Amino-terminal-derived JNK Fragment Alters Expression and Activity of c-Jun, ATF2, and p53 and Increases H2O2-induced Cell Death
2000; Elsevier BV; Volume: 275; Issue: 22 Linguagem: Inglês
10.1074/jbc.m910045199
ISSN1083-351X
AutoresThomas Buschmann, Zhimin Yin, Anindita Bhoumik, Ze’ev A. Ronai,
Tópico(s)Retinoids in leukemia and cellular processes
ResumoThe stress-activated protein kinase JNK plays an important role in the stability and activities of key regulatory proteins, including c-Jun, ATF2, and p53. To better understand mechanisms underlying the regulation of JNK activities, we studied the effect of expression of the amino-terminal JNK fragment (N-JNK; amino acids 1–206) on the stability and activities of JNK substrates under nonstressed growth conditions, as well as after exposure to hydrogen peroxide. Mouse fibroblasts that express N-JNK under tetracycline-off (tet-off) inducible promoter exhibited elevated expression of c-Jun, ATF2, and p53 upon tetracycline removal. This increased coincided with elevated transcriptional activities of p53, but not of c-Jun or ATF2, as reflected in luciferase activities ofp21Waf1/Cip1 -Luc, AP1-Luc, andJun2-Luc, respectively. Expression of N-JNK in cells that were treated with H2O2 impaired transcriptional output as reflected in a delayed and lower level of c-Jun-, limited ATF2-, and reduced p53-transcriptional activities. N-JNK elicited an increase in H2O2-induced cell death, which is p53-dependent, because it was not seen in p53 null cells yet could be observed upon coexpression of p53 and N-JNK. The ability to alter the activity of ATF2, c-Jun, and p53 and the degree of stress-induced cell death by a JNK-derived fragment identifies new means to elucidate the nature of JNK regulation and to alter the cellular response to stress. The stress-activated protein kinase JNK plays an important role in the stability and activities of key regulatory proteins, including c-Jun, ATF2, and p53. To better understand mechanisms underlying the regulation of JNK activities, we studied the effect of expression of the amino-terminal JNK fragment (N-JNK; amino acids 1–206) on the stability and activities of JNK substrates under nonstressed growth conditions, as well as after exposure to hydrogen peroxide. Mouse fibroblasts that express N-JNK under tetracycline-off (tet-off) inducible promoter exhibited elevated expression of c-Jun, ATF2, and p53 upon tetracycline removal. This increased coincided with elevated transcriptional activities of p53, but not of c-Jun or ATF2, as reflected in luciferase activities ofp21Waf1/Cip1 -Luc, AP1-Luc, andJun2-Luc, respectively. Expression of N-JNK in cells that were treated with H2O2 impaired transcriptional output as reflected in a delayed and lower level of c-Jun-, limited ATF2-, and reduced p53-transcriptional activities. N-JNK elicited an increase in H2O2-induced cell death, which is p53-dependent, because it was not seen in p53 null cells yet could be observed upon coexpression of p53 and N-JNK. The ability to alter the activity of ATF2, c-Jun, and p53 and the degree of stress-induced cell death by a JNK-derived fragment identifies new means to elucidate the nature of JNK regulation and to alter the cellular response to stress. mitogen-activated protein kinase extracellular signal-regulated kinase stress-activated protein kinase hemagglutinin tetracycline whole cell extract polyacrylamde gel electrophoresis adenosine 5′-O-(thiotriphosphate) fluorescence-activated cell sorter The family of mitogen-activated protein kinases (MAPK)1 consists of evolutionarily conserved proteins, which play a central role in development and growth as well as in the protection of cells from stress and DNA damage (1.Errede B. Levin B.E. Curr. Opin. 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Unlike ERKs, which are the best studied MAPKs, the nature of JNK domains and their respective functions remain unclear. As an approach to elucidate the nature of JNK domains involved in targeting ubiquitination and kinase activities, we have monitored the cellular changes elicited by a 206-amino acid amino-terminal fragment of JNK (N-JNK). This fragment contains the kinase domain and, based on its homology with ERK2, is expected to mediate association with JNK substrates. Furthermore, due to the lack of a carboxyl-terminal domain, N-JNK is not expected to be subject to intramolecular inhibition, which is a common mode of regulation for stress kinases (59.Barila D. Superti-Furga G. Nat. Genet. 1998; 18: 280-282Crossref PubMed Scopus (184) Google Scholar). By analogy to ERK2, dimerization of JNK molecules may be impaired upon expression of N-JNK. Two-hybrid screening using the amino-terminal domain of JNK as bait has identified multiple clones that possess the carboxyl-terminal domain of JNK, thereby indicating association between the amino- and carboxyl-terminal regions of this protein. 2A. Bhoumik, L. Broday, S. Fuchs, and Z. Ronai, unpublished observations. This result suggests that JNK may be found either as a dimer or that JNK may exhibit intramolecular inhibition in which the carboxyl-terminal region loops back onto the amino-terminal domain. Either possibility was shown to exist for other MAPK family members (39.Cobb M.H. Prog. Biophys. Mol. Biol. 1999; 71: 479-500Crossref PubMed Scopus (762) Google Scholar), and is expected to play an important role in the regulation of JNK activities. Using a tetracycline (tet)-off-inducible system we demonstrate the effect of N-JNK on its substrates (c-Jun, ATF2, and p53) under normal growing conditions and after exposure to external stress. The nature of N-JNK effects before and after stress and the possible use of N-JNK to further elucidate the regulation of JNK activities are discussed. The mouse fibroblast cell line NIH 3T3 cells that stably express the pSV40-Hyg plasmid (CLONTECH) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (Life Technologies, Inc.). Cells were grown at 37 °C with 5% CO2. The pTet-N-JNK was constructed by subcloning the cDNA of the amino-terminal 206 amino acids of wild type JNK1 into the tet-regulated promoter of the pUHD-10-3 vector. Cell clones that stably express both constructs were selected in 600 μg/ml geneticin in the presence of hygromycin (100 μg/ml) 24 h after DOTAP transfection with the ptet-N-JNK-UHD-10-3 construct. Cells inducible with N-JNK-tet were maintained in Dulbecco's modified Eagle's medium containing fetal bovine serum (10%), hygromycin (100 μg/ml), and geneticin (400 μg/ml). To maintain suppression of N-JNK expression, tet was added to the medium every 3 days (to a final concentration of 1 μg/ml). Proteins were prepared from cells as described previously (60.Adler V. Fuchs S.Y. Kim J. Kraft A. King M.P. Pelling J. Ronai Z. Cell Growth Differ. 1995; 6: 1437-1446PubMed Google Scholar). In all cases, buffer contained a mixture of proteases (1 μg/ml pepstatin, leupeptin, and aprotinin) and the phosphatase inhibitors (sodium vanadate 1 mm; sodium fluoride 5 mm). Antibodies to c-Jun, phospho-c-Jun, ATF-2, phospho-ATF-2, and phospho-JNK were purchased from New England BioLabs. Polyclonal antibodies to JNK or p53 were generated using bacterially expressed JNK or p53 as antigen. Monoclonal JNK antibodies (clone 333) (PharMingen) and monoclonal antibodies to p53, clone pAb421 (Oncogene Science) were purchased. Immunoprecipitations were carried out using 1 mg of whole cell extracts (WCE) and 2 μg of the respective antibodies, for 16 h at 4 °C. Protein G beads (Life Technologies, Inc.) were added (15 μl) for 30 min at room temperature before washes were carried out in phosphate-buffered saline/0.5 m LiCl supplemented with Tween 100 (0.5%). Immunoprecipitated material was subjected to Western blot analysis. Immunoblot analysis was performed using 100 μg of WCE separated on SDS-polyacrylamide gel electrophoresis (PAGE) followed by electrotransfer to a nitrocellulose membrane. Ponceau staining was carried out to confirm equal loading followed by blocking (5% non-fat milk) and reaction with the respective antibodies. Reactions were visualized using chemiluminesence (ECL) reagents (Amersham Pharmacia Biotech). Transfected cells in the growing phase were treated for 1 h with 100 μmH2O2 by taking the medium from the culture dish and mixing it with freshly diluted H2O2. Medium containing H2O2 was immediately applied to the fibroblasts (107 cells). Protein kinase assays were carried out using fusion proteins, GST-Jun or GST-ATF-2 (60.Adler V. Fuchs S.Y. Kim J. Kraft A. King M.P. Pelling J. Ronai Z. Cell Growth Differ. 1995; 6: 1437-1446PubMed Google Scholar), as substrates. Purity of bacterially produced c-Jun and ATF-2 was confirmedvia silver-stained SDS-PAGE. Immunokinase reactions were carried out on immunoprecipitated kinase using antibodies to JNK, which was then incubated with the respective substrate, in a soluble form, in the presence of kinase buffer (20 mm HEPES, pH 7.6, 1 mm EGTA, 1 mm dithiothreitol, 2 mmMgCl2, 2 mm MnCl2, 5 mmNaF, 1 mm NaVO3, 50 mm NaCl) at 37 °C for 15 min. The protein G beads were pelleted and washed extensively with PBST (150 mm NaCl, 16 mmsodium phosphate, pH 7.5, 1% Triton X-100, 2 mm EDTA, 0.1% β-MeOH, 0.2 mm phenylmethylsulfonyl fluoride, and 5 mm benzamidine) before they were incubated with [γ-32P]ATP (50 cpm/fmol) in the presence of kinase buffer. Following extensive washing, the phosphorylated substrate was boiled in SDS sample buffer and the eluted proteins were run on a 10% SDS-polyacrylamide gel. The gel was dried, and phosphorylation of the c-Jun or ATF-2 substrate was determined by autoradiography. In all cases the buffers used contained a mixture of protease and phosphatase inhibitors (10.Adler V. Schaffer A. Kim J. Dolan L.R. Ronai Z. J. Biol. Chem. 1995; 270: 26071-26077Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 47.Adler V. Yin Z. Fuchs S.Y. Benezra M. Rosario L. Tew K.D. Pincus M.R. Sardana M. Henderson C.J. Wolf C.R. Davis R. Ronai Z. EMBO J. 1999; 18: 1321-1334Crossref PubMed Scopus (966) Google Scholar, 60.Adler V. Fuchs S.Y. Kim J. Kraft A. King M.P. Pelling J. Ronai Z. Cell Growth Differ. 1995; 6: 1437-1446PubMed Google Scholar). The in vitroubiquitination assay (see Ref. 49.Fuchs S.Y. Xie B. Adler V.A. Fried V.A. Davis R.J. Ronai Z. J. Biol. Chem. 1997; 272: 32163-32168Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar for details) was performed using 50 μg of whole cell lysates prepared from N-JNK-expressing cells (maintained with or without tetracycline) that were incubated on ice with bacterially expressed (6x)his-tagged human p53 (5 μg) bound to NTA beads for 45 min. After extensive washes (four times with 1 ml of kinase buffer (49.Fuchs S.Y. Xie B. Adler V.A. Fried V.A. Davis R.J. Ronai Z. J. Biol. Chem. 1997; 272: 32163-32168Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar)), the substrate-bound beads were equilibrated with 1× ubiquitination buffer (50 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 0.5 mm dithiothreitol, 2 mm NaF, and 3 μm okadaic acid) and incubated in the same buffer supplemented with 2 mm ATP, 10 mm creatine phosphate, 0.02 unit of creatine phosphokinase, 2 μg of hemagglutinin (HA)-tagged ubiquitin (49.Fuchs S.Y. Xie B. Adler V.A. Fried V.A. Davis R.J. Ronai Z. J. Biol. Chem. 1997; 272: 32163-32168Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), 1.5 mmATPγS (Sigma; a reagent included to block degradation, thus enabling monitoring of the degree of ubiquitination) and 33% reticulocyte lysate (v/v) in a total volume of 30 μl at 37 °C for 5 min. The reaction was stopped by adding 0.5 ml of 8 m urea in sodium phosphate buffer (pH 6.3) with 0.1% Nonidet P-40. The beads were washed, and the protein moiety was eluted and resolved on 8% SDS-PAGE followed by electrotransfer onto nitrocellulose filters. Nitrocellulose filters were blocked with 5% non-fat milk and probed with HA11 antibody (BabCo). Respective luciferase constructs were cotransfected with βGal vector controlled by the same promoter. Transcriptional analysis was carried out on proteins prepared at time points indicated under “Results.” Transcriptional analysis of Jun/ATF2 was carried out using the 5xJun2 or AP1-driven luciferase construct as previously reported (61.van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (571) Google Scholar). Analysis of p53 transcription was carried out using the p21-luc construct. In all cases, values were normalized with respect to transfection efficiency based on levels of βGal activities. Data shown represent three independent experiments performed in duplicate. Analysis of cell death was carried out as described by Kumar et al. (62.Kumar S. Kinoshita M. Noda M. Copeland N.G. Jenkins N.A. Genes Dev. 1994; 8: 1613-1626Crossref PubMed Scopus (588) Google Scholar). Briefly, triplicates of >5,000 cells per measurement, at the time points indicated under “Results,” were subjected to trypan blue as a vital stain which identifies dead cells. In all cases, analysis was performed at least three times. Analysis of cell death in transfected 10.1 cells (constructs: p53-HA in pcDNA3; JNK1–206 in pcDNA3) was carried out using PI staining. Flow cytometric analysis (20,000 cells per assay) was performed on a fluorescence-activated cell sorter (FACS; Calibur flow cytometer, Becton Dickinson). Data analysis was carried out using the FACS Desk software. To elucidate the possible contribution of JNK domains to its cellular activities, we analyzed the effects of a JNK fragment, which represents the amino-terminal portion (amino acids 1–206) of JNK (N-JNK), and includes the kinase domain. The N-JNK fragment was cloned into the tet-off-inducible expression vector, which was transfected into mouse fibroblasts. Following drug selection, we identified cell clones in which expression of amino-terminal JNK is induced upon tetracycline removal. Immunoblot analysis allows detection of N-JNK expression as early as 1 h after tet removal followed by a time-dependent increase, which reaches its maximal expression levels after 12 h (Fig.1 a). Expression of N-JNK increased the level of endogenous JNK expression (compare time 0 intet− versus tet+ in Fig.1 b) but not the degree of JNK phosphorylation (Fig.1 b). Expression of N-JNK did not cause major changes in JNK expression and activity following exposure to H2O2 (Fig. 1 b). To test the effect of the N-JNK fragment on JNK substrates, we first monitored changes in the expression, phosphorylation, and activity of c-Jun and ATF2. Under nonstressed growth conditions, expression of N-JNK led to increased expression of c-Jun (4-fold; compare time 0 in tet− and tet+ lanes of Fig. 2 a,upper panel) and ATF2 (2-fold; Fig. 2 b,upper panel) based on densitometric analysis of three experiments. Increased expression of N-JNK also caused elevated basal phosphorylation levels of c-Jun (23-fold) and ATF2 (40-fold) (Fig.2 a, compare time 0 in tet+ with tet− lanes). Because JNK targets the ubiquitination of nonphosphorylated forms of its associated proteins (48.Fuchs S.Y. Dolan L.R. Davis R. Ronai Z. Oncogene. 1996; 13: 1529-1533Google Scholar), elevated phosphorylation is expected to confer respective increases in the expression levels of this protein. Next, we assessed the changes in the expression and activities of c-Jun and ATF2 in cells exposed to stress, using hydrogen peroxide as a model. Treatment with H2O2 increased phosphorylation of c-Jun and ATF2. Although phosphorylation of c-Jun was 9-fold higher 1 h after H2O2treatment, it was otherwise similar to that seen in the absence of N-JNK expression. Duration of ATF2 phosphorylation is shorter upon N-JNK expression, because ATF2 phosphorylation is three times lower 4 h after H2O2 treatment (Fig.2 b, compare tet+ and tet− lanes at the 4-h time point). These data suggest that the effects of N-JNK are more pronounced in nonstressed cells where N-JNK increases expression and phosphorylation levels of both ATF2 and c-Jun. Importantly, immunoprecipitation of N-JNK using HA antibodies from hydrogen peroxide tet− cells followed by solid phase kinase reaction using GST-Jun as a substrate did not reveal Jun phosphorylation, whereas immunoprecipitation using antibodies to JNK (allowing detection of endogenous JNK activity) as a positive control revealed such activity (data not shown). This observation suggests that, although the N-JNK fragment contains the kinase domain, it does not elicit kinase activities. To test whether N-JNK expression would alter transcriptional activity of Jun/ATF2, cells were transfected with Jun2-Luciferase or AP1-Luciferase constructs, which contain the target sequence for Jun/ATF2 or c-Jun/c-Fos heterodimers, respectively. Expression of N-JNK caused a modest decrease (25%) in AP1-Luc activity (time 0 in Fig.2 c), and it increased (33%) activity of Jun2-Luc (Fig.2 d). Within 4 h after H2O2treatment, there was a noticeable increase in both AP1-Luc (70%) and Jun2-Luc (110%) activities. Removal of tet−, which enables the expression of N-JNK in hydrogen peroxide-treated cells, caused a delayed and a modest increase (40%) in AP1-Luc activities after 4 h (Fig. 2 c). Furthermore, at this time point after treatment with hydrogen peroxide, the degree of AP1-driven transcription was 30% lower than the level seen in cells that do not express N-JNK (Fig.2 c). This observation suggests that expression of N-JNK attenuates the degree of AP1-mediated transcription under conditions that are known to increase c-Jun phosphorylation and transactivation. Analysis of Jun2-Luc activities in hydrogen peroxide treated cells revealed a 2-fold increase within the first 4 h, which was also attenuated in the presence of N-JNK expression. In fact, N-JNK-expressing cells maintained the same level of transactivation seen under nonstressed conditions, which was 30% higher than the basal levels of Jun2-Luc activity (Fig. 2 d). This result indicates that, in N-JNK-expressing cells, activities of c-Jun and ATF2 are limited following exposure to stress in the form of hydrogen peroxide. Such change is expected to alter the cellular response to stress and damage. To test whether N-JNK associates with c-Jun or ATF2, proteins prepared from control or hydrogen peroxide-treated cells that were maintained in the presence or absence of tet were subjected to immunoprecipitation using antibodies to HA (which recognizes the HA-tagged N-JNK) followed by immunoblot using antibodies to either c-Jun or ATF2. This analysis did not identify association between ATF2 or c-Jun and N-JNK (Fig.2 e). To further explore cellular effects of the amino-terminal JNK fragment, we elucid
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