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The Evi-1 oncoprotein inhibits c-Jun N-terminal kinase and prevents stress-induced cell death

2000; Springer Nature; Volume: 19; Issue: 12 Linguagem: Inglês

10.1093/emboj/19.12.2958

1460-2075

Mineo Kurokawa,

Ubiquitin and proteasome pathways

Article15 June 2000free access The Evi-1 oncoprotein inhibits c-Jun N-terminal kinase and prevents stress-induced cell death Mineo Kurokawa Mineo Kurokawa Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Kinuko Mitani Kinuko Mitani Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Tetsuya Yamagata Tetsuya Yamagata Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Tokiharu Takahashi Tokiharu Takahashi Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Koji Izutsu Koji Izutsu Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Seishi Ogawa Seishi Ogawa Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Tetsuo Moriguchi Tetsuo Moriguchi Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502 Japan Search for more papers by this author Eisuke Nishida Eisuke Nishida Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502 Japan Search for more papers by this author Yoshio Yazaki Yoshio Yazaki Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Hisamaru Hirai Corresponding Author Hisamaru Hirai Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Mineo Kurokawa Mineo Kurokawa Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Kinuko Mitani Kinuko Mitani Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Tetsuya Yamagata Tetsuya Yamagata Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Tokiharu Takahashi Tokiharu Takahashi Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Koji Izutsu Koji Izutsu Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Seishi Ogawa Seishi Ogawa Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Tetsuo Moriguchi Tetsuo Moriguchi Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502 Japan Search for more papers by this author Eisuke Nishida Eisuke Nishida Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502 Japan Search for more papers by this author Yoshio Yazaki Yoshio Yazaki Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Hisamaru Hirai Corresponding Author Hisamaru Hirai Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan Search for more papers by this author Author Information Mineo Kurokawa1, Kinuko Mitani1, Tetsuya Yamagata1, Tokiharu Takahashi1, Koji Izutsu1, Seishi Ogawa1, Tetsuo Moriguchi2, Eisuke Nishida2, Yoshio Yazaki1 and Hisamaru Hirai 1 1Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan 2Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, 606-8502 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:2958-2968https://doi.org/10.1093/emboj/19.12.2958 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Evi-1 encodes a nuclear protein involved in leukemic transformation of hematopoietic cells. Evi-1 possesses two sets of zinc finger motifs separated into two domains, and its characteristics as a transcriptional regulator have been described. Here we show that Evi-1 acts as an inhibitor of c-Jun N-terminal kinase (JNK), a class of mitogen-activated protein kinases implicated in stress responses of cells. Evi-1 physically interacts with JNK, although it does not affect its phosphorylation. This interaction is required for inhibition of JNK. Evi-1 protects cells from stress-induced cell death with dependence on the ability to inhibit JNK. These results reveal a novel function of Evi-1, which provides evidence for inhibition of JNK by a nuclear oncogene product. Evi-1 blocks cell death by selectively inhibiting JNK, thereby contributing to oncogenic transformation of cells. Introduction Evi-1 was first identified as a gene existing in a common locus of retroviral integration in myeloid tumors in AKXD mice (Mucenski et al., 1988). This gene encodes a 145 kDa nuclear-localized protein, which possesses seven and three repeats of zinc finger motifs separated into two clusters (Morishita et al., 1988, 1990). The human Evi-1 gene is located on chromosome 3q26, and rearrangements involving this region often activate Evi-1 expression in myeloid leukemia and myelodysplasia (Morishita et al., 1992b; Levy et al., 1994; Ogawa et al., 1996a; Peeters et al., 1997), although its expression is barely detectable in normal bone marrow and peripheral blood. In t(3;21)(q26;q22), found in cases with blastic crisis of chronic myelogenous leukemia, we have reported that Evi-1 is fused to the AML1 gene and is transcriptionally activated as the AML1–Evi-1 chimera (Mitani et al., 1994). Many lines of evidence suggest a critical role for Evi-1 in t(3;21) leukemogenesis (Tanaka et al., 1994, 1995; Kurokawa et al., 1995, 1998a,b). Elevated expression of Evi-1 also occurs without cytogenetically evident translocations in some myeloid malignancies (Russell et al., 1994; Ogawa et al., 1996b). These facts indicate that Evi-1 has a pivotal role in malignant transformation of hematopoietic cells as a dominant oncogene. Thus far, characteristics of Evi-1 as a transcriptional regulator have been described (Lopingco and Perkins, 1996; Bartholomew et al., 1997). We reported that Evi-1 elevates intracellular AP-1 activity and stimulates the c-fos promoter with dependence on the second zinc finger domain (Tanaka et al., 1994). With regard to the biological effects of Evi-1, it is known that overexpressed Evi-1 can disturb hematopoietic cell differentiation (Morishita et al., 1992a; Kreider et al., 1993). We have reported that Evi-1 causes cellular transformation when overexpressed in Rat-1 fibroblast cells (Kurokawa et al., 1995) and that it antagonizes the growth-inhibitory effects of transforming growth factor-β (TGF-β) by inhibiting Smad3 (Kurokawa et al., 1998a,b). Available evidence suggests that Evi-1 potentially possesses abilities for growth promotion and differentiation block in some types of cells. Mitogen-activated protein (MAP) kinase cascades are universal signal transduction modules that are used in a wide variety of biological response mechanisms (Davis, 1993). In vertebrates, at least three such pathways have been identified, which activate different MAP kinase classes, known as ERK, JNK and p38 (Treisman, 1996; Robinson and Cobb, 1997). Among MAP kinases, JNK (also known as SAPK) is activated preferentially by extracellular stress stimuli including UV light, γ-radiation, osmotic shock, protein synthesis inhibitors, tumor necrosis factor-α (TNF-α) and interleukin-1 (Derijard et al., 1994; Kyriakis et al., 1994; Kharbanda et al., 1995; Chen et al., 1996a; Fanger et al., 1997). Whereas ERK signaling is generally involved in the control of cell proliferation and differentiation, recent evidence suggests that the JNK pathway may play an important role in triggering apoptosis in response to cellular stresses (Xia et al., 1995; Chen et al., 1996a; Verheij et al., 1996; Zanke et al., 1996). JNK activation requires phosphorylation at two residues, Thr183 and Tyr185, by MKK4 (also called SEK1 or MEK4) (Sanchez et al., 1994; Derijard et al., 1995; Lin et al., 1995) or MKK7 (Holland et al., 1997; Moriguchi et al., 1997; Tournier et al., 1997), which are dual specificity protein kinases. Like ERKs, the activated JNKs translocate into the nucleus where they phosphorylate transcription factors such as c-Jun (Derijard et al., 1994) and strongly augment their transcriptional activity. While several nuclear transcription factors exhibit alterations in their function depending on phosphorylation by activated MAP kinases, regulatory mechanisms that modify the activities of MAP kinases in the nucleus are not well understood. In the present study, we demonstrate that Evi-1 suppresses JNK activity, thus preventing cellular death induced by stress stimuli. Identification of Evi-1 as a regulator of JNK activity provides a novel function for a nuclear oncoprotein, and proposes a novel mechanism through which Evi-1 contributes to malignant transformation of cells. Results Evi-1 inhibits JNK activity To examine the potential role of Evi-1 in regulating JNK signal transduction, we evaluated the effect of Evi-1 on the kinase activity of JNK. Hemagglutinin (HA)-tagged JNK1 was expressed either alone or with Evi-1 in COS7 cells. The cells were exposed to UV, and JNK1 activity was measured by immunoprecipitation followed by in vitro kinase assay using GST–c-Jun as a substrate. In the absence of Evi-1, phosphorylation of GST–c-Jun by JNK1 is enhanced efficiently after UV stimulation (Derijard et al., 1994) (Figure 1A). However, Evi-1 significantly suppressed JNK1 activity when they were co-expressed. JNK is also known to be activated in response to the protein synthesis inhibitor anisomycin, sorbitol-induced osmolarity changes and treatment with TNF-α. Consistent with previous observations, these reagents effectively activated JNK1 expressed in COS7 cells (Figure 1B). All of these effects were again inhibited when Evi-1 was present, indicating that Evi-1 can suppress JNK activity induced by a wide variety of stimuli. To test whether Evi-1 can inhibit JNK in vitro, Flag-tagged Evi-1 was expressed in COS7 cells, recovered on protein G–Sepharose conjugated with the anti-Flag antibody (M2; Sigma) and eluted with the Flag peptide. Then it was added to the precipitates including HA-JNK1 in increasing doses, and JNK1 activity was measured. As shown in Figure 1C, Flag-Evi-1 inhibited JNK activity in a dose-dependent manner. These results suggest that JNK inhibition by Evi-1 does not require intricate pathways that are possible only in intact cells, although they do not preclude the need for accessory molecules. Kinase assays were also performed with p38, another mammalian MAP kinase that can be activated by cellular stresses (Han et al., 1994), and with ERK. In contrast to JNK1, Evi-1 did not inhibit the activity of p38 (Figure 1D). The kinase activity of ERK was also little affected by Evi-1 (Figure 1E). These results indicate that Evi-1 specifically inhibits JNK activity. Figure 1.(A and B) Effects of Evi-1 on JNK activation by cellular stresses. (A) pSRα-HA-JNK1 was transfected into COS7 cells either alone or together with pME18S-Evi-1, and the cells were treated with 60 J/m2 UV. HA-JNK1 was isolated by immunoprecipitation, and its activity was measured by phosphorylation of the substrate GST–c-Jun(1–79) using SDS–PAGE (top). Expression of JNK1 and Evi-1 is shown (middle and bottom). (B) COS7 cells transfected with pSRα-HA-JNK1 either alone or together with pME18S-Evi-1 were treated with 0.5 M sorbitol, 10 μg/ml anisomycin or 5 nM TNFα. HA-JNK1 was isolated by immunoprecipitation with 12CA5, and subjected to the kinase assay (top). Expression of HA-JNK1 was monitored (bottom). (C) Evi-1 inhibits JNK1 activity in vitro. COS7 cells introduced with HA-JNK1 were either left untreated or treated with 60 J/m2 UV. HA-JNK1 was immunoprecipitated, incubated with increasing doses of Flag-Evi-1 isolated from COS7 cells and subjected to the kinase assay (top). Expression of HA-JNK1 was monitored (bottom). (D and E) Activities of p38 and ERK in the presence of Evi-1. HA-p38 or ERK was expressed in COS7 cells either alone or together with Evi-1, and the cells were treated with 0.7 M NaCl or FCS plus epidermal growth factor (EGF), respectively. HA-p38 or ERK was immunoprecipitated with 12CA5 or αC92, and subjected to kinase assays using ATF2 or MBP as a substrate (top). Expression of each protein was monitored (middle and bottom). Download figure Download PowerPoint Next, we tested the effect of Evi-1 on endogenous JNK activity. We introduced Evi-1 into Rat-1 cells by retroviral infection and exposed them to UV. As shown in Figure 2A, JNK1 activity was significantly suppressed in the Rat-1 cells stably expressing Evi-1. We investigated further whether the naturally expressed Evi-1 also inhibits JNK activity. MOLM-1 cells, a human megakaryoblastoid cell line that uniquely expresses a truncated form of Evi-1 (Ogawa et al., 1996a), were treated with an antisense oligonucleotide complementary to the sequence encoding the N-terminus of the first zinc finger domain of Evi-1. As shown in Figure 2B, endogenous Evi-1 expression in MOLM-1 cells was reduced effectively by treatment with the antisense oligonucleotide, as compared with the corresponding sense oligonucleotide. Expression of endogenous JNK1 remained unchanged in the presence of either oligonucleotide. We determined the UV-induced activity of endogenous JNK1 in these cells, and found that JNK1 activity was restored when expression of endogenous Evi-1 was repressed (Figure 2B). Similar experiments were performed with HEC1B cells, a human endometrial carcinoma cell line that expresses Evi-1 at a high level (Morishita et al., 1990). When Evi-1 expression was reduced by antisense inhibition with different doses of oligonucleotide, the endogenous JNK1 activity was recovered with dependence on expression levels of Evi-1 (Figure 2C). Taking these results together, we can conclude that Evi-1 inhibits endogenous JNK activity in both non-hematopoietic and hematopoietic cells. We next examined whether Evi-1 affects phosphorylation of endogenous substrates as a result of JNK inhibition. To this end, we introduced Evi-1 into NIH 3T3 cells, and evaluated the amount of phosphorylated c-Jun in these cells. Phosphorylation of c-Jun was clearly detected in response to UV stimulation in the mock-infected cells using the antibody specific to phosphorylated c-Jun (Figure 2D). This phosphorylation was reduced significantly in the Evi-1-expressing cells, while the amount of endogenous c-Jun was little affected regardless of Evi-1 expression or UV stimulation. These results suggest that Evi-1 can reduce phosphorylation of the endogenous substrate for JNK in vivo. Figure 2.Evi-1 inhibits endogenous JNK activity. (A) Evi-1 was introduced into Rat-1 cells by retroviral infection, and the cells were treated with 300 J/m2 UV. Endogenous JNK1 was immunoprecipitated with anti-JNK1, and the kinase assay was performed (top). Samples from the parental and the mock-infected Rat-1 cells are shown (lanes 1 and 2). Expression of endogenous JNK1 and retrovirally introduced Evi-1 was monitored (middle and bottom). (B) MOLM-1 cells were treated with 5 μg of the sense (S) or antisense (AS) oligonucleotide for Evi-1, and then treated with 100 J/m2 UV. Endogenous JNK1 was then immunoprecipitated with anti-JNK1 and subjected to the kinase assay (top). Expression of endogenous Evi-1 and JNK1 was determined (middle and bottom). (C) HEC1B cells were treated with 0.1 (+) or 5 μg (++) of the sense or antisense oligonucleotide for Evi-1, and treated with 100 J/m2 UV. Then endogenous JNK1 was immunoprecipitated with anti-JNK1 and subjected to the kinase assay (top). Expression of endogenous Evi-1 and JNK1 was determined (middle and bottom). (D) Evi-1 was introduced into NIH 3T3 cells by retroviral infection, and the cells were treated with 80 J/m2 UV. Whole-cell extracts were subjected to SDS–PAGE and immunoblotted with the phospho-c-Jun antibody, anti-c-Jun, anti-Evi-1 or anti-JNK1 as indicated. Download figure Download PowerPoint Evi-1 interferes with the interaction between JNK and its substrate Extracellular stimuli activate a class of JNK kinases, which in turn phosphorylate and activate JNK. To examine whether Evi-1 interrupts these activation processes, we evaluated phosphorylation of JNK1 in the presence of Evi-1 using the phospho-specific JNK antibody. As shown in Figure 3A, JNK1 was phosphorylated effectively by UV stimulation. This phosphorylation was not eliminated by the presence of Evi-1. We next examined the binding of JNK1 to c-Jun immobilized on glutathione–Sepharose in either the absence or presence of Evi-1 by a pull-down assay. It was observed that JNK1 from either unstimulated or UV-stimulated cells bound equally to GST–c-Jun (Figure 3B). This binding was reduced significantly by concomitant expression of Evi-1. These results point to a mechanism for JNK inhibition in which Evi-1 interrupts the interaction between JNK and its substrates. To preclude the possibility that Evi-1 may act as a substrate for JNK1 and thus compete with other substrates, we analyzed phosphorylation of endogenous Evi-1 in either untreated or UV-treated HEC1B cells using metabolic labeling with [32P]phosphate. UV exposure of HEC1B cells did not induce phosphorylation of Evi-1 even when JNK1 was overexpressed (data not shown), suggesting that Evi-1 is an authentic inhibitor of JNK rather than a tightly bound substrate. Figure 3.(A) Phosphorylation of JNK1 is not inhibited by Evi-1. pSRα-HA-JNK1 was transfected into COS7 cells either alone or together with pME18S-Evi-1, and the cells were treated with 60 J/m2 UV. Whole-cell extracts were subjected to SDS–PAGE and immunoblotted with the phospho-specific JNK antibody (top). Expression of HA-JNK1 and Evi-1 was monitored (middle and bottom). (B) Effect of Evi-1 on the interaction between JNK1 and c-Jun. HA-JNK was expressed in COS7 cells with or without Evi-1. The cells were either left untreated or stimulated with 60 J/m2 UV. Whole-cell extracts were incubated with GST or GST–c-Jun(1–79), and bound JNK1 was detected by immunoblotting with 12CA5 (top). Expression of HA-JNK1, Evi-1 and GST proteins was monitored as indicated. Download figure Download PowerPoint Evi-1 interacts with JNK through the first zinc finger domain To explore the mechanism by which Evi-1 affects JNK activity, we examined whether Evi-1 physically interacts with JNK, using a pull-down assay. As shown in Figure 4A, bacterially produced GST–JNK2 immobilized on glutathione–Sepharose associated with Evi-1 that was expressed in COS7 cells. We found that GST–JNK2 also bound to Evi-1ΔZF8–10, a mutant that lacks the second zinc finger domain (ZF8–10) (Figure 4A). Although ZF8–10 is known to be responsible for the ability of Evi-1 to induce intracellular AP-1 activity (Tanaka et al., 1994), these results indicate that ZF8–10 is dispensable for the interaction between Evi-1 and JNK. These interactions are thought to be direct, as demonstrated by the pull-down assay using GST–JNK2 protein and in vitro translated Evi-1 (Figure 4B). We found that Evi-1(1–252), a C-terminally truncated mutant that consists mainly of the first zinc finger domain (ZF1–7) (Figure 4C), can associate with GST–JNK2 (Figure 4D). As shown in Figure 4E, a pull-down assay using Evi-1 mutants that harbor specific deletions within this domain revealed that Evi-1ΔZF1–7, a mutant lacking the entire ZF1–7 region, failed to interact with GST–JNK2. These results indicate that ZF1–7 is responsible for Evi-1–JNK binding. Figure 4.(A) Binding of full-length Evi-1 (FL) and Evi-1ΔZF8–10 to GST–JNK2 (top, lanes 4 and 5). Inputs of whole-cell extracts from COS7 cells transfected with the indicated Evi-1 constructs are shown (lanes 7 and 8). GST fusion proteins are shown in the bottom panel. (B) Binding of [35S]methionine-labeled Evi-1 that was synthesized in vitro (IVTT-Evi-1) to GST–JNK2 (top, lane 4). Input of IVTT-Evi-1 is indicated (lane 6). GST fusion proteins are shown in the bottom panel. (C) Structures of full-length Evi-1 and of its deletion mutants. Zinc finger motifs are numbered 1–10. (D) The pull-down assay for binding of Evi-1(1–252) to GST–JNK2 (top, lane 4). Input of whole-cell extracts from COS7 cells transfected with Evi-1(1–252) is shown (lane 6). GST fusion proteins are shown in the bottom panel. (E) The pull-down assay for binding of the Evi-1 mutants to GST–JNK2 (top). Expression of each Evi-1 mutant in COS7 cells was monitored (middle) and GST fusion proteins are shown (bottom). (F) The first zinc finger of Evi-1 is required for inhibition of JNK1 activity. pSRα-HA-JNK1 was transfected into COS7 cells either alone or with the Evi-1 FL, Evi-1ΔZF1–7 or Evi-1ΔZF8–10. The cells were either left untreated or treated with 60 J/m2 UV, and HA-JNK1 was immunoprecipitated with 12CA5, followed by the kinase assay (top). Expression of HA-JNK1 and the Evi-1 mutants is shown. Download figure Download PowerPoint To determine the functional consequences of the Evi-1–JNK interaction, we examined the effect of Evi-1 mutants on JNK activity. As shown in Figure 4F, Evi-1ΔZF1–7, which cannot bind to JNK, failed to suppress UV-activated JNK1, while Evi-1ΔZF8–10, which can bind to JNK, inhibited JNK1 as effectively as full-length Evi-1. These results indicate that inhibition of JNK activity is dependent on the interaction with Evi-1 through ZF1–7. To test the in vivo interaction between JNK and Evi-1, we performed a co-precipitation assay using HEC1B cells. As shown in Figure 5A, Evi-1 was co-precipitated with JNK1, both of which were endogenously expressed in HEC1B cells. We also observed that the amount of Evi-1 co-precipitated with JNK1 was elevated in response to UV stimulation. To clarify the effect of JNK1 phosphorylation on Evi-1–JNK1 binding, the purified form of ZF1–7 (GST–ZF1–7) was incubated with HA-JNK1-containing COS7 cell extracts in vitro, and subsequently ZF1–7-bound JNK1 was detected by a pull-down assay. In contrast to the results of the co-precipitation assay, GST–ZF1–7 bound equally to JNK1 derived from either unstimulated or UV-stimulated cells (Figure 5B). Using immunofluorescent labeling of HEC1B cells, we observed that Evi-1 was consistently nuclear regardless of UV stimulation (data not shown). In contrast, JNK1, which was predominantly cytoplasmic without UV stimulation, almost translocated completely into the nucleus upon UV stimulation and co-localized with Evi-1 in the nucleus. We found a small fraction of JNK1 in the nucleus even in the absence of UV stimulation, however, which may contribute to constitutive binding of Evi-1 and JNK1 detected in unstimulated cells (Figure 5A). Available evidence suggests that the interaction of Evi-1 and JNK is reinforced upon UV stimulation as a result of UV-induced translocation of JNK into the nucleus, where the two proteins can co-localize. Figure 5.(A) The constitutive interaction between Evi-1 and JNK1, and further stimulation by UV exposure. HEC1B cells were either left untreated or treated with 100 J/m2 UV. Whole-cell extracts were immunoprecipitated with anti-JNK1, and bound Evi-1 was detected by immunoblotting with anti-Evi-1 (top). Expression of JNK1 and Evi-1 is monitored as indicated. (B) Evi-1 binds to both inactive and activated JNK. COS7 cells were transfected with HA-JNK1 and then either left untreated or treated with 60 J/m2 UV. Whole-cell extracts were incubated with GST or GST–ZF1–7, and bound JNK1 was detected by immunoblotting with 12CA5. HA-JNK1 and GST proteins were monitored as indicated. (C) JNK1, but not p38, binds to the first zinc finger domain of Evi-1. Whole-cell extracts from COS7 cells introduced with HA-JNK1 or HA-p38 were subjected to a pull-down assay using GST or GST–Evi-1(1–252) as indicated (top). Input of whole-cell extracts from HA-JNK1- or HA-p38-expressing COS7 cells is shown (lanes 8 and 9). GST fusion proteins are shown at the bottom. Download figure Download PowerPoint To confirm the specificity of the binding of Evi-1 to JNK, we tested whether Evi-1 can interact with p38 by a pull-down assay. As shown in Figure 5C, no interaction was detected between GST–ZF1–7 and p38, whereas the GST–ZF1–7–JNK1 binding was clearly recognized. These results are consistent with the fact that Evi-1 effectively inhibits the activity of JNK1, but not that of p38. Evi-1 protects cells from stress-induced cell death with dependence on its ability to inhibit JNK The JNK signaling pathway is implicated in apoptotic cell death induced by UV (Chen et al., 1996b), Fas ligand (FasL) (Gulbins et al., 1995) and TNF-α (Kolesnick and Golde, 1994). To test the effect of Evi-1 on these apoptotic responses, we performed transient transfection cell protection assays using 293 cells. The 293 cells were co-transfected with the β-galactosidase expression plasmid plus the effector plasmid for Evi-1, Evi-1ΔZF1–7, or various controls as indicated in Figure 6A. Each transfection was performed in duplicate for treatments with or without UV exposure and then assayed for β-galactosidase activity. A decrease in the number of β-galactosidase-expressing cells in comparison with the control group was used as an indicator of cell death (Chen et al., 1996b; Liu et al., 1996). JNK1(APF), a dominant-negative form of JNK1 (Derijard et al., 1994), blocked UV-induced cell death (Figure 6A), in agreement with the previous report (Chen et al., 1996b). A kinase-dead form of MKK7 [MKK7(KL)] (Moriguchi et al., 1997) and Bcl-xL, a strong anti-apoptotic protein (Boise et al., 1993), also suppressed cell death effectively. Under these conditions, Evi-1 blocked cell death significantly after UV exposure (Figure 6A and B). In contrast, the Evi-1ΔZF1–7 mutant that can neither interact with nor inhibit JNK did not show any effect on cell survival. We performed similar assays using HEC1B cells and found that suppression of Evi-1 expression by treatment with the antisense oligonucleotide significantly enhanced UV-induced cell death (Figure 6C and D). These results suggest that Evi-1 prevents UV-mediated apoptosis by inhibiting JNK. Figure 6.Blocking of UV-induced cell death by Evi-1. (A) The 293 cells were transfected in duplicate with pSRα-βgal and the indicated plasmids. Then the cells were either left untreated or treated with 60 J/m2 UV, and stained for β-galactosidase expression. The number of blue cells in five randomly chosen fields was determined, and the data shown are averages of three separate experiments. (B) Colorimetric staining of vector (pME18S)- (a and b) or pME18S-Evi-1- (c and d) transfected cells either left untreated (a and c) or treated with UV (b and d). Scale bar = 150 μm. (C) HEC1B cells were treated with 5 μg of the sense or antisense oligonucleotide for Evi-1. Then the cells were either left untreated or treated with 100 J/m2 UV, and stained for β-galactosidase expression. The number of blue cells in five randomly chosen fields was determined, and the data shown are averages of three separate experiments. (D) Colorimetric staining of the sense (a and b) or antisense (c and d) oligonucleotide-transfected cells either left untreated (a and c) or treated with UV (b and d). Scale bar = 150 μm. Download figure Download PowerPoint A recent study has revealed that stress activation of JNK promotes up-regulation of FasL expression in T lymphocytes, which is one of the mechanisms that potentially cause apoptosis (Faris et al., 1998). To examine whether Evi-1 affects JNK-induced up-regulation of FasL, Jurkat cells stably expressing full-length Evi-1 or Evi-1ΔZF1–7 were exposed to UV and subsequently analyzed for FasL expression as determined by flow cytometry. We observed a significant increase in the FasL expression level in response to UV stimulation in the mock-transfected cells (Figure 7A). In contrast, the Evi-1-expressing cells failed to show distinct FasL induction. Remarkably, Evi-1ΔZF1–7 has lost the ability to prevent FasL up-regulation. These results indicate that Evi-1 represses UV-induced FasL up-regulation in T lymphocytes with dependence on its ability to inhibit JNK. The DNA fragmentation assay showed that the UV-induced cell death was inhibited effectively in the Evi-1-expressing Jurkat cells (Figure 7B), in parallel with FasL expression. To confirm further the effect of Evi-1 on FasL gene expression, we performed the transcriptional response assay using a FasL promoter reporter plasmid (CD95L-486) (Faris et al., 1998). Transient transfection of CD