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

2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês

10.1074/jbc.m207951200

ISSN

1083-351X

Autores

Seiji Inoshita, Kohsuke Takeda, Takiko Hatai, Yoshio Terada, Makoto Sano, Junichi Hata, Akihiro Umezawa, Hidenori Ichijo,

Tópico(s)

NF-κB Signaling Pathways

Resumo

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 1). The mobility of both bands was delayed by H2O2 treatment in a time-dependent manner (Fig. 1 A, top, lanes 2–6). The treatment of cell lysates prepared from H2O2-stimulated (Fig. 1 A,top, lane 7) and unstimulated (data not shown) PAE cells with λ-protein phosphatase resulted in the acceleration of the mobility of those bands. These findings suggest that endogenous Mcl-1 is partially phosphorylated under non-stressed conditions and that additional phosphorylation occurred after H2O2 treatment. To identify the kinase responsible for Mcl-1 phosphorylation in response to H2O2, we examined the activation state of three classes of MAPK, namely, ERK, JNK, and p38, which are all 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, 31Guyton K.Z. Liu Y. Gorospe M., Xu, Q. Holbrook N.J. J. Biol. Chem. 1996; 271: 4138-4142Abstract Full Text Full Text PDF PubMed Scopus (1134) Google Scholar). The kinetics of activation of JNK correlated with the extent of mobility of Mcl-1 (Fig. 1 A), suggesting that JNK might be involved in the H2O2-induced phosphorylation of Mcl-1. To examine which MAPK can phosphorylate Mcl-1 directly, we carried out an in vitro kinase assay using recombinant Mcl-1 as the substrate. JNK and p38 strongly phosphorylated Mcl-1in vitro, whereas the phosphorylation of Mcl-1 by ERK was marginal (Fig. 1 B). The co-transfection of ASK1, a MAPKKK that activates JNK and that p38 MAPK cascades in vivo, strongly enhanced the phosphorylation of Mcl-1 by JNK and p38 (Fig.1 C, top, lanes 1–5). ASK1 itself phosphorylated Mcl-1 very weakly (Fig. 1 C, top,lane 6). These findings suggested that Mcl-1 may serve as a specific substrate for JNK and p38 at least in vitro. We next examined whether Mcl-1 could be phosphorylated by JNK and p38 in mammalian cells. When Mcl-1 was co-transfected with JNK or p38 alone, the phosphorylation of Mcl-1 was undetectable as determined by the band shift analysis (Fig. 1 D, lanes 2 and4). In contrast, Mcl-1 became a shifted doublet by the co-expression of the activated allele of ASK1 (ASK1ΔN) together with JNK or p38 (Fig. 1 D, lanes 3 and 5). Phosphatase treatment shifted the doublet down to the basal status (Fig. 1 D, lanes 6–10), indicating that activated JNK and p38 could phosphorylate Mcl-1 in vivo. A sequence comparison of human and mouse Mcl-1 revealed that Mcl-1 possesses two conserved sites, Ser-121 and Thr-163, in humans that conforms to the consensus motif for the substrate of JNK and p38 (Fig.2 A). These sites are located in the PEST (proline, glutamic acid, serine, and threonine) domain of Mcl-1 (Fig. 2 A) and correspond to the so-called unstructural loop region in Bcl-2, which regulates the anti-apoptotic function of Bcl-2 (32Chang B.S. Minn A.J. Muchmore S.W. Fesik S.W. Thompson C.B. EMBO J. 1997; 16: 968-977Crossref PubMed Scopus (263) Google Scholar). To examine which sites are phosphorylated by JNK or p38, we constructed three alanine substitution mutants of Mcl-1 (S121A, T163A, and S121A/T163A). The Myc-tagged wild-type (WT) or alanine-substituted mutant of Mcl-1 was co-transfected with JNK or p38 plus ASK1ΔN. Cells were metabolically labeled with [32P]orthophosphate and analyzed by autoradiography after immunoprecipitation using anti-Myc antibody. WT and single alanine substitution mutants (S121A and T163A) of Mcl-1 were clearly phosphorylated by the co-expression of activated JNK and p38 (Fig.2 B, top, lanes 5–7 and9–11). In contrast, little phosphorylation was detected in the double-alanine mutant (S121A/T163A) of Mcl-1 (Fig. 2 B,top, lanes 8 and 12). These findings suggested that when overexpressed, activated JNK and p38 can phosphorylate both Ser-121 and Thr-163 of Mcl-1 and that these two amino acids are the major phosphorylation sites of Mcl-1 in vivo. To investigate the involvement of Ser-121 and Thr-163 in oxidative stress-induced phosphorylation of Mcl-1 as observed in Fig.1 A, we generated PAE cell clones stably expressing Myc-tagged WT and S121A/T163A mutant of Mcl-1. When these cells were treated with H2O2, the activations of endogenous JNK and p38 were clearly observed in both cells in a time-dependent manner (Fig. 2 C,middle and bottom panels). In parallel with JNK activation, gel mobility of Mcl-1 was retarded in WT but not in S121A/T163A mutant-expressing cells (Fig. 2 C,top), and the retardation was canceled by treatment with λ-protein phosphatase (Fig. 2 C, lane 7). We have examined three independently selected clones of WT and mutant Mcl-1 and obtained essentially the same results in independent clones (data not shown). These results suggest that both Ser-121 and Thr-163 of Mcl-1 are phosphorylated in response to oxidative stress. The overexpression of either activated JNK or p38 phosphorylated Mcl-1in vivo (Figs. 1 D and 2 B), and both kinases were activated by H2O2 treatment (Figs.1 A and 2 C). However, time course analysis indicated that the activation of JNK coincided with Mcl-1 phosphorylation following H2O2 stimulation much better than that of p38 (Figs. 1 A and 2 C). To examine which signaling pathway is physiologically required for Mcl-1 phosphorylation in response to H2O2, we used the p38 inhibitor SB203580 and a recombinant adenovirus encoding dominant negative MKK4. Although p38 was specifically inactivated by SB203580 (data not shown), the treatment of PAE cells with SB203580 before H2O2 stimulation did not alter H2O2-induced Mcl-1 mobility shift (Fig.3 A). In contrast, the expression of the dominant negative MKK4 significantly reduced the gel mobility shift of Mcl-1 upon H2O2 treatment (Fig. 3 B). JNK but not p38 activation was specifically reduced by adenovirus encoding dominant negative MKK4. Taken together, Mcl-1 appears to be phosphorylated mainly via the JNK pathway in response to oxidative stress. Finally, we assessed the functional importance of Mcl-1 phosphorylation in oxidative stress-induced apoptosis. To this end, the susceptibility to H2O2-induced apoptosis was examined in PAE clones stably expressing WT and those expressing S121A/T163A Mcl-1. PAE clones were treated with 0.5 mmH2O2 for 3 h, and cell death was determined by the trypan blue exclusion assay (Fig.4). WT Mcl-1 conferred only minimal resistance compared with the vector control. However, S121A/T163A Mcl-1 showed substantially stronger anti-apoptotic activity than WT Mcl-1 following H2O2 treatment. We have examined three independently selected clones of WT and mutant Mcl-1 and obtained essentially the same results in independent clones (data not shown). These data indicated that eliminating phosphorylation sites increased anti-apoptotic activity of Mcl-1. In other words, Mcl-1 appears to be negatively regulated through phosphorylation of Ser-121 and Thr-163 by JNK following H2O2 stimulation. In this study, we demonstrated that Mcl-1 was phosphorylated at Ser-121 and Thr-163 through the JNK pathway and inactivated following H2O2 treatment. We also demonstrated that both JNK and p38 could phosphorylate Mcl-1 in vitro, whereas ERK induced little phosphorylation. A recent study also suggested using an ERK inhibitor that ERK was involved in 12-O-tetradecanoylphorbol-13-acetate-induced Mcl-1 phosphorylation (33Domina A.M. Smith J.H. Craig R.W. J. Biol. Chem. 2000; 275: 21688-21694Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Further investigations will be needed to determine whether the ERK pathway is also involved in Mcl-1 phosphorylation depending on stimuli. We identified two phosphorylation sites, which regulate the anti-apoptotic function of Mcl-1 in response to H2O2. Although human Mcl-1 possesses five potential phosphorylation sites that can be phosphorylated by JNK and p38, we could not detect any phosphorylation in the S121A/T163A mutant, suggesting that Ser-121 and Thr-163 are the main sites to undergo H2O2-induced phosphorylation. Bcl-2 has been shown to be phosphorylated by JNK in at least four serine/threonine sites. Especially, Ser-70 and Ser-87 play an important role in negatively regulating the anti-apoptotic function 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). Recent work suggested that the number of phosphorylated sites of Bcl-2 appeared to depend on the intensity of kinase activation. We detected another shift of gel mobility of the S121A/T163A mutant of Mcl-1 when it was co-expressed with JNK and ASK1ΔN in 293 cells (data not shown). It remains to be determined whether other phosphorylation sites contribute to the regulation of the anti-apoptotic activity of Mcl-1. The mechanisms by which phosphorylation of Bcl-2 regulates anti-apoptotic function are poorly understood. Several studies have shown that phosphorylated Bcl-2 does not heterodimerize with Bax, and thus, apoptosis is promoted by an increase in the amount of free Bax (34Basu A. Haldar S. Int. J. Oncol. 1998; 13: 659-664PubMed Google Scholar, 35Haldar S. Basu A. Croce C.M. Cancer Res. 1998; 58: 1609-1615PubMed Google Scholar). We could not detect any changes in the interaction of Bax and Mcl-1 before or after the phosphorylation of Mcl-1 (data not shown). However, in the phosphorylation sites of Mcl-1 located in the PEST motif, there was no difference in the half-life of WT and S121A/T163A mutant of Mcl-1 after H2O2stimulation (data not shown). Further studies will be needed to elucidate the mechanism of phosphorylation-mediated inactivation of Mcl-1. The JNK signaling pathway is essential for exocytotoxic stress-induced apoptosis in neurons and UV-induced apoptosis in mouse embryonic fibroblast (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, 36Yang D.D. Kuan C.Y. Whitmarsh A.J. Rincon M. Zheng T.S. Davis R.J. Rakic P. Flavell R.A. Nature. 1997; 389: 865-870Crossref PubMed Scopus (1109) Google Scholar). It seems that activated JNK acts on mitochondria and induces apoptosis through the release of cytochrome c(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, 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). The mechanism of cytochrome c release by JNK is not known at all. Although the Bcl-2 family is a potential target of JNK that regulates cytochrome c, several discrepancies have been pointed out. For example, Bcl-2 phosphorylation has been suggested to increase rather than decrease anti-apoptotic function (37Ito T. Deng X. Carr B. May W.S. J. Biol. Chem. 1997; 272: 11671-11673Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). Stimuli that cause JNK-induced apoptosis such as UV do not necessarily cause 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 our study, Mcl-1 was phosphorylated by JNK and its anti-apoptotic function decreased. Phosphorylation and inactivation of Mcl-1 thus may be one of the mechanisms by which JNK induces apoptosis in response to oxidative stress. We thank Y. Tsujimoto and S. Shimizu for valuable comments. We are grateful to H. Okita for providing plasmids and antibodies. We also thank all the members of Cell Signaling Laboratory for critical comments.

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