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JNK- and p38 Kinase-mediated Phosphorylation of Bax Leads to Its Activation and Mitochondrial Translocation and to Apoptosis of Human Hepatoma HepG2 Cells

2006; Elsevier BV; Volume: 281; Issue: 30 Linguagem: Inglês

10.1074/jbc.m510644200

ISSN

1083-351X

Autores

Bong‐Jo Kim, Seung‐Wook Ryu, Byoung‐Joon Song,

Tópico(s)

Nuclear Receptors and Signaling

Resumo

Mitochondrial translocation of pro-apoptotic Bax prior to apoptosis is well established after treatment with many cell death stimulants or under apoptosis-inducing conditions. The mechanism of mitochondrial translocation of Bax is, however, still unknown. The aim of this work was to investigate the mechanism of Bax activation and mitochondrial translocation to initiate apoptosis of human hepatoma HepG2 and porcine kidney LLC-PK1 cells exposed to various cell death agonists. Phosphorylation of Bax by JNK and p38 kinase activated after treatment with staurosporine, H2O2, etoposide, and UV light was demonstrated by the shift in the pI value of Bax on two-dimensional gels and confirmed by metabolic labeling with inorganic [32P]phosphate in HepG2 cells. Specific inhibitors of JNK and p38 kinase significantly inhibited Bax phosphorylation and mitochondrial translocation and apoptosis of HepG2 cells. A specific small interfering RNA to MAPKK4 (the upstream protein kinase of JNK and p38 kinase) markedly decreased the levels of MAPKK4 and MAPKK3/6, blocked the activation of JNK or p38 kinase, and inhibited Bax phosphorylation. However, the negative control small interfering RNA did not cause these changes. Confocal microscopy of various Bax mutants showed differential rates of mitochondrial translocation of Bax before and after staurosporine treatment. Among the Bax mutants, T167D did not translocate to mitochondria after staurosporine exposure, suggesting that Thr167 is a potential phosphorylation site. In conclusion, our results demonstrate, for the first time, that Bax is phosphorylated by stress-activated JNK and/or p38 kinase and that phosphorylation of Bax leads to mitochondrial translocation prior to apoptosis. Mitochondrial translocation of pro-apoptotic Bax prior to apoptosis is well established after treatment with many cell death stimulants or under apoptosis-inducing conditions. The mechanism of mitochondrial translocation of Bax is, however, still unknown. The aim of this work was to investigate the mechanism of Bax activation and mitochondrial translocation to initiate apoptosis of human hepatoma HepG2 and porcine kidney LLC-PK1 cells exposed to various cell death agonists. Phosphorylation of Bax by JNK and p38 kinase activated after treatment with staurosporine, H2O2, etoposide, and UV light was demonstrated by the shift in the pI value of Bax on two-dimensional gels and confirmed by metabolic labeling with inorganic [32P]phosphate in HepG2 cells. Specific inhibitors of JNK and p38 kinase significantly inhibited Bax phosphorylation and mitochondrial translocation and apoptosis of HepG2 cells. A specific small interfering RNA to MAPKK4 (the upstream protein kinase of JNK and p38 kinase) markedly decreased the levels of MAPKK4 and MAPKK3/6, blocked the activation of JNK or p38 kinase, and inhibited Bax phosphorylation. However, the negative control small interfering RNA did not cause these changes. Confocal microscopy of various Bax mutants showed differential rates of mitochondrial translocation of Bax before and after staurosporine treatment. Among the Bax mutants, T167D did not translocate to mitochondria after staurosporine exposure, suggesting that Thr167 is a potential phosphorylation site. In conclusion, our results demonstrate, for the first time, that Bax is phosphorylated by stress-activated JNK and/or p38 kinase and that phosphorylation of Bax leads to mitochondrial translocation prior to apoptosis. Programmed cell death or apoptosis is an important cellular process that eliminates unwanted cells during normal development or damaged cells after removal of trophic factors or exposure to toxic chemicals. Recent studies have demonstrated that a variety of apoptosis-stimulating agents cause translocation of pro-apoptotic Bax and BH3 (Bcl-2 homology 3)-only proteins such as Bim and truncated Bid to mitochondria from the cytoplasm to initiate mitochondrion-dependent apoptosis through changing mitochondrial permeability (1Kroemer G. Reed J.C. Nat. Med. 2000; 6: 513-519Crossref PubMed Scopus (2785) Google Scholar, 2Adams J.M. Cory S. Trends Biochem. Sci. 2001; 26: 61-66Abstract Full Text Full Text PDF PubMed Scopus (815) Google Scholar, 3Jaeschke H. LeMasters J.J. Gastroenterology. 2003; 125: 1246-1257Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar). Apoptosis is reported to be stimulated by staurosporine (STS) 2The abbreviations used are: STS, staurosporine; JNK, c-Jun N-terminal kinase; mAb, monoclonal antibody; MAPKK, mitogen-activated protein kinase kinase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; XTT, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt; siRNA, small interfering RNA; PVDF, polyvinylidene difluoride; GFP, green fluorescent protein; MEF, mouse embryonic fibroblast. (4Hsu Y.-T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1032) Google Scholar, 5Wolter K.G. Hsu Y.-T. Smith C.L. Nechushtan A. Xi X.-G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1578) Google Scholar, 6Wei M.C. Zong W.-X. Cheng E.H.-Y. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3373) Google Scholar); irradiation (4Hsu Y.-T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1032) Google Scholar); dexamethasone (4Hsu Y.-T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1032) Google Scholar); removal of interleukin-3 (7Gross A. Jockel J. Wei M.C. Korsmeyer S.J. EMBO J. 1998; 17: 3878-3885Crossref PubMed Scopus (968) Google Scholar), interleukin-7 (8Khaled A.R. Kim K. Hofmeister R. Muegge K. Durum S.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14476-14481Crossref PubMed Scopus (219) Google Scholar), or nerve growth factor (9Putcha G.V. Deshmukh M. Johnson Jr., E.M. J. Neurosci. 1995; 19: 7476-7485Crossref Google Scholar); vitamin E succinate (10Yu W. Sanders B.G. Kline K. Cancer Res. 2003; 63: 2483-2491PubMed Google Scholar); various chemotherapeutic agents such as etoposide (11Boldt S. Weidle U.H. Kolch W. 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Chem. 2004; 279: 21085-21095Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar) prevents Bax translocation to mitochondria and the subsequent apoptosis, possibly through activation of the phosphatidylinositol 3-kinase- and Akt/protein kinase B-related cell survival pathway (15Tsuruta F. Masuyama N. Gotoh Y. J. Biol. Chem. 2002; 277: 14040-14047Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). This pathway was recently shown to promote phosphorylation of Bax at Ser184, followed by its retention in the cytoplasm, thus preventing Bax translocation to mitochondria (14Gardai S.J. Hildeman D.A. Frankel S.K. Whitlock B.B. Frasch S.C. Borregaard N. Marrack P. Bratton D.L. Henson P.M. J. Biol. Chem. 2004; 279: 21085-21095Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 16Xin M. Deng X. J. Biol. Chem. 2005; 280: 10781-10789Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). These results and other reports described below indicate that Bax and other pro-apoptotic proteins may be retained by their interacting proteins present in the cytoplasm in normal physiological states. Bax-interacting proteins identified so far are Bcl-2 and its homologous proteins (17Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar, 18Sato T. Hanada M. Bodrug S. Irie S. Iwama N. Boise L. Thompson C.B. Golemis E. Fong L. Wang H.-G. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9238-9242Crossref PubMed Scopus (594) Google Scholar), adenine nucleotide translocator (19Marzo I. Brenner C. Zamzami N. Jurgensmeier J.M. Susin S.A. Vieira H.L. Prevost M.C. Xie Z. Matsuyama S. Reed J.C. Kroemer G. Science. 1998; 281: 2027-2031Crossref PubMed Scopus (1058) Google Scholar), voltage-dependent anion channel protein (20Shimizu S. Matsuoka Y. Shinohara Y. Moneda Y. Tsujimoto Y. J. Cell Biol. 2001; 152: 237-250Crossref PubMed Scopus (332) Google Scholar), humanin (21Guo B. Zhai D. Cabezas E. Welsh K. Nouraini S. Satterthwait A.C. Reed J.C. Nature. 2003; 423: 456-461Crossref PubMed Scopus (481) Google Scholar), Ku70 (22Sawada M. Sun W. Hayes P. Leskov K. Boothman D.A. Matsuyama S. Nat. Cell Biol. 2003; 5: 320-329Crossref PubMed Scopus (318) Google Scholar), 14-3-3 (23Nomura M. Shimizu S. Sugiyama T. Narita M. Ito T. Matsuda H. Tsujimoto Y. J. Biol. Chem. 2003; 278: 2058-2065Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 24Tsuruta F. Sunayama J. Mori Y. Hattori S. Shimizu S. Tsujimoto Y. Yoshioka K. Masuyama N. Gotoh Y. EMBO J. 2004; 23: 1889-1899Crossref PubMed Scopus (454) Google Scholar), heat shock protein Hsp60 (25Kirchhoff S.R. Gupta S. Knowlton A.A. Circulation. 2002; 105: 2899-2904Crossref PubMed Scopus (250) Google Scholar), cofilin (26Chua B.T. Volbracht C. Tan K.O. Li R. Yu V.C. Li P. Nat. Cell Biol. 2003; 5: 1083-1089Crossref PubMed Scopus (256) Google Scholar), Mcl-1 (27Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (520) Google Scholar), protein kinase Cϵ (28McJilton M.A. Van Sikes C. Wescott G.G. Wu D. Foreman T.L. Gregory C.W. Weidner D.A. Harris-Ford O. Morgan Lasater A. Mohler J.L. Terrian D.M. Oncogene. 2003; 22: 7958-7968Crossref PubMed Scopus (99) Google Scholar), Asc (29Ohtsuka T. Ryu H. Minamishima Y.A. Macip S. Sagara J. Nakayama K.I. Aaronson S.A. Lee S.W. Nat. Cell Biol. 2004; 6: 121-128Crossref PubMed Scopus (206) Google Scholar), etc. Despite the characterization of the Bax-retaining proteins (17Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar, 18Sato T. Hanada M. Bodrug S. Irie S. Iwama N. Boise L. Thompson C.B. Golemis E. Fong L. Wang H.-G. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9238-9242Crossref PubMed Scopus (594) Google Scholar, 19Marzo I. Brenner C. Zamzami N. Jurgensmeier J.M. Susin S.A. Vieira H.L. Prevost M.C. Xie Z. Matsuyama S. Reed J.C. Kroemer G. Science. 1998; 281: 2027-2031Crossref PubMed Scopus (1058) Google Scholar, 20Shimizu S. Matsuoka Y. Shinohara Y. Moneda Y. Tsujimoto Y. J. Cell Biol. 2001; 152: 237-250Crossref PubMed Scopus (332) Google Scholar, 21Guo B. Zhai D. Cabezas E. Welsh K. Nouraini S. Satterthwait A.C. Reed J.C. Nature. 2003; 423: 456-461Crossref PubMed Scopus (481) Google Scholar, 22Sawada M. Sun W. Hayes P. Leskov K. Boothman D.A. Matsuyama S. Nat. Cell Biol. 2003; 5: 320-329Crossref PubMed Scopus (318) Google Scholar, 23Nomura M. Shimizu S. Sugiyama T. Narita M. Ito T. Matsuda H. Tsujimoto Y. J. Biol. Chem. 2003; 278: 2058-2065Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 24Tsuruta F. Sunayama J. Mori Y. Hattori S. Shimizu S. Tsujimoto Y. Yoshioka K. Masuyama N. Gotoh Y. EMBO J. 2004; 23: 1889-1899Crossref PubMed Scopus (454) Google Scholar, 25Kirchhoff S.R. Gupta S. Knowlton A.A. Circulation. 2002; 105: 2899-2904Crossref PubMed Scopus (250) Google Scholar, 26Chua B.T. Volbracht C. Tan K.O. Li R. Yu V.C. Li P. Nat. Cell Biol. 2003; 5: 1083-1089Crossref PubMed Scopus (256) Google Scholar, 27Nijhawan D. Fang M. Traer E. Zhong Q. Gao W. Du F. Wang X. Genes Dev. 2003; 17: 1475-1486Crossref PubMed Scopus (520) Google Scholar, 28McJilton M.A. Van Sikes C. Wescott G.G. Wu D. Foreman T.L. Gregory C.W. Weidner D.A. Harris-Ford O. Morgan Lasater A. Mohler J.L. Terrian D.M. Oncogene. 2003; 22: 7958-7968Crossref PubMed Scopus (99) Google Scholar, 29Ohtsuka T. Ryu H. Minamishima Y.A. Macip S. Sagara J. Nakayama K.I. Aaronson S.A. Lee S.W. Nat. Cell Biol. 2004; 6: 121-128Crossref PubMed Scopus (206) Google Scholar), it is largely unknown how Bax is activated and then translocated to mitochondria to initiate apoptosis after exposure to cell death stimulants. The aim of this study was to investigate the mechanism of activation and mitochondrial translocation of pro-apoptotic Bax to initiate apoptosis. Our results provide a novel mechanism by which JNK- and p38 mitogen-activated protein kinase (p38 kinase)-mediated phosphorylation of Bax leads to its activation prior to mitochondrial translocation and induction of apoptosis upon exposure to cell death stimulants such as STS and H2O2. Materials—Mouse anti-Bax monoclonal antibody (mAb) B9, anti-Hsp60 mAb, rabbit polyclonal antibody specific to MAPKK3/6 (catalog no. sc-13069), major upstream kinases of p38 kinase, and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-Bax mAbs 6A7 and 2D2 (which recognize activated and total (native + activated) Bax, respectively), CHAPS, STS, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI), Me2SO, Hoechst 33342, and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt (XTT; catalog no. Tox-2) were obtained from Sigma. Antibody to cytochrome c was from BD Biosciences. A specific small interfering RNA (siRNA) to MAPKK4 (SEK1; catalog no. 51333), the upstream protein kinase of JNK and p38 kinase (30Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5045) Google Scholar), and a nonspecific negative control No. 1 siRNA (catalog no. 4635) were obtained from Ambion, Inc. (Austin, TX). Other materials not listed here were as described previously (31Suh S. Hood B.L. Kim B.-J. Conrads T.P. Veenstra T.D. Song B.-J. Proteomics. 2004; 4: 3401-3412Crossref PubMed Scopus (69) Google Scholar, 32Soh Y. Jeong K.S. Lee I.J. Bae M.A. Kim Y.C. Song B.-J. Mol. Pharmacol. 2000; 58: 535-541Crossref PubMed Scopus (115) Google Scholar, 33Bae M.A. Pie J.E. Song B.-J. Mol. Pharmacol. 2001; 60: 847-856PubMed Google Scholar, 34Bae M.A. Song B.-J. Mol. Pharmacol. 2003; 63: 401-408Crossref PubMed Scopus (121) Google Scholar). Cell Culture, Treatments, and Measurement of Cell Viability— Human hepatoma HepG2 cells and porcine kidney LLC-PK1 cells (purchased from American Type Culture Collection, Manassas, VA) were maintained in minimal essential medium with Earle's salts, 10% (v/v) heat-inactivated fetal bovine serum, 2 mm glutamine, and antibiotics as described (31Suh S. Hood B.L. Kim B.-J. Conrads T.P. Veenstra T.D. Song B.-J. Proteomics. 2004; 4: 3401-3412Crossref PubMed Scopus (69) Google Scholar, 32Soh Y. Jeong K.S. Lee I.J. Bae M.A. Kim Y.C. Song B.-J. Mol. Pharmacol. 2000; 58: 535-541Crossref PubMed Scopus (115) Google Scholar, 33Bae M.A. Pie J.E. Song B.-J. Mol. Pharmacol. 2001; 60: 847-856PubMed Google Scholar, 34Bae M.A. Song B.-J. Mol. Pharmacol. 2003; 63: 401-408Crossref PubMed Scopus (121) Google Scholar). LLC-PK1 or HepG2 cells (grown in 96-well plates at 2 × 104 cells/well for 1 day) were incubated with 1 μm or 2 μm STS, respectively, in minimal essential medium with Earle's salts and 1% fetal bovine serum for the indicated times. Cell viability was determined by XTT reduction. Alternatively, cell death rates were determined by staining with DAPI or Hoechst 33342 (32Soh Y. Jeong K.S. Lee I.J. Bae M.A. Kim Y.C. Song B.-J. Mol. Pharmacol. 2000; 58: 535-541Crossref PubMed Scopus (115) Google Scholar, 33Bae M.A. Pie J.E. Song B.-J. Mol. Pharmacol. 2001; 60: 847-856PubMed Google Scholar, 34Bae M.A. Song B.-J. Mol. Pharmacol. 2003; 63: 401-408Crossref PubMed Scopus (121) Google Scholar). More than 300 cells in three different areas for each sample were counted by fluorescence microscopy. Data are presented as the means ± S.D. of four measurements for each group, repeated three times (unless indicated otherwise). Another batch of HepG2 cells was exposed to UV light at 8 or 16 J/m2 using a Stratalinker 1800 UV cross-linker (Stratagene) and incubated for an additional 24 or 48 h before cell harvest. Transient transfection of HepG2 cells with each siRNA (50 nm) was performed as described previously (33Bae M.A. Pie J.E. Song B.-J. Mol. Pharmacol. 2001; 60: 847-856PubMed Google Scholar, 34Bae M.A. Song B.-J. Mol. Pharmacol. 2003; 63: 401-408Crossref PubMed Scopus (121) Google Scholar). Immunoprecipitation and Two-dimensional Gel Analysis— Freshly harvested HepG2 and LLC-PK1 cells were homogenized with hypotonic buffer (50 mm Tris-HCl (pH 7.4), 1 mm NaF, 100 μm sodium orthovanadate, and protease inhibitor mixture) with 1% CHAPS. Anti-Bax antibody B9 or 2D2 was used to immunoprecipitate Bax. CHAPS-solubilized proteins (1 mg/sample) were initially incubated with 50 μl of protein G-agarose (50% suspension) to remove nonspecific binding proteins by following a previously published method (21Guo B. Zhai D. Cabezas E. Welsh K. Nouraini S. Satterthwait A.C. Reed J.C. Nature. 2003; 423: 456-461Crossref PubMed Scopus (481) Google Scholar). The remaining proteins were then incubated with 3 μg of anti-Bax mAb B9 or 2D2 for 2 h with constant agitation. Protein G-agarose was added to each tube and further incubated for an additional 1 h to facilitate immunoprecipitation. Proteins bound to protein G-agarose were washed three times with 1× phosphate-buffered saline and 1% CHAPS, which does not cause conformational change (4Hsu Y.-T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1032) Google Scholar), to remove nonspecifically bound proteins. After the final centrifugation, the proteins bound to anti-Bax antibodies and agarose beads were dissolved in two-dimensional gel buffer (8 m urea, 50 mm dithiothreitol, 2% CHAPS, and 0.5% immobilized pH gradient buffer (pH 3-10)) 30 min before isoelectrofocusing on dry immobilized pH gradient strips (nonlinear gradient of pH 3-10) as described (31Suh S. Hood B.L. Kim B.-J. Conrads T.P. Veenstra T.D. Song B.-J. Proteomics. 2004; 4: 3401-3412Crossref PubMed Scopus (69) Google Scholar) and subjected to immunoblot analysis. Preparation of Subcellular Fractions and Immunoblot Analysis—Freshly harvested HepG2 or LLC-PK1 cells were used to determine the relative distribution of Bax or cytochrome c in the cytosol and mitochondria prepared by differential centrifugation (31Suh S. Hood B.L. Kim B.-J. Conrads T.P. Veenstra T.D. Song B.-J. Proteomics. 2004; 4: 3401-3412Crossref PubMed Scopus (69) Google Scholar). Cells pretreated with STS or H2O2 (0.3 mm) were washed twice with 1× phosphate-buffered saline, resuspended in isotonic STE buffer (0.25 m sucrose, 50 mm Tris-HCl (pH 7.4), 1 mm EDTA, 1 mm NaF, 0.1 mm sodium orthovanadate, and protease inhibitor mixture) on ice for 10 min, and homogenized, and cell debris and nuclear fractions were removed by centrifugation at 700 × g for 5 min. The supernatant fraction was then subjected to centrifugation at 16,100 × g for 20 min to prepare the cytosolic and mitochondrial fractions. The mitochondrial pellets were washed once with STE buffer to minimize contamination of the cytosolic proteins. After centrifugation of the washed pellets, the mitochondrial pellets were dissolved in hypotonic buffer (50 mm Tris-HCl, pH 7.4) containing 1.0% CHAPS. Cytosolic and solubilized mitochondrial proteins were separated on SDS-polyacrylamide gels, transferred to Immobilon polyvinylidene difluoride (PVDF) membranes, and then incubated with specific antibody to each target protein. The antigen detected by the primary antibody was visualized with the appropriate secondary antibody conjugated to horseradish peroxidase for enhanced chemiluminescence detection (31Suh S. Hood B.L. Kim B.-J. Conrads T.P. Veenstra T.D. Song B.-J. Proteomics. 2004; 4: 3401-3412Crossref PubMed Scopus (69) Google Scholar, 32Soh Y. Jeong K.S. Lee I.J. Bae M.A. Kim Y.C. Song B.-J. Mol. Pharmacol. 2000; 58: 535-541Crossref PubMed Scopus (115) Google Scholar, 33Bae M.A. Pie J.E. Song B.-J. Mol. Pharmacol. 2001; 60: 847-856PubMed Google Scholar, 34Bae M.A. Song B.-J. Mol. Pharmacol. 2003; 63: 401-408Crossref PubMed Scopus (121) Google Scholar). Metabolic Labeling and Autoradiography—Intact HepG2 cells grown in culture dishes (150-mm diameter) were metabolically labeled overnight with [32P]orthophosphoric acid (1 mCi/culture dish; specific activity of >9000 Ci/mmol; PerkinElmer Life Sciences) in phosphate-free Dulbecco's modified Eagle's medium (catalog no. 11971, Invitrogen) with 5% fetal bovine serum and antibiotics. HepG2 cells were then treated with STS (2 μm) for an additional 8 h before cell harvest and quick freezing in dry ice. Frozen cells were extracted with hypotonic buffer with 1% CHAPS. CHAPS-solubilized proteins (1 mg of protein each) were subjected to immunoprecipitation with anti-Bax mAb B9 or 2D2 for further two-dimensional gel analyses, transfer to Immobilon PVDF membranes, and autoradiography. Site-directed Mutagenesis and Confocal Microscopic Analysis—Based on the structural prediction of phosphorylation sites in Bax, we prepared various Bax mutants using the QuikChange® site-directed mutagenesis kit (Stratagene) following the manufacturer's direction. In addition, some Bax mutants were prepared on a contract basis (GenScript Corp., Piscataway, NJ). The correct nucleotide sequence of each Bax mutant was confirmed by DNA sequencing (data not shown) prior to further analysis. Green fluorescent protein (GFP)-fused wild-type Bax (5Wolter K.G. Hsu Y.-T. Smith C.L. Nechushtan A. Xi X.-G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1578) Google Scholar) was kindly provided by Dr. Richard J. Youle (National Institutes of Health, Bethesda, MD) and used as a positive control. The plasmid for GFP-P168A Bax (35Schinzel A. Kaufmann T. Schuler M. Martinalbo J. Grubb D. Borner C. J. Cell Biol. 2004; 164: 1021-1032Crossref PubMed Scopus (127) Google Scholar) was kindly provided by Dr. Christoph Borner (Institute for Molecular Medicine and Cell Research, Freiburg, Germany) and used as a negative control. Mouse embryonic fibroblast (MEF) cells from Bax/Bak double knock-out mice (36Zong W.-X. Lindsten T. Ross A.J. MacGregor G.R. Thompson C.B. Genes Dev. 2001; 15: 1481-1486Crossref PubMed Scopus (715) Google Scholar), kindly provided by Dr. Craig B. Thompson (University of Pennsylvania, Philadelphia, PA), were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and transfected with each mutant cDNA. Confocal microscopy of transfected MEF cells with various Bax mutants was performed before and after treatment with STS for 2 or 4 h by the method as described (5Wolter K.G. Hsu Y.-T. Smith C.L. Nechushtan A. Xi X.-G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1578) Google Scholar). Statistical Analysis—Most of the experimental data shown were repeated more than three times, unless indicated otherwise. Statistical analysis was performed by Student's t test, and p < 0.05 was considered significant. Mitochondrial Translocation of Bax and Apoptosis upon Exposure to STS—We treated human hepatoma HepG2 or porcine kidney LLC-PK1 cells with STS as a cell death stimulant to study the mechanism for translocation of Bax to mitochondria and apoptosis. Consistent with the previous reports (4Hsu Y.-T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3668-3672Crossref PubMed Scopus (1032) Google Scholar, 5Wolter K.G. Hsu Y.-T. Smith C.L. Nechushtan A. Xi X.-G. Youle R.J. J. Cell Biol. 1997; 139: 1281-1292Crossref PubMed Scopus (1578) Google Scholar, 6Wei M.C. Zong W.-X. Cheng E.H.-Y. Lindsten T. Panoutsakopoulou V. Ross A.J. Roth K.A. MacGregor G.R. Thompson C.B. Korsmeyer S.J. Science. 2001; 292: 727-730Crossref PubMed Scopus (3373) Google Scholar), our results showed that STS caused apoptosis of both HepG2 and LLC-PK1 cells. The morphology of STS-treated HepG2 and LLC-PK1 cells showed cell shrinkage, rounding, and partial detachment, with the lobulated appearance of apoptotic cells and condensed DNA stained with DAPI dye (data not shown). STS significantly increased the rates of cell death determined at 24 h in both cells (Fig. 1A). Translocation of Bax to mitochondria (detected by anti-Bax mAb 2D2) and release of mitochondrial cytochrome c to the cytoplasm started to take place at 8 h after STS exposure, whereas cytosolic Bax detected by mAb 2D2 began to decline (Fig. 1, B and C). Increased translocation of Bax to mitochondria and release of mitochondrial cytochrome c were evident in HepG2 cells at 16 h, whereas the levels of actin and Hsp60 (used as loading controls for the cytoplasm and mitochondria, respectively) were comparable in all samples analyzed. We also observed similar patterns of mitochondrial translocation of Bax and cytochrome c release from mitochondria to the cytoplasm after HepG2 and LLC-PK1 cells were treated with H2O2 for different times (Fig. 2).FIGURE 2Increased translocation of activated Bax to mitochondria after exposure to H2O2. HepG2 (A) and LLC-PK1 (B) cells (grown in culture dishes 150 mm in diameter) were exposed to 0.3 and 0.1 mm H2O2, respectively, for the indicated times before cell harvest. Equal amounts of cytosolic and mitochondrial proteins (20 μg/well) isolated from HepG2 and LLC-PK1 cells were separated on 12% SDS-polyacrylamide gels and then subjected to immunoblot (IB) analysis for various target proteins. Cyto C, cytochrome c.View Large Image Figure ViewerDownload Hi-res image Download (PPT) JNK- or p38 Kinase-dependent Bax Translocation and Cytochrome c Release after STS Treatment—Because of the well established role of JNK and p38 kinase in apoptosis caused by various cell death stimuli (32Soh Y. Jeong K.S. Lee I.J. Bae M.A. Kim Y.C. Song B.-J. Mol. Pharmacol. 2000; 58: 535-541Crossref PubMed Scopus (115) Google Scholar, 33Bae M.A. Pie J.E. Song B.-J. Mol. Pharmacol. 2001; 60: 847-856PubMed Google Scholar, 34Bae M.A. Song B.-J. Mol. Pharmacol. 2003; 63: 401-408Crossref PubMed Scopus (121) Google Scholar, 37Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2037) Google Scholar, 38Lei K. Nimnual A. Zong W.-X. Kennedy N.J. Flavell R.A. Thompson C.B. Bar-Sagi D. Davis R.J. Mol. Cell. Biol. 2002; 22: 4929-4942Crossref PubMed Scopus (443) Google Scholar, 39Kamata H. Honda S. Maeda S. 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We used SP600125 (1,9-pyrazoloanthrone), a specific inhibitor of JNK, and SB203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole), a selective inhibitor of p38 kinase, to effectively inhibit the activation of JNK and p38 kinase, respectively. When these inhibitors were used at 5 or 10 μm, we observed only partial inhibition of the respective kinase in HepG2 cells. However, nearly complete inhibition of each kinase was observed in the presence of the respective inhibitor at 20 μm or above. Therefore, in subsequent studies, we used inhibitors at 20 μm. This concentration is similar to or less than that used by other investigators (40Ghatan S. Larner S. Kinoshita Y. Hetman M. Patel L. Xia Z. Youle R.J. Morrison R.S. J. Cell Biol. 2000; 150: 335-347Crossref PubMed Scopus (364) Google Scholar, 42Nadkarni V. Gabbay K.H. Bohren K.M. Sheikh-Hamad D. J. Biol. Chem. 1999; 274: 20185-20190Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 43Li D. 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