GTC
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Genes to Cells (2008) 13, 411-419. doi:10.1111/j.1365-2443.2008.01177.x
© 2008 Blackwell Publishing or its licensors
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fujii, Y.
Right arrow Articles by Koyasu, S.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Fujii, Y.
Right arrow Articles by Koyasu, S.

ERK5 is involved in TCR-induced apoptosis through the modification of Nur77

 Yasushi Fujii1,2, Satoshi Matsuda1,3, Gensuke Takayama1 and Shigeo Koyasu1,3,*

1 Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
2 Department of Pediatrics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
3 Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo 102-0081, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Nur77 is a nuclear orphan steroid receptor that has been implicated in negative selection when immature T cells are strongly activated through interaction with self peptide-MHC complexes. The expression of Nur77 in thymocytes and T cell lines leads to apoptosis in a manner dependent on its transcriptional activity. It is well established that Nur77 function is negatively regulated by post-translational modification. Here we demonstrate that the MAPK-induced phosphorylation of Nur77 during T cell activation plays a critical role in the induction of apoptosis. Upon T cell receptor (TCR) stimulation, ERK5 (also known as big MAP kinase 1, BMK1), a member of the MAPK family, phosphorylates Nur77, leading to its transcriptional activation. In contrast, the activation of the ERK2 signaling pathway failed to activate Nur77 although ERK2 is also able to phosphorylate Nur77. Furthermore, the blockade of ERK5 signaling pathway suppressed TCR-induced cell death. These results indicate that ERK5 regulates Nur77 function through its phosphorylation.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Apoptosis or programmed cell death is essential for the development and homeostasis of T cells. In the thymus, CD4+CD8+ double positive (DP) thymocytes bearing T cell receptor (TCRs) that fail to recognize the self MHC molecules die rapidly through a process termed death by neglect, while the recognition of self MHC structures with bound peptide can trigger either functional differentiation (positive selection) or apoptosis (negative selection) of DP cells. If positively selected, immature DP thymocytes develop into mature single positive (SP) T cells expressing either CD4 or CD8. DP thymocytes bearing TCRs that strongly react with relatively abundant thymic self-antigens undergo negative selection, leading to the clonal deletion of potentially autoreactive T cells (von Boehmer 2004).

MAPK family members are evolutionarily conserved signaling molecules, which play a critical role in transducing extracellular signals to the nucleus. They include ERK1/2, JNKs, p38 MAPKs and ERK5 (also termed big MAP kinase 1, BMK1) (Nishida & Gotoh 1993; Cobb & Goldsmith 1995; Schaeffer & Weber 1999). These pathways have significant roles in mediating signals triggered by cytokines, growth factors and environmental stresses, and are involved in proliferation, differentiation and apoptosis in many cell types. Several studies have demonstrated that intracellular signals through different MAPK cascades selectively regulate T cell development in the thymus: the ERK1/2 pathway is involved in positive selection and the p38 and/or JNK pathways in negative selection (Rincon et al. 1998; Sugawara et al. 1998; Diehl et al. 2000). It has also been reported that the duration and strength of ERK1/2 activation regulate both positive and negative selection (Mariathasan et al. 2001). However, the molecular targets of MAPK cascades during thymic selection remain obscure.

Nur77 (also known as NGFI-B in rat and TR3 in human), an orphan nuclear steroid receptor, plays a critical role in negative selection (Winoto & Littman 2002; Hsu et al. 2004). The expression of a dominant-negative Nur77 blocks activation-induced cell death in T-cell hybridomas as well as negative selection in the thymus of transgenic mice (Zhou et al. 1996). Conversely, transgenic mice that express wild-type Nur77 exhibit enhanced apoptosis and a reduction in both thymocyte numbers and the proportion of DP thymocytes (Calnan et al. 1995). Thus, Nur77 likely plays an important role in T cell apoptosis.

TCR-mediated Nur77 expression requires an increase in intracellular calcium concentration (Woronicz et al. 1995). The Nur77 promoter has two calcium-regulated consensus binding sites for myocyte enhancer factor-2 (MEF2) (Woronicz et al. 1995). These observations implicate MEF2, originally discovered as a transcription factor for muscle-specific gene expression, as a calcium-dependent transcription factor for Nur77 expression. Recent findings further indicate that a TCR-induced increase in intracellular calcium concentration leads to the dissociation of MEF2 from Cabin1 as a result of the competitive binding of activated calmodulin to Cabin1, resulting in MEF2 binding to the Nur77 promoter (Youn et al. 1999). In addition to the calcium–MEF2 pathway, MAPK signaling pathways are also involved in the induction of Nur77 in excitable cells such as muscle and nerve cells (van den Brink et al. 1999; Sakaue et al. 2001). It is thus likely that Nur77 function is regulated through a MAPK pathway in addition to the MEF2 pathway during TCR-mediated apoptosis.

Here we demonstrate that Nur77 is phosphorylated through the ERK5 pathway. It has been shown that Akt-mediated phosphorylation of Nur77 inhibits its DNA binding activity (Masuyama et al. 2001). In contrast, ERK5-mediated phosphorylation is indispensable for the positive regulation of Nur77 function, as the inhibition of ERK5 pathway results in the blockade of TCR-mediated apoptosis. These results indicate that ERK5 plays an essential role in TCR-mediated apoptosis presumably through the post-translational modification of Nur77.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Regulation of Nur77 function through its phosphorylation

TCR stimulation results in the activation-induced cell death of a murine T-cell hybridoma, DO11.10 cells (Liu et al. 1994; Woronicz et al. 1994). The same stimulation induced the transient expression of Nur77 in these cells (Fig. 1A) (Winoto & Littman 2002; Hsu et al. 2004). Similar to TCR stimulation, simultaneous stimulation with PMA and A23187 [GenBank] that can mimic TCR signals (Koyasu et al. 1987) induced Nur77 expression, reaching a peak at 3 h after stimulation (data not shown). The stimulation of cells with A23187 [GenBank] alone induced the expression of Nur77 to a level comparable to PMA and A23187 [GenBank] stimulation while stimulation with PMA alone failed to induce Nur77 expression (Fig. 1B, upper panel). This is in line with the previous observation that the expression level of Nur77 is regulated through the Ca2+-induced Cabin1–MEF2 pathway (Youn et al. 1999). To our surprise, however, treatment with A23187 [GenBank] alone caused apoptosis in DO11.10 cells only marginally, whereas simultaneous stimulation with PMA was required to fully induce apoptosis (Fig. 1B, lower panel). It should be noted that the electrophoretic mobility of Nur77 is somewhat slower when stimulated with PMA and A23187 [GenBank] compared to stimulation with A23187 [GenBank] alone (compare lanes 3 and 4), raising the possibility that the PMA-induced modification of Nur77 is involved in its ability to cause apoptosis. Given that the phosphorylation status of proteins often affects their electrophoretic mobility, we examined whether Nur77 is phosphorylated during T cell activation. Nur77 induced via simultaneous stimulation with PMA and A23187 [GenBank] was incubated with alkaline phosphatase in the presence or absence of a phosphatase inhibitor. Treatment with alkaline phosphatase resulted in the disappearance of the more slowly migrating Nur77 protein bands while the addition of a phosphatase inhibitor to the reaction canceled the effect of alkaline phosphatase (Fig. 1C). These results strongly suggest that Nur77 is phosphorylated during T cell activation presumably through PMA-sensitive signaling pathways.


Figure 1
View larger version (33K):
[in this window]
[in a new window]

  		Figure 1  Nur77 is phosphorylated during T cell activation. (A) DO11.10 cells were stimulated with plate-bound anti-CD3{varepsilon} (145-2C11) mAb for the indicated times. Cell lysates (corresponding to 2 x 106 cells) were then obtained and subjected to immunoblot analysis with an anti-Nur77 mAb (upper panel) or an anti-{alpha}-tubulin mAb (lower panel) as a loading control. (B) DO11.10 cells were stimulated with 5 ng/mL PMA (P) and/or 200 ng/mL A23187 (I) and subjected to immunoblot analysis using the anti-Nur77 mAb (3 h after stimulation, upper panel) or the anti-{alpha}tubulin mAb as a loading control (middle panel) as well as cell death assay by a dye-exclusion method (20 h after stimulation, lower panel). As for death assay, three independent experiments were performed and data are presented as means ± SD. Closed and open arrowheads indicate Nur77 bands corresponding to a slower migrating form and a faster migrating form, respectively. (C) DO11.10 cells were stimulated with 5 ng/mL PMA and 200 ng/ml A23187 for 3 h. The cell lysates were obtained without phosphatase inhibitors, followed by incubation with 50 U/mL calf intestine alkaline phosphatase (PPase) in the presence or absence of 100 mM β-glycerophosphate (Inhibitor) at 37 °C for 30 min. Closed and open arrowheads indicate Nur77 bands corresponding to a phosphorylated form and a non-phosphorylated form, respectively.

 
MAPK signaling pathway is responsible for Nur77 phosphorylation

Given that PMA treatment activates a variety of MAPK family members, we investigated the effects of MAPK inhibitors on PMA-induced Nur77 phosphorylation. Since p38 and/or JNK pathways are implicated in negative selection in the thymus (Rincon et al. 1998; Sugawara et al. 1998; Diehl et al. 2000), we initially expected that Nur77 phosphorylation would be suppressed in the presence of SB203580. Although SB203580 is a well-known inhibitor for p38, it has been reported that SB203580 is also able to inhibit JNK activity at a concentration higher than 10 µM (Chen et al. 1998). Contrary to our expectation, SB203580 had little effect on Nur77 phosphorylation during T cell activation (Fig. 2A). In contrast, the treatment of cells with PD98059 as well as U0126, both of which are well-known inhibitors for the ERK1/2 cascade (Pang et al. 1995; DeSilva et al. 1998), suppressed the phosphorylation of Nur77, which is demonstrated by disappearance of the slowly migrating bands (Fig. 2A).


Figure 2
View larger version (48K):
[in this window]
[in a new window]

  		Figure 2  Effects of MAPK inhibitors on Nur77 phosphorylation. (A) DO11.10 cells were pretreated with 50 µM PD98059 (PD), 10 µM U0126 (U), 10 µM SB203580 (SB) or 100 ng/ml CsA for 1 h, followed by stimulation with 5 ng/mL PMA (P) and 200 ng/mL A23187 (I) for 3 h. Cell lysates were subjected to immunoblot analysis with the anti-Nur77 mAb (upper panel) and an anti-HSP90 antibody as a loading control (lower panel). Solid and open arrowheads indicate mobility of the bands corresponding to hyper- and hypo-phosphorylated Nur77, respectively. (B) DO11.10 cells were pretreated with U0126 at the indicated concentrations or 100 ng/mL CsA for 1 h, followed by stimulation with 5 ng/mL PMA and 200 ng/mL A23187 (P + I) for 3 h. The cells were subjected to immunoblot analysis with the anti-Nur77 mAb (upper panel) and the anti-HSP90 antibody as a loading control (lower panel). (C) DO11.10 cells were pretreated with 50 µM PD98059 (PD), 10 µM U0126 (U) or 100 ng/mL CsA for 1 h, followed by stimulation with 5 ng/mL PMA (P) and/or 200 ng/mL A23187 (I) for 10 h. The cells were then subjected to DNA fragmentation assay. (D) DO11.10 cells were pretreated with 10 µM U0126 (U) or 50 µM PD98059 (PD) for 1 h, followed by stimulation with 5 ng/mL PMA and 200 ng/mL A23187 (P + I) for 16 h. The percentages of apoptotic cells were then evaluated by annexin-V staining. Three independent experiments were performed and data are presented as means ± SD.

 
As previously reported (Yazdanbakhsh et al. 1995), treatment with cyclosporin A (CsA), a potent inhibitor of the calcium–calcineurin pathway, led to the marked reduction of Nur77 expression (Fig. 2A). Interestingly, CsA had little effect on the phosphorylation status of Nur77 as demonstrated by the existence of the slowly migrating bands (Fig. 2B). At concentrations ranging from 2.5 to 10 µM, U0126 had, if any, a marginal effect on Nur77 expression while blocking Nur77 phosphorylation (Fig. 2B). However, Nur77 expression during T cell activation was partially inhibited in the presence of a higher concentration (25 µM) of U0126, suggesting that an U0126-sensitive signaling pathway(s) is also involved in the Nur77 expression (Fig. 2B). As shown in Fig. 2C, DNA fragmentation, an indicator of apoptosis, caused by simultaneous stimulation with PMA and A23187 [GenBank] was blocked by the pretreatment of cells with CsA. Moreover, pretreatment with either U0126 or PD98059 also led to nearly complete inhibition of DNA fragmentation under conditions where the expression level of Nur77 was maintained at a level comparable to that without inhibitors. The suppressive effect of U0126 as well as PD98059 on DO11.10 was confirmed by FACS analysis using annexin-V as an indicator for apoptosis (Fig. 2D).

Although PD98059 and U0126 are highly selective inhibitors of the ERK1/2 signaling pathway (Pang et al. 1995; DeSilva et al. 1998), the ERK5 signaling pathway is also sensitive to these reagents (Kamakura et al. 1999). This was indeed the case with DO11.10 cells (Fig. 3). Pretreatment of the cells with PD98059 suppressed both ERK2 and ERK5 activation in response to PMA and A23187. [GenBank] Furthermore, U0126 reduced ERK2 and ERK5 activation to a nearly basal level (Fig. 3A,B). When we compared the kinetics of ERK1/2 and ERK5 activation, we found that ERK5 activation was more sustained in comparison with ERK2 (Fig. 3A,C). We also noted that stimulation with PMA and A23187 [GenBank] failed to activate ERK1 in DO11.10 cells where ERK1 was expressed to a level comparable to ERK2 (Fig. 3A and data not shown). On the other hand, CsA had no effect on ERK2 or ERK5 activation while apoptosis was inhibited.


Figure 3
View larger version (47K):
[in this window]
[in a new window]

  		Figure 3  ERK5 as well as ERK2 signaling pathways are involved in Nur77 phosphorylation. (A) DO11.10 cells were pretreated with 100 ng/mL CsA, 50 µM PD98059 or 10 µM U0126 for 1 h, followed by stimulation with 5 ng/mL PMA and 200 ng/mL A23187 (P + I) for the indicated times. ERK1/2 activity was estimated by immunoblot analysis with an anti-phospho-ERK1/2 mAb. Closed and open arrowheads indicate positions corresponding to phosphorylated ERK2 and phosphorylated ERK1, respectively. (B) DO11.10 cells were pretreated with 100 ng/mL CsA, 50 µM PD98059 (PD) or 10 µM U0126 (U) for 1 h, followed by stimulation with 5 ng/mL PMA and 200 ng/mL A23187 (P + I) for 10 min. The cell lysates were then subjected to immunoprecipitation with an anti-ERK5 antibody, followed by an in vitro kinase assay using MBP as a substrate. (C) DO11.10 cells were stimulated with 5 ng/mL PMA and 200 ng/mL A23187 for the indicated times. The cell lysates were immunoprecipitated with an anti-ERK5 antibody, and assayed for ERK5 activity. This experiments is a representative of two. (D) DO11.10 cells were stimulated with 5 ng/mL PMA and 200 ng/mL A23187 for 10 min, followed by immunoprecipitation with a control IgG (mock), the anti-ERK2 antibody, and the anti-ERK5 antibody. The immunoprecipitates were incubated with GST-Nur77 in the presence of [{gamma}-32P] ATP, and 32P incorporation was quantified on a BAS2000. (E) COS7 cells were transfected with an expression vector for GFP-fused Nur77 along with the indicated combination of MAPKK-MAPK. Cell lysates were obtained after 36-h incubation, and subjected to immunoblot analysis with an anti-GFP mAb (upper panel) and an anti-HSP90 antibody (lower panel). Closed and open arrowheads indicate Nur77 bands corresponding to a hyper-phosphorylated form and a hypo-phosphorylated form, respectively.

 
These data raise the possibility that the ERK5 and/or ERK2 signaling pathways are involved in Nur77 phosphorylation. Consistent with this idea, we found that mouse Nur77 contains 12 potential MAPK phosphorylation sites, which are evolutionally conserved among mouse, rat and human species. Indeed, ERK5 as well as ERK2 directly phosphorylated recombinant mouse Nur77 protein in vitro (Fig. 3D). We thus examined whether ERK5 and/or ERK2 are able to phosphorylate Nur77 in vivo by using COS7 cells, which are resistant to Nur77-induced apoptosis, to avoid secondary effects caused by the apoptotic process. It is well established that MAPK is efficiently activated in the presence of its cognate upstream activator, MAPKK (Nishida & Gotoh 1993). The activation of either ERK2 (by co-transfection with ERK2 and {Delta}SESE, a constitutively active form of MEK1) or ERK5 (by co-transfection with ERK5 and MEK5(D), a constitutively active form of MEK5) led to Nur77 phosphorylation as demonstrated by the appearance of more slowly migrating bands (Fig. 3E). In contrast, the co-expression of inactive MEK1 (referred to here as SASA) and MEK5 (referred to here as MEK5(A)) failed to induce Nur77 phosphorylation. These results collectively indicate that ERK5 and ERK2 are capable of phosphorylating Nur77 in vitro and in vivo.

ERK5-mediated phosphorylation is important for Nur77 function

We next examined whether MAPK-mediated phosphorylation affects Nur77 function. Previous reports have demonstrated that the translocation of Nur77 to mitochondria results in cytochrome c release, which causes activation of the caspase 9/caspase 3 cascade and leads to apoptosis in some cell lines such as LNCaP cells (Li et al. 2000). However, TCR-induced Nur77 exclusively localized in the nucleus in DO11.10 cells (data not shown), which is consistent with a previous observation that the transcriptional activity of Nur77 correlated with its potential to cause apoptosis in T cells (Kuang et al. 1999). In addition, MAPK is known to translocate into the nucleus once activated (Cobb & Goldsmith 1995; Schaeffer & Weber 1999). It is thus likely that MAPK-mediated phosphorylation regulates the transcriptional activity of Nur77 in the nucleus. In fact, without MAPK-mediated phosphorylation, Nur77 had very low transcriptional activity whereas the activation of ERK5 pathway augmented the transcriptional activity of Nur77 (Fig. 4A). Interestingly, the activation of ERK2 pathway had a marginal effect on Nur77 activation. These data demonstrate that ERK5-mediated, but not ERK2-mediated phosphorylation is sufficient to activate Nur77. We also found that the activation of ERK5 pathway augmented A23187 [GenBank] -induced apoptosis in DO11.10 cells (Fig. 4B). In addition, the expression of a dominant negative form of ERK5 (referred to here as dnERK5), which interferes the interaction between endogenous ERK5 and its substrate (Nakaoka et al. 2003), suppressed Nur77 activation induced by simultaneous stimulation with PMA and A23187 [GenBank] (Fig. 4C). It is thus likely that ERK5 plays a critical role in Nur77-mediated apoptosis, presumably through increasing transcriptional activity of Nur77. In silico analysis <http://mbs.cbrc.jp/research/db/TFSEARCH.html> suggests that Thr residue at 145 located within the transcriptional activation domain is a candidate for ERK5-mediated phosphorylation site. However, a mutant form of Nur77 where Ala was substituted for Thr 145 had similar transcriptional activity to wild-type Nur77 when over-expressed in DO11.10 cells (data not shown). The functional phosphorylation site(s) by ERK5 in Nur77 is now under investigation.


Figure 4
View larger version (41K):
[in this window]
[in a new window]

  		Figure 4  ERK5-mediated phosphorylation of Nur77 is required to cause apoptosis. (A) DO11.10 cells were transfected with NBRE-luc and phRL-TK along with the indicated combination of MAPKK-MAPK. Luciferase activities were measured at 7 h after stimulation with 200 ng/mL A23187 according to the manufacturer's instructions (Promega). (B) DO11.10 cells were transfected with pEGFP alone (control) or MEK5(D) along with pEGFP-ERK5 (active). The cells were then stimulated with 200 ng/mL A23187 for 16 h. The percentages of apoptotic cells among GFP-positive cells were evaluated by annexin-V staining. Three independent experiments were performed and data are presented as means ± SD. (C) DO11.10 cells were transfected with NBRE-luc and phRL-TK along with or without the indicated amounts of the expression vector for dnERK5. Luciferase activities were measured at 7 h after stimulation with 5 ng/mL PMA and 200 ng/mL A23187. (D) DO11.10 cells were stably transfected with expression vectors for the indicated constructs. The expression level of ERK5 was indicated by immunoblot analysis with the anti-ERK5 antibody (upper panel) and the anti-ERK2 antibody served as loading control (middle panel). The transfectants were stimulated with plate-bound 145-2C11 for 12 h, and assayed for DNA fragmentation (lower panel). Shown are percentages of apoptotic cells evaluated by annexin-V staining at 16 h after stimulation.

 
To examine whether the activation of ERK5 pathway is required for Nur77-induced apoptosis, we utilized a functional knockdown approach. Although Sohn et al. have reported the functional siRNA sequence for murine ERK5 (Sohn et al. 2005), we failed to reproduce their result via lentiviral vector-mediated introduction (data not shown). We thus chose more classical antisense approach to knockdown endogenous ERK5 in DO11.10 cells. Consistent with our expectation, the introduction of antisense ERK5 construct into DO11.10 cells reduced the expression level of endogenous ERK5, rendering the cells resistant to TCR-induced apoptosis (Fig. 4D, compare lanes 1 and 2). Furthermore, DO11.10 cells expressing dnERK5 were also resistant to TCR-induced apoptosis (Fig. 4D, compare lanes 2 and 3). We thus conclude that ERK5 is an essential component for Nur77-mediated T cell apoptosis.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
ERK5, also known as BMK1, is a member of the MAPK family and is activated by a wide range of extracellular stimuli such as mitogens (Kato et al. 1997, 2000; English et al. 1999; Kamakura et al. 1999) and stress (Abe et al. 1996). It has been shown that ERK5 plays a critical role in a variety of physiological processes such as the differentiation of skeletal muscle cells, cardiac development, vascular maturation/angiogenesis and neural differentiation (Dinev et al. 2001; Regan et al. 2002; Nishimoto et al. 2005). However, whether ERK5 is involved in the TCR signaling pathway has been obscure. The results presented here suggest that the phosphorylation of Nur77 presumably mediated through ERK5 signaling pathway is required for its function in causing apoptosis during T cell activation. It is of interest to note that one of the best-characterized substrates of ERK5 is MEF2C (Kato et al. 1997), which has been shown to be involved in TCR-induced Nur77 induction (Youn et al. 1999). Consistently, the blockade of ERK5 activation with a higher dose of U0126 resulted in the partial inhibition of Nur77 expression during T cell activation (Fig. 2B). Furthermore, it has been reported that the C-terminal domain of ERK5 when over-expressed augments Nur77 gene expression (Kasler et al. 2000). These results collectively suggest that the ERK5 pathway affects Nur77 function through two distinct mechanisms, gene expression and post-translational modification.

We hypothesize that the ERK5–Nur77 pathway functions as a signal integrator to quantify the strength of TCR engagement and direct the cell fate of immature T cells determining whether or not the cells will die. This concept is in line with an observation that Nur77 is induced even during positive selection where a weak signal is transduced into the nucleus (Masuyama et al. 2001). Interestingly, it has been shown that the pretreatment of fetal thymocytes with PD98059 resulted in the blockade of negative selection, leading to the conclusion that ERK1/2 pathways are involved in negative selection (Mariathasan et al. 2000). However, based on our observation, this could be explained by the inhibition of Nur77 function through suppressing ERK5 pathway in PD98059-treated cells. Studies with conditional knockout mice recently established (Hayashi et al. 2004) will clarify the contribution of the ERK5 pathway to negative selection under physiological conditions.

It is yet to be determined why ERK2 activation had only a slight effect on the transcriptional activation of Nur77 although the activation of ERK2 pathway resulted in Nur77 phosphorylation to a level comparable to the ERK5 pathway (Fig. 3D,E). One possible mechanism is that ERK5 activation is sustained while ERK2 activation is transient and such sustained activation of ERK5 is required for the post-translational modification of Nur77 (Fig. 3A,C) as is the case for c-Fos stabilization during IL-6/gp130 stimulation (Sasaki et al. 2006). Alternatively, since ERK5 and ERK2 phosphorylate distinct subsets of transcription factors in the nucleus (Kamakura et al. 1999), it is possible that Nur77 phosphorylation mediated by ERK2, which may occur on a site(s) distinct from that phosphorylated by ERK5, rather inhibits Nur77 function. In accordance with this hypothesis, Katagiri et al. have reported that the activation of conventional Ras/MAPK cascade, which is presumably mediated through ERK1/2, resulted in the phosphorylation of Nur77 at Ser105, leading to nuclear export of Nur77 (Katagiri et al. 2000). This would be consistent with the observation demonstrating that the ERK1/2 pathway plays a critical role in positive selection (Sugawara et al. 1998): ERK1/2-mediated phosphorylation suppresses Nur77 function leading to survival and differentiation of DP cells.

In summary, present results show that ERK5 plays an important role in T cell apoptosis by enhancing the transcriptional activity of Nur77 through phosphorylation. Our results also suggest that ERK5 and ERK1/2 play distinct roles in the regulation of Nur77 and T cell fate during thymic development.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell culture and transfection

The DO11.10 mouse T-cell hybridoma (Haskins et al. 1983) and COS7 cells were maintained in RPMI 1640 medium containing 10% FCS, penicillin–streptomycin, 10 mM Hepes buffer solution and 50 µM β-mercaptoethanol (Invitrogen, Carlsbad, CA). For electroporation, DO11.10 cells (1 x 107) suspended in Opti-MEM (Invitrogen) were mixed with 25 µg of plasmid DNA, and electroporated with a 250-V pulse at 960 µF on a Gene Pulser apparatus (Bio-Rad, Hercules, CA). COS7 cells were transfected with Superfect transfection reagent (Qiagen, Valencia, CA) according to the manufacturer's instructions.

Antibodies and reagents

The antibodies and inhibitors used in this study include anti-Nur77 mAb (BD Bioscience, Franklin Lakes, NJ), anti-GFP mAb (Clontech, Palo Alto, CA), anti-ERK2 polyclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti-ERK5 polyclonal antibody and anti-{alpha}tubulin mAb (Sigma, St. Louis, MO), anti-phospho-ERK1/2 mAb and PD98059 (Cell Signaling, Beverly, MA), anti-HSP90 polyclonal antibody (Yonezawa et al. 1988), U0126 and SB203580 (Calbiochem, San Diego, CA). Anti-CD3{varepsilon} mAb (145-2C11) was purified from the culture supernatant of a hybridoma 145-2C11. Phorbol-12-myristate 13-acetate (PMA) was purchased from Sigma, and calcium ionophore A23187 [GenBank] was from Calbiochem.

Constructs

The Nur77 construct was provided by Dr B. A. Osborne (University of Massachusetts, Amherst, MA). An expression vector for GFP-fused mouse Nur77 (pEGFP-Nur77) was constructed by subcloning a PCR fragment of Nur77 into the pEGFP-C1 vector (Clontech). A Nur77-responsive luciferase reporter plasmid (NBRE-luc) (Katagiri et al. 1997) was provided by Drs Y. Katagiri and G. Guroff (National Institutes of Health, Bethesda, MD). Expression vectors for MAPKs (pSR{alpha}HA-ERK2 and pSR{alpha}HA-ERK5) and MAPKKs (pSR{alpha}HA-SASA for a dominant-negative form of MEK1, pSR{alpha}HA-{Delta}SESE for a constitutively active form of MEK1, pSR{alpha}-MEK5(A) for a dominant-negative form of MEK5, and pSR{alpha}-MEK5(D) for a constitutively active form of MEK5) were provided by Dr E. Nishida (Kyoto University, Kyoto, Japan). Dominant negative ERK5 (referred to here as dnERK5), where Thr219 and Tyr221 were replaced with Ala and Phe, respectively, was generated by a PCR-based method and subcloned into pcDNA3.1 (Invitrogen). A cDNA fragment of mouse ERK5 was subcloned into pcDNA3.1 in a reverse direction to knockdown endogenous ERK5.

In vitro kinase assay

Prior to the kinase assay for ERK5, DO11.10 cells were cultured in RPMI1640 containing 0.5% FCS for 4 h. After stimulation, cells were washed once with ice-cold PBS, and lysed in a lysis buffer solution (20 mM Tris–HCl, pH 7.5, 2 mM EGTA, 25 mM β-glycerophosphate, 1% Triton X-100, 2 mM dithiothreitol, 1 mM vanadate, 1 mM phenylmethylsulfonyl fluoride and 1% aprotinin), followed by centrifugation at 15 000 g for 30 min. The lysates were incubated for 2 h at 4 °C with an anti-ERK5 antibody along with protein A-Sepharose beads (Amersham Bioscience, Uppsala, Sweden). After washing 3 times with Tris-buffered saline containing 500 mM NaCl, the resulting immunoprecipitates were divided into two aliquots: one was used for the kinase assay, and the other for immunoblotting to evaluate the efficiency of immunoprecipitation. The immune complex was incubated at 30 °C for 30 min with a reaction buffer solution (20 mM Tris–Cl, pH 7.5, 10 mM MgCl2 and 100 µM cold ATP along with 7.4 kBq of [{gamma}-32P]ATP) containing 10 µg of myelin basic protein (MBP) or GST-Nur77 as a substrate. In some experiment, GST-Nur77 was also incubated with the immunoprecipitates by an anti-ERK2 antibody. After SDS-PAGE, 32P incorporated into the substrate was quantified on an image analyzer (BAS2000, Fujifilm, Tokyo, Japan).

DNA fragmentation assay

DNA fragmentation was detected as described previously (Hirt 1967). Briefly, DO11.10 cells (1 x 106) were incubated at room temperature for 1 h in a fragmentation buffer solution (10 mM Tris–HCl, pH 8.0, 10 mM EDTA and 0.6% SDS). NaCl was then added to a final concentration of 1 M and the samples incubated at 4 °C overnight. Nuclear debris was then spun down for 30 min at 15 000 g at 4 °C. The DNA fraction in the supernatant was prepared by QIAquick PCR purification kit (Qiagen), followed by incubation with 200 µg/mL RNase A at 37 °C for 2 h. Samples were then electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining.

Annexin-V staining

After stimulation, DO11.10 cells were incubated with annexin-V-APC (BD Bioscience) in the presence of 1 mM CaCl2 for 20 min at 4 °C, followed by washing with PBS containing 1 mM CaCl2. Apoptotic cells were defined by APC-positive cells on a FACSCalibur.

Luciferase assay

To examine the transcriptional activation of Nur77, we employed luciferase assay system using NBRE-luc as a reporter (Katagiri et al. 1997). DO11.10 cells were transiently co-transfected with expression vectors for MAPKK and MAPK along with NBRE-luc in combination with pRL-TK (Promega, Madison, WI) for normalization by electroporation at 250 V, 960 µF. Luciferase activities in cell lysates were measured in triplicate on a luminometer (LB9507; Berthold, Bad Wildbad, Germany), using the Dual-Luc assay system (Promega).


   Acknowledgements
 
We thank Dr L. K. Clayton for critical reading of the manuscript. This work was supported by a grant from the Mitsubishi Foundation, a Keio University Special Grant-in-Aid for Innovative Collaborative Research Project, a Grant-in-Aid for Scientific Research for young scientist (16790293 to S. M.) from the Japan Society for the Promotion of Science, a Grant-in-Aid for Scientific Research on Priority Areas (14021110 to S. K. and 16043248 to S. M.), a National Grant-in-Aid for the Establishment of a High-Tech Research Center in a private University, and a Scientific Frontier Research Grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Eisuke Nishida

aPresent address: Department of Cell Signaling, Institute of Biomedical Science, Kansai Medical University, Moriguchi 570-8506, Japan

* Correspondence: Email: koyasu{at}sc.itc.keio.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Abe, J., Kusuhara, M., Ulevitch, R.J., Berk, B.C. & Lee, J.D. (1996) Big mitogen-activated protein kinase 1 (BMK1) is a redox-sensitive kinase. J. Biol. Chem. 271, 16586–16590.[Abstract/Free Full Text]

von Boehmer, H. (2004) Stimulation of the T-cell receptor: receptor-controlled checkpoints in T-cell development. Adv. Immunol. 84, 201–238.[Medline]

van den Brink, M.R., Kapeller, R., Pratt, J.C., Chang, J.H. & Burakoff, S.J. (1999) The extracellular signal-regulated kinase pathway is required for activation-induced cell death of T cells. J. Biol. Chem. 274, 11178–11185.[Abstract/Free Full Text]

Calnan, B.J., Szychowski, S., Chan, F.K., Cado, D. & Winoto, A. (1995) A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection. Immunity 3, 273–282.[CrossRef][Medline]

Chen, C.-Y., Del Gatto-Konczak, F., Wu, Z. & Karin, M. (1998) Stabilization of interleukin-2 mRNA by the c-Jun NH2-terminal kinase pathway. Science 280, 1945–1949.[Abstract/Free Full Text]

Cobb, M.H. & Goldsmith, E.J. (1995) How MAP kinases are regulated. J. Biol. Chem. 270, 14843–14846.[Free Full Text]

DeSilva, D.R., Jones, E.A., Favata, M.F., Jaffee, B.D., Magolda, R.L., Trzaskos, J.M. & Scherle, P.A. (1998) Inhibition of mitogen-activated protein kinase kinase blocks T cell proliferation but does not induce or prevent anergy. J. Immunol. 160, 4175–4181.[Abstract/Free Full Text]

Diehl, N.L., Enslen, H., Fortner, K.A., Merritt, C., Stetson, N., Charland, C., Flavell, R.A., Davis, R.J. & Rincon, M. (2000) Activation of the p38 mitogen-activated protein kinase pathway arrests cell cycle progression and differentiation of immature thymocytes in vivo. J. Exp. Med. 191, 321–334.[Abstract/Free Full Text]

Dinev, D., Jordan, B.W.M., Neufeld, B., Lee, J.D., Lindemann, D., Rapp, U.R. & Ludwig, S. (2001) Extracellular signal regulated kinase 5 (ERK5) is required for the differentiation of muscle cells. EMBO Rep. 2, 829–834.[CrossRef][Medline]

English, J.M., Pearson, G., Hockenberry, T., Shivakumar, L., White, M.A. & Cobb, M.H. (1999) Contribution of the ERK5/MEK5 pathway to Ras/Raf signaling and growth control. J. Biol. Chem. 274, 31588–31592.[Abstract/Free Full Text]

Haskins, K., Kubo, R., White, J., Pigeon, M., Kappler, J. & Marrack, P. (1983) The major histocompatibility complex-restricted antigen receptor on T cells. I. Isolation with a monoclonal antibody. J. Exp. Med. 157, 1149–1169.[Abstract/Free Full Text]

Hayashi, M., Kim, S.W., Imanaka-Yoshida, K., Yoshida, T., Abel, E.D., Eliceiri, B., Yang, Y., Ulevitch, R.J. & Lee, J.D. (2004) Targeted deletion of BMK1/ERK5 in adult mice perturbs vascular integrity and leads to endothelial failure. J. Clin. Invest. 113, 1138–1148.[CrossRef][Medline]

Hirt, B. (1967) Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365–369.[CrossRef][Medline]

Hsu, H.C., Zhou, T. & Mountz, J.D. (2004) Nur77 family of nuclear hormone receptors. Curr. Drug Targets Inflamm. Allergy 3, 413–423.

Kamakura, S., Moriguchi, T. & Nishida, E. (1999) Activation of the protein kinase ERK5/BMK1 by receptor tyrosine kinases. Identification and characterization of a signaling pathway to the nucleus. J. Biol. Chem. 274, 26563–26571.[Abstract/Free Full Text]

Kasler, H.G., Victoria, J., Duramad, O. & Winoto, A. (2000) ERK5 is a novel type of mitogen-activated protein kinase containing a transcriptional activation domain. Mol. Cell. Biol. 20, 8382–8389.[Abstract/Free Full Text]

Katagiri, Y., Hirata, Y., Milbrandt, J. & Guroff, G. (1997) Differential regulation of the transcriptional activity of the orphan nuclear receptor NGFI-B by membrane depolarization and nerve growth factor. J. Biol. Chem. 272, 31278–31284.[Abstract/Free Full Text]

Katagiri,Y., Takeda, K., Yu, Z.X., Ferrans, V.J., Ozato, K. & Guroff, G. (2000) Modulation of retinoid signaling through NGF-induced nuclear export of NGFI-B. Nat. Cell Biol. 2, 435–440.[CrossRef][Medline]

Kato, Y., Chao, T.H., Hayashi, M., Tapping, R.I. & Lee, J.D. (2000) Role of BMK1 in regulation of growth factor-induced cellular responses. Immunol. Res. 21, 233–237.[CrossRef][Medline]

Kato, Y., Kravchenko, V.V., Tapping, R.I., Han, J., Ulevitch, R.J. & Lee, J.D. (1997) BMK1/ERK5 regulates serum-induced early gene expression through transcription factor MEF2C. EMBO J. 16, 7054–7066.[CrossRef][Medline]

Koyasu, S., Suzuki, G., Asano, Y., Osawa, H., Diamantstein, T. & Yahara, I. (1987) Signals for activation and proliferation of murine T lymphocyte clones. J. Biol. Chem. 262, 4689–4695.

Kuang, A.A., Cado, D. & Winoto, A. (1999) Nur77 transcription activity correlates with its apoptotic function in vivo. Eur. J. Immunol. 29, 3722–3728.[CrossRef][Medline]

Li, H., Kolluri, S.K., Gu, J., et al. (2000) Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3. Science 289, 1159–1164.[Abstract/Free Full Text]

Liu, Z.G., Smith, S.W., McLaughlin, K.A., Schwartz, L.M. & Osborne, B.A. (1994) Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene nur77. Nature 367, 281–284.[CrossRef][Medline]

Mariathasan, S., Ho, S.S., Zakarian, A. & Ohashi, P.S. (2000) Degree of ERK activation influences both positive and negative thymocyte selection. Eur. J. Immunol. 30, 1060–1068.[CrossRef][Medline]

Mariathasan, S., Zakarian, A., Bouchard, D., Michie, A.M., Zuniga-Pflucker, J.C. & Ohashi, P.S. (2001) Duration and strength of extracellular signal-regulated kinase signals are altered during positive versus negative thymocyte selection. J. Immunol. 167, 4966–4973.[Abstract/Free Full Text]

Masuyama, N., Oishi, K., Mori, Y., Ueno, T., Takahama, Y. & Gotoh, Y. (2001) Akt inhibits the orphan nuclear receptor Nur77 and T-cell apoptosis. J. Biol. Chem. 276, 32799–32805.[Abstract/Free Full Text]

Nakaoka, Y., Nishida, K., Fujio, Y., Izumi, M., Terai, K., Oshima, Y., Sugiyama, S., Matsuda, S., Koyasu, S., Yamauchi-Takihara, K., Hirano, T., Kawase, I. & Hirota, H. (2003) Activation of gp130 transduces hypertrophic signal through interaction of scaffolding–docking protein Gab1 with tyrosine phosphates SHP2 in cardiomyocytes. Circ. Res. 93, 221–229.[Abstract/Free Full Text]

Nishida, E. & Gotoh, Y. (1993) The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem. Sci. 18, 128–131.[CrossRef][Medline]

Nishimoto, S., Kusakabe, M. & Nishida, E. (2005) Requirement of the MEK5–ERK5 pathway for neural differentiation in Xenopus embryonic development. EMBO Rep. 6, 1064–1069.[CrossRef][Medline]

Pang, L., Sawada, T., Decker, S.J. & Saltiel, A.R. (1995) Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J. Biol. Chem. 270, 13585–13588.[Abstract/Free Full Text]

Regan, C.P., Li, W., Boucher, D.M., Spatz, S., Su, M.S. & Kuida, K. (2002) Erk5 null mice display multiple extraembryonic vascular and embryonic cardiovascular defects. Proc. Natl. Acad. Sci. USA 99, 9248–9253.[Abstract/Free Full Text]

Rincon, M., Whitmarsh, A., Yang, D.D., Weiss, L., Derijard, B., Jayaraj, P., Davis, R.J. & Flavell, R.A. (1998) The JNK pathway regulates the in vivo deletion of immature CD4+CD8+ thymocytes. J. Exp. Med. 188, 1817–1830.[Abstract/Free Full Text]

Sakaue, M., Adachi, H., Dawson, M. & Jetten, A.M. (2001) Induction of Egr-1 expression by the retinoid AHPN in human lung carcinoma cells is dependent on activated ERK1/2. Cell Death Differ. 8, 411–424.[CrossRef][Medline]

Sasaki, T., Kojima, H., Kishimoto, R., Ikeda, A., Kunimoto, H. & Nakajima, K. (2006) Spatiotemporal regulation of c-Fos by ERK5 and the E3 ubiquitin ligase UBR1, and its biological role. Mol. Cell 24, 63–75.[CrossRef][Medline]

Schaeffer, H.J. & Weber, M.J. (1999) Mitogen-activated protein kinases: Specific messages from ubiquitous messengers. Mol. Cell. Biol. 19, 2435–2444.[Free Full Text]

Sohn, S.J., Li, D., Lee, L.K. & Winoto, A. (2005) Transcriptional regulation of tissue-specific genes by ERK5 mitogen-activated protein kinase. Mol. Cell. Biol. 25, 8553–8566.[Abstract/Free Full Text]

Sugawara, T., Moriguchi, T., Nishida, E. & Takahama, Y. (1998) Differential roles of ERK and p38 MAPK pathways in positive and negative selection of T lymphocytes. Immunity 9, 565–574.[CrossRef][Medline]

Winoto, A. & Littman, D.R. (2002) Nuclear hormone receptors in T lymphocytes. Cell 109, S57–S66.[CrossRef][Medline]

Woronicz, J.D., Calnan, B., Ngo, V. & Winoto, A. (1994) Requirement for the orphan steroid receptor Nur77 in apoptosis of T-cell hybridomas. Nature 367, 277–281.[CrossRef][Medline]

Woronicz, J.D., Lina, A., Calnan, B.J., Szychowski, S., Cheng, L. & Winoto, A. (1995) Regulation of the Nur77 orphan steroid receptor in activation-induced apoptosis. Mol. Cell. Biol. 15, 6364–6376.[Abstract/Free Full Text]

Yazdanbakhsh, K., Choi, J.W., Li, Y., Lau, L.F. & Choi, Y. (1995) Cyclosporin A blocks apoptosis by inhibiting the DNA binding activity of the transcription factor Nur77. Proc. Natl. Acad. Sci. USA 92, 437–441.[Abstract/Free Full Text]

Yonezawa, N., Nishida, E., Sakai, H., Koyasu, S., Matsuzaki, F., Iida, K. & Yahara, I. (1988) Purification and characterization of the 90-kDa heat-shock protein from mammalian tissues. Eur. J. Biochem. 177, 1–7.[Medline]

Youn, H.D., Sun, L., Prywes, R. & Liu, J.O. (1999) Apoptosis of T cells mediated by calcium-induced release of the transcription factor MEF2. Science 286, 790–793.[Abstract/Free Full Text]

Zhou,T., Cheng, J., Yang, V., Wang, Z., Liu, C., Su, X., Bluethmann, H. & Mountz, J.D. (1996) Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self-reactive T cells. J. Exp. Med. 183, 1879–1892.[Abstract/Free Full Text]

Received: 23 June 2007
Accepted: 17 January 2008




This article has been cited by other articles:


Home page
 J. Immunol.Home page
A. Wang, J. Rud, C. M. Olson Jr., J. Anguita, and B. A. Osborne
Phosphorylation of Nur77 by the MEK-ERK-RSK Cascade Induces Mitochondrial Translocation and Apoptosis in T Cells
J. Immunol., September 1, 2009; 183(5): 3268 - 3277.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fujii, Y.
Right arrow Articles by Koyasu, S.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Fujii, Y.
Right arrow Articles by Koyasu, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE ADVANCED SEARCH TABLE OF CONTENTS