Distinct Domains for Anti- and Pro-apoptotic Activities of IEX-1
2006; Elsevier BV; Volume: 281; Issue: 22 Linguagem: Inglês
10.1074/jbc.m600054200
ISSN1083-351X
AutoresShen Li, Jinjin Guo, Cynthia Santos‐Berríos, Mei X. Wu,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoIEX-1 (immediate early response gene X-1) is a stress-inducible gene. Its overexpression can suppress or enhance apoptosis dependent on the nature of stress, yet the polypeptide does not possess any of the functional domains that are homologous to those present in well characterized effectors or inhibitors of apoptosis. This study using sequence-targeting mutagenesis reveals a transmembrane-like integrated region of the protein to be critical for both pro-apoptotic and anti-apoptotic functions. Substitution of the key hydrophobic residues with hydrophilic ones within this region impairs the capacity IEX-1 to positively and negatively regulate apoptosis. Mutations at N-linked glycosylation and phosphorylation sites or truncation of the C terminus of IEX-1 also abrogated its potential to promote cell survival. However, distinguished from the transmembrane-like domain, these mutants preserved pro-apoptotic activity of IEX-1 fully. On the contrary, mutation of nuclear localization sequence, despite its importance in apoptosis, did not impede IEX-1-mediated cell survival. Strikingly, all the mutants that lose their anti-apoptotic ability are unable to prevent acute increases in production of intracellular reactive oxygen species (ROS) at the initial onset of apoptosis, whereas those mutants that can sustain anti-death function also control acute ROS production as sufficiently as wild-type IEX-1. These findings suggest a critical role of IEX-1 in regulation of intracellular ROS homeostasis, providing new insight into the mechanism underlying IEX-1-mediated cell survival. IEX-1 (immediate early response gene X-1) is a stress-inducible gene. Its overexpression can suppress or enhance apoptosis dependent on the nature of stress, yet the polypeptide does not possess any of the functional domains that are homologous to those present in well characterized effectors or inhibitors of apoptosis. This study using sequence-targeting mutagenesis reveals a transmembrane-like integrated region of the protein to be critical for both pro-apoptotic and anti-apoptotic functions. Substitution of the key hydrophobic residues with hydrophilic ones within this region impairs the capacity IEX-1 to positively and negatively regulate apoptosis. Mutations at N-linked glycosylation and phosphorylation sites or truncation of the C terminus of IEX-1 also abrogated its potential to promote cell survival. However, distinguished from the transmembrane-like domain, these mutants preserved pro-apoptotic activity of IEX-1 fully. On the contrary, mutation of nuclear localization sequence, despite its importance in apoptosis, did not impede IEX-1-mediated cell survival. Strikingly, all the mutants that lose their anti-apoptotic ability are unable to prevent acute increases in production of intracellular reactive oxygen species (ROS) at the initial onset of apoptosis, whereas those mutants that can sustain anti-death function also control acute ROS production as sufficiently as wild-type IEX-1. These findings suggest a critical role of IEX-1 in regulation of intracellular ROS homeostasis, providing new insight into the mechanism underlying IEX-1-mediated cell survival. A coordinated balance between cell survival and apoptosis is essential for embryonic development, tissue homeostasis, and cellular responses to various types of stress (1Herr I. Debatin K.M. Blood. 2001; 98: 2603-2614Crossref PubMed Scopus (686) Google Scholar). Defects in this balance may contribute to a variety of diseases, including cancers, autoimmune disorders, and aberrant embryonic development (2Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6191) Google Scholar). Programmed cell death occurs in mitochondrion-dependent and independent pathways, and the former accounts for most forms of apoptosis in response to cellular stress, loss of survival factors, and developmental cues (3Green D.R. Evan G.I. Cancer Cell. 2002; 1: 19-30Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 4Kadenbach B. Arnold S. Lee I. Huttemann M. Biochim. Biophys. Acta. 2004; 1655: 400-408Crossref PubMed Scopus (191) Google Scholar). The mitochondrial pathway triggers cell death as a consequence of alteration in mitochondrial membrane permeability induced by apoptotic effectors like oxidative stress and signaling-mediated translocation of Bax, Bad, Bid, or Bim, leading to the release of cytochrome c and other proteins contained in the mitochondrial intermembrane space (5Newmeyer D.D. Ferguson-Miller S. Cell. 2003; 112: 481-490Abstract Full Text Full Text PDF PubMed Scopus (1086) Google Scholar, 6Ricci J.E. Waterhouse N. Green D.R. Cell Death Differ. 2003; 10: 488-492Crossref PubMed Scopus (95) Google Scholar). Substantial evidence indicates that prior to an irreparable loss of mitochondrial structural integrity, apoptotic effectors often stimulate an acute increase in the mitochondrial membrane potential Δψm that facilitates the formation of reactive oxygen species (ROS), 2The abbreviations used are: ROS, reactive oxygen species; TM, transmembrane; WT, wild type; GFP, green fluorescent protein; CHO, Chinese hamster ovary; ER, endoplasmic reticulum; Ab, antibody; DCF, 2′,7′-dichlorofluorescein; CM-H2DCFDA, 5-(and-6)-chloromethyl-2′,7′-dichlorofluorescein diacetate, acetyl ester; TNF, tumor necrosis factor; ERK, extracellular signal-regulated kinase. the amplitude of which determines a cell to die by apoptosis or to adapt (4Kadenbach B. Arnold S. Lee I. Huttemann M. Biochim. Biophys. 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Conceivably, prevention of ROS production in the initial phase of apoptosis is essential to guard the integrity of mitochondrial membrane, protecting cells from undergoing apoptosis. IEX-1 (immediate early response gene X-1), also known as IER3, p22/PRG1, Dif-2, or the mouse homologue gly96, is a stress-inducible gene (12Pietzsch A. Buchler C. Aslanidis C. Schmitz G. Biochem. Biophys. Res. Commun. 1997; 235: 4-9Crossref PubMed Scopus (46) Google Scholar, 13Kondratyev A.D. Chung K.N. Jung M.O. Cancer Res. 1996; 56: 1498-1502PubMed Google Scholar, 14Schafer H. Lettau P. Trauzold A. Banasch M. Schmidt W.E. Pancreas. 1999; 18: 378-384Crossref PubMed Scopus (31) Google Scholar, 15Charles C.H. Yoon J.K. Simske J.S. Lau L.F. Oncogene. 1993; 8: 797-801PubMed Google Scholar). It can be rapidly induced in various cells by irradiation, viral infection, inflammatory cytokines, chemical carcinogens, growth factors, and hormones under the control of transcription factors such as NF-κB/rel complexes, p53, Sp1, c-Myc, and Ap-1 (12Pietzsch A. Buchler C. Aslanidis C. Schmitz G. Biochem. Biophys. Res. Commun. 1997; 235: 4-9Crossref PubMed Scopus (46) Google Scholar, 13Kondratyev A.D. Chung K.N. Jung M.O. Cancer Res. 1996; 56: 1498-1502PubMed Google Scholar, 14Schafer H. Lettau P. Trauzold A. Banasch M. Schmidt W.E. Pancreas. 1999; 18: 378-384Crossref PubMed Scopus (31) Google Scholar, 15Charles C.H. Yoon J.K. Simske J.S. Lau L.F. Oncogene. 1993; 8: 797-801PubMed Google Scholar, 16Huang Y.H. Wu J.Y. Zhang Y. Wu M.X. Oncogene. 2002; 21: 6819-6828Crossref PubMed Scopus (45) Google Scholar, 17Domachowske J.B. Bonville C.A. Mortelliti A.J. Colella C.B. Kim U. Rosenberg H.F. J. Infect. Dis. 2000; 181: 824-830Crossref PubMed Scopus (45) Google Scholar, 18Kumar R. Kobayashi T. Warner G.M. Wu Y. Salisbury J.L. Lingle W. Pittelkow M.R. Biochem. Biophys. Res. Commun. 1998; 253: 336-341Crossref PubMed Scopus (42) Google Scholar, 19Im H.J. Craig T.A. Pittelkow M.R. Kumar R. Oncogene. 2002; 21: 3706-3714Crossref PubMed Scopus (23) Google Scholar). Like other immediate early response genes, IEX-1 plays a pivotal role in cell survival under conditions of stress (20Wu M.X. Ao Z. Prasad K.V. Wu R. Schlossman S.F. Science. 1998; 281: 998-1001Crossref PubMed Google Scholar, 21Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (92) Google Scholar, 22Mittal A. Papa S. Franzoso G. Sen R. J. Immunol. 2006; 176: 2183-2189Crossref PubMed Scopus (35) Google Scholar). In vivo targeted expression of IEX-1 to lymphocytes protects activated T cells from apoptosis, giving rise to an extended immune response after antigen stimulation and predisposing to a lupus-like autoimmune disease and T cell lymphoma in mice (23Zhang Y. Schlossman S.F. Edwards R.A. Ou C.N. Gu J. Wu M.X. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 878-883Crossref PubMed Scopus (88) Google Scholar, 24Zhang Y. Finegold M.J. Porteu F. Kanteti P. Wu M.X. Oncogene. 2003; 22: 6845-6851Crossref PubMed Scopus (30) Google Scholar). Gene-targeted deletion of IEX-1 caused hypertension and cardiac hypertrophy in mice, but the underlying mechanisms remain elusive (25Sommer S.L. Berndt T.J. Frank E. Patel J.B. Redfield M.M. Dong X. Griffin M.D. Grande J.P. van Deursen J.M. Sieck G.C. Romero J.C. Kumar R. J. Appl. Physiol. 2005; 100: 707-716Crossref PubMed Scopus (24) Google Scholar). In the present study, we identify distinct as well as overlapping functional regions for anti- and pro-apoptotic activities of IEX-1. Substitution of three key hydrophobic residues with hydrophilic ones in the transmembrane (TM)-like sequence is sufficient to abrogate effects of IEX-1 on inhibition and promotion of apoptosis. Additionally, N-linked glycosylation and phosphorylation as well as the C-terminal sequence of IEX-1, are all crucial for anti-apoptosis activity of IEX-1, but these structures appear to exert no discernible role in pro-apoptosis. On the contrary, mutation of nuclear localization sequence, despite its importance in apoptosis, did not impede IEX-1-mediated cell survival (26Kruse M.L. Arlt A. Sieke A. Grohmann F. Grossmann M. Minkenberg J. Folsch U.R. Schafer H. J. Biol. Chem. 2005; 280: 24849-24856Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). IEX-1-mediated cell survival is found to correlate well with its ability of reducing intracellular ROS formation either at basal levels or immediately after apoptotic stimulation. These findings, in line with IEX-1 localization in mitochondria, suggest a key role for IEX-1 in regulation of ROS homeostasis in cells. Mutagenesis—Sequence-targeting mutagenesis of human IEX-1 was carried out according to a PCR overlap extension technique using primers containing the expected mutations. The primers were forward 5′-ATGGCACACTCTCGCAGCGCACACCC-3′ and reverse 5′-TAGAAGGCGGCCGGGTGTTGCT-3′ for generating IEX-1 C2AC7A mutant; forward 5′-ACCGAGCACGCAGTGCACGCGTTCTCTACC-3′ and reverse 5′-GGTAGAGAACGCGTGCACTGCGTGCTCGGTG-3′ for K60AR63A mutant; and forward 5′-CTAACACTCACCAGCGTCTTCTGTCAAATCACCATGGC-3′ and reverse 5′-GCCATGGTGATTTGACAGAAGACGCTGGTGAGTGTTAG-3′ for transmembrane segment mutation (TM-mutant). To substitute N-glycosylated asparagine at position 133, the following primers were employed to insert an NheI restriction enzyme site: forward 5′-GGGTACCATGTGTCACTCTCGAAGCTGTCA-3′ and reverse 5′-AAGTGCTAGCAAAGGGCTCGAGGACGGG-3′ for amplification of cDNA sequence 1–414; and forward 5′-TTGCTAGCACTTCGGAGCCCTCGGACTACGCT-3′ and reverse 5′-CGGATCCGAAGGCGGCCGGGTGTTG-3′ for sequence 402–471. The two PCR products were then fused in pBluescript using an NheI restriction enzyme site. Truncation of 18 residues at the C terminus was obtained by amplification of IEX-1 cDNA sequence ranging from 1 to 414 by PCR. All the IEX-1 mutants were verified by DNA sequencing. T18A mutant was a kind gift from Dr. F. Pörteu (Universite Rene Descartes, Paris, France) (21Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (92) Google Scholar). Wild-type (WT) IEX-1 and its variants were cloned into mammalian cell-expression plasmid pcDNA3 (Invitrogen). For localization studies, WT IEX-1 and its TM-mutant were cloned in-frame into pEGFP-N3 (Clontech) to generate GFP fusion proteins. Transfection and Apoptosis Assays—Chinese Hamster Ovary (CHO) and p65KO3T3 cells were seeded into gelatinized 12-well plates and cultured overnight in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C with 5% CO2. p65KO3T3 is a mouse embryonic fibroblast cell line derived from NF-κB p65-knock out mice (27Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2935) Google Scholar). The cells were transfected in duplicate with various constructs, along with a LacZ-expressing reporter plasmid at a 4:1 ratio of IEX-1 construct to reporter plasmid, using Polyfect® transfection reagent (Qiagen). After 36 h, the cells were treated with apoptosis inducers for indicated times, followed by fixation of the cells with 2% formaldehyde and 0.5% glutaraldehyde in phosphate-buffered saline for 5 min at room temperature, and stained with 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) overnight at 37 °C. Percentages of apoptosis were blindly evaluated by counting a minimum of 200 lacZ-positive cells in three randomly selected areas of each well in duplicate samples using an inverted microscope. Apoptotic cells were determined by characteristic morphology of apoptosis (20Wu M.X. Ao Z. Prasad K.V. Wu R. Schlossman S.F. Science. 1998; 281: 998-1001Crossref PubMed Google Scholar). Alternatively, relative caspase-3 activities, indicative of apoptosis, were measured in cell lysate using a caspase-3 fluorometric substrate. Briefly, the cells were transfected with WT IEX-1 or its variant constructs and stimulated as above, followed by lysis of the cells on ice for 30 min in a cell lysis buffer (50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mm NaCl, and 1 mm EDTA) supplemented with 1% protease inhibitor mixture (Sigma). After centrifugation at 13,000 × g for 5 min at 4 °C, the supernatant (50 μl) was incubated in triplicate for 10 min at room temperature with a caspase-3 fluorometric substrate (Upstate, Charlottesville, VA) at a final concentration of 50 μm in the lysis buffer. The caspase-3 fluorometric substrate, acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin was hydrolyzed by active caspase-3 specifically, releasing fluorescent 7-amido-4-methylcoumarin that was measured at 0 and 10 min, with the fluoroMax-3 spectrometer using 380/460 nm as excitation/emission wavelength. A blank was measured in parallel without substrate and subtracted from all the measurements. Isolation of Mitochondria—293T cells stably transfected with either WT IEX-1 or TM-mutant were suspended on ice for 10 min in homogenizing buffer (H buffer; 210 mm mannitol, 70 mm sucrose, 1 mm EDTA, 5 mm HEPES, and 1:100 protein inhibitor mixture, pH 7.4) and Dounce-homogenized. The resultant homogenate was centrifuged at 600 × g for 10 min at 4 °C to remove nuclei and unbroken cells. The remaining supernatant was spun at 7,000 × g for 15 min at 4 °C to obtain a crude mitochondrial pellet and an ER-enriched supernatant as described (28Bogenhagen D. Clayton D.A. J. Biol. Chem. 1974; 249: 7991-7995Abstract Full Text PDF PubMed Google Scholar). The mitochondrion-enriched pellet was washed twice by centrifugation at 7,000 × g for 15 min at 4 °C, re-suspended in H buffer, and loaded onto a continuous sucrose gradient from 0.8 m to 1.5 m in H buffer, followed by centrifugation at 35,000 rpm for 2 h at 4°C using an SW41-Ti rotor (L-90K Ultracentrifuge, Beckman Coulter). The visible mitochondrial band in the sucrose gradient was carefully collected and subjected to two more rounds of sucrose gradient fractionation as above. After a final centrifugation, samples were collected consecutively from the bottom of the centrifuge tube at 0.5 ml per fraction. Each fraction was diluted with 1 ml of H buffer, pelleted by centrifugation as above, and analyzed by Western blotting using a polyclonal antibody (Ab) specific for IEX-1. Detection of Intracellular ROS—Intracellular ROS were assayed by measuring intracellular oxidation of 2′,7′-dichlorofluorescein (DCF) as described (29Carter W.O. Narayanan P.K. Robinson J.P. J. Leukoc. Biol. 1994; 55: 253-258Crossref PubMed Google Scholar). The substrate is 5-(and-6)-chloromethyl-2′,7′-dichlorofluorescein diacetate, acetyl ester (CM-H2DCFDA, Invitrogen). It is nonfluorescent until removal of the acetate groups by intracellular oxidation and thus serves as a cell-permeate indicator for ROS. To measure intracellular ROS, the substrate was added directly to cell culture at a final concentration of 5 μm and incubated for 15 min at 37 °C followed by two washes with phosphate-buffered saline. DCF fluorescence intensity was measured randomly at six different fields per well with a Spectra MAX GEMINI EM microplate fluorometer using 488/525 nm as excitation/emission wavelength, and the average fluorescence intensity was given by the instrument. Background controls were cells transfected with the same construct without substrate loading, and two wells for each construct were measured in parallel. Western Blot Analysis—Cells transfected with indicated constructs were lysed in cell-lysis buffer and centrifuged to remove nuclei and cell debris. Approximately 50 μg of proteins of whole cell lysate were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and immunoblotted with a polyclonal Ab specifically recognizing IEX-1, which was raised in rabbits against IEX-1 peptide sequence 51–75 (30Feldmann K.A. Pittelkow M.R. Roche P.C. Kumar R. Grande J.P. Histochem. Cell Biol. 2001; 115: 489-497Crossref PubMed Scopus (41) Google Scholar). IEX-1 and its mutants were detected by incubation of the membrane with horseradish peroxidase-linked goat anti-rabbit Ab. The x-ray films were developed using a SuperSignal West Pico Kit (Pierce). The membrane was stripped and re-probed by anti-glyceraldehyde-3-phosphate dehydrogenase Ab or anti-cytochrome C Ab (BD Sciences) for protein loading controls. Confocal Laser Scanning Microscopy—To determine intracellular localization of IEX-1, MCF-7 and CHO cells on gelatinized coverslips were transfected with GFP vector control, IEX-1-GFP, or TM-mu-GFP construct for 36 h and examined directly by confocal laser-scanning microscopy. To determine localization of IEX-1 in mitochondria, cells expressing GFP, IEX-1-GFP, or TM-mu-GFP were counterstained for 30 min at 37 °C in complete culture medium with MitoTracker red580 (Molecular Probes Inc.), a red fluorescence specific for mitochondria, at a concentration of 50 nm, followed by five washes with phosphate-buffered saline. The stained samples were mounted and analyzed on a Leica TCS4D confocal laser scanning microscope equipped with Leica Confocal Software Version 2.5. Statistical Analysis—Two-tailed Student's t tests were used to analyze the statistical significance of experimental samples compared with relevant controls. Target Mutagenesis of IEX-1—IEX-1 does not share any of the functional domains significantly homologous with those presented in well characterized pro- or anti-apoptotic effectors, despite its pivotal role in regulation of apoptosis. To delineate functional domains of IEX-1, a series of mutants were made on the basis of residue charge, hydrophobicity, conservation, and demonstrated functional significance in other systems, as depicted in Fig. 1A. A C2A/C7A mutant has an alanine substitution in the place of two cysteines at positions 2 and 7, on the assumption that they may be involved in formation of an intra- or inter-peptide disulfide bond and are required for IEX-1-mediated regulation of apoptosis. IEX-1 contains a polybasic sequence HRKRSRR, spanning amino residues from 58 to 64, respectively. The sequence matches a canonical nuclear localization sequence motif, K(K/R)X(K/R) (13Kondratyev A.D. Chung K.N. Jung M.O. Cancer Res. 1996; 56: 1498-1502PubMed Google Scholar, 26Kruse M.L. Arlt A. Sieke A. Grohmann F. Grossmann M. Minkenberg J. Folsch U.R. Schafer H. J. Biol. Chem. 2005; 280: 24849-24856Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 31Williams C.L. Cell. Signal. 2003; 15: 1071-1080Crossref PubMed Scopus (158) Google Scholar). Targeting Lys and Arg at positions 60 and 63 for Ala replacement generated a K60A/R63A mutant. Like Bcl-2 and Bcl-xL, IEX-1 has a TM-like integrated region at positions 86–101 that can potentially target the protein to the ER, Golgi, and the mitochondria as predicted by the program PSORT. In an attempt of disrupting intracellular localization of IEX-1, three hydrophobic residues at positions 90, 93, and 99 were converted to hydrophilic Thr or Ser, respectively, within the TM-like region, and the mutant is referred to as the "TM-mutant" (Fig. 1A). In addition, a previous study identified an N-linked glycosylation site at position 133 of the protein, which is highly conserved in mammals (15Charles C.H. Yoon J.K. Simske J.S. Lau L.F. Oncogene. 1993; 8: 797-801PubMed Google Scholar). To test its role in the function of IEX-1, Asn at position 133 was substituted with Ala by introducing an NheI restriction enzyme site. The replacement also resulted in substitution of a leucine with a serine residue at position 134, thus generating a two-site mutation, a N133A/L134S mutant (Fig. 1A). Finally, 18 residues at the C terminus were truncated to make a Δ139–156 deletion mutant. These variants, along with WT IEX-1, were transfected into 293T cells, CHO cells, or p65KO3T3 cells. The relative levels of protein expression were assessed 36 h later. It was found that protein expression levels of these IEX-1 variants, except for Δ138–156 mutant, were comparable to those of WT IEX-1 in 293T cells (data not shown), CHO cells (Fig. 1B) and p65KO3T3 cells (data not shown), indicating that these mutations did not adversely affect the stability of the protein. Truncation of the C terminus appeared to stabilize the polypeptide or to allow one of the IEX-1 isoforms accumulating at a high level (Fig. 1B, lane 7). Considering the fact that IEX-1 is a protein with a high turnover rate, its half-life being only 15–20 min (15Charles C.H. Yoon J.K. Simske J.S. Lau L.F. Oncogene. 1993; 8: 797-801PubMed Google Scholar), the finding is somewhat surprising. IEX-1 expressed as multiple protein bands in SDS-PAGE, and their sizes varied slightly with cell lines (293T versus CHO cells), with prominent protein bands at 28, 23–25, and 18–20 kDa, respectively, in agreement with previous observations (15Charles C.H. Yoon J.K. Simske J.S. Lau L.F. Oncogene. 1993; 8: 797-801PubMed Google Scholar, 24Zhang Y. Finegold M.J. Porteu F. Kanteti P. Wu M.X. Oncogene. 2003; 22: 6845-6851Crossref PubMed Scopus (30) Google Scholar, 30Feldmann K.A. Pittelkow M.R. Roche P.C. Kumar R. Grande J.P. Histochem. Cell Biol. 2001; 115: 489-497Crossref PubMed Scopus (41) Google Scholar). The TM-like Domain Is Required for Both Anti- and Pro-apoptosis—To determine effects of IEX-1 mutations on cell survival, WT IEX-1 and its variant constructs were transfected, along with or without a β-galactosidase-expressing reporter plasmid, into CHO cells, followed by stimulation of the transfectants for 6 h with staurosporin. Apoptosis was evaluated by counting apoptotic cells among β-galactosidase-positive cells (Fig. 2) or measurement of caspase-3 activity (Fig. 3). Staurosporin induced ∼50% of cell death in vector-transfected cells over background controls (Fig. 2A). The staurosporin-triggered apoptosis was diminished by half in cells expressing WT IEX-1, an observation similar to the early investigation (21Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (92) Google Scholar). Expression of C2A/C7A and K60A/R63A mutants of IEX-1 also prevented cells from staurosporin-induced cell death to a degree comparable to WT IEX-1 (Fig. 2A). The result suggests that the two cysteines at positions 2 and 7 as well as the nuclear localization signal are not required for anti-apoptotic activity of IEX-1. In contrast, CHO cells expressing the TM-mutant underwent apoptosis indistinguishable from the cells transfected with a control plasmid (Fig. 2A). Deletion of the entire TM segment, ranging from 86 to101 residues, yielded a similar result (data not shown). These data suggest a critical role for the TM-like region in IEX-1-regulated cell survival. As a positive mutation control, we included IEX-1 T18A mutant in the assay, in which a threonine at position 18, a potential ERK phosphorylation site, was replaced by alanine (21Garcia J. Ye Y. Arranz V. Letourneux C. Pezeron G. Porteu F. EMBO J. 2002; 21: 5151-5163Crossref PubMed Scopus (92) Google Scholar). As reported previously, expression of T18A mutant was also ineffective in preventing cells from apoptosis triggered by staurosporin. Similar results were obtained when these transfectants were treated with varying concentrations of H2O2 (Fig. 2B) or when p65KO3T3 cells were transfected with these IEX-1 variants and stimulated by TNF-α (Fig. 2C).FIGURE 3Effects of IEX-1 and its variants on caspase-3 activation. CHO cells were transfected with indicated constructs and stimulated in triplicate for 6 h with or without 400 nm staurosporin (STS) (A) or 1 mm H2O2 (B). At the end of stimulation, cells were harvested, lysed, and reacted with a caspase-3-specific fluorometric substrate. Fluorescence intensity was measured immediately (0 min) or 10 min after addition of the substrate by FluoroMax-3 spectrometer using 380/460 nm as excitation/emission wavelength. A blank was measured in parallel without substrate and subtracted from all the measurement. Relative caspase-3 activity is expressed as percentage increases (means ± S.D.) in fluorescence intensity at 10 min relative to 0 time point in the cells transfected with the same construct. **, statistical significance (p < 0.01) in the presence versus absence of overexpressed IEX-1 or its variants after apoptotic stimulation. One representative result of three independent experiments performed is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The described percentages of cell death were consistent with increased caspase-3 activity that detects apoptotic cell death in a more objective manner as compared with counting apoptotic cells in the basis of their morphology. As shown in Fig. 3A, staurosporin treatment increased caspase-3 activity in CHO cells by ∼60% in vector-transfected cells as compared with dimethyl sulfoxide solvent-containing background controls. The increment was, however, reduced to 20% or <20% over background after expressing WT IEX-1, C2A/C7A, or K60A/R63A mutants (Fig. 3A). Mutation of the TM-like domain in IEX-1 again severely impaired its ability to prevent caspase-3 activation induced by staurosporin. This also was true for H2O2-induced caspase-3 activation in CHO cells (Fig. 3B) as well as TNF-α-stimulated caspase-3 activation in p65KO3T3 cells (data not shown). These results indicate that the TM-like domain is indispensable for IEX-1-mediated protection against apoptosis induced by both intrinsic and extrinsic stimuli. Apart from anti-death effects, IEX-1 has also been shown to facilitate apoptosis under conditions of serum deprivation (32Grobe O. Arlt A. Ungefroren H. Krupp G. Folsch U.R. Schmidt W.E. Schafer H. FEBS Lett. 2001; 494: 196-200Crossref PubMed Scopus (35) Google Scholar). To see whether these mutations had any effect on pro-apoptotic activity of IEX-1, CHO cells were transfected with various IEX-1 mutants, along with or without a β-galactosidase-expressing reporter plasmid. After 36 h of transfection, the cells were cultured in serum-free medium or media containing only 0.1% and 0.5% serum. Overexpression of WT IEX-1 enhanced apoptosis significantly under serum deprivation as shown by increases in the percentages of apoptotic cells (Fig. 4) or in caspase-3 activation (data not shown). In addition, the apoptosis-enhancing effect of IEX-1 remained intact while two cysteines at positions 2 and 7 were mutated. Strikingly, the TM-mutant not only failed to prevent cells from apoptosis (Figs. 2 and 3) but also was unable to influence cell death or caspase-3 activation under serum starvation, when compared with WT IEX-1 (Fig. 4A and data not shown). The finding stresses an indispensable role for the TM-like domain in regulation of both anti- and pro-apoptotic activities of IEX-1. Although important in anti-apoptotic activity, T18A mutant, similar to its WT counterpart, significantly enhanced apoptosis upon serum withdrawal, suggesting that ERK-induced phosphorylation at position 18 is not essential for the observed pro-apoptotic activity of IEX-1. Perhaps the ERK signaling pathway does not work appropriately under this condition. Conversely, a K60A/R63A mutant with an altered nuclear localization sequence showed no apoptosis-enhancing effects over background (Fig. 4A), in contrast to its insignificance in cell survival, confirming early investigation (26Kruse M.L. Arlt A. Sieke A. Grohmann F. Grossmann M. Minkenberg J. Folsch U.R. Schafer H. J. Biol. Chem. 2005; 280: 24849-24856Abstract Full Text Full Text P
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