ASK1-Signaling Promotes c-Myc Protein Stability during Apoptosis

Biochemical and Biophysical Research Communications 281, 1313–1320 (2001) doi:10.1006/bbrc.2001.4498, available online at http://www.idealibrary.com on

ASK1-Signaling Promotes c-Myc Protein Stability during Apoptosis Kohji Noguchi,* ,† ,1 Akiko Kokubu,† Chifumi Kitanaka,† Hidenori Ichijo,‡ and Yoshiyuki Kuchino† ,§ ,1 *Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan; †Biophysics Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan; ‡Department of Cell Signaling, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan; and §CREST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan

Received February 17, 2001

We previously reported that JNK is involved in the regulation of c-Myc-mediated apoptosis triggered by UV irradiation and anticancer drug treatment. Here we show that ASK1 is an upstream regulator for c-Mycmediated apoptosis triggered by UV, and we found a direct role for Ser-62 and Ser-71 in the regulation of protein stability and function of c-Myc. The ASK1-JNK pathway enhanced the protein stability of c-Myc through phosphorylation at Ser-62 and Ser-71, which was required for c-Myc-dependent apoptosis by ASK1signaling. Interestingly, ASK1-signaling attenuated the degradation of ubiquitinated c-Myc without affecting the ubiquitination process. Together, these findings indicate that the ASK1-JNK pathway promotes the proapoptotic activity of c-Myc by modulating c-Myc protein stability through phosphorylation at Ser-62 and Ser-71. © 2001 Academic Press

The Myc protein is a short-lived, unstable nuclear phosphorylated protein (1, 2), and the expression and activity of c-Myc are strictly controlled at many levels, including transcription, mRNA stability, translation and protein stability (reviewed in Ref. 3). It was recently revealed that the rapid degradation of c-Myc protein is governed by the ubiquitin-dependent proteasome system and an N-terminal domain containing Myc-boxes is required for the degradation (4 –9). A Abbreviations used: JNK, c-jun N-terminal kinase; ASK1, apoptosis signal regulating kinase 1; MAPK, mitogen-activated protein kinase; ERK2, extracellular signal-regulated kinase 2; UV, ultraviolet; EGFP, enhanced green fluorescence protein; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol. 1 To whom correspondence and reprint requests may be addressed. Fax: ⫹81-3-5285-1272. E-mail: [email protected] or ykuchino@ ncc.go.jp.

functional association with the phosphorylation of c-Myc at Thr-58 and Ser-62 has been suggested for the cell proliferation and the cell cycle regulation (10 –17). Concerning cell proliferation, c-Myc is phosphorylated at Ser-62 by ERK2, a mitogenic MAPK, and the Rasactivated Raf-MAPK pathway induces accumulation of c-Myc (18). These studies raise the possibility that the phosphorylation at Thr-58 and/or Ser-62 contributes to the stabilization of c-Myc for the oncogenic and the cell proliferative activity (8, 9), although the role of these residues in the ubiquitination-dependent proteolysis is remained. Many studies have demonstrated that the deregulated expression of c-myc sensitizes cells to apoptotic stimuli (reviewed in Refs. 19, 20). The precise effect of c-myc expression on cell sensitization during apoptosis is still unclear, but we have shown that c-Myc protein is directly phosphorylated by JNK at Ser-62 and Ser71, and this phosphorylation is required for cell sensitization to UV- and Taxol-triggered apoptosis (21, 22). However, it remains to be learned what molecule upstream of JNK initiates c-Myc-mediated apoptosis triggered by UV, and how the phosphorylation contributes to the c-Myc activity is unknown. A new MAP kinase kinase kinase, ASK1 that activates both the MKK4/7JNK and MKK3/6-p38 MAP kinase pathways is shown to play pivotal roles in tumor necrosis factor- and stress stimuli-induced apoptosis (23–25). Interestingly, JNK-mediated phosphorylation of substrate such as c-Jun and p53 renders the protein stable and functional (26, 27), and a direct relationship has been suggested between the stress-responsive phosphorylation/de-phosphorylation cascade and the targeting of the substrate proteins by the ubiquitin-proteasome system (28). These observations strongly suggest a possible role of ASK1-signaling in the regulation of c-Mycactivity during apoptosis.

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To elucidate any interaction between the stressactivated ASK1-signaling and the regulation of c-Myc, here we investigated the functional role of ASK1signaling for the regulation of c-Myc through phosphorylation and stabilization during apoptosis. We found that the phosphorylation at both Ser-62 and Ser-71 induced by the ASK1-JNK pathway stimulated the accumulation of c-Myc, which affected the apoptosispromoting activity. Furthermore, the phosphorylation at both Ser-62 and Ser-71 by ASK1-signaling attenuated the degradation of ubiquitinated c-Myc. These findings suggest that the ASK1-signaling modulates the protein stability of c-Myc for the regulation of c-Myc-mediated apoptosis. MATERIALS AND METHODS Materials. pc3GF, pc3GFhcmyc, pc3GFhcmyc(S62A/S71A), pflaghcmyc, pflag-hcmyc(S62A/S71A), pcDNA3EGFP, and pflag-jnk were constructed essentially as described before (21, 22). To obtain c-myc (S62E/S71E) mutant cDNA, mutagenesis of human c-myc cDNA was performed with PCR-based QuikChange site-Directed Mutagenesis kits (Stratagene, La Jolla, CA) according to the instruction manual. In brief, synthetic DNA primers (5⬘-CTGCTGCCCACCCCGCCCCTGGAGCCTA-GCCGCCGCTCCGGGCTCTGCGAGCCCTCCTACGTTGCGGTCAC-3⬘ and 5⬘-GTGACCGCAACGTA-GGAGGGCTCGCAGAGCCCGGAGCGGC-GGCTAGGCTCCAGGGGCG-GGGTGGGCAGCAG-3⬘) were used in the PCR with template pcDNA3hcmyc(S62A/ S71A) plasmid. pcDNA3/His-ubiquitin was kindly provided by Dr. Akihiro Tomida (University of Tokyo, Japan). To construct GST-E6, E6 cDNA was cloned into pGEX2T by RT-PCR method from HeLa cDNA using synthetic DNA primers (5⬘-GAAGATCTATGGCGCGCTTTGAGGATCC-3⬘ and 5⬘-GAAGATCTT-ATACTTGTGTTTCTCTGCG-3⬘). All PCR-generated cDNA clones were fully sequenced and subcloned into each plasmid. pcDNA3/ASK1, pcDNA3/⌬⌵ASK1, and pcDNA3/ASK1(KM) were described before (29). pFC-MEK1(S218E/ S222E, ⌬32–51) was purchased from Stratagene and pCEP4 from Invitrogen (CH Groningen, The Netherlands). GST-c-Myc (1–181), GST-c-Jun (1–92) and anti-phospho c-Myc(Ser71) polyclonal antibody were prepared as described (21, 22). Anti-c-Myc polyclonal antibody (06340) was purchased from Upstate biotechnology (Lake Placid, NY) and anti-c-Myc monoclonal antibody (9E10) from Calbiochem–Novabiochem (La Jolla, CA). Anti-Flag M2 antibody was purchased from Eastman Kodak (Rochester, NY) and antiphospho c-Myc (Thr58/Ser62) antibody was from New England Biolabs Inc. (Beverly, MA). Anti-JNK1(C-17), anti-phospho JNK (G-7), anti-HA (Y-11), anti-p27 Kip (C-19) and control rabbit antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). MG132 (carbobenzoxyl-leucyl-leucyl-leucinal) was purchased from Peptide Institute Inc. (Osaka, Japan). G418 was from Life Technologies (Grand Island, NY). Cell culture. Mouse embryonic fibroblast NIH3T3 cells, human embryonic kidney transformed 293T cells and human cervical carcinoma HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui, Tokyo) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cytosystems, Castle Hill, Australia). Human wild type and S62A/S71A c-myc expressing NIH3T3 clone CM-9 and S6271A-13 cells were established before (21). To establish ASK1expressing stable NIH3T3 clones, NIH3T3 cells were transfected with each ASK1-expressing plasmid, selected in the presence of G418 (200 ␮g/ml) for 2 weeks, and cloned. Expression of each HAASK1s was confirmed by Western blot analysis using anti-HA (Y-11) antibody.

Pulldown and in-gel kinase assay. 293T cells were transfected with each plasmid using SuperFect reagents (Qiagen GmbH, Germany). After two days, cells were lysed in Hepes-binding buffer (20 mM Hepes–KOH, pH 7.6, 50 mM NaCl, 2.5 mM MgCl 2, 0.05% Triton X-100, 0.1 mM EDTA, 1 mM PMSF, 20 ␮M MG132, 1 ␮g/ml leupeptin, 1 ␮M pepstatin, 20 ␮g/ml phosphoramidon, 20 mM NaF, 20 mM ␤-glycerophosphate, 1 mM Na 3VO 4, 1 mM DTT). Cell extracts (from 2 ⫻ 10 6 cells) were rocked with GST-c-Myc (1–181) protein (30 ␮g)/GSH-Sepharose beads for 2 h at 4°C. Beads were washed with Hepes-binding buffer (1 ml ⫻ four times), and bound proteins were subjected to in-gel kinase assay. In-gel kinase assay was performed as described using GST-c-Jun (200 ␮g/ml) as a polymerized substrate (21). Kinase activities were analyzed with a BAS2000 Bio-Image analyzer (Fujix, Tokyo). Detection of phosphorylated c-Myc and phosphorylated JNK. For the detection of phosphorylated c-Myc in vivo, 293T cells were cotransfected with pc3GFhcmyc (3 ␮g) and each ASK1-expressing plasmid (1 ␮g). Two days after transfection, cells were suspended in cytosolic buffer (0.2% NP-40, 20 mM Tris–HCl pH 7.6, 137 mM NaCl, 20 mM NaF, 20 mM ␤-glycerophosphate, 1 mM Na 3VO 4, 1.5 mM MgCl 2, 1 mM PMSF, 20 ␮M MG132, 10 ␮g/ml leupeptin, 1 ␮M pepstatin) and the particle nuclear pellet was lysed with 1 ⫻ Laemmli sample buffer then sonicated. Cells were lysed by each buffer (same volume) and an equal volume of each lysate was resolved by 10% SDS–PAGE and transferred to filters. Filters were blocked with 5% low-fat milk/Tris-buffered saline (TBS)– 0.1% Tween 20 (TBST) at room temperature, and phosphorylation of Thr-58/Ser-62 or Ser-71 of c-Myc was detected using anti-phospho c-Myc(Thr58/Ser62) antibody (␣-P-T58/S62) and anti-phospho c-Myc(Ser71) antibody (␣-P-S71). For the detection of JNK, cells after treatment were once washed with phosphate-buffered saline (PBS) and frozen with liquid nitrogen. They were then lysed in 1 ⫻ Laemmli sample buffer containing phosphatase inhibitors (20 mM NaF, 1 mM Na 3VO 4, 20 mM ␤-glycerophosphate), heat-denatured, and sonicated. Equal amounts of protein were resolved by 12% SDS– PAGE, transferred and hybridized with first antibody such as antiphospho JNK (G-7). First antibodies diluted with 3% BSA/TBS were used for an overnight incubation at 4°C and after hybridization with secondary antibodies conjugated with horseradish peroxide, signals were detected with ECL detection reagent (Amersham–Pharmacia Biotech, Buckinghamshire, England). Western blot analysis. NIH3T3 cells after treatment were lysed in the radioimmunoprecipitation assay (RIPA)-S buffer (20 mM Tris–HCl pH 7.6, 137 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF) and cleared lysates were obtained by centrifugation. In the case of 293T cells for the detection of high-molecular-weight shifted c-Myc, cells were lysed in urea buffer (8 M urea, 100 mM sodium phosphate, pH 8, 10 mM imidazole). Equal amounts of protein were resolved by 8 or 10% SDS–PAGE and transferred to nitrocellulose membrane. Filters were blocked in 5% low-fat milk/PBS– 0.1% Tween 20 (PBST) and hybridized with first antibodies (06340 for mouse c-Myc in 3% low-fat milk/PBS, 9E10 for human c-Myc, anti-JNK1 (C-17) for JNK, and anti-HA (Y-11) for HA-ASK1 in 5% low-fat milk/PBST). After hybridization with secondary antibodies conjugated with horseradish peroxidase, immunocomplex was detected with ECL detection reagent. In vitro ubiquitination of c-Myc. For in vitro ubiquitin conjugation assay, 35S-methionine-labeled c-Myc protein was prepared by in vitro transcription/translation with rabbit reticulocyte lysate (Promega, Madison, WI) in a 50-␮l volume. Ubiquitin conjugation assay was carried out in a 30-␮l reaction mix (8 ␮l of translation mix, 40 mM Tris–Cl, pH 7.6, 5 mM MgCl 2, 2 mM DTT, 5 mM ATP, 0.2 mM MG132, 0.2 mg/ml ubiquitin, 20 ␮g/ml ubiquitin-aldehyde, 5 ␮l of untreated rabbit reticulocyte lysate, with or without recombinant GST-fused protein) at 37°C for 30 min, and stopped by adding 10 ␮l of 4 ⫻ Laemmli sample buffer. Samples were resolved by 10% SDS– PAGE and analyzed by BAS2000.

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Pulse-chase analysis of c-Myc protein. One day after transfection, 293T cells were equally divided into five wells, and the next day, cells were pulse-labeled with 35S-methionine (200 ␮Ci/ml) for 1 h. Pulselabeled cells were harvested and lysed in RIPA-M buffer (1% NP-40, 0.25% sodium deoxycholate, 20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 2 mM EDTA, 1 mM PMSF, 1 ␮g/ml leupeptin, 1 ␮M pepstatin). Cell lysates obtained by centrifugation were diluted with three volumes of TBS and precleared by control rabbit Ig/protein G4FF beads for 1 h at 4°C. c-Myc protein was immunoprecipitated from precleared lysate with anti-c-Myc antibody (06340) for 3 h at 4°C, and then beads were washed with HS buffer (1% NP-40, 25 mM Tris–Cl, pH 7.5, 420 mM NaCl, 2 mM EDTA, 1 mM PMSF) (1 ml ⫻ five times) followed by D buffer (1% NP-40, 25 mM Tris–Cl, pH 7.5, 2 mM EDTA, 1 mM PMSF) (one time). Samples were resolved by SDS–PAGE and analyzed by BAS2000.

RESULTS

Next, we investigated a functional role of ASK1 for c-Myc-mediated apoptosis triggered by UV. Transient expression assay in HeLa cells showed that c-Mycmediated apoptosis triggered by UV was suppressed by dominant-negative ASK1(KM) (Fig. 1D). Likewise, transient expression of wild-type ASK1 induced apoptosis in wild-type c-Myc-expressing CM-9 cells without UV irradiation, but not in either parental NIH3T3 or non-phosphorylated mutant c-Myc (S62A/S71A)expressing S6271A-13 cells (Fig. 1E). In addition, we compared the effect of another kinase, MEK1 that activates the ERK-pathway, on c-Myc-expressing NIH3T3 cell lines because ERK could phosphorylate c-Myc at Ser-62. In contrast to ASK1, constitutivelyactive MEK1(S218E/S222E, ⌬32-51) did not induce apoptosis (Fig. 1E). These observations indicated that both Ser-62 and Ser-71 residues of c-Myc are required for the ASK1 signaling-induced apoptosis in NIH3T3 cells.

ASK1-Signaling Induces Phosphorylation of c-Myc at Ser-62 and Ser-71

ASK1-Signaling Enhances c-Myc Protein Level Dependent on Ser-62 and Ser-71

The activation of ASK1 in turn leads to activation of the JNK/p38-pathway (23), and our recent findings demonstrated that c-Myc protein is phosphorylated at both Ser-62 and Ser-71 by JNK (21). Here we examined the involvement of ASK1 in UV-induced JNK activation. We introduced flag-JNK with or without a kinaseinactive and dominant negative form HA-ASK1 (KM) (29) into 293T cells, and measured the activation of flag-JNK after UV irradiation (Fig. 1A). As expected, coexpression of a dominant-negative HA-ASK1 (KM) remarkably suppressed the activation of JNK by UV irradiation (Fig. 1A). To demonstrate that ASK1signaling activates JNK bound to c-Myc, we performed a pull down assay with GST-c-Myc (1–181). This experiment confirmed that p46 and p54 JNK activated by wild-type ASK1 and N-terminal region-deleted constitutively-active ⌬NASK1 but not kinase-inactive ASK1 (KM), were coprecipitated with GST-c-Myc (1– 181) (Fig. 1B). Moreover, we studied whether the ASK1-JNK-signaling pathway induces c-Myc phosphorylation. To detect the phosphorylation of c-Myc, we compared the phosphorylation status of c-Myc by Western blotting analysis with phospho-c-Myc specific antibodies in the presence of ASK1 derivatives such as wild-type ASK1, ⌬NASK1, and ASK1 (KM) (Fig. 1C). The result clearly showed that the phosphorylation of c-Myc at Ser-62 and Ser-71 was enhanced in the nuclear fraction in the presence of wild-type ASK1 and ⌬NASK1, but not ASK1 (KM). In addition, we also found that the phosphorylation of cytosolic c-Myc protein was weakly affected by ASK1-signaling. These observations indicated that the ASK1-signaling is linked to the nuclear c-Myc protein through phosphorylation.

During experiments, we noticed that the expression level of c-Myc protein increased at 1 h after UV irradiation in NIH3T3 cells. Then, to examine the effect of phosphorylation at Ser-62 and Ser-71 on the accumulation of c-Myc induced by UV irradiation, we compared the protein level of wild type c-Myc and nonphosphorylated mutant c-Myc (S62A/S71A) after UV irradiation. This experiment showed that the protein expression level of mutant c-Myc (S62A/S71A) was little increased by UV irradiation unlike that of wild-type c-Myc (Fig. 2A). Here, we noticed that the transient accumulation of wild-type c-Myc, but not c-Myc (S62A/ S71A), at 1 h after UV irradiation was accompanied by the appearance of high-molecular-weight shifted c-Myc. Interestingly, we found that coexpression of ASK1 induced an increase of high-molecular-weight shifted c-Myc in transient transfection assay (Fig. 2B). To examine the role of phosphorylation at Ser-62 and Ser-71 on the high-molecular-weight shifted c-Myc, we constructed a phosphorylated analogous mutant c-Myc (S62E/S71E) because replacement of Ser residues with Glu can mimic the effect of phosphorylation on c-Myc (14). We found that the expression of the shifted c-Myc (S62E/S71E) to be increased compared with that of wild-type c-Myc (Fig. 2C). These results indicated that the high-molecular-weight shifted c-Myc was induced by the phosphorylation at Ser-62 and Ser-71. Collectively, these observations suggested that the phosphorylation of c-Myc at Ser-62 and Ser-71 induced by ASK1signaling was involved in the accumulation of intact and modified c-Myc protein. However, northern blot analysis indicated that the increase of c-myc mRNA was smaller than that of c-Myc protein (data not shown). Thus, posttranscriptional/translational modi-

Apoptosis assay. Transient apoptosis assay was performed as described (21). In brief, cells were transfected with each expression plasmid with marker plasmid pcDNA3EGFP. At 40 h posttransfection, cells were fixed and the EGFP-positive cells were examined for normal or apoptotic morphology under a fluorescence microscope.

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FIG. 1. ASK1-signaling activates c-Myc-mediated apoptosis through phosphorylation of c-Myc at Ser-62 and Ser-71. (A) UV irradiation activates JNK through ASK1. 293T cells were cotransfected with pflag-jnk1 (0.1 ␮g) in the presence or absence of pcDNA3/HA-ASK1 (KM) (0.9 ␮g). After 2 days, cells were irradiated or not with UVC (100 J/m 2) followed by a further 10 min culture. Then, flag-tagged JNK1 was immunoprecipitated with anti-Flag M2 antibody, and subjected to in vitro kinase assay using GST-c-Jun (1–92). The expression of Flag-tagged JNK1 and HA-tagged ASK1 (KM) in 293T cells detected by Western blot analysis and the kinase activity of Flag-JNK1 are demonstrated (top panels). Negative control sample (no plasmid was transfected) was also subjected to same treatment (left sample). Kinase activity of Flag-JNK1 was analyzed with BAS 2000 Bio-Image analyzer (Fujix), and activation ratio obtained from the result of two independent experiments is shown (bottom graph). (B) Activation of JNK was detected by in-gel kinase assay. GST-c-Myc (1–181) bound JNK was precipitated from each ASK1-transfected 293T cell lysate by pull-down assay, and resolved on 11% SDS–polyacrylamide gel polymerized with GST-c-Jun (1–92). Arrowheads indicate activated p46 and p54 JNK. A representative result of in-gel kinase assay is shown. (C) Phosphorylation of c-Myc at Ser-62 and Ser-71 detected by phospho-c-Myc specific antibodies. c-myc-expressing plasmid (3 ␮g) was cotransfected with wild-type ASK1- or, kinase-negative ASK1(KM)-expressing plasmid (1 ␮g) in 293T cells. After 24 h, cytosolic and particle cell lysates were prepared, and phosphorylated c-Myc was detected by subsequent Western blot analysis using anti-phospho c-Myc(Ser71) polyclonal antibody (␣-P-S71) (top panel), anti-phospho c-Myc(Thr58/Ser62) polyclonal antibody (␣-P-T58/S62) (middle panel), and anti-c-Myc monoclonal antibody (9E10) (bottom panel). (D) UV irradiation induced c-Myc-dependent apoptosis through the ASK1-pathway. HeLa cells were cotransfected by pc3GFhcmyc plasmid (2.5 ␮g) with pcDNA3/HA-ASK1(KM) or control plasmid (2.5 ␮g), and after 2 days culture, were irradiated with UVC (30 J/m 2). At 4-h after irradiation, cells were fixed and cell morphologies of EGFP-positive cells were examined under a fluorescent microscope. More than 300 cells were analyzed and % apoptotic cell death was determined from the results of two independent 1316

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fications might have occurred on the c-Myc protein after UV irradiation.

tively-active ⌬NASK1 was introduced into NIH3T3 cells and stably transfected clones 3T3(⌬N) cells were isolated. The expression of ⌬NASK1 protein and the activation of the JNK-pathway were confirmed by Western blotting with anti-HA and anti-phospho JNK antibodies (Fig. 3A). Then, we investigated the ubiquitination status of c-Myc in vivo and in vitro. First, parental NIH3T3 cells along with constitutively active ASK1 expressing 3T3(⌬N) cells were treated with a reversible proteasome inhibitor, MG132, and subsequently, the accumulation kinetics of ubiquitinated c-Myc was compared in these two cell lines by Western blot analysis. It was found that the accumulation kinetics of the high-molecular-weight polyubiquitinated c-Myc was similar in the parental NIH3T3 and the 3T3(⌬N) cells (Fig. 3B). Second, to examine the effect of Ser-62 and Ser-71 on the ubiquitination process, in vitro translated c-Myc proteins such as wild-type, S62E/S71E and S62A/S71A were used for in vitro ubiquitin conjugation assay in a rabbit reticulocyte lysate system (Fig. 3C). This experiment demonstrated that the wild-type-, S62E/S71E- and S62A/S71A-c-Myc were ubiquitinated at comparable level in the presence or absence of papillomavirus oncoprotein E6 that is known to promote ubiquitination of c-Myc (5). These results indicated that the ubiquitination of c-Myc appeared to be independent of both ASK1-signaling and phosphorylation of c-Myc at Ser-62 and Ser-71. Next, to analyze the degradation kinetics of the ubiquitinated c-Myc protein, NIH3T3 and 3T3(⌬N) cells were pretreated with MG132 for 6 h and then the inhibitor was washed out to restart the degradation of ubiquitinated c-Myc protein by proteasome. Here, we found that the degradation kinetics of ubiquitinated c-Myc were significantly attenuated in 3T3(⌬N) cells compared with parental NIH3T3 cells (Fig. 3D, as seen in lanes 3–5 and lanes 8 –10). This result indicated that the degradation of ubiquitinated c-Myc was suppressed by ASK1-signaling, and thus, the highmolecular-weight shifted c-Myc protein observed in Fig. 2 would be product of an accumulated ubiquitinated c-Myc triggered by the phosphorylation of Ser-62 and Ser-71.

ASK1-Signaling Attenuates the Degradation of Ubiquitinated c-Myc

Effect of Ser-62 and Ser-71 on c-Myc Protein Stability

As the shifted c-Myc appeared to have been modified by ubiquitination, our results suggested that the phosphorylation is involved in the regulation of c-Myc by the ubiquitin-proteasome system. To examine the association between ASK1-signaling and the posttranscriptional/ translational modification of c-Myc, HA-tagged constitu-

The results above indicated that the phosphorylation of c-Myc at Ser-62 and Ser-71 caused the attenuation of c-Myc protein degradation. To clarify the significance of ASK1-signaling-induced phosphorylation of c-Myc to the protein stability, we analyzed the degradation kinetics of c-Myc protein in the presence and absence of

FIG. 2. Ser-62 and Ser-71 are required for c-Myc protein accumulation and mobility shift. (A) Wild-type c-Myc- and nonphosphorylated mutant c-Myc (S62A/S71A)-expressing NIH3T3 cells (CM-9 and S6271A-13 clones, respectively) were irradiated with UV (100 J/m 2), and cell lysates from equal numbers of cells were prepared at the time indicated. The expression of c-Myc protein was detected by Western blot analysis using anti-c-Myc antibody (␣-c-Myc, 9E10). (B) ASK1-signaling induced high-molecular-weight shifted c-Myc. pc3GFhcmyc plasmid (1.5 ␮g) and pcDNA3/His-ubiquitin (2 ␮g) were cotransfected into 293T cells in the presence or absence of pflag-jnk (1.5 ␮g) and pcDNA3/HA-ASK1 (0.1 ␮g). At 20 h posttransfection, cell lysates were prepared and the effect of ASK1-JNK pathway on c-Myc protein mobility shift was examined by Western blot analysis using ␣-c-Myc (9E10). High-molecular-weight shifted c-Myc was shown as High-Mw c-Myc. (C) High-molecular-weight shifted c-Myc (S62E/S71E). 293T cells were cotransfected with pcDNA3/Hisubiquitin (2.5 ␮g) and each pc3GFhcmyc (2.5 ␮g). At 40 h posttransfection, c-Myc protein mobility shift was examined by Western blot analysis as above. High-molecular-weight shifted c-Myc was shown as High-Mw c-Myc.

experiments. (E) ASK1-pathway induced c-Myc-mediated apoptosis dependent on Ser-62 and Ser-71. Parental NIH3T3, wild-type c-myc expressing CM-9 and mutant c-myc(S62A/S71A) expressing S6271A-13 cells were cotransfected by pcDNA3EGFP plasmid (2 ␮g) with pcDNA3/HA-ASK1, pFC-MEK1 (S218E/S222E, ⌬32–51) or control plasmid (3 ␮g). After 2-days culture in the presence of 10% serumcontaining medium, cells were fixed and cell morphologies of EGFP-positive cells were photographed under a fluorescent microscope (top panel, magnification ⫻ 100). EGFP-positive cells were analyzed and % apoptotic cell death was determined as above (bottom graph). Data show the means and standard deviations from the results of two independent experiments. 1317

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ASK1 in 293T cells by pulse-chase labeling experiments. We found that the degradation of c-Myc protein was attenuated early on when ASK1 was coexpressed (Fig. 4A, left side). In contrast, that of mutated c-Myc (S62A/S71A) was not changed despite the ASK1 coexpression (Fig. 4A, right side). These results indicated that the ASK1-signaling attenuated the degradation of c-Myc through phosphorylation at Ser-62 and Ser-71. In addition, to confirm the effect of phosphorylation at Ser-62 and Ser-71 on c-Myc protein stability, we compared the protein stability of the wild-type, S62E/ S71E, and S62A/S71A c-Myc proteins. It was found that the degradation of c-Myc (S62E/S71E) was attenuated, but that of c-Myc (S62A/S71A) was rather potentiated, compared to that of wild-type c-Myc, especially in the early phase (Fig. 4B). These results indicated that the phosphorylation at Ser-62 and Ser-71 affected the protein stability of c-Myc. DISCUSSION

FIG. 3. ASK1-signaling affects the degradation of ubiquitinated c-Myc but not the ubiquitination process. (A) ASK1-expressing NIH3T3 cell clones. The expression of HA-tagged constitutivelyactive ⌬NASK1 was shown by Western blot analysis in transfected 3T3(⌬N) cells (left panel). The activated JNK (P-JNK) and total amounts of JNK1 (JNK) were also detected by Western blot analysis using anti-phospho JNK (G-7) and anti-JNK (C-17) antibody (right panel). (B) Accumulation kinetics of ubiquitinated c-Myc. NIH3T3 and constitutively-active ASK1-expressing 3T3(⌬N) cells were treated with a proteasome inhibitor, MG132 (20 ␮M), and the accumulation of ubiquitinated c-Myc was examined by Western blot analysis using ␣-c-Myc (06340). Asterisks indicate high-molecularweight polyubiquitinated c-Myc protein. (C) In vitro ubiquitin conjugation assay. In vitro translated 35S-methionine labeled c-Myc (wildtype, S62E/S71E, and S62A/S71A) was used to examine the effect of Ser-62 and Ser-71 on the ubiquitination process. Ubiquitination in the presence or absence of GST-E6 (2.5 ␮g) was performed at 37°C for 30 min, and a negative control reaction was run at 0°C. Polyubiquitinated c-Myc protein is indicated as Ub-c-Myc. (D) Degradation of ubiquitinated c-Myc. NIH3T3 and 3T3(⌬N) cells were pretreated with the proteasome inhibitor MG132 (20 ␮M) for 6 h without serum, and then the inhibitor was washed out following further incubation

The c-Myc protein is a short-lived nuclear phosphorylated transcriptional factor involved in the cell cycle progression, cell transformation and apoptosis (30). The c-Myc protein is phosphorylated at multiple sites including Thr-58, Ser-62, and Ser-71 in vivo (17), with the phosphorylation of both Ser-62 and Ser-71 by JNK shown to be important for the proapoptotic activity of c-Myc (21). Here we demonstrated the importance of Ser-62 and Ser-71 to the stability of protein that is under the control of a stress-activated ASK1-JNK pathway. ASK1 is a mediator of a death receptor such as Fas or TNF receptor through interaction with Daxx and TRAF2, and known to be activated by various stress stimuli (31). Previous studies showed that the ASK1-signaling functions as an apoptotic signal through activation of the JNK- and p38 MAPKpathways which leads to the caspase activation during apoptosis triggered by Fas-, TNF-, Taxol- and cisplatin (23–25). However, the mechanism activating caspasecascade downstream of the JNK-pathway during stress-triggered ASK1-mediated apoptosis is not fully understood. We showed in this study that Ser-62 and Ser-71 of c-Myc were also involved in the ASK1mediated apoptosis. Based on these observations, we presume that the ASK1-signaling stabilizes c-Myc protein via the JNK-pathway to stimulate the proapoptotic activity of c-Myc.

of the cells with serum-free medium. After removal of MG132, ubiquitinated c-Myc protein was detected by Western blotting. Cell lysates prepared at time 0 without MG132-treatment were also examined (lanes 1 and 6). Asterisks indicate high-molecular-weight ubiquitinated-c-Myc. As a control, the result of Western blot analysis of p27 Kip is shown (bottom panel).

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tations of c-Myc in the N-terminal region and protein stability (8, 9). The phosphorylation of Ser-62 in Mycbox I by a mitogenic MAPK appears to enhance the stability of c-Myc in quiescent cell (18). These observations raise the possibility that the phosphorylation in the N-terminal region of c-Myc interferes with the negative regulation by the proteasome system, contributing to the growth promoting ability of c-Myc. However, inconsistent results have been reported concerning the role of Thr-58 and Ser-62 in the functional activity of c-Myc (17), and unfortunately, the effect of Thr-58 and Ser-62 on the ubiquitinational regulation of c-Myc has not been examined in those studies. We did not observe any significant difference between single alanine substituted forms such as c-Myc (T58A), c-Myc (S62A), and wild-type c-Myc regarding the ubiquitination level in normal growing 293T cells (data not shown). Thus, further study is required to clarify the molecular mechanism behind the effect of phosphorylation at Thr-58 and Ser-62 on the proteolytic processing of c-Myc in growing cells. Our findings suggest that the ASK1-signaling may change susceptibility of the c-Myc protein for the proteasome system through phosphorylation at Ser-62 and Ser-71, and the ASK1-signaling-induced c-Myc protein accumulation thus must be an important mechanism for ASK1-induced apoptosis. We suspect that the accumulation mechanism of proapoptotic proteins such as c-Myc may be an attractive target for apoptosis-driving cancer chemotherapy in c-Mycoverexpressing tumors, and our study will provide insight into the development of molecular-based anticancer drugs with respect to both stress-activated kinasecascade and proteasomal regulation of proapoptotic proteins. FIG. 4. ASK1-signaling modulates protein stability of c-Myc. (A) The degradation kinetics of c-Myc protein was analyzed by pulsechase experiments. 293T cells were cotransfected with pc3GFhcmyc or pc3Gfhcmyc (S62A/S71A) plasmid (5 ␮g) and pcDNA3/HA-ASK1 or pcDNA3 (0.2 ␮g). After 48 h, cells were pulse-labeled with [ 35S]methionine for 1 h. Cell lysates were prepared at the time indicated. 35 S-labeled c-Myc protein was immunoprecipitated (IP) by anti-c-Myc polyclonal antibody (06340), resolved by SDS–PAGE, dried, and analyzed with a BAS2000 Bio-Image analyzer. Quantitative results by BAS2000 analysis are shown in the bottom graph, and % of remaining radioactivity present for c-Myc at each time points is demonstrated. (B) Protein stability of wild-type c-Myc, c-Myc (S62E/S71E) and c-Myc (S62A/S71A) was analyzed by pulse-chase experiments as described for A.

ACKNOWLEDGMENTS We thank Drs. A. Tomida, H. Seimiya, N. Fujita, T. Mashima, and Z. Chen for providing reagents and valuable suggestions. This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan for Cancer Research (to K.N. and to Y.K.) and in part by a Grant-in-Aid from the Ministry of Health and Welfare of Japan for the Second-Term Comprehensive 10-Year Strategy for Cancer Control (to Y.K.).

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