myristoylation was evaluated by metabolic labeling in transfected cells. For these analyses, a FLAG-tag was introduced at the C terminus of these constructs. As shown in Fig. 1A, lane 2, transfection of COS-1 cells with cDNA coding for FLAG-tagged tGelsolin gave rise to a 44-kDa protein band with the expected molecular mass (42-kDa tGelsolin plus 2-kDa linker and FLAG-tag). The 44-kDa protein band was efficiently N-myristoylated, as determined by [3H]myristic acid labeling (Fig. 1A, lane 6). When Gly2 of tGelsolin-FLAG was replaced with Ala (tGelsolinG2A-FLAG), no incorporation of [3H]myristic acid into this mutant was observed despite the effective expression of this protein, as shown in Fig. 1A, lanes 3 and 7. Transfection of cDNAs coding for full-length gelsolin (gelsolin-FLAG) and the N-terminal fragment of gelsolin (N-gelsolin-FLAG) gave rise to protein bands with the expected molecular mass (88 and 44 kDa, respectively). [3H]Myristic acid incorporation into these proteins was not observed (Fig. 1A, lanes 1, 4, 5, and 8). As shown in Fig. 1B, the efficiency of [3H]myristic acid incorporation ([3H]myristic acid labeling/Western blotting) into tGelsolin-FLAG (lanes 3 and 6) was comparable with that into tActin-FLAG (lanes 2 and 5) and Gi1α-TNF-FLAG (lanes 1 and 4) having the N-myristoylation motif of Gi1α protein at its N terminus (20Utsumi T. Sato M. Nakano K. Takemura D. Iwata H. Ishisaka R. J. Biol. Chem. 2001; 276: 10505-10513Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), indicating that tGelsolin-FLAG is efficiently N-myristoylated.Induction of Apoptosis Induces the Generation of Posttranslationally N-Myristoylated tGelsolin—To determine whether the intracellular generation of N-myristoylated tGelsolin is induced by caspase-mediated cleavage of gelsolin during apoptosis, COS-1 cells transfected with FLAG-tagged gelsolin were treated with etoposide or staurosporine, apoptosis-inducing agents, and the generation of N-myristoylated tGelsolin was examined by Western blotting and [3H]myristic acid labeling. As shown in Fig. 2A, lanes 1, 2, 4, 6, 7, and 9, the generation of N-myristoylated tGelsolin was induced by the treatment of cells with 200 nm etoposide or 2 μm staurosporine. The induction of the generation of the N-myristoylated tGelsolin was completely inhibited by pretreatment of the cells with 100 μm Z-VAD-fmk, a caspase inhibitor, before addition of etoposide or staurosporine (Fig. 2A, lanes 3, 5, 8, and 10). To determine whether the generation of posttranslationally N-myristoylated tGelsolin was observed on endogenous gelsolin during apoptosis, HeLa cells were treated with staurosporine, and the generation of N-myristoylated tGelsolin was examined. As shown in Fig. 2B, the generation of N-myristoylated tGelsolin was induced by the treatment of cells with 2 μm staurosporine (lanes 2 and 5), and this induction was completely inhibited by pretreatment of the cells with 100 μm Z-VAD-fmk (lanes 3 and 6). These results strongly indicate that the generation of N-myristoylated tGelsolin is induced by caspase-mediated cleavage of gelsolin during apoptosis.FIGURE 2Induction of apoptosis induces the generation of N-myristoylated tGelsolin. A, COS-1 cells transfected with cDNA coding for gelsolin-FLAG were incubated with 200 nm etoposide or 2 μm staurosporine (STS) at 37 °C for 18 h in 1 ml of DMEM with 5% FCS containing [3H]myristic acid (100 μCi/ml). For treatment with Z-VAD-fmk, the cells were pretreated with 100 μm Z-VAD-fmk for 3 h before the addition of etoposide or staurosporine. Left panel, total cell lysates were analyzed by Western blotting using anti-FLAG antibody. Right panel, following immunoprecipitation with anti-FLAG antibody, the labeled proteins were analyzed by SDS-PAGE and fluorography. B, HeLa cells were incubated with 2 μm staurosporine at 37 °C for 10 h in 1 ml of DMEM with 5% FCS containing 200 μCi of [3H]myristic acid. After incubation, the cells were harvested and lysed with RIPA buffer, and gelsolin and tGelsolin were immunoprecipitated with anti-gelsolin C-terminal fragment antibody. For the treatment with Z-VAD-fmk, the cells were pretreated with 100 μm Z-VAD-fmk for 3 h before addition of staurosporine. Left panel, the samples were analyzed by Western blotting using anti-gelsolin C-terminal antibody. Right panel, the samples were analyzed by SDS-PAGE and fluorography.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Exogenously Expressed tGelsolin Does Not Localize to Mitochondria— Previous studies showed that both N-myristoylated tBID and tActin colocalize with mitochondria in an N-myristoylation-dependent manner (6Zha J. Weiler S. Oh K.-J. Wei M.C. Korsmeyer S.J. Science. 2000; 290: 1761-1765Crossref PubMed Scopus (472) Google Scholar, 7Utsumi T. Sakurai N. Nakano K. Ishisaka R. FEBS Lett. 2003; 539: 37-44Crossref PubMed Scopus (106) Google Scholar). These results indicate that posttranslational N-myristoylation might function as a mitochondrial-targeting signal. To examine whether the exogenously expressed N-myristoylated tGelsolin colocalizes with mitochondria or not, immunofluorescence staining coupled with staining with MitoTracker, a mitochondria-specific dye, was performed. As observed previously, exogenously expressed tActin colocalized with mitochondria, as shown in Fig. 3, a and b. In contrast, the distribution of exogenously expressed tGelsolin detected by immunofluorescence staining were distinct from that of MitoTracker, indicating that tGelsolin does not colocalize with mitochondria (Fig. 3, c and d). To determine whether the exogenously expressed tGelsolin colocalizes with endoplasmic reticulum or not, tGelsolin-FLAG was coexpressed with" @default.
• W2065269093 created "2016-06-24" @default.
• W2065269093 creator A5014580667 @default.
• W2065269093 creator A5089190393 @default.
• W2065269093 date "2006-05-01" @default.
• W2065269093 modified "2023-10-08" @default.
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