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JNK-dependent Phosphorylation of c-Jun on Serine 63 Mediates Nitric Oxide-induced Apoptosis of Neuroblastoma Cells

2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês

10.1074/jbc.m310415200

1083-351X

Lei Li, Zhiwei Feng, Alan G. Porter,

Synthesis and Characterization of Heterocyclic Compounds

c-Jun NH2-terminal kinases (JNKs) potentiate transcriptional activity of c-Jun by phosphorylating serines 63 and 73. Moreover, JNK and c-Jun can modulate apoptosis. However, an involvement of nitric oxide (NO)-induced phosphorylation of c-Jun on Ser-63 and Ser-73 in apoptosis has not been explored. We report that in SH-Sy5y neuroblastoma cells, NO induced apoptosis following JNK activation and phosphorylation of c-Jun almost exclusively on Ser-63. Importantly, NO-induced apoptosis and caspase-3 activity were inhibited in cells stably transformed with dominant-negative c-Jun in which Ser-63 is mutated to alanine (S63A), but not in cells transformed with dominant-negative c-Jun (S73A). Ser-63 of c-Jun (but not Ser-73) was required for NO-induced, c-Jun-dependent transcriptional activity. NO-induced apoptosis, Ser-63 phosphorylation of c-Jun, and caspase-3 activity were all inhibited in SH-Sy5y cells transformed with dominant-negative jnk. A caspase-3 inhibitor prevented apoptosis but not c-Jun phosphorylation. In a different neuroblastoma cell line, NO-induced Ser-63 phosphorylation of c-Jun and apoptosis were blocked by a specific JNK inhibitor. We conclude that NO-inducible apoptosis is mediated by JNK-dependent Ser-63 phosphorylation of c-Jun upstream of caspase-3 activation in neuroblastoma cells. c-Jun NH2-terminal kinases (JNKs) potentiate transcriptional activity of c-Jun by phosphorylating serines 63 and 73. Moreover, JNK and c-Jun can modulate apoptosis. However, an involvement of nitric oxide (NO)-induced phosphorylation of c-Jun on Ser-63 and Ser-73 in apoptosis has not been explored. We report that in SH-Sy5y neuroblastoma cells, NO induced apoptosis following JNK activation and phosphorylation of c-Jun almost exclusively on Ser-63. Importantly, NO-induced apoptosis and caspase-3 activity were inhibited in cells stably transformed with dominant-negative c-Jun in which Ser-63 is mutated to alanine (S63A), but not in cells transformed with dominant-negative c-Jun (S73A). Ser-63 of c-Jun (but not Ser-73) was required for NO-induced, c-Jun-dependent transcriptional activity. NO-induced apoptosis, Ser-63 phosphorylation of c-Jun, and caspase-3 activity were all inhibited in SH-Sy5y cells transformed with dominant-negative jnk. A caspase-3 inhibitor prevented apoptosis but not c-Jun phosphorylation. In a different neuroblastoma cell line, NO-induced Ser-63 phosphorylation of c-Jun and apoptosis were blocked by a specific JNK inhibitor. We conclude that NO-inducible apoptosis is mediated by JNK-dependent Ser-63 phosphorylation of c-Jun upstream of caspase-3 activation in neuroblastoma cells. In the central nervous system, NO 1The abbreviations used are: NOnitric oxideCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidDNdominant-negativeJNKc-Jun NH2-terminal kinaseJunAAdominant-negative c-JunMAPmitogen-activated proteinNCAMneural cell adhesion moleculeSNPsodium nitroprussideDEVDAsp-Glu-Val-Asp.1The abbreviations used are: NOnitric oxideCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidDNdominant-negativeJNKc-Jun NH2-terminal kinaseJunAAdominant-negative c-JunMAPmitogen-activated proteinNCAMneural cell adhesion moleculeSNPsodium nitroprussideDEVDAsp-Glu-Val-Asp. generation results in part from successive events of enhanced glutamate release, N-methyl-d-aspartate receptor stimulation, Ca2+ influx, and NO synthase activation (1Ankarcrona M. Dypbukt J.M. Bonfoco E. Zhivotovsky B. Orrenius S. Lipton S.A. Nicotera P. Neuron. 1995; 15: 961-973Abstract Full Text PDF PubMed Scopus (1682) Google Scholar, 2Leist M. Nicotera P. Exp. 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Neurol. 1995; 8: 480-486Crossref PubMed Scopus (104) Google Scholar). Such damage in turn triggers downstream signal transduction pathways, which lead to apoptosis or necrosis (6Kroncke K.D. Fehsel K. Kolb-Bachofen V. Nitric Oxide. 1997; 1: 107-120Crossref PubMed Scopus (469) Google Scholar). However, the death pathways that are activated in neurons in response to massive NO production are not well understood. Because NO can stimulate the activity of the transcription factor AP-1 in neurons (8Bogdan C. Trends Cell Biol. 2001; 11: 66-75Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, 9Haby C. Lisovoski F. Aunis D. Zwiller J. J. Neurochem. 1994; 62: 496-501Crossref PubMed Scopus (147) Google Scholar), one such death pathway might involve the AP-1-dependent regulation of cell death or survival genes.c-Jun, a prominent member of the AP-1 transcriptional factor family, has been implicated in the regulation of a wide range of biological processes including apoptosis, which it can promote or counteract, depending on the tissue, the developmental stage and the nature of the death stimulus (10Leppa S. Bohmann D. Oncogene. 1999; 18: 6158-6162Crossref PubMed Scopus (437) Google Scholar, 11Shaulian E. Karin M. Nat. Cell Biol. 2002; 4: E131-E136Crossref PubMed Scopus (2164) Google Scholar). Its transcriptional activities are regulated by changes in the level of c-jun expression as well as post-translational modifications of the c-Jun protein. In particular, phosphorylation of Ser-63 and Ser-73 in the NH2-terminal transactivation domain of c-Jun, which is mediated primarily by the c-Jun NH2-terminal kinases (JNKs) (12Morton S. Davis R.J. McLaren A. Cohen P. EMBO J. 2003; 22: 3876-3886Crossref PubMed Scopus (228) Google Scholar), substantially enhances the activity of c-Jun as a transcriptional factor (13Pulverer B.J. Kyriakis J.M. Avruch J. Nikolakaki E. Woodgett J.R. Nature. 1991; 353: 670-674Crossref PubMed Scopus (1187) Google Scholar, 14Smeal T. Binetruy B. Mercola D.A. Birrer M. Karin M. Nature. 1991; 354: 494-496Crossref PubMed Scopus (696) Google Scholar). c-Jun NH2-terminal phosphorylation on Ser-63 and Ser-73 can be either pro- or antiapoptotic (15Behrens A. Sibilia M. Wagner E.F. Nat. Genet. 1999; 21: 326-329Crossref PubMed Scopus (594) Google Scholar, 16Wisdom R. Johnson R.S. Moore C. EMBO J. 1999; 18: 188-197Crossref PubMed Scopus (517) Google Scholar). c-Jun phosphorylation is thought to be required for the antiapoptotic function of c-Jun during hepatogenesis (17Ganiatsas S. Kwee L. Fujiwara Y. Perkins A. Ikeda T. Labow M.A. Zon L.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6881-6886Crossref PubMed Scopus (176) Google Scholar). The precise role of c-Jun phosphorylation in genotoxin-induced apoptosis (UV irradiation, DNA-damaging agents) is controversial, but c-Jun phosphorylation is proapoptotic in neurons subjected to kainate, a low potassium concentration, or nerve growth factor deprivation (15Behrens A. Sibilia M. Wagner E.F. Nat. Genet. 1999; 21: 326-329Crossref PubMed Scopus (594) Google Scholar, 18Harada J. Sugimoto M. Jpn. J. Pharmacol. 1999; 79: 369-378Crossref PubMed Scopus (113) Google Scholar, 19Le Niculescu H. Bonfoco E. Kasuya Y. Claret F.X. Green D.R. Karin M. Mol. Cell. Biol. 1999; 19: 751-763Crossref PubMed Scopus (439) Google Scholar, 20Yamagishi S. Yamada M. Ishikawa Y. Matsumoto T. Ikeuchi T. Hatanaka H. J. Biol. Chem. 2001; 276: 5129-5133Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Bim, Hrk, and Fas ligand are among the proteins whose up-regulation upon neurotrophin withdrawal is transcriptionally controlled, at least in part, by c-Jun phosphorylation (19Le Niculescu H. Bonfoco E. Kasuya Y. Claret F.X. Green D.R. Karin M. Mol. Cell. Biol. 1999; 19: 751-763Crossref PubMed Scopus (439) Google Scholar, 21Harris C.A. Johnson Jr., E.M. J. Biol. Chem. 2001; 276: 37754-37760Abstract Full Text Full Text PDF PubMed Google Scholar, 22Whitfield J. Neame S.J. Paquet L. Bernard O. Ham J. Neuron. 2001; 29: 629-643Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar).The mitogen-activated protein (MAP) kinases include the JNKs and the p38 MAP kinases, which are activated by diverse cellular stress including inflammatory cytokines, heat shock, and UV irradiation (10Leppa S. Bohmann D. Oncogene. 1999; 18: 6158-6162Crossref PubMed Scopus (437) Google Scholar, 23Canman C.E. Kastan M.B. Nature. 1996; 384: 213-214Crossref PubMed Scopus (173) Google Scholar, 24Ichijo H. Oncogene. 1999; 18: 6087-6093Crossref PubMed Scopus (472) Google Scholar). JNK and p38 activities have been implicated in cell death associated with glutamate excitotoxicity (25Chung K.C. Shin S.W. Yoo M. Lee M.Y. Lee H.W. Choe B.K. Ahn Y.S. J. Neurochem. 2000; 75: 9-17Crossref PubMed Scopus (20) Google Scholar, 26Kawasaki H. Morooka T. Shimohama S. Kimura J. Hirano T. Gotoh Y. Nishida E. J. Biol. Chem. 1997; 272: 18518-18521Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Two previous reports suggested that NO activates p38 MAP kinase, triggering significant apoptosis in neuronal cells (27Cheng A. Chan S.L. Milhavet O. Wang S. Mattson M.P. J. Biol. Chem. 2001; 276: 43320-43327Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 28Ghatan 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 (362) Google Scholar). JNK-mediated c-Jun phosphorylation is important for apoptosis of starved neuronal cells (19Le Niculescu H. Bonfoco E. Kasuya Y. Claret F.X. Green D.R. Karin M. Mol. Cell. Biol. 1999; 19: 751-763Crossref PubMed Scopus (439) Google Scholar), and the JNK3 isoform is required for kainate-induced cytotoxicity in the central nervous system (29Yang 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 (1112) Google Scholar). Mouse fibroblasts derived from jnk1–/– jnk2–/– double knock-out embryos that lack all JNK activity are less sensitive to apoptosis induced by UV irradiation. The brains of these embryos exhibit altered morphologies because of deregulated apoptosis, which surprisingly is increased in some brain regions but decreased in others (30Kuan C.Y. Yang D.D. Samanta Roy D.R. Davis R.J. Rakic P. Flavell R.A. Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar). Thus, much evidence suggests that c-Jun phosphorylation is often but not always proapoptotic, particularly in neuronal cells.We reported recently that in SH-Sy5y cells, the constitutive activity of c-Jun/AP-1 in the absence of detectable AP-1 DNA binding is required for the expression of the neural cell adhesion molecule NCAM-140 (31Feng Z. Li L. Ng P.Y. Porter A.G. Mol. Cell. Biol. 2002; 22: 5357-5366Crossref PubMed Scopus (42) Google Scholar). This basal c-Jun/AP-1-dependent synthesis of NCAM-140 counteracts NO-induced apoptosis. Here, we investigated whether NO induces c-Jun phosphorylation and regulates apoptosis through the JNK-c-Jun pathway, as do other cellular stressors. Notably, we found that JNK-dependent phosphorylation of c-Jun on Ser-63 promotes NO-induced apoptosis of neuroblastoma cells. A speculative model is proposed which can account for the pro- and antiapoptotic action of c-Jun/AP-1 within a single neuroblastoma cell.EXPERIMENTAL PROCEDURESMaterials and Plasmid Constructions—The human neuroblastoma cell lines SH-Sy5y and SHEP were obtained from Eva Feldman (University of Michigan, Ann Arbor) and from Evelyne Goillot (Laboratoire d'Immunologie, Centre Leon Berard, Lyon, France), respectively. The polyclonal antibodies against phospho-c-Jun, phospho-JNK, c-Jun, and JNK were from Cell Signaling Technology. The antibody against actin was from Santa Cruz Biotechnology, Inc. DEVD-afc and z-DEVD-fmk were obtained from BACHEM. Lipofectin and Opti-MEM were purchased from Invitrogen. The cell proliferation reagent WST-1 was purchased from Roche Applied Science. The D-TAT and D-JNKI1 peptides were from Alexis Biochemicals (Switzerland). The dual luciferase assay kit and β-galactosidase assay kit were from Promega. All the other reagents used in this research were from Sigma.The plasmid encoding dominant-negative c-Jun (denoted JunAA) was obtained from Dan Mercola (Sidney Kimmel Cancer Center, San Diego). The plasmids bearing the dominant-negative S63A or S73A mutations in c-Jun were constructed by PCR-based mutagenesis based on the plasmid JunAA. The primers used were: S63A (forward), 5′-GCT CAA GCT GGC GTC TCC CGA GCT GG-3′; S63A (reverse), 5′-CCA GCT CGG GAG ACG CCA GCT TGA GC-3′; S73A (forward,) 5′-CCT CCT CAC CTC TCC CGA CG-3′; S73A (reverse), 5′-CGT CGG GAG AGG TGA GGA GG-3′, respectively. The Gal4-c-Jun transactivator and Gal4-luciferase reporter plasmids were purchased from Stratagene. The modified Gal4-c-Jun plasmids (S63A or S73A) were constructed by cloning the transactivation domain of c-Jun (amino acids 1–221) bearing either the S63A or S73A mutations into the pFA-CMV vector from Stratagene. The pGL3-AP1 reporter plasmid and RPL-TK plasmid were kindly provided by S. Dhakshinamoorthy (Institute of Molecular and Cell Biology, Singapore). The plasmid encoding dominant-negative (DN)-JNK1 was provided by Roger J. Davis (Howard Hughes Medical Institute, University of Massachusetts Medical School, Worcester), and the plasmid encoding DN-JNK2 was provided by Dr. Shengcai Lin (Institute of Molecular and Cell Biology, Singapore).Cell Culture and Transfection—Both SH-Sy5y and SHEP cells were maintained in Dulbecco's modified medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. The substrate that ensures cell plating is presumed to be extracellular matrix proteins, either exogenous or derived from the bovine serum. Normally the cells were split after 48 h in culture. The JunAA, S63A, S73A, DN-jnk1, and DN-jnk2, plasmids were transfected into SH-Sy5y cells using Lipofectin following the manufacturer's instructions. The stably transfected cells were selected and maintained in medium with 100 μg/ml hygromycin (JunAA, S63A, S73A), 500 μg/ml G418 (DN-jnk1), and 100 μg/ml zeosin (DN-jnk2).Reporter Assays—For the Gal4-c-Jun reporter assay, 50 ng of Gal4-c-Jun activator plasmids (wild type, S63A, S73A, or JunAA), 1 μg of Gal4-luciferase reporter plasmid, and 10 ng of β-galactosidase plasmid were cotransfected into SH-Sy5y cells in 6-well plates (1–2 × 105 cells/plate). 40 h later, cells were treated with 2 mm SNP for the indicated times. Medium was removed, and cells were washed three times with ice-cold phosphate-buffered saline. Cells were harvested with 400 μl of reporter lysis buffer (provided in the β-galactosidase assay kit) and subjected to a short spin. 20 μl of supernatant was added to 100 μl of luciferase substrate, and the luciferase activity was measured immediately in a TD-20e luminometer. An aliquot of the same sample was used to determine β-galactosidase activity for normalizing luciferase activity obtained above. For the AP-1 reporter assay, 1 μg of pGL3-AP1 reporter plasmid and 10 ng of RPL-TK plasmid were cotransfected in S63A or S73A or vector control cells in 6-well plates. 40 h later, cells were treated with 2 mm SNP for the indicated times and harvested as described above. Lysates were diluted 10 times, and firefly luciferase activity was measured by adding 10 μl of diluted lysate and 100 μl of firefly luciferase substrate. The Renilla luciferase activity, as internal control, was measured by adding Stop&Glo solution in the same tube.Cell Death Assays—To measure cell death by WST-1 or lactate dehydrogenase release assay, cells (1–2 × 104/well) were plated in 96-well plates and treated with SNP for up to 15 h. WST-1 was added to the culture medium at a 1:10 dilution and incubated at 37 °C for 1 h or until the color of the medium turned red (incubation time can vary according to the cell number in the culture). The absorbance was measured at a wavelength of 420 nm. To carry out the lactate dehydrogenase release assay, the supernatants of the cells were collected, and the cell layer was lysed with an equal volume of lysis buffer (Dulbecco's modified Eagle's medium plus 0.1% Triton X-100). Lactate dehydrogenase activity in the supernatant and the lysate was quantitated. The cytotoxicity was calculated as percentage of lactate dehydrogenase release by the ratio of supernatant/(lysate + supernatant).Caspase Activity Assay—The activity of caspase-3-like proteases was measured using microtiter plates as described previously (28Ghatan 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 (362) Google Scholar, 32Hentze H. Schmitz I. Latta M. Krueger A. Krammer P.H. Wendel A. J. Biol. Chem. 2002; 277: 5588-5595Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) with modifications. After SNP treatment, the cells were lysed in lysis buffer (20 mm Hepes, pH 7.5, 250 mm sucrose, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, and 1 μg/ml each pepstatin, leupeptin, and aprotinin), and the lysate was stored at –80 °C. The samples were diluted 1:10 with reaction buffer (60 μm fluorogenic substrate DEVD-afc in 50 mm Hepes, pH 7.4, 1% sucrose, 0.1% CHAPS, 10 mm dithiothreitol) in a final volume of 100 μl and incubated at 37 °C for 30 min. Released afc was measured kinetically with a fluorescent spectrophotometer set at excitation wavelength of 400 nm and emission wavelength of 505 nm. For normalization, protein concentrations of the corresponding samples were estimated simultaneously by using the BCA reagents from Pierce Chemical Company. 1 microunit/mg corresponds to 1 (μmol/mg) × min.Western Blot Analysis—105 cells were lysed in 200 μl of SDS sample buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromphenol blue, and 50 mm freshly added dithiothreitol). Sonication was needed to shear the genomic DNA and reduce the viscosity of the lysate. The sonicated lysate was then heated at 99 °C for 5 min and subjected to centrifugation at 14,000 rpm for 5 min at 4 °C. 50 μl of the supernatants was separated on 0.1% SDS and 12% polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore). Detection of bands was performed using the Phototope®-horseradish peroxidase Western Blot Detection System (Cell Signaling).Peptide Inhibition Assay—The inhibition assay was carried out in SHEP neuroblastoma cells. D-TAT and D-JNKI1 peptides were added at final concentrations of 20, 50, and 100 μm into the medium. After 24 h, the medium was refreshed with peptides, and SNP was added at a final concentration of 2 mm. At various times thereafter, cell death and c-Jun phosphorylation were measured as described above.RESULTSNO Induces JNK Activation, c-Jun Phosphorylation on Ser-63, and Apoptosis in SH-Sy5y Cells—Various NO donors have been used widely to study oxidative stress and cellular responses by mimicking endogenous NO generation (33Brune B. von Knethen A. Sandau K.B. Cell Death Differ. 1999; 6: 969-975Crossref PubMed Scopus (264) Google Scholar). SH-Sy5y neuroblastoma cells are highly sensitive to cell death induced by various NO donors including SNP at concentrations in the range 0.5–2.5 mm, and the mode of cell death under these conditions is apoptosis (28Ghatan 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 (362) Google Scholar, 31Feng Z. Li L. Ng P.Y. Porter A.G. Mol. Cell. Biol. 2002; 22: 5357-5366Crossref PubMed Scopus (42) Google Scholar, 34Oh-Hashi K. Maruyama W. Yi H. Takahashi T. Naoi M. Isobe K. Biochem. Biophys. Res. Commun. 1999; 263: 504-509Crossref PubMed Scopus (121) Google Scholar). As described previously (31Feng Z. Li L. Ng P.Y. Porter A.G. Mol. Cell. Biol. 2002; 22: 5357-5366Crossref PubMed Scopus (42) Google Scholar), a time-dependent increase in cell death was observed beginning around 8 h after the addition of SNP to the SH-Sy5y cells (Fig. 1A), and this increase correlated with the appearance of significant JNK activity at 6–7.5 h after the addition of SNP (Fig. 1B, upper panel).Because JNKs are the main upstream kinases for c-Jun NH2-terminal phosphorylation (12Morton S. Davis R.J. McLaren A. Cohen P. EMBO J. 2003; 22: 3876-3886Crossref PubMed Scopus (228) Google Scholar), we next tested whether the Ser-63 and Ser-73 residues of c-Jun were phosphorylated after SNP treatment of SH-Sy5y cells by using phosphoserine 63- and phosphoserine 73-specific antibodies. A strong and sustained c-Jun phosphorylation on Ser-63 was observed at 6–8 h after the addition of SNP, whereas phosphorylation on Ser-73 was virtually undetectable (Fig. 1C). In contrast, UV irradiation of SH-Sy5y cells resulted in similar levels of Ser-63 and Ser-73 phosphorylation (Fig. 1C). Thus, JNK activation and selective phosphorylation of c-Jun on Ser-63 both occurred around the onset of NO donor-induced apoptosis. Ser-63 phosphorylation and the death of SH-Sy5y cells both occurred at 1.5 and 2 mm SNP. Concentrations of SNP lower than 1.5 mm neither induced cell death nor elicited Ser-63 phosphorylation of c-Jun (data not shown), indicating that c-Jun is phosphorylated only at toxic concentrations of SNP. Excessive concentrations of SNP higher than 2.5 mm resulted in detectable Ser-73 phosphorylation that closely correlated with the onset of appreciable necrosis, indicating that predominant Ser-63 phosphorylation is an apoptosis-related phenomenon (data not shown).NO-induced Apoptosis Is Blocked in S63A and JunAA Stable Cells but Not in S73A Stable Cells—To investigate whether c-Jun phosphorylation contributes to NO-induced apoptosis, we stably transfected SH-Sy5y cells with plasmids encoding various dominant-negative forms of c-Jun. In one form, Ser-63 was mutated to alanine (denoted S63A), and in another Ser-73 was mutated to alanine (denoted S73A). In a third form, both Ser-63 and Ser-73 were mutated to alanines (JunAA), which compromises the ability of c-Jun to transactivate target genes (15Behrens A. Sibilia M. Wagner E.F. Nat. Genet. 1999; 21: 326-329Crossref PubMed Scopus (594) Google Scholar).To exclude the possibility that highly overexpressed S63A, S73A, or JunAA might quench JNK activity by sequestering JNK in an abortive complex, we chose for further analysis independent clones in which S63A (Fig. 2A, top panel) or JunAA (Fig. 2A, lower panel) or S73A (data not shown) are expressed at levels only slightly in excess of endogenous c-Jun. Normal phosphorylation of endogenous c-Jun on Ser-63 in response to UV irradiation was still observed in two independent clones of S63A stable cells (Fig. 2B); and as expected, the endogenous Ser-73 phosphorylation of c-Jun in response to UV irradiation was more intense in S63A stable cells compared with the vector control cells (Fig. 2C). Analogous results were obtained in UV irradiation-treated S73A cells (data not shown). In addition, NO-induced phosphorylation of endogenous c-Jun on Ser-63 still occurred in two independent JunAA clones (Fig. 2D). These data indicate that the expression of c-Jun mutated to S63A and/or S73A did not compromise endogenous JNK activity.Fig. 2Stable expression of S63A or JunAA does not inhibit endogenous c-Jun phosphorylation in SH-Sy5y cells.A, expression levels of the exogenous c-Jun proteins in the stable cell lines compared with vector control (V.C.). Upper panel, two clones of S63A. Lower panel, two clones of JunAA. B, two independent clones of S63A stable cell and the vector control cell were treated with 100 J/m2 UV irradiation, and the cells were harvested after 1 h. Total proteins in the cell lysates were fractionated on 0.1% SDS and 12% polyacrylamide gels and subjected to immunoblot analysis using an antibody against phosphoserine 63. C, two independent clones of S63A stable cell and the vector control cell line were treated with 100 J/m2 UV irradiation, and total proteins were prepared as in B for immunoblot analysis using an antibody against phosphoserine 73. D, two independent clones of JunAA stable cell and the vector control cell line were treated with 2 mm SNP for 10 h. Total proteins were prepared as in B for immunoblot analysis using an antibody against phosphoserine 63. Actin was visualized as a loading control in all three panels.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then compared the sensitivities of the above three different stable cells and vector control cells to NO donors and UV radiation. At any concentrations of SNP which were sufficient to induce apoptosis, several independent clones of S63A and JunAA stable cells showed markedly increased resistance to cell death compared with vector control cells (Fig. 3A). Importantly, various S73A stable cell lines failed to show resistance to NO compared with vector control cells (Fig. 3A). In contrast, neither S63A nor S73A stable cells were resistant to UV irradiation-induced cell death, whereas JunAA cells only showed a marginal increase in resistance to UV irradiation (Fig. 3B). These data provide evidence that Ser-63 phosphorylation of c-Jun is important in NO-induced, but not UV irradiation-induced cell death.Fig. 3Stable expression of S63A and JunAA increases resistance of SH-Sy5y cells to NO.A, S63A, S73A, and JunAA stable cells and vector control (V.C.) cells were treated with SNP for 15 h at the different indicated concentrations. The percentage of dead cells was measured. Values are the mean ± S.D. determined from three independent clones of S63A, S73A, or JunAA, each in triplicate. B, S63A, S73A, and JunAA stable cells and vector control cells were treated with UV irradiation at the different indicated doses. After 24 h, the percentage of dead cells was measured. Values are the mean ± S.D. determined from three independent clones of S63A, S73A, or JunAA, each in triplicate.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Ser-63 of c-Jun Is Required for c-Jun- and AP-1-mediated Transactivation in Response to NO—Dual phosphorylation of Ser-63 and Ser-73 has been found previously to lead to c-Jun-dependent transactivation (14Smeal T. Binetruy B. Mercola D.A. Birrer M. Karin M. Nature. 1991; 354: 494-496Crossref PubMed Scopus (696) Google Scholar), and accordingly mutation of both serines reduces the ability of c-Jun to transactivate target genes (15Behrens A. Sibilia M. Wagner E.F. Nat. Genet. 1999; 21: 326-329Crossref PubMed Scopus (594) Google Scholar). Because we found that NO caused c-Jun phosphorylation predominantly on Ser-63, and since S63A and JunAA stable cells showed markedly increased resistance to cell death, we next asked whether Ser-63 phosphorylation alone can potentiate c-Jun and AP-1 transactivation. Using a Gal4-c-Jun reporter system, we found that Gal4-c-Jun (wild type) as well as Gal4-c-Jun (S73A) were transactivated up to 4-fold in SH-Sy5y cells upon NO stimulation (Fig. 4A). However, Gal4-c-Jun (S63A) and Gal4-c-Jun (JunAA) were completely inactive in transactivation (Fig. 4A). In parallel experiments, transient transfections with AP-1 reporter plasmids revealed that NO-induced AP-1 activation of up to ∼2.7-fold occurred in SH-Sy5y and S73A cells but was absent in S63A cells (Fig. 4B). Thus, our combined data from the c-Jun and AP-1 reporter assays indicate that the presence of Ser-63 (but not Ser-73) is required for NO-induced c-Jun/AP-1 transactivation. These results also indicate that S63A and the JunAA constructs function as dominant-negatives by inhibiting gene transcription mediated by endogenous c-Jun.Fig. 4Ser-63 of c-Jun is required for c-Jun- and AP-1-mediated transactivation in response to NO.A, different Gal4-c-Jun constructs bearing the unmodified (WT) c-Jun sequence, or S63A, S73A, or JunAA mutations in the transactivation region of c-Jun, were cotransfected with a luciferase reporter plasmid together with β-galactosidase into SH-Sy5y cells. 40 h later, the cells were treated with 2 mm SNP for the indicated times and harvested. Luciferase activity was measured, and β-galactosidase activity was also measured as an internal control. B, the AP-1 reporter plasmid was cotransfected with the RPL-TK plasmid into S63A, S73A, and vector control (V-AA) stable cells. 40 h later, the cells were treated with 2 mm SNP for the indicated times and harvested. Firefly luciferase activity was measured, and Renilla luciferase activity was measured as an internal control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Caspase-3 Contributes to NO-induced Cell Death and Is Inhibited in S63A Stable Cells—Caspase-3 was found to be important for NO-induced apoptosis of SH-Sy5y cells because prevention of caspase-3 activity by