Phosphorylation and Inactivation of Myeloid Cell Leukemia 1 by JNK in Response to Oxidative Stress

Abstract

Oxidative stress induces JNK activation, which leads to apoptosis through mitochondria-dependent caspase activation. However, little is known about the mechanism by which JNK alters mitochondrial function. In this study, we investigated the role of phosphorylation of myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the Bcl-2 family, in oxidative stress-induced apoptosis. We found that JNK phosphorylated Ser-121 and Thr-163 of Mcl-1 in response to stimulation with H2O2 and that transfection of unphosphorylatable Mcl-1 resulted in an enhanced anti-apoptotic activity in response to stimulation with H2O2. JNK-dependent phosphorylation and thus inactivation of Mcl-1 may be one of the mechanisms through which oxidative stress induces cellular damage. Oxidative stress induces JNK activation, which leads to apoptosis through mitochondria-dependent caspase activation. However, little is known about the mechanism by which JNK alters mitochondrial function. In this study, we investigated the role of phosphorylation of myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the Bcl-2 family, in oxidative stress-induced apoptosis. We found that JNK phosphorylated Ser-121 and Thr-163 of Mcl-1 in response to stimulation with H2O2 and that transfection of unphosphorylatable Mcl-1 resulted in an enhanced anti-apoptotic activity in response to stimulation with H2O2. JNK-dependent phosphorylation and thus inactivation of Mcl-1 may be one of the mechanisms through which oxidative stress induces cellular damage. Oxidative stress has been implicated in the pathogenesis of several abnormal conditions and diseases including ischemia, cancer, and diabetes mellitus (1Carden D.L. Granger D.N. J. Pathol. 2000; 190: 255-266Crossref PubMed Scopus (1374) Google Scholar, 2Muller I. Jenner A. Bruchelt G. Niethammer D. Halliwell B. Biochem. Biophys. Res. Commun. 1997; 230: 254-257Crossref PubMed Scopus (119) Google Scholar, 3Guigliano O. Ceriello A. Diabetes Care. 1996; 19: 257-267Crossref PubMed Scopus (1677) Google Scholar). A recent study suggests that stress-activated protein kinases such as JNK 1The abbreviations used are: JNK, c-Jun NH2-terminal kinase; ASK, apoptosis signal-regulating kinase; MAPK, mitogen-activated protein kinase; MAPKKK, MAPK kinase kinase; Mcl-1, myeloid cell leukemia 1; PAE, porcine aortic endothelial; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; DTT, dithiothreitol; GST, glutathioneS-transferase; WT, wild type 1The abbreviations used are: JNK, c-Jun NH2-terminal kinase; ASK, apoptosis signal-regulating kinase; MAPK, mitogen-activated protein kinase; MAPKKK, MAPK kinase kinase; Mcl-1, myeloid cell leukemia 1; PAE, porcine aortic endothelial; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; DTT, dithiothreitol; GST, glutathioneS-transferase; WT, wild typeand p38 play important roles in triggering apoptosis in response to various cellular stressors including oxidative stress. We have shown that oxidative stress-induced sustained activation of JNK and p38 is required for apoptosis (4Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO J. 2001; 2: 222-228Crossref Scopus (983) Google Scholar). Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK), specifically mediates the sustained activation of JNK/p38 and apoptosis in response to oxidative stress (5Ichijo 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 (1989) Google Scholar, 6Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2045) Google Scholar). ASK1-dependent apoptosis is mediated by the release of cytochrome c from the mitochondria followed by caspase 9 activation (7Hatai T. Matsuzawa A. Inoshita S. Mochida Y. Kuroda T. Sakamaki K. Kuida K. Yonehara S. Ichijo H. Takeda K. J. Biol. Chem. 2000; 275: 26576-26581Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). It has also been reported that JNK is required for UV-induced release of cytochromec and that new gene expression is not required for this process (8Tournier C. Hess P. Yang D.D., Xu, J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1532) Google Scholar). These reports indicate that JNK induces apoptosis in part through the mitochondria-dependent caspase activation. However, the molecular mechanism by which activated JNK induces mitochondrial dysfunction is unclear. The members of the Bcl-2 family play pivotal roles in cellular decision to undergo apoptosis. Bcl-2 has been reported to be phosphorylated by JNK in response to different stimuli (9Yamamoto K. Ichijo H. Korsmeyer S.J. Mol. Cell. Biol. 1999; 19: 8469-8478Crossref PubMed Scopus (905) Google Scholar, 10Maundrell K. Antonsson B. Magnenat E. Camps M. Muda M. Chabert C. Gillieron C. Boschert U. Vial-Knecht E. Martinou J.C. Arkinstall S. J. Biol. Chem. 1997; 272: 25238-25242Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 11Thomas A. Giesler T. White E. Oncogene. 2000; 19: 5259-5269Crossref PubMed Scopus (91) Google Scholar). Although the significance of phosphorylation of Bcl-2 is controversial, it was suggested that phosphorylation by JNK within the unstructured loop region of Bcl-2 decreases its anti-apoptotic activity (9Yamamoto K. Ichijo H. Korsmeyer S.J. Mol. Cell. Biol. 1999; 19: 8469-8478Crossref PubMed Scopus (905) Google Scholar, 10Maundrell K. Antonsson B. Magnenat E. Camps M. Muda M. Chabert C. Gillieron C. Boschert U. Vial-Knecht E. Martinou J.C. Arkinstall S. J. Biol. Chem. 1997; 272: 25238-25242Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 12Ojala P.M. Yamamoto K. Castanos-Velez E. Biberfeld P. Korsmeyer S.J. Makela T.P. Nat. Cell Biol. 2000; 2: 819-825Crossref PubMed Scopus (142) Google Scholar). Anti-apoptotic Bcl-2 family proteins thus may be potential mediators of JNK-induced apoptosis. However, little is known about the relation between JNK and the other anti-apoptotic members of the Bcl-2 family in the context of oxidative stress-induced apoptosis signaling. The myeloid cell leukemia 1 (Mcl-1) (13Kozopas K.M. Yang T. Buchan H.L. Zhou P. Craig R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3516-3520Crossref PubMed Scopus (871) Google Scholar), also known as EAT (14Umezawa A. Maruyama T. Inazawa J. Imai S. Takano T. Hata J. Cell Struct. Funct. 1996; 21: 143-150Crossref PubMed Scopus (19) Google Scholar), is an anti-apoptotic Bcl-2 family member. Mcl-1 plays an important role in the development of various carcinomas (15Shigemasa K. Katoh O. Shiroyama Y. Mihara S. Mukai K. Nagai N. Ohama K. Jpn. J. Cancer Res. 2002; 93: 542-550Crossref PubMed Scopus (70) Google Scholar, 16Zhang B. Gojo I. Fenton R.G. Blood. 2002; 99: 1885-1893Crossref PubMed Scopus (343) Google Scholar, 17Zhou P. Levy N.B. Xie H. Qian L. Lee C.Y. Gascoyne R.D. Craig R.W. Blood. 2001; 97: 3902-3909Crossref PubMed Scopus (161) Google Scholar). Similar to other Bcl-2 family members, Mcl-1 localizes in the mitochondrion as well as in other intracellular membranes (18Yang T. Kozopas K.M. Craig R.W. J. Cell Biol. 1995; 128: 1173-1184Crossref PubMed Scopus (269) Google Scholar) and can associate with other pro-apoptotic family members (19Bodrug S.E. Aime-Sempe C. Sato T. Krajewski S. Hanada M. Reed J.C. Cell Death Differ. 1995; 2: 173-182PubMed Google Scholar). Mcl-1 differs from Bcl-2 and Bcl-XL in structure (13Kozopas K.M. Yang T. Buchan H.L. Zhou P. Craig R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3516-3520Crossref PubMed Scopus (871) Google Scholar), in its short half-life (13Kozopas K.M. Yang T. Buchan H.L. Zhou P. Craig R.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3516-3520Crossref PubMed Scopus (871) Google Scholar), in the regulation of its promoter (20Townsend K.J. Trusty J.L. Traupman M.A. Eastman A. Craig R.W. Oncogene. 1998; 17: 1223-1234Crossref PubMed Scopus (97) Google Scholar, 21Townsend K.J. Zhou P. Qian L. Bieszczad C.K. Lowrey C.H. Yen A. Craig R.W. J. Biol. Chem. 1999; 274: 1801-1813Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 22Wang J.M. Chao J.R. Cen W. Kuo M.L. Yen J.J. Yang-Yen H.F. Mol. Cell. Biol. 1999; 19: 6195-6206Crossref PubMed Google Scholar), and in its ability to protect cells from a variety of cytotoxic stimuli (23Zhou P. Qian L. Kozopas K.M. Craig R.W. Blood. 1997; 89: 630-643Crossref PubMed Google Scholar, 24Reynolds J.E., Li, J. Craig R.W. Exp. Cell Res. 1996; 225: 430-436Crossref PubMed Scopus (127) Google Scholar). Little is known regarding posttranslational modification and regulation of Mcl-1. In this study, we investigated the potential involvement of phosphorylation regulation of Mcl-1. HEK293 cells were grown under 5% CO2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 4.5 g/liter glucose, and 100 units/ml penicillin. Porcine aortic endothelial (PAE) cells were grown under 5% CO2 in F12 medium (Invitrogen) supplemented with 10% fetal bovine serum, 10 mm HEPES, and 100 units/ml penicillin. Transfection with various constructs in pGEX-Neo was performed using 2 μg of plasmid and 8 μl of Tfx 50 (Promega). Transfected cells were selected in the presence of 1 mg/ml Geneticin for 2 weeks, and drug-resistant single-cell colonies were chosen and maintained in growth medium containing 0.4 mg/ml Geneticin. Rabbit polyclonal antibody to Mcl-1 was purchased from BD Biosciences. Phospho-JNK (Thr-183/Tyr-185) and p38 (Thr-180/Tyr-182) were purchased from New England Biolabs. Phospho-ERK (Thr-183/Tyr-185) was purchased from Promega. The antibodies to Myc tag (clone 9E10), HA tag (clone 3F10), and FLAG tag were purchased from Calbiochem, Roche Molecular Biochemicals, and Sigma, respectively. SB203580 was purchased from Calbiochem. Cells were lysed in a lysis buffer containing 150 mm NaCl, 50 mm Tris-HCl, pH 8.0, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, and 1.5% aprotinin. Cell extracts were clarified by centrifugation and resolved on SDS-PAGE followed by electroblotting onto polyvinylidene difluoride membrane. After blocking with 5% skim milk in Tris-buffered saline with Tween 20 (150 mm NaCl, 50 mm Tris-HCl, pH 8.0, and 0.05% Tween 20), the membranes were probed with antibodies. The antibody-antigen complexes were detected using the ECL system (Amersham Biosciences). A cDNA clone containing the full-length of the Mcl-1 coding region was inserted into pcDNA3.0 vector. To replace Ser-121 and/or Thr-163 with Ala, a PCR-based site-directed mutagenesis method was used. The Myc tag was inserted at the NH2 termini of wild type and mutant Mcl-1. pcDNA3-HA-ERK, pcDNA3-HA-JNK, pcDNA3-HA-p38, pcDNA3-HA-ASK1, pcDNA3-HA-ASK1ΔN, and pcDNA3-FLAG-ASK1 have been described previously (6Saitoh M. Nishitoh H. Fujii M. Takeda K. Tobiume K. Sawada Y. Kawabata M. Miyazono K. Ichijo H. EMBO J. 1998; 17: 2596-2606Crossref PubMed Scopus (2045) Google Scholar, 26Nishitoh H. Saitoh M. Mochida Y. Takeda K. Nakano H. Rothe M. Miyazono K. Ichijo H. Mol. Cell. 1998; 2: 389-395Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar, 27Morita K. Saitoh M. Tobiume K. Matsuura H. Enomoto S. Hideki Nishitoh H. Ichijo H. EMBO J. 2001; 20: 6028-6036Crossref PubMed Scopus (245) Google Scholar, 28Matsuura H. Nishitoh H. Takeda K. Matsuzawa A. Amagasa T. Ito M. Yoshioka K. Ichijo H. J. Biol. Chem. 2002; 277: 40703-40709Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Recombinant adenovirus was constructed as described elsewhere (29Saito I. Oya Y. Yamamoto K. Yuasa T. Shimojo H. J. Virol. 1985; 54: 711-719Crossref PubMed Google Scholar, 30Miyake S. Makimura M. Kanegae Y. Harada S. Sato Y. Takamori K. Tokuda C. Saito I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324Crossref PubMed Scopus (786) Google Scholar). MKK4 cDNA was subcloned in pcDNA3 by PCR. Lys-116 was replaced by Arg using a PCR-based site-directed mutagenesis method. Green fluorescent protein-tagged MKK4 mutant cDNA was subcloned into the SwaI site of pAdex1pCAw cassette cosmid. Each cosmid bearing the expression unit and adenovirus DNA-terminal protein complex was cotransfected into the E1 transcomplementing 293 cell clone. The recombinant adenoviruses generated by homologous recombination were isolated, and high titer stocks of recombinant adenoviruses were grown in 293 cells and purified. Nearly 100% infection of PAE cells by recombinant adenoviruses can be achieved at a m.o.i. of 100 as determined by green fluorescent protein fluorescence (data not shown). A cDNA encoding the human Mcl-1 protein corresponding to amino acids 31–229 was inserted into the pGEX-2T expression vector (Amersham Biosciences). Mcl-1-GST protein was induced in Escherichia coli BL21 cells by adding 0.5 mm isopropyl-β-d-thiogalactopyranoside and purified with glutathione-Sepharose 4B (Amersham Biosciences). The immune complex kinase assay was done as described previously (26Nishitoh H. Saitoh M. Mochida Y. Takeda K. Nakano H. Rothe M. Miyazono K. Ichijo H. Mol. Cell. 1998; 2: 389-395Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). The indicated plasmids were co-transfected into 293 cells by Tfx 50 (Promega). Cells were lysed in a lysis buffer containing 150 mm NaCl, 20 mm Tris-HCl, pH 7.5, 5 mm EGTA, 1% Triton X-100, 1% deoxycholate, 12 mm β-glycerophosphate, 50 mm NaF, 1 mm sodium orthovanadate, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, and 1.5% aprotinin. Cell extracts were clarified by centrifugation, and the supernatants were immunoprecipitated with anti-HA antibody using protein A-Sepharose (Zymed Laboratories Inc.). The beads were washed twice with washing buffer (150 mm NaCl, 20 mmTris-HCl, pH 7.5, 5 mm EGTA, and 1 mm DTT), and then incubated with GST-Mcl-1 as the substrate for 20 min at 30 °C in 30 μl of kinase buffer (20 mm Tris-HCl, pH 8.0, 20 mm MgCl2, and 0.3 μCi of [γ-32P]ATP). The kinase reaction was stopped by adding SDS sample buffer and analyzed by SDS-PAGE and a Fuji BAS2000 Image analyzer. Cells were lysed in a lysis buffer for phosphatase treatment containing 150 mm NaCl, 10 mm Tris-HCl, pH 7.5, 1 mm EDTA, 1% Nonidet P-40, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, and 1.5% aprotinin. Cellular debris was removed by centrifugation. Lysates were incubated with or without 2 units/μl of λ-protein phosphatase (New England Biolabs) according to the instructions provided by the manufacturer. The reaction was terminated by adding SDS sample buffer and boiling for 3 min. Cells were incubated in phosphate-free medium containing 0.1% fetal bovine serum and 10 mm HEPES, pH 7.0, at 37 °C for 3 h. [32P]Orthophosphate (Amersham Biosciences) was then added at a final concentration of 1 mCi/ml, and labeling was continued at 37 °C for 3 h. The cells were transferred onto ice and washed twice with ice-cold phosphate-buffered saline and then lysed and immunoprecipitated with anti-Myc antibody and analyzed by SDS-PAGE. Cells were stimulated with 0.5 mm H2O2 containing F12 medium for 3 h. Cell viability was measured by the trypan blue (Sigma) dye exclusion method. Cells were trypsinized, centrifuged, resuspended in phosphate-buffered saline, and counted using a hemocytometer after dilution in trypan blue. Blue cells were considered as dead cells. To investigate whether Mcl-1 is regulated by phosphorylation in response to oxidative stress, PAE cells were exposed to H2O2 and the electrophoretic mobility of Mcl-1 was assessed by immunoblotting analysis. We detected endogenous Mcl-1 of PAE cells as a double band under non-stressed conditions (Fig.1 A, top,lane The mobility of was by H2O2 treatment in a 1 A, The treatment of cell from 1 and (data not PAE cells with λ-protein phosphatase resulted in the of the mobility of These that endogenous Mcl-1 is phosphorylated under non-stressed conditions and that phosphorylation after H2O2 To the kinase for Mcl-1 phosphorylation in response to we the activation of of MAPK, ERK, JNK, and which known to be activated by H2O2 (4Tobiume K. Matsuzawa A. Takahashi T. Nishitoh H. Morita K. Takeda K. Minowa O. Miyazono K. Noda T. Ichijo H. EMBO J. 2001; 2: 222-228Crossref Scopus (983) Google Scholar, Y. Xu, J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The of activation of JNK with the of mobility of Mcl-1 1 that JNK be in the phosphorylation of Mcl-1. To which MAPK can Mcl-1 we an in kinase assay using recombinant Mcl-1 as the JNK and p38 phosphorylated the phosphorylation of Mcl-1 by was 1 The of a that JNK and that p38 MAPK in enhanced the phosphorylation of Mcl-1 by JNK and p38 (Fig.1 phosphorylated Mcl-1 1 top,lane These suggested that Mcl-1 may as a substrate for JNK and p38 at in We whether Mcl-1 be phosphorylated by JNK and p38 in cells. Mcl-1 was co-transfected with JNK or p38 the phosphorylation of Mcl-1 was as determined by the band 1 2 In Mcl-1 a by the of the activated of with JNK or p38 1 3 and treatment the to the 1 that activated JNK and p38 Mcl-1 in A of human and Mcl-1 that Mcl-1 Ser-121 and in that to the for the substrate of JNK and p38 These in the and of Mcl-1 2 and to the loop region in which the anti-apoptotic of Bcl-2 EMBO J. 1997; PubMed Scopus Google Scholar). To which phosphorylated by JNK or we constructed of Mcl-1 and The or mutant of Mcl-1 was co-transfected with JNK or p38 Cells were with and analyzed by after using anti-Myc and and of Mcl-1 were phosphorylated by the of activated JNK and p38 In little phosphorylation was detected in the mutant of Mcl-1 2 8 and These suggested that activated JNK and p38 can Ser-121 and Thr-163 of Mcl-1 and that amino acids the phosphorylation of Mcl-1 in To investigate the involvement of Ser-121 and Thr-163 in oxidative stress-induced phosphorylation of Mcl-1 as in A, we generated PAE cell and mutant of Mcl-1. cells were with the of endogenous JNK and p38 were in cells in a 2 and In with JNK activation, mobility of Mcl-1 was in not in cells 2 and the was by treatment with λ-protein phosphatase 2 We have selected of and mutant Mcl-1 and the in (data not shown). These that Ser-121 and Thr-163 of Mcl-1 phosphorylated in response to oxidative stress. The of activated JNK or p38 phosphorylated 1 and 2 and kinases were activated by H2O2 treatment A and 2 However, indicated that the activation of JNK with Mcl-1 phosphorylation H2O2 stimulation that of p38 1 A and 2 To which is required for Mcl-1 phosphorylation in response to we used the p38 SB203580 and a recombinant adenovirus encoding Although p38 was specifically by SB203580 (data not the treatment of PAE cells with SB203580 H2O2 stimulation not Mcl-1 mobility In the expression of the MKK4 the mobility of Mcl-1 H2O2 treatment 3 JNK not p38 activation was specifically by adenovirus encoding Mcl-1 to be phosphorylated the JNK in response to oxidative stress. we assessed the of Mcl-1 phosphorylation in oxidative stress-induced apoptosis. To this the to apoptosis was in PAE and Mcl-1. PAE were with 0.5 for 3 and cell was determined by the trypan blue exclusion assay Mcl-1 with the vector However, Mcl-1 anti-apoptotic activity Mcl-1 H2O2 We have selected of and mutant Mcl-1 and the in (data not shown). These indicated that phosphorylation anti-apoptotic activity of Mcl-1. In other Mcl-1 to be regulated through phosphorylation of Ser-121 and Thr-163 by JNK H2O2 In this study, we that Mcl-1 was phosphorylated at Ser-121 and Thr-163 through the JNK and H2O2 We also that JNK and p38 Mcl-1 in induced little A recent study also suggested using an that was in Mcl-1 phosphorylation Craig R.W. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). be to whether the is also in Mcl-1 phosphorylation on We phosphorylation which the anti-apoptotic of Mcl-1 in response to H2O2. Although human Mcl-1 potential phosphorylation that can be phosphorylated by JNK and we not phosphorylation in the that Ser-121 and Thr-163 the to undergo Bcl-2 has been shown to be phosphorylated by JNK in at and play an important role in the anti-apoptotic of Bcl-2 (9Yamamoto K. Ichijo H. Korsmeyer S.J. Mol. Cell. Biol. 1999; 19: 8469-8478Crossref PubMed Scopus (905) Google Scholar, 11Thomas A. Giesler T. White E. Oncogene. 2000; 19: 5259-5269Crossref PubMed Scopus (91) Google Scholar). suggested that the of phosphorylated of Bcl-2 to on the of kinase activation. We detected of mobility of the mutant of Mcl-1 it was with JNK and in 293 cells (data not shown). It to be determined whether other phosphorylation to the regulation of the anti-apoptotic activity of Mcl-1. The mechanisms by which phosphorylation of Bcl-2 anti-apoptotic have shown that phosphorylated Bcl-2 not with and apoptosis is by an in the of A. S. J. 1998; Google Scholar, S. A. Cancer Res. 1998; Google Scholar). We not in the of and Mcl-1 or after the phosphorylation of Mcl-1 (data not shown). However, in the phosphorylation of Mcl-1 in the was in the half-life of and mutant of Mcl-1 after (data not shown). be to the mechanism of inactivation of Mcl-1. The JNK is for stress-induced apoptosis in and UV-induced apoptosis in (8Tournier C. Hess P. Yang D.D., Xu, J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1532) Google Scholar, C.Y. M. Davis R.J. P. Flavell R.A. 1997; PubMed Scopus Google Scholar). It that activated JNK on mitochondria and induces apoptosis through the release of cytochrome T. Matsuzawa A. Inoshita S. Mochida Y. Kuroda T. Sakamaki K. Kuida K. Yonehara S. Ichijo H. Takeda K. J. Biol. Chem. 2000; 275: 26576-26581Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, C. Hess P. Yang D.D., Xu, J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1532) Google Scholar). The mechanism of cytochrome c release by JNK is not known at Although the Bcl-2 family is a potential of JNK that cytochrome several have been Bcl-2 phosphorylation has been suggested to anti-apoptotic T. B. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). that JNK-induced apoptosis such as not Bcl-2 phosphorylation (8Tournier C. Hess P. Yang D.D., Xu, J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1532) Google Scholar). In study, Mcl-1 was phosphorylated by JNK and its anti-apoptotic and inactivation of Mcl-1 thus may be one of the mechanisms by which JNK induces apoptosis in response to oxidative stress. We Y. and S. for We to H. for plasmids and antibodies. We also the members of Cell for