ResearchArticle

DNA Damage–Dependent Translocation of B23 and p19ARF Is Regulated by the Jun N-Terminal Kinase Pathway

Orli Yogev,1 Keren Saadon,1 Shira Anzi,1 Kazushi Inoue,2 and Eitan Shaulian1

1Department of Experimental Medicine and Cancer Research, Hebrew University Medical School, Jerusalem, Israel and 2Departments of Pathology/Cancer Biology, Wake Forest University Health Sciences, Winston-Salem, North Carolina

Abstract arrest or apoptosis. However, increased tumor development in The dynamic behavior of the nucleolus plays a role in the triple knockout mice nullizygous for ARF, p53, and Mdm2 showed detection of and response to DNA damage of cells. Two that ARF acts also in a p53-independent manner (11). Some of the nucleolar proteins, p14ARF/p19ARF and B23, were shown to p53-independent activity is attributed to its ability to reduce rRNA translocate out of the nucleolus after exposure of cells to DNA- processing (12, 13) and inhibit oncogene-induced transcription damaging agents. This translocation affects multiple cellular (14, 15). functions, such as DNA repair, proliferation, and survival. In ARF augments p53 activity mainly in response to oncogenic this study, we identify a pathway and scrutinize the mecha- stress. Its expression is up-regulated in response to deregulated nisms leading to the translocation of these proteins after oncogenic activity due to elevated transcription governed by several transcription factors, such as E2F (16), c-myc (17), AP-1 exposure of cells to DNA-damaging agents. We show that redistribution of B23 and p19ARF after the exposure to (18), or by oncogenic Ras (19). A second mechanism regulating ARF activity involves the control of its subcellular localization (3). The genotoxic stress occurs preferentially when the c-Jun-NH2- kinase (JNK) pathway is activated and is inhibited when the nucleolar localization of ARF raised a model suggesting that ARF JNK pathway is impaired. The stress-induced translocation of sequesters Mdm2 to the nucleolus and thereby releases p53 from alternative reading frame (ARF) is JNK dependent and its inhibitory effects (11). However, later work (20) suggested that mediated by two activator proteins, c-Jun and JunB. Thr91 ARF may also posses growth-suppressing activity when it is located and Thr93 of c-Jun are required for the translocation, but the in the nucleoplasm. Moreover, recent studies have actually transcriptional activity of c-Jun is dispensable. Instead, c-Jun suggested that retention of ARF in the nucleolus inhibits its interacts with B23 in a dose-dependent manner. c-Jun itself is activity (3, 21), whereas its redistribution to the nucleoplasm enables extended interaction with Hdm2 (3). Furthermore, this last excluded from the nucleolus in a JNK-dependent manner. Hence, we suggest that c-Jun translocates B23 and ARF from study showed that ARF is redistributed in the nucleus in response the nucleolus after JNK activation by means of protein to DNA damage (3). interactions. In senescent cells, JNK activity and c-Jun levels ARF localization is mostly governed by interactions with the are reduced concomitantly with ARF nucleolar accumulation, abundant nucleolar protein B23 (22, 23). B23 functions as a and UV radiation does not cause the translocation of ARF. molecular chaperone that shuttles between the nucleus and the cytoplasm. It affects cellular proliferation, resistance to DNA [Cancer Res 2008;68(5):1398–406] damage–dependent cell death, and maintenance of genomic stabi- lity, in addition to its activities in ribosome biogenesis and rRNA Introduction processing (24). Its interactions with ARF direct ARF to the A growing body of evidence attributes a new role to the nucleolus. In acute myeloid leukemia cells containing cytoplasmic nucleolus, extending beyond its traditional activity of ribosome mutants of B23, ARF is cytoplasmic as well (25, 26), and in cells production, stress response (1, 2). Among the proteins that deficient in B23, ARF is excluded from the nucleolus (27). comprise it, some are important regulators of the cell cycle and Independently of ARF, B23 interacts with two key proteins in cell ARF stress responses. The localization of two of these proteins, p14 / cycle regulation, Hdm2 and p53 (5, 28). Both of these interactions ARF p19 and B23 (nucleophosmin, numartin, or NO38), is regulated seem to lead to p53 stabilization (5, 28), in agreement with this by genotoxic stress (3–5). hypothesized role of the protein in the maintenance of genomic ARF ARF The human p14 /mouse p19 are important tumor sup- stability. Moreover, increased expression of B23 has been ARF pressors. Knockout of p19 underscored its importance in cancer associated with improved DNA repair (29), and B23 1–deficient ARF prevention, as p19 -deficient animals are more prone to cancer cells exhibit increased H2AXg phosphorylation (27). Its redistribu- (6). The tumor suppressor effects of alternative reading frame tion from the nucleolus in response to cytotoxic drugs and (ARF) are mainly relayed by regulation of the p53 pathway (7, 8). genotoxic stresses, such as the inhibition of RNA polymerase I and ARF interacts and inactivates Mdm2, thereby increasing the II or exposure to DNA intercalating agents or UV radiation, further stability and activity of the tumor suppressor p53 (9, 10). Hence, supports its role in the response to DNA damage (3–5, 30, 31). It increased expression of ARF results in a p53-dependent growth seems that the translocation from the nucleolus is an important regulatory step that controls the effects of B23 on DNA repair. Several members of the AP-1 transcription factor family are Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). highly responsive to genotoxic stress. c-Jun and JunB are strongly Requests for reprints: Eitan Shaulian, Department of Experimental Medicine and induced by DNA-damaging agents such as UV radiation (32). Cancer Research, Hebrew University Medical School, Ein Karem, Jerusalem 91120, Induction of c-Jun by DNA damage is mostly dependent on its Israel. Phone: 972-2-6757617; Fax: 972-2-6414583; E-mail: [email protected]. I2008 American Association for Cancer Research. upstream kinase, the c-Jun amino-terminal kinase (JNK; refs. 33, doi:10.1158/0008-5472.CAN-07-2865 34). However, JNK-independent inductions of c-Jun and JunB were

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. JNK Regulates ARF and B23 Nuclear Distribution also observed (35). JNK phosphorylates c-Jun at Ser63 and Ser73 point. We defined redistribution of ARF and B23 when cells exhibiting and Thr91 and Thr93, and regulates its transcriptional capacity normal staining of 4¶,6-diamidino-2-phenylindole (DAPI) did not exhibit and ability to interact with other proteins (36–38). Induction of visible nuclear foci of ARF and B23. In all cases, the fraction of cells c-Jun is important for UV-driven apoptosis (39). Reports on the expressing nucleolar ARF after treatment was scored. The Y axis title and the figure legends specify the 100% reference point of each experiment. effects of JNK on JunB are mixed. Although early reports suggested Each experiment was repeated at least thrice. that JunB does not possess JNK phosphorylation sites (40), a later 102 104 Immunoprecipitations and immunoblotting. For immunoprecipita- report showed phosphorylation of Thr and Thr by JNK (41). tions, cell were lysed with NP40 lysis buffer [50 mmol/L Tris-HCl (pH 8.0), However, the significance of the induction of JunB by UV is not 120 mmol/L NaCl, 0.5% NP40, 1 mmol/L EDTA plus proteinase inhibitors clear. (1:200 Cocktail III; Calbiochem), 0.1 mmol/L sodium orthovanadate, and Currently, the pathways and mechanisms that regulate the 0.1 mmol/L sodium fluoride]. Two milligrams of total cell extract were used redistribution of B23 and ARF after exposure to DNA-damaging per reaction, and standard immunoprecipitation protocols were used agents are unknown. In light of the importance of these throughout. Nuclear extracts were used for detection of JunB by immu- translocations, we explored the molecular mechanisms that noblotting analysis. Each experiment was repeated at least thrice. regulate them. We show that activation of the JNK pathway is required for dispersal of B23 and p19ARF from the nucleolus. The stress-induced translocation of ARF is mediated by JunB and c-Jun Results whose phosphorylation at Thr91 and Thr93 is required for the JNK activity regulates B23 and p19ARF distribution. The translocation. Moreover, we show that B23 directly interacts with recent studies describing the translocation of the nucleolar c-Jun, and that JNK-dependent translocation of c-Jun translocates proteins B23 and p14ARF after exposure of cells to genotoxic associated B23. We also show that when JNK activity is reduced stresses (3–5) prompted us to examine whether signaling speci- due to senescence, UV radiation does not trigger the translocation ficity exists in the redistribution of these proteins. To examine the of ARF. phenomena in relatively normal cells, we irradiated mouse embryo fibroblasts (MEF; passage 3) with 30 J/m2 UV and 20 Gy of ionizing ARF Materials and Methods radiation and then stained for p19 and B23 at 3 h after irradiation. As previously reported, in untreated cells, the majority Cell lines and tissue culture. All the cells were grown in DMEM of p19ARF colocalized with B23 in the nucleoli (Fig. 1A), but the supplemented with 10% fetal bovine serum. The cell lines used in this work ARF À À À À have been previously described as follows: c-jun / p53 / by Shaulian et al. nucleolar staining of p19 and B23 was significantly reduced (39), 63/73 Ala by Behrens et al. (42), and 91/93 Ala by Laderoute et al. (43). after exposure to UV radiation. Interestingly, no change in the ARF Plasmids. Expression vectors for c-jun, junB, and junD were described distribution of p19 and B23 was observed after exposure of the previously (44). cDNAs for wild-type c-Jun and 63/73Ala were cloned into cells to ionizing radiation. To determine whether the reduction in pcDNA3 to generate cytomegalovirus (CMV)-derived expression vectors. the staining after exposure to UV radiation reflects a reduction in CMV 91/93 Ala was obtained from G. Li and R. Johnson (University of the levels of p19ARF or B23 that anchors p19ARF to the nucleolus, California, San Diego, California). The conversion of Arg 272 and Lys 273 to we measured the level of both proteins before and 3 h after glutamic acids was performed using the QuikChange site-directed UV exposure. Within this time frame, the changes in the levels of mutagenesis kit II, according to the manufacturer’s instructions (Stra- p19ARF and B23 were minor (Fig. 1B). This suggests that the tagene). Expression vectors for green fluorescent protein (GFP)-fibrillarin reduced nucleolar staining reflects changes in p19ARF distribution and JDP2 were obtained from Y. Shav-Tal and A. Aronheim (Bar Ilan ARF University, Israel and the Technion-Israel Institute of Technology, Israel, and not a change in the total amounts of p19 or B23. respectively). ShRNA for JunB was previously described (18). One of the most significant differences between UV and ionizing Antibodies. Polyclonal anti–c-Jun antibody (H-79; Santa Cruz Biotech) radiations is the pathways that they activate. Specifically, there is was used for c-Jun immunoprecipitation, immunofluorescence staining, and a difference in their abilities to activate the JNK pathway (39). immunoblotting. Monoclonal antibodies KM-1 (Santa Cruz Biotech) and C-J Therefore, we explored a possible correlation between JNK 4C4 (Abcam) were used to detect phospho-Ser63 and phospho-Thr91-c-Jun, activation and translocation of p19ARF in response to the different ARF respectively by immunoblotting. Polyclonal anti–p19 ab80 (Abcam) genotoxic stresses that activate JNK. To this end, we exposed ARF was used for p19 staining and immunoblotting. Monoclonal mouse MEFs to 30 J/m2 UV, 20 Gy ionizing radiation, 50 Amol/L cisplatin anti-B23 FC-61991 (Zymed; Invitrogen) was used for immunostaining, (CDDP), or 50 Amol/L mitomycin C (MMC) and examined their immunoprecipitation, and immunoblotting. Monoclonal anti-actin C4 (MP ability to activate JNK and induce p19ARF translocation. JNK Biomedical) was used for immunoblotting. For detection of JunB, polyclonal activation was determined by measuring the extent of Thr183/ anti-JunB N-17 (Santa Cruz Biotech) was used. HA-Tag 6E2 (Cell Signaling 185 Technology) was used for the detection of HA-tagged proteins by Tyr phosphorylation, which is known to activate it (reviewed by immunofluorescence. For detection of JNK activation, anti–phospho- ref. 45). Thr183/Tyr185 JNK (Cell Signaling Technology) was used. Total JNK levels As depicted in Fig. 1C, UV is the most efficient activator of JNK were determined using the monoclonal antibody G151-333 (BD Biosciences and ionizing radiation is the weakest one. At the concentration PharMingen). examined, MMC activates JNK better than ionizing radiation but Immunofluorescence staining. Cells were plated on coverslips and less efficiently than CDDP. The stress-dependent translocation by usually harvested 3 h after UV irradiation. Cells were fixed by a 30-min the different agents was closely correlated to the level of JNK incubation in 4% paraformaldehyde at room temperature. After washes activation. Nucleolar staining was retained in 76% of ionizing with PBS, cells were incubated with the primary antibody for 1 h at room radiation–treated cells, 47% of MMC-treated cells, 18% of CDDP- temperature in PBS containing 3% bovine serum albumin and 0.3% triton. The slides were then washed with PBS with 0.1% Tween and incubated with treated cells, and in only 15% of the UV-treated cells (Fig. 1D). fluorescently labeled secondary antibody for 40 min at room temperature. To directly examine the involvement of JNK in the distribution of ARF ARF Slides were mounted using dibutylpthalate xylene mounting solution (Fluka p19 and B23, we used two cellular models. As p19 expression Biochemika). Images were visualized using an Olympus fluorescence is elevated in p53-deficient cells, it is easily detected by staining. À À microscope. At least 200 randomly selected cells were counted at each Irradiation of p53 / fibroblasts with 30J/m2 UV almost completely www.aacrjournals.org 1399 Cancer Res 2008; 68: (5). March1, 2008

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Figure 1. Translocation of p19ARF from the nucleolus correlates with JNK activation. A, early passage MEFs were irradiated with 30 J/m2 UV or 20 Gy of ionizing radiation and stained for p19ARF, B23, andwith DAPI 3 h after irradiation. Pictures were taken through a fluorescent microscope. B, the protein levels of p19ARF in the irradiated cells were determined 3 h after irradiation with 30 J/m2 UV. Actin servedas a loadingcontrol. C, early passage MEFs were irradiated with 30 J/m2 UV or 20 Gy of ionizing radiation or treated with MMC or CDDP andharvestedat the indicated times (hours after treatment). The level of phosphorylatedJNK was determined using anti–phospho-Thr183/ Tyr185-JNK antibody. JNK1 was used as a loading control. D, p19ARF localization was determined by immunostaining the cells treatedwith UV, ionizing radiation, MMC, andCDDP; andthe portion of cells retaining nucleolar p19ARF at 4 h after treatment was plotted. Nucleolar ARF,the fraction of cells expressing nucleolar ARF from the total cell population.

dispersed both B23 and p19ARF from the nucleolus (Fig. 2A–B). mediate p19ARF redistribution. The plethora of JNK substrates However, a preincubation with 100 Amol/L of JNK inhibitor complicates this task; however, AP-1 genes are clear candidates. As SP600125 at 1 h before treatment inhibited the translocation of depicted in Figs. 2 and 3B, UV induces c-Jun and JunB in a kinetic p19ARF from the nucleolus, and the percentage of cells in which that correlates with the p19ARF redistribution. c-Jun is a primary nucleolar p19ARF was detected increased >15-fold, from 3.98 to JNK substrate. Therefore, we initially examined whether the status 61.8 (Fig. 2A–B). As depicted in Fig. 2C, the significant increase of c-Jun affects the redistribution of p19ARF after exposure to UV. À À À À À À in p19ARF staining in the nucleolus is not a result of any increase in To that end, we irradiated p53 / c-jun+/+ and p53 / c-jun / its overall quantity. In fact, SP600125 treatment slightly reduced the mouse fibroblasts with 30 J/m2 and examined p19ARF localization levels of p19ARF. Significant repression of c-Jun phosphorylation 3 h later. As depicted in Fig. 3A, p19ARF was also dispersed from À À À À served as a control for the inhibitor activity. To corroborate the the nucleoli of p53 / c-jun / cells. However, the translocation notion that JNK controls p19ARF and B23 nuclear localization, we of p19ARF was more extensive in c-jun–expressing cells, as only À À also took a genetic approach and examined ARF translocation 4% of the p53 / c-jun+/+ cells were stained for nucleolar ARF, after UV exposure in JNK+/+ mouse fibroblasts and JNK 1 and 2 whereas nucleolar staining of ARF was observed in 49% of the À À À À À À double-knockout fibroblasts (JNK1,2 / ), in which c-Jun is not p53 / c-jun / (Fig. 3A). Preincubation with the JNK inhibitor phosphorylated by JNK in response to UV, and its induction is increased the fraction of cells stained for nucleolar p19ARF by À À À À À À relatively limited (Supplementary Fig. S1A). As depicted in Fig. 2D, 64% and 27% in p53 / c-jun+/+ and p53 / c-jun / cells, in the absence of JNK expression, the nucleolar staining of both respectively, suggesting that c-Jun is required for p19ARF trans- B23 and p19ARF is morphologically distinct, the nucleoli are smaller, location, but an additional factor contributes as well. Thus, and more B23 is located in the nucleoplasm. However, UV we examined other AP-1 proteins that may be involved in irradiation does not trigger the export of p19ARF and B23 from p19ARF translocation. We knocked down JunB expression with the nucleolus in these cells; thus proving that JNK activity is specific small hairpin RNA (shRNA; ref. 18) in low-passage involved in dispersing them from the nucleolus. Moreover, the MEFs and examined the redistribution of p19ARF after UV À À dispersal of p19ARF in JNK1,2 / after UV exposure was comparable irradiation (Fig. 3C–D). Indeed, repression of JunB expression with that of and JNK+/+ cells treated with SP600125. Both were also inhibited the translocation of p19ARF from the nucleolus in significantly higher than in SP600125-untreated, UV-irradiated MEFs (Fig. 3D). Hence, we conclude that JunB cooperates with JNK+/+ cells (Supplementary Fig. S1B). c-Jun to translocate p19ARF. To test if the effect of c-Jun and c-Jun and JunB mediate the JNK-dependent redistribution of JunB on p19ARF translocation is additive, we knocked down junB À À À À p19ARF by UV. The results presented above show that the JNK in p53 / c-jun / cells to generate c-jun, junB–deficient cells. pathway regulates the translocation of p19ARF from the nucleolus. junB shRNA significantly reduced the levels of JunB (Supple- À À À À Therefore, it was of interest to identify what JNK substrates mentary Fig. S2A). Irradiation of p53 / c-jun / reduced the

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. JNK Regulates ARF and B23 Nuclear Distribution portion of cells expressing nucleolar p19ARF to 14%, and Fig. S2B). In these cells, the pretreatment with SP600125 only inhibition of the JNK pathway prevented translocation and mildly increased the proportion of cells exhibiting nucleolar increased the fraction of cells expressing nucleolar p19ARF to 84% p19ARF. This result suggests that the effects of c-Jun and JunB (Supplementary Fig. S2B). In contrast, the translocation of are additive. À À À À p19ARF in p53 / c-jun / shjunB cells was significantly reduced, Overexpression of Jun proteins translocates p19ARF from the retaining nucleolar p19ARF in 64% of the cells (Supplementary nucleolus. UV radiation activates multiple pathways and triggers intensive transcriptional responses. To examine the net effects of Jun proteins on the redistribution of p19ARF, we transfected À À À À p53 / c-jun / cells with Ha-tagged c-jun, junB, junD, or JDP2 and then localized p19ARF in the transfected cells. As depicted in Fig. 4A, all Jun proteins dispersed p19ARF from the nucleolus. However, significant differences in their potency were observed. c-Jun redistributed p19ARF most efficiently, and only 33% of the transfected cells retained nucleolar p19ARF. JunB was 1.5-fold less efficient and JunD was relatively inefficient, with p19ARF retained in the nucleoli of 77% of the transfected cells (Fig. 4A). Jun specificity in p19ARF redistribution is supported by the fact that in addition to GFP, JDP2, another JNK target, did not redistribute p19ARF when overexpressed under the same conditions (Fig. 4A). The reduced nucleolar staining of p19ARF is not due to reduction in its level, as transduction of c-Jun by viral expression in these cells did not repress p19ARF expression (Supplementary Fig. S3). Two clusters in c-Jun are phosphorylated by JNK after exposure of cells to UV, Ser63 and Ser73, and Thr91 and Thr93 (Fig. 4B). We determined which JNK phosphorylation sites are required for c-Jun–dependent redistribution of p19ARF by transfecting wild-type c-jun or mutants, in which JNK phosphorylation clusters were À À substituted to alanines (Ser63/73Ala and Thr91/93Ala) into p53 / À À c-jun / , and subsequent examination of p19ARF localization (Fig. 4C, left). Interestingly, the canonical JNK phosphorylation sites, Ser63 and Ser73, were dispensable for p19ARF redistribution as Ser63/73Ala dispersed p19ARF comparably to wild-type c-Jun. On the other hand, Thr91 and Thr93 are required as the Thr91/93Ala mutant is impaired in its ability to disperse p19ARF. To avoid any possible transfection artifacts, we also examined the localization of p19ARF in mouse fibroblasts in which Ser63/73Ala or Thr91/93Ala were substituted for the endogenous wild-type c-jun (42, 43). In agreement with the lack of ability of Thr91/93Ala mutant to redistribute p19ARF when overexpressed, p19ARF was not signifi- cantly dispersed in the Thr91/93Ala cells 3 h after exposure to UV, whereas in cells expressing wild-type c-Jun, a significant redistri- bution was observed (Fig. 4C, right). The p19ARF was redistributed less in Ser63/73Ala cells probably due to the lower level of c-Jun induction in these cells shortly after UV exposure (Supplementary Fig. S4A; ref. 42). These results suggest that Thr91 and Thr93 of c-Jun are important for redistribution of p19ARF. c-Jun is a transcription factor that regulates the expression of numerous genes (32). To examine whether the transcriptional activity of c-Jun is required for p19ARF redistribution, we generated a transcriptionally inactive c-Jun mutant by replacing two basic residues, Arg272 and Lys273, in its basic DNA-binding domain with two glutamic acids to Figure 2. Export of p19ARF from the nucleolus is JNK dependent. A, p53À/À generate a mutant incapable of binding DNA. Its ability to induce mouse fibroblasts were irradiated with 30 J/m2 UV. Control fibroblasts were the transcription of AP-1–controlled reporter gene (3XTRE-Luc) left untreated. Some of the cells were preincubated with a JNK inhibitor was compared with that of wild-type c-Jun (Supplementary SP600125 (SP) 1 h before irradiation. The cells were than stained for p19ARF andB23. B, quantitative analysis of p19ARF dispersal, which is shown in A. Fig. S4B). Unlike wild-type c-Jun, which enhances the transcription Y-axis, percentage of cells containing nucleolar p19ARF from the total cell of the reporter gene in a dose-dependent manner and whose tran- population. Empty bars, SP600125-treatedcells; black bars, untreatedcells. C, protein extracts were preparedfrom p53 À/À fibroblasts treatedas indicatedin A; scriptional capacity is augmented by coexpression of its upstream andthe levels of p19 ARF, B23, c-Jun, andphospho-c-Jun were determinedby MAP3K, MEKK1, the mutant 272/273E did not activate transcrip- immunoblotting with specific antibodies. Actin was used as a loading control. +/+ À/À 2 tion (Supplementary Fig. S4B). Nevertheless, its transfection into D, JNK andJNK1,2 double-knockouts were exposed to 30 J/m UV, À/À À/À ARF andp19 ARF andB23 localization was determinedby immunostaining p53 c-jun culminated in the dispersal of p19 from the 3 h after irradiation. c, control. nucleolus, comparable with that of wild-type c-Jun (Fig. 4D). 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Figure 3. c-Jun andJunB are required for UV-induced p19ARF translocation. A, p53À/Àc-jun +/+ (striped bars) and p53À/À c-jun À/À (black bars) mouse fibroblasts were exposedto 30 J/m 2 UV, with or without preincubation with JNK inhibitor (SP). The cells were stainedfor p19 ARF 3 h after UV exposure, andthe percentage of cells containing nucleolar p19ARF at that time is plottedabove. The fraction of cells containing nucleolar p19ARF in the untreatedcontrol was consideredas100%. B, JunB protein levels were determined by immunoblotting in MEFs exposedto UV andthen harvestedat the indicatedtimes (hours after treatment). Actin was usedas a loading control. C, MEFs were infected with a retrovirus-expressing shRNA against junB or the vector virus alone, and selected. Six days after infection, the cells were exposedto 30 J/m 2 UV, andthe level of JunB at 3 h after irradiation was determined by immunoblotting. Lamin A andB servedas the loadingcontrols. D, the MEFs whose JunB levels are exhibitedin C were also stainedfor p19 ARF, andthe percentage of cells containing nucleolar p19ARF is plotted. Dark bars, cells expressing the vector; empty bars, cells expressing shRNA for junB.

result suggests that the transcriptional activity of c-Jun is observed in extracts from p19ARF-deficient cells (Supplementary dispensable for the redistribution of p19ARF. To further corroborate Fig. S5). These results suggest that c-Jun interacts with B23 and this notion, we examined the induction of the c-jun gene, which is that this interaction is not dependent on JNK phosphorylation. subject to autoregulation by the endogenous Jun protein and can Indirect interactions between c-Jun and p19ARF were observed in therefore serve as an indicator for its activity, in wild-type and NIH3T3 cells, containing metallothionein promoter–regulated Thr91/93Ala-expressing cells after exposure to UV (Supplementary p19ARF 24 h after induction with ZN2+. Immunoprecipitation of Fig. S4C). Again, the ability to regulate gene expression does not p19ARF revealed the presence of B23 as well as low levels of c-Jun in correlate with the ability to redistribute p19ARF,asc-jun mRNA the precipitate, thus, suggesting the presence of a complex expression increased to the same extent in both cell lines 3 h after containing p19ARF/B23 and c-Jun (Fig. 5B, left). The hypothesis exposure to 30 J/m2 UV (Supplementary Fig. S4C), whereas p19ARF that c-Jun directly displaces p19ARF from the complex with B23 was was dispersed only in wild-type c-jun–expressing cells (Fig. 4C). directly tested by transfecting HEK293 cells with expression vectors c-Jun interacts with B23 in a JNK-independent manner and for B23, p19ARF, and c-Jun and examining the amounts of p19ARF changes its subnuclear localization in a JNK-dependent that coprecipitated with B23 in the presence of increasing amounts manner. As the transcriptional activity of c-Jun is dispensable of c-Jun. As depicted in Fig. 5B (right), B23 was precipitated even for redistribution of p19ARF, we tested whether its interactions with in nontransfected cells, and comparable high levels were precip- other proteins contribute to the ability of c-Jun to disperse p19ARF itated by the antibody after transfection. Increasing the amount of from the nucleolus. We explored two possible interactions: transfected c-Jun resulted in an increase in the association between interactions between c-Jun and p19ARF and interactions of c-Jun B23 and c-Jun (Fig. 5B, right). Surprisingly, p19ARF interactions with B23. Coimmunoprecipitation experiments were performed to with B23 increased rather than decreased, in parallel to the determine the validity of the hypothetical interactions. We directly increase in B23 to c-Jun interactions (Fig. 5B, right). These results examined interactions of endogenous, not overexpressed, proteins show that c-Jun does not prevent p19ARF from interacting with À À in p53 / fibroblasts that were exposed to UV (or left untreated) B23. Therefore, we examined the alternative possibility that JNK and harvested 3 h later. In addition, we preincubated the same cells might control the subnuclear localization of c-Jun and, thus, the with SP600125 to determine the dependency of the interactions on localization of interacting B23 as well. It should be mentioned that JNK activity. In these experiments, immunoprecipitates of c-Jun the localization of several AP-1 proteins was previously found to be did not contain p19ARF (data not shown). However, precipitated regulated by UV (46). We, therefore, stained untreated and UV- À À c-Jun coprecipitated B23 reproducibly (Fig. 5A). The weak inter- irradiated JNK+/+ and JNK1,2 / mouse fibroblasts for c-Jun to actions observed between c-Jun and B23 in untreated cells were detect JNK-dependent changes in its subnuclear localization after strengthened in UV-irradiated cells. However, these interactions do UV exposure (Fig. 5C). In JNK+/+ mouse fibroblasts, c-Jun is not correlate with c-Jun phosphorylation by JNK, as preincubation distributed throughout the nucleus, including in the nucleolus with SP600125, which significantly reduced c-Jun phosphorylation where B23 is concentrated. After exposure to UV, however, c-Jun is after UV exposure, actually increased the association of c-Jun with excluded from certain areas suspected to be the nucleoli (Fig. 5C), B23 (compare UV À SP600125 to UV + SP600125). These inter- in correlation with B23 dispersal. To determine whether the actions are independent of p19ARF expression as they were also unstained areas are indeed the nucleoli, we transfected the JNK+/+

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. JNK Regulates ARF and B23 Nuclear Distribution mouse fibroblasts with an expression vector for GFP-tagged reduced without the application of experimental manipulation, fibrillarin, which is often used as a nucleolar marker, and detected we simultaneously examined JNK activity and the location of its expression relative to c-Jun in UV-irradiated cells (Fig. 5C, p19ARF in early and late passage MEFs before and after exposure to bottom). This analysis confirmed that the areas from which c-Jun is UV radiation. As depicted in Fig. 6A, both basal and UV-induced excluded are indeed the nucleoli. c-Jun staining is also observed in JNK-dependent c-Jun and phospho–c-Jun levels are higher in early À À the entire nuclear area of untreated JNK1,2 / cells. Moreover, passage MEFs. Concordantly, in early passage MEFs, p19ARF was regions of intense c-Jun staining that coreside with the nucleolar efficiently translocated from the nucleoli after UV exposure. In B23 were also observed (Fig. 5C, arrows). However, unlike in JNK- contrast, and in agreement with the hypothesized effects of low expressing cells, c-Jun was not excluded from the nucleolus of JNK activity, little dispersal of p19ARF from the nucleoli of late irradiated JNK-deficient cells. Quantitative analysis revealed a good passage MEFs was observed after UV exposure (Fig. 6B). This result correlation between c-Jun exclusion and the export of B23 from the shows that low JNK activity can result in the prevention of p19ARF nucleoli (Fig. 5D). These results suggest that JNK-dependent redistribution after UV exposure. translocation of c-Jun is correlated with the exclusion of B23 from the nucleolus. Additional experiments showed that B23 is redistri- buted after UV exposure also in cells expressing Ser63/73Ala c-Jun. Discussion However, it is not dispersed in the Thr91/93Ala cells (Supplementary The nucleolus functions in the stress response (1, 2, 4) and Fig. S6). redistribution of B23 and p19ARF may relay this response (3, 4). In p19ARF is not dispersed from the nucleoli of senescent cells. this study, we define for the first time a pathway and a mechanism ARF levels increase during progressive passages of explanted MEFs that underlies B23 and p19ARF redistribution. Both are redistributed in culture. JNK activity, on the other hand, is reduced in senesced after the exposure of cells to DNA damage, but this redistribution is fibroblasts, resulting in defective stress responses in these cells selective and occurs efficiently when the JNK pathway is activated. (47). To show the importance of the JNK pathway for p19ARF UV radiation, the most potent JNK activator, triggers the redistribution in a physiologic system in which JNK activity is redistribution of p19ARF and B23 most effectively, whereas exposure

Figure 4. Redistribution of p19ARF by Jun proteins. A, p53À/Àc-jun À/À cells were transfectedwith expression vectors coding for HA-tagged c-jun, junB, junD, or with expression vectors coding for GFP or JDP2. Transfectedcells were identifiedby staining against the HA tag at 36 h after transfection, andthe localization of p19 ARF in Jun-expressing cells was determined using coimmunofluorescent staining. The proportion of cells containing nucleolar p19ARF after GFP transfection was regarded as 100%. Bands above the bars exhibit the level of each of the transfectedproteins as determinedby immunoblotting. B, c-jun +/+ fibroblasts were preincubatedwith JNK inhibitor or not, exposedto 30 J/m 2 UV, andharvested at the indicated times (h). The levels of phospho-Thr91 (p Thr 91) and phospho-Ser63 (pSer63) as well as the total level of c-Jun were determined by immunoblotting. Glyceraldehyde-3- phosphate dehydrogenase (GAPDH) servedas a loadingcontrol. C, p53À/À c-jun À/À cells were transfectedwith plasmids coding for HA-tagged wild-type (wt) andmutatedc-Jun, as indicated( left). The cells were stainedandsubjectedto microscopic determination of p19ARF localization as in A. Levels of transfected c-Jun proteins exhibitedabove were determined by immunoblotting. Right, p19ARF localization was determined in mouse fibroblasts expressing the indicated c-Jun before or 3 h after exposure to 30 J/m2 UV. The fraction of cells expressing nucleolar ARF from the total cell population is presented. D, expression vectors for wild-type c-jun,c-jun mutant 272/273E, andfor GFP were transfectedto p53À/À c-jun À/À cells. Costaining for c-Jun and p19ARF was usedto determine the localization of the last-in Jun-transfected cells. The proportion of cells containing nucleolar p19ARF after GFP transfection was regarded as 100%.

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Figure 5. c-Jun interacts with B23 in a JNK-independent manner and is excluded from the nucleolus in a JNK-dependent manner. A, p53À/À fibroblasts were either exposedto 30 J/m 2 UV or not, in the presence or absence of JNK inhibitor SP600125. Endogenous c-Jun was immunoprecipitated from cells harvested 3 h after irradiation, and the levels of immunoprecipitated c-Jun and coprecipitated B23 were detected by immunoblotting using specific antibodies. The right part of the panel shows the level, Ser63 phosphorylatedc-Jun, andB23 in the extracts subjectedto immunoprecipitation. NC, negative control immunoprecipitate from UV-exposed cells that were incubatedwith irrelevant antibody. B, p19ARF was precipitatedfrom extracts of control or UV-irradiatedNIH3T3containing metallothionein promoter–regulatedp19 ARF, 24 h after induction with ZN2+, and levels of coprecipitated endogenous B23 and c-Jun were determined using specific antibodies (left). Right, HEK293 cells were cotransfectedwith constant levels of B23 andp19 ARF (2 and4 Ag, respectively) andthe indicatedamounts of c-Jun (in Ag). Twenty-four hours after transfection, the cells were harvested, and B23 was immunoprecipitated with a specific antibody. The levels of precipitated B23, c-Jun, and p19ARF were determined by immunoblotting with a specific antibody. C, JNK+/+ andJNK1,2 À/À mouse fibroblasts were exposedto UV andstainedfor B23 andc-Jun 3 h after irradiation. Bottom, JNK+/+ cells were transfectedwith an expression vector for GFP-fibrillarin. The cells were irradiatedat24 h after transfection andthen stainedfor c-Jun at 3 h after irradiation, using a specific antibody. D, quantitative analysis of nucleolar c-Jun (left graph) andB23 ( right graph) before and3 h after UV exposure. Black bars, staining in JNK+/+ cells; white bars, staining in JNK1,2À/À cells. to ionizing radiation, which is a poor activator of JNK, does not the redistribution (Fig. 3A and D), and combined inhibition of both result in efficient p19ARF redistribution. Furthermore, inhibition of prevents the redistribution after exposure to UV almost as JNK activity by the synthetic inhibitor SP600125, or in cells efficiently as inhibition of JNK activity, suggesting that most of deficient in JNK, efficiently prevented the redistribution of p19ARF the effect is mediated by these proteins (Supplementary Fig. S2). and B23, thus, proving that the DNA damage-dependent redistri- Inhibition of the JNK-dependent activity of JunD may also be bution is JNK-dependent. However, our data suggest that although required for a complete inhibition of p19ARF redistribution. This the JNK pathway is a major regulator of DNA damage–dependent notion is supported by two experimental findings: transient localization of p19ARF and B23, it is not the only one. We show that expression of JunD into mouse fibroblasts resulted in moderate impairment of JNK activity prevents only 60% to 80% of the UV- redistribution of the endogenous p19ARF (Fig. 4A). In addition, a dependent redistribution p19ARF (Figs. 2–3). In addition, at later previous study reported an intense nucleolar staining of p19ARF in À À times after UV-irradiation, p19ARF may exit the nucleolus in a JNK- junD / MEFs (48). This was intuitively attributed to the ability of independent manner (data not shown). Hence, we suggest that the JunD to down-regulate p19ARF expression (48). Alternatively, the JNK-dependent redistribution is the early response to the stress. strong inhibition of translocation when JNK activity is impaired Out of the plethora of JNK substrates, the Jun proteins regulate may also stem from direct JNK phosphorylation of either p19ARF or the JNK-dependent B23 and p19ARF translocations. p19ARF redistri- B23 as a precedence for the involvement of JNK in the trafficking bution in the absence of c-Jun or JunB is reduced, demonstrating of other nucleolar proteins was described in other systems (49). that both are required for the redistribution. However, reduced This notion, however, is not supported by the fact that in cells expression of each of these individual proteins partially prevented expressing c-Jun mutated at the JNK phosphorylation sites

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Thr91/93 no translocation is observed after UV exposure. We also on transcription initiation factor-IA (49), JNK is involved in the showed the requirement of the JNK pathway for the distribution of shuttling of the c-Jun–associated B23. In contrast to the situation p19ARF in a physiologically relevant system of senesced cells, in in JNK-expressing cells, c-Jun is not excluded from the nucleoli which JNK activity and c-Jun levels are low, and p19ARF was not of JNK-deficient cells. This is in perfect correlation with B23 translocated after UV treatment (Fig. 6A and B). translocation (Fig. 5C–D). The possibility that the nucleolus could To study the mechanism of the redistribution, we focused our be totally disrupted after irradiation is not valid as fibrillarin, often attention on the c-Jun protein, due to the presence of extensive used as nucleolar marker, is concentrated at nucleolar-shaped data and reagents related to its modulation by JNK. Interestingly, structures from which c-Jun is excluded after UV exposure. In our results show for the first time a specific role for the phos- addition, when p19ARF was ectopically overexpressed to high levels, phorylation at Thr91/93. These sites are phosphorylated by UV, with UV irradiation caused only partial redistribution of p19ARF (data a kinetic similar to that of the phosphorylation of Ser63/73 (Fig. 4B). not shown), thus, suggesting that p19ARF redistribution is a However, as of today, no biological activity has been assigned to stoichiometric process. In this respect, the involvement of at least this phosphorylation. Using both cell lines expressing knock-in two UV-inducible Jun proteins can provide a reasonable stoichio- c-Jun mutants and transient overexpression experiments, we show metric relationship to explain the B23 translocation. A recent study that the presence of Thr91/93 is important for the redistribution, by Liuet al. (46) offered a possible mechanism by which c-Jun whereas the activity of Ser63/73 is dispensable. We also show that localization might be changed by UV radiation; Jun homodimers c-Jun–dependent transcriptional regulation of target genes is not are concentrated in nucleolar-like structures; and upon exposure to involved in the redistribution of p19ARF.DNA-bindingand UV, c-Jun interacts with activating transcription factor 2 and transcriptionally inactive mutant c-Jun (272/273E) can efficiently changes its subnuclear position. redistribute p19ARF (Fig. 4D). Instead, we identified a more direct A recent model suggested that upon UV exposure, p14ARF mechanism: c-Jun interacts with B23. Surprisingly, nonphosphory- dissociates from B23 and interacts with Hdm2 (3). B23 itself is also lated c-Jun interacts with B23 better than phosphorylated c-Jun translocated to the nucleoplasm after UV exposure by a mechanism (Fig. 5A). Therefore, our results suggest that the interactions obscure before this study (4, 5). Whether B23-associated p19ARF is between B23 and c-Jun are JNK-independent. Hence, the activity translocated along with c-Jun and the dissociation occurs at later of JNK may be important for augmenting the reservoir of c-Jun stage is currently studied. Our model suggests that the induction of (32, 34, 50). The interaction between B23 and c-Jun is p19ARF- the AP-1 pathway is the initial and essential step. We suggest that independent (Supplementary Fig. S5) and does not enhance the the JNK activity elevates c-Jun levels, thus, increasing the dissociation between B23 and p19ARF (Fig. 5B). On the other hand, association between c-Jun and B23, and that the phosphorylation we show that the translocation of Jun and B23 out of the nucleolus of Thr91/93 by JNK enhances translocation of c-Jun and the is JNK dependent. Therefore, we suggest that, just as for the effects associated B23 out of the nucleolus. As B23 anchors p19ARF to

Figure 6. UV does not affect p19ARF localization in senescedcells. A, levels of c-Jun andSer 63-phosphorylatedc-Jun were examinedin passage 2 andpassage 6 MEFs by immunoblotting with specific antibodies. Actin was usedas a loadingcontrol. B, early passage (P2) andlate passage ( P6) MEFs were irradiated with UV and the localization of p19ARF was detected by fluorescent immunostaining. C and D, a model describing the activity of the JNK pathway in stress-induced translocation of B23 and ARF. C, cells not exposedto UV. D, UV-irradiated cells.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2008 American Association for Cancer Research. Cancer Research the nucleolus, its translocation will inevitably result in the exit Grant support: Grant number 0397583 from the Israeli Science Foundation and ARF grant number 0394234 from the Public Committee for Allocation of Estate Funds, of p19 from the nucleolus as illustrated in our model (Fig. 6C Ministry of Justice, Israel. and D). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acknowledgments We thank G. Li, R. Johnson, C. Sherr, and A. Behrens for cell lines; Y. Shav-Tal, A. Aroneim, and M.Oren for plasmids; and G. Peters for critical reading and Received 7/26/2007; revised 12/4/2007; accepted 12/27/2007. comments.

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