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Endocrinology Vol. 148, No. 1 34-44
Copyright © 2007 by The Endocrine Society

Regulation of the Orphan Receptor TR3 Nuclear Functions by c-Jun N Terminal Kinase Phosphorylation

Bo Liu, Jia-fa Wu, Yan-yan Zhan, Hang-zi Chen, Xiao-yan Zhang and Qiao Wu

Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian Province, China

Address all correspondence and requests for reprints to: Qiao Wu, Ph.D., Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian, China. E-mail: xgwu{at}xmu.edu.cn.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The orphan receptor TR3 functions in the nucleus as a transcription factor to negatively or positively regulate gene expression. c-Jun N-terminal kinase (JNK) phosphorylation plays an important role in modulating the nuclear functions of TR3. Although TR3 is the phosphorylation target of JNK, the regulatory mechanism of JNK on TR3 functions remains to be elucidated. Here we showed that JNK activator anisomycin induced TR3 phosphorylation through JNK1 rather than p38 and ERK signals, which is mediated by its upstream factors MAPK kinase 4 and MAPK kinase 7. We also identified the exact phosphorylation site of JNK to be serine 95 at the N terminus of TR3, around which a classical JNK phosphorylation motif exists. Furthermore, we demonstrated that TR3 phosphorylation by JNK coincided with its ubiquitination and degradation, resulting in the loss of its mitogenic activity. Finally, we showed that JNK-induced phosphorylation blocked the DNA binding property of TR3 and hence diminished its transactivation activity. Taken together, our findings revealed a novel cross talk between TR3 and JNK signal pathway and shed light on the mechanism of JNK phosphorylation-dependent regulation on TR3 nuclear functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TR3 (ALSO CALLED NGFI-B or Nur77) is an orphan member of the steroid/thyroid receptor superfamily (1, 2). It functions in the nucleus as a transcription factor to negatively or positively regulate gene expression, which is closely related to the alteration of cellular phenotype in response to various stimuli (3, 4). Our previous study demonstrated that TR3 heterodimerizes with retinoid X receptor (RXR), binds to the retinoic acid (RA) response element (RARE) and activates the retinoic acid receptor (RAR)-ß promoter in response to RXR-selective retinoids, which is critical to inhibit cell growth and induce apoptosis in both trans-RA-sensitive and trans-RA-resistant breast cancer cell lines (5). In addition, we also found that TR3 exerts its effect on RARE through forming complexes with another orphan receptor COUP-TF that binds to the RARß promoter and is required for efficient RARß expression, resulting in disruption of COUP-TF stability on RA responsiveness of RARE (6). TR3 is an immediate early gene transiently induced by serum, growth factors, and nerve growth factors (1, 7). When induced by growth factors, TR3 remains transcriptionally inactive due to posttranslational modification (8). This is consistent with the observation that expression of TR3 mRNA does not necessarily correlate with induction of apoptosis (9). Recently we and others showed that mitochondrial targeting of TR3, but not its DNA binding and transactivation, is essential for its proapoptotic effect in response to phorbol ester 12-O-tetradecanoyl-13-phorbol acetate or nerve growth factor (9, 10, 11). Therefore, the diverse biological activities of TR3 may be regulated through distinct pathways at transcriptional and posttranscriptional levels.

Regulation of TR3 activities involves its phosphorylation. TR3 is heavily phosphorylated in vivo on multiple sites at its amino terminus, which is primarily responsible for its transactivation activity, whereas its carboxyl terminus is devoid of phosphorylation sites (12). Phosphorylation of TR3 on Ser350 negatively regulates its function. Ser350 resides within the domain required for specific binding of TR3 to DNA, and its phosphorylation results in reduction of both the DNA binding and transcriptional activities of TR3 in PC12 cells (8, 13). In vivo phosphorylation of Ser350 occurs in T cells as well as other cells (12, 14, 15). More recently it has been demonstrated that transfection of dominant-negative Akt or treatment with a phosphatidylinositol 3-kinase inhibitor accelerates mitogen-activated protein/ERK kinase (MEKK)-1-induced TR3 nuclear export through regulatory pathway of TR3 phosphorylation (16). We also found that the interaction of TR3 with Akt is a prerequisite for Akt to phosphorylate TR3 in the nuclear and cytoplasm (our unpublished data). Therefore, phosphorylation of TR3 by various kinases seems to antagonize its biological functions.

Many proteins required phosphorylation before ubiquitination, which creates docking sites recognized by specific E3 ligases (17, 18). Most nuclear hormone receptors are phosphoproteins, and the phosphorylation process is a potent regulatory event for nuclear receptor function. Depending on the nature of each receptor, phosphorylation positively or negatively regulates receptors’ DNA binding, dimerization, coactivator recruitment, and transactivation (19, 20). For example, phosphorylation of RAR at the N-terminal transactivation function domain augments ligand-induced transactivation (21). Conversely, RXR phosphorylation inhibits its transactivation properties (22).

The c-Jun N-terminal kinase (JNK) is activated by a variety of cellular signals including growth factors, inflammatory cytokines, and environmental stress (23). Multiple signaling pathways are associated with JNK activation. Activated JNK phosphorylates a variety of targeting proteins that belong to transcription factors and nuclear proteins, such as c-Jun, activating transcription factor-2, p53, phosphorylated mothers against decapentaplegic-3, and nuclear factor of activated T cells-4. It has been reported that the effect of MEKK1 is likely mediated by its activation on JNK, which in turn phosphorylates TR3 and inhibits TR3 DNA binding and transactivation activities (24). TR3 phosphorylation by JNK is essential but not sufficient to induce TR3 nuclear export (16). Although the JNK pathway has been shown to be involved in the regulation of TR3 phosphorylation (24), the exact phosphorylation site and the regulatory mechanism are still largely unknown. In the present study, we showed that JNK activator anisomycin could induce TR3 phosphorylation through the JNK (mainly JNK1) but not p38 and ERK pathways, which is regulated by its upstream factors, MAPK kinase (MKK)-4 and MKK7. We also identified the phosphorylation site of JNK on TR3 as serine 95, which belongs to a classical JNK phosphorylation motif (S/P). We further demonstrated that phosphorylation by JNK promoted TR3 degradation through the ubiquitin/proteasome pathway, which led to inhibition of TR3 DNA binding and transactivation activity, and finally resulted in the loss of TR3 mitogenic activity on cell proliferation. Taken together, our study provides the better understanding on the regulation of TR3 nuclear functions by JNK phosphorylation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
We purchased JNK activator (anisomycin, 10 µmol/liter; Sigma, St. Louis, MO), JNK inhibitor (JNK inhibitor II, 20 µmol/liter; Calbiochem, La Jolla, CA), MG132 (N-Cbz-Leu-Leu-Leu-al, 20 µ mol/liter; Sigma), ALLN (25 µmol/liter, N-acetyl-Leu-Leu-Norleu-al; Sigma), ALLM (25 µmol/liter, N-acetyl-Leu-Leu-Met-al; Calbiochem), and Z-VAD-FMK (25 µmol/liter; Sigma).

Cell culture and transfection
The human embryonic kidney (HEK) 293T cell line was maintained in DMEM containing 10% fetal bovine serum and antibodies (1 mmol/liter glutamine and 100 µg/ml penicillin). 293T cells were transfected using the calcium phosphate method. The total amount of DNA plasmid was kept constant by adding the respective empty vector plasmid DNA to the transfection mixtures. After transfection for 24 h, cells were treated with JNK activator or JNK inhibitor and harvested at different time points as required and then subjected to different experiments.

Ubiquitination assay
Cells were transfected with green fluorescent protein (GFP)-TR3, GFP-TR3 mutants, or His-Ubi expression vector and then lysed in Ni-agarose lysis buffer (50 mmol/liter NaH2PO4, 300 m mol/liter NaCl, 5 mmol/liter imidazole, 0.05% Tween 20, 100 µg N-ethylmaleimide per milliliter, and complete protease inhibitors). His-ubiquitin-conjugated proteins were purified by nickel chromatography (Ni-NTA-agarose; QIAGEN, Valencia, CA). The beads were washed successively 10 times with Ni-agarose wash buffer (50 mmol/liter NaH2PO4, 300 mmol/liter NaCl, 10 mmol/liter imidazole, 0.05% Tween 20). To reduce nonspecific binding to beads, nickel binding proteins were resuspended in 2x sample buffer supplemented with 200 mmol/liter imidazole and heated for 10 min at 100 C before being subjected to Western blotting with anti-GFP antibody.

In vitro phosphorylation assay
TR3 or its mutant TR3/S95A was cloned into a pGEX-4T-1 vector respectively (Amersham Pharmacia, Uppsala, Sweden). The glutathione-S-transferase (GST)-fusion protein was isolated according to the manufacturer’s recommendations. Purified GST-TR3 and GST-TR3/S95A were incubated with 200 ng JNK (Stratagene, La Jolla, CA) with the following modifications: 20 µCi [{gamma}-32P]ATP, 2 µM ATP, 8 mM 3[N-morpholino]propanesulfonic acid (pH 7.0), and 0.2 mM EDTA. The reaction was performed at 30 C for 30 min and then electrophoresed on a 10% polyacrylamide gel. The gel was dried and exposed to x-ray film.

EMSA
Nuclear protein was prepared and analyzed by EMSA as described previously (25). IRDye800-labeled TR3 oligonucleotides were purchased from LI-COR (Express Primers; Lincoln, NE). Briefly, EMSA binding reactions were performed by incubating 3 µg of nuclear extract with the annealed oligonucleotides according to the manufacturer’s instructions. The reaction mixture was subjected to electrophoresis on a 5% native gel and analyzed by an Odyssey infrared imaging system (LI-COR).

Western blot analysis
Cell extracts were prepared. About 50 µg of protein were electrophoresed on approximately 8–10% denaturing gel and electroblotted onto a nitrocellulose membrane. The membrane was incubated with different antibodies required at 4 C overnight, followed by adding corresponding secondary antibody at room temperature approximately or 3–4 h. An enhanced chemiluminescence kit (Pierce, Rockford, IL) was used to detect the antibody reactivity according to the manufacturer’s instruction.

Luciferase assay
Cells were transfected with TR3-Luc reporter plasmid (NurRE) and ß-galactosidase (ß-gal) expression vector in the absence or presence of different protein expression vectors as indicated in the figures using the calcium phosphate method. After transfection, luciferase activity was normalized for transfection efficiency using corresponding ß-gal activity. The ratios of luciferase to ß-gal activity were used as indicators of p53 transcriptional activity.

5-Bromo-2'-deoxyuridine (BrdU) assay
Cells were transfected with expression vectors as required and treated with or without JNK activator and then incubated with BrdU (20 µmol/liter; Sigma) for 2 h. After washing with PBS, cells were fixed with 4% paraformaldehyde for 30 min at 4 C and then incubated with saponin (0.1%; Sigma) for another 10 min. The cells were washed twice with PBS containing 0.1% saponin and resuspended in PBS containing 30 µg DNase I. After incubation with anti-BrdU antibody for 1 h, cells were given two PBS washes and then incubated with phycoerythrin-linked antimouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Finally, cells were washed with PBS and analyzed by flow cytometer (Beckman Coulter, Fullerton, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Activation of JNK induces TR3 phosphorylation
To investigate whether JNK signal is involved in the regulation of orphan receptor TR3, we treated TR3 expression vector transfected HEK 293T cells with either a potent JNK activator (anisomycin) (26, 27, 28) or a JNK inhibitor (JNK inhibitor II) (16, 24). Cell lysates were then prepared and subjected to Western blotting analysis. Treatment of cells with JNK activator for 1 h resulted in an obvious up-shift of the TR3 band (Fig. 1AGo, upper panel), whereas treatment of JNK inhibitor did not induce such band shift (Fig. 1AGo, lower panel). We further incubated the cell lysates with calf intestinal alkaline phosphatase (CIAP) that catalyzes protein dephosphorylation (29). We found that CIAP could effectively diminish the JA-induced band shift of TR3 (Fig. 1AGo, upper panel). On the other hand, pretreatment of cells with okadaic acid (OA), a general inhibitor of protein phosphatases (30), slightly enhanced the up-shift of TR3 (Fig. 1AGo, upper panel). Together these results suggested that the mobility change of TR3 is due to phosphorylation. In the control experiments, neither CIAP nor OA showed effects on the gel mobility of TR3 when cells were treated with JNK inhibitor (Fig. 1AGo, lower panel). Thus, we concluded that activation of the JNK signaling pathway induces the phosphorylation of TR3.


Figure 1
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  		FIG. 1. Effect of JNK on TR3 phosphorylation in HEK293T cells. A, JNK induced phosphorylation of TR3. GFP-TR3 expression vector-transfected cells were treated with JNK activator (JA) anisomycin or JNK inhibitor II (JI) for 1 h. Expression of GFP-TR3 was determined by Western blotting using anti-GFP antibody; {alpha}-tubulin expression was served as control for similar loading of proteins in each lane. To characterize the up-shifted band, the cell lysates were preincubated with calf intestinal alkaline phosphatase (CIAP, 0.5 µmol/liter) for 30 min, or cells were pretreated with OA (5 nmol/liter) for 30 min. B, JNK activator-induced phosphorylation of TR3 was mediated by JNK rather than p38 and ERK. Cells transfected with GFP-TR3 were treated with JNK activator for 1 h with or without SB203580, PD98059, or JI. Expression of GFP-TR3 was determined by Western blotting using anti-GFP antibody, and the specificities of effect of JI, SB203580, and PD98059 were determined by using antibodies against phosphor-c-Jun, phosphor-p38 and phosphor-ERK. C, MKK4/7 was involved in regulation of TR3 phosphorylation induced by JNK. Cells were transfected with dominant-negative MKK4 (MKK4-DN) or MKK7 (MKK7-DN) together with GFP-TR3 expression vector and then treated with JNK activator, CIAP, or OA as described above. TR3 expression was examined by Western blotting. D, JNK1 but not JNK2 was critical for TR3 phosphorylation. Cells were transfected with dominant-negative JNK1 (JNK1-AF) and JNK2 (JNK2-AF) in the absence or presence of JA. Cell lysates were probed with anti-GFP, -Flag, and -hemagglutinin (HA), respectively, to detect the expressions of TR3, JNK1-AF, and JNK2-AF.

 
Because anisomycin can activate JNK (16, 27) and p38 (31) but not ERKs (16, 27), we further investigated whether activation of p38 or ERK is also responsible for TR3 phosphorylation. Although JNK inhibitor could efficiently block the anisomycin-induced phosphorylation of TR3, the p38-specific inhibitor SB203580 and the ERK-specific inhibitor PD98059 failed to do so (Fig. 1BGo). Therefore, phosphorylation of TR3 was mainly mediated by the activation of JNK rather than p38 and ERK.

Kinases MKK4 and MKK7 are two known upstream activators of JNK (32). To address the role of MKK in TR3 phosphorylation, two MKK dead mutants, MKK4-DN and MKK7-DN (33), were coexpressed with TR3 in 293T cells, respectively, to suppress the endogenous JNK activity. As shown in Fig. 1CGo, these two mutants could inhibit JNK-mediated TR3 phosphorylation induced by anisomycin, even cells that were pretreated with OA to prevent dephosphorylation. Therefore, the MKK signal is indeed involved in the JNK-induced TR3 phosphorylation. JNK has been shown to consist of several isoforms, including JNK1 and JNK2 (34). We used catalytically inactive forms JNK1 (JNK1-AF) and JNK2 (JNK2-AF) that could act as dominant-negative mutants and thus inhibit activation of endogenous JNK (35, 36) to determine the involvement of each isoform in TR3 phosphorylation. The results showed that JNK1-AF, but not JNK2-AF, could markedly block the effect of JNK activator on inducing TR3 phosphorylation (Fig. 1DGo). Taken together, these results demonstrated that induction of TR3 phosphorylation by JNK activator is mainly mediated through the activation of JNK1, which is regulated by the MKK4/MKK7 signaling pathway.

JNK phosphorylates TR3 on Ser95
Next, we went on to determine the exact phosphorylation site of JNK on TR3. We first determined which domain of TR3 is responsive to JNK activation. Serial deletion mutants of TR3 were constructed and expressed in 293T cells, respectively (Fig. 2AGo). The phosphorylation status of each mutant (reflected by gel mobility) in response to JNK activator was then examined. Although both the C-terminal-truncated mutant TR3{Delta}C574 and the N-terminal-truncated mutant TR3{Delta}N50 [lacking amino acids (a.a.) 1–50] were phosphorylated, we found that further removal of another 48 a.a. from the N terminus totally abolished the phosphorylation (Fig. 2BGo), indicating that the region between a.a. 51 and 99 contains the phosphorylation site(s) of JNK.


Figure 2
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  		FIG. 2. Characterization of the exact phosphorylation site of JNK on TR3. A, Schematic representation of TR3 deletion mutants and their expressions. Different TR3 deletion mutants were transfected into 293T cells, respectively, and their expressions were determined by Western blotting. B and C, Phosphorylation of different TR3 deletion mutants by JNK. 293T cells were transfected with different GFP-TR3 mutants as indicated and then treated with JNK activator for 2 h or pretreated with CIAP and OA as described in Fig. 1AGo. The TR3 expression was analyzed by Western blotting. DBD, DNA binding domain; LBD, ligand binding domain; TAD, transactivation domain.

 
To identify the exact JNK phosphorylation residue(s) on TR3, we constructed more TR3 deletion mutants, including TR3{Delta}N74, TR3{Delta}N84, and TR3{Delta}N92 (Fig. 2AGo), for further analysis. All these mutants could still be phosphorylated by JNK activator-activated JNK (Fig. 2BGo), suggesting that the JNK phosphorylation site(s) should be within the region of a.a. 93–98 on TR3. Sequence analysis of this region (Fig. 2AGo) revealed a classical JNK phosphorylation motif (S/P) at position 95–96 (37). To confirm that Ser95 is indeed the phosphorylation site of JNK, we mutated this residue into alanine (TR3/S95A) to mimic its constitutively unphosphorylated status and then transfected this construct into 293T cells. As expected, the mutation of S95A totally abolished the phosphorylation induced by JNK activator (Fig. 2CGo, upper panel). In contrast, mutation of a nearby serine into alanine (TR3/S100A) did not affect the phosphorylation of TR3 (Fig. 2CGo, lower panel). Together these results demonstrated that Ser95 on TR3 is the phosphorylation target of JNK.

Phosphorylation of TR3 by JNK triggers its degradation
Next, we examined the phosphorylation status of endogenous TR3 in gastric cancer cell line BGC-823. Consistent with the results seen in 293T cells, JNK activator also induced the phosphorylation of TR3 in BGC-823 cells (Fig. 3AGo). Moreover, we noted that the expression level of endogenous TR3 reduced upon extended treatment of JNK activator. When cells were treated with JNK activator for 4 h, TR3 became hardly detected, indicating that TR3 phosphorylation may trigger its degradation. The JNK activator-induced TR3 degradation was largely blocked when cells were pretreated with JNK inhibitor (Fig. 3AGo), further confirming that the degradation is phosphorylation dependent. Similar to endogenous TR3 in BGC-823 cells, exogenous TR3 expressed in 293T cells was also degraded upon the treatment of JNK activator, and such degradation could also be repressed by JNK inhibitor (Fig. 3BGo, upper panel). The observed degradation turned out to be TR3 specific because expression of GFP alone showed no obvious degradation under the same condition (Fig. 3BGo, lower panel).


Figure 3
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  		FIG. 3. JNK induced degradation of TR3 through the ubiquitination/proteasome pathway. A and B, Degradation of endogenous and exogenous TR3 in JNK activator (JA)-treated gastric cancer BGC-823 cells and 293T cells. Cells were transfected with GFP-TR3 expression vector and then treated with JNK activator for different times as indicated, with or without pretreated JNK inhibitor (JI) for 30 min. The expression of TR3 was analyzed by Western blotting, and the expression of GFP was monitored as a control. EGFP, Enhanced GFP. C, Effect of different inhibitors on degradation of TR3 induced by JNK. GFP-TR3 expression vector transfected 293T cells were pretreated with different inhibitors, including MG132, ALLN, ALLM, and Z-VAD for 30 min. After further incubation with JNK activator for another 4 h, cell extracts were prepared. The expression of TR3 was analyzed by Western blotting. D, Effect of JNK activator on TR3 ubiquitination. His-ubiquitin and GFP-TR3 were coexpressed in 293T cells. TR3 ubiquitination was monitored in immunoblots performed on nickel-agarose beads purified proteins. The upper panel was probed with GFP antibody to identify any TR3-associated tagged ubiquitin. The lower panel was analyzed using antitubulin antibody to demonstrate similar loading of proteins in each lane. WB, Western blot. E and F, JNK-induced degradation and ubiquitination in different TR3 mutants. 293T cells were transfected with different TR3 mutants and then treated with JNK activator as indicated. TR3 expression and ubiquitination were analyzed as described above.

 
To determine which pathway is involved in JNK-induced TR3 degradation, 293T cells transfected with TR3 were pretreated with MG132 (a potent cell permeable proteasome inhibitor), ALLN (an inhibitor for calpains, cathepsins, and proteasomal proteolysis), ALLM (a potential inhibitor for calpains and cathepsins but not for proteasome), or Z-VAD-FMK (a caspase inhibitor) for 30 min, respectively, and followed by treatment with JNK activator for 4 h. As shown in Fig. 3CGo, the JNK activator-dependent degradation of TR3 was specifically blocked by MG132 and ALLN but not by ALLM and Z-VAD-FMK, indicating that JNK-induced TR3 degradation requires the activation of the proteasome pathway. To further assess whether this degradation is associated with a ubiquitin-dependent mechanism, ubiquitin and TR3 expression vectors were cotransfected into 293T cells. Cell lysates were prepared after the treatment of JNK activator for 4 h, and ubiquitin was purified with nickel chromatography and then immunoblotted with appropriate antibody. As shown in Fig. 3DGo, high-molecular-weight bands representing ubiquitinated forms of TR3 were detected in samples that cotransfected TR3 with ubiquitin but not in samples that transfected with TR3 alone. Moreover, the ubiquitinated bands of TR3 were enhanced in the presence of MG132. Together, these results suggest that the degradation of TR3 induced by JNK is through the ubiquitination/proteasome pathway.

We further investigated the role of JNK phosphorylation in the degradation of TR3. For this purpose, the TR3 deletion mutant TR3{Delta}N92, which has been shown to be phosphorylated by JNK, was transfected into 293T cells. Treatment of the cells with JNK activator did induce the degradation of TR3{Delta}N92 in a time-dependent manner (Fig. 3EGo). In the presence of MG132, the JNK activator-induced ubiquitination bands of TR3{Delta}N92 were further enhanced (Fig. 3FGo). In contrast, exogenous expressed TR3{Delta}N98, the mutant that could not be phosphorylated by JNK, did not show any level of degradation, even when the cells had been treated with JNK activator for 4 h (Fig. 3EGo). When coexpressed with ubiquitin, its ubiquitinated bands did not display any enhancement in the presence of MG132 (Fig. 3FGo). Similar results were observed with TR3/S95A that was not phosphorylated by JNK (Fig. 3Go, E and F). Clearly these data demonstrate that JNK phosphorylation results in TR3 degradation via the ubiquitination/proteasome pathway.

TR3 loses its mitogenic effect upon phosphorylation
TR3 exerts its effect on cell proliferation by functioning as a nuclear transcription factor in the nucleus of lung cancer cells (24). In agreement with this observation, we also found that TR3 has mitogenic effects in 293T cells as detected by BrdU assay. GFP-TR3-transfected cells showed an obvious increase in BrdU incorporation than the GFP-transfected cells (Fig. 4AGo), suggesting that TR3 functions as a mitogenic factor in cell growth through stimulating DNA synthesis of 293T cells. However, when the transfected cells were treated with JNK activator, the BrdU incorporation rate decreased, indicating that JNK phosphorylation may impair the mitogenic activity of TR3. We further used different mutants of TR3 (TR3{Delta}N98 TR3{Delta}C574, and TR3/S95A) to characterize the relationship between phosphorylation of TR3 and DNA synthesis in the presence of JNK activator. As shown in Fig. 4Go, JNK activator could decrease BrdU incorporation in cells transfected with TR3{Delta}C574 (Fig. 4BGo) but not TR3{Delta}N98 (Fig. 4CGo) and TR3/S95A (Fig. 4DGo), indicating an important effect by JNK-induced phosphorylation on TR3 mitogenic activity. These results thus further support that impairing of the nuclear mitogenic activity of TR3 is correlated with its phosphorylation.


Figure 4
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  		FIG. 4. JNK inhibited TR3-mediated mitogenic activity. TR3 and its mutation expression vectors were transfected into 293T cells as indicated. After treatment with JNK activator (JA) for 1 h, the cells were maintained in BrdU containing medium for 2 h. The cells were then identified by flow cytometry.

 
JNK phosphorylation represses the DNA binding and transactivation activity of TR3
The loss of mitogenic effect of phosphorylated TR3 suggests that the transcriptional activity of TR3 may be repressed upon its phosphorylation by JNK. Therefore, we examined the transcriptional activity of TR3 in response to JNK activator by monitoring luciferase reporter gene that contains a TR3 homodimer-binding site (NurRE) in its promoter region (38). Transfection of the TR3 expression vector into 293T cells strongly induced the transcriptional activity of reporter gene in a dose-dependent manner (Fig. 5AGo). As expected, JNK activator treatment obviously decreased the transcriptional activity of TR3 in a time-dependent manner (Fig. 5BGo). To exclude the possibility that reduction of transcriptional activity of TR3 is due to decreased amount of TR3 by JNK activator, MG132 was added to protect TR3 from degradation. Under this circumstance, an inhibition of TR3 transcriptional activity by JNK activator was still observed in a time-dependent fashion (Fig. 5BGo), suggesting a direct correlation between transcriptional activity and phosphorylation of TR3 induced by JNK activator. In addition, introducing dominant-negative MKK4 or MKK7 disrupted the effect of JNK on decreasing TR3 transcriptional activity (Fig. 5BGo). Next, we investigated the transactivation activities of different TR3 mutants. As shown in Fig. 5CGo, the transactivational function of TR3{Delta}C574, but not that of the point mutants TR3/S95A, was impaired by JNK activator treatment, suggesting a correlation between TR3 phosphorylation and transactivation activity. Moreover, cotransfection of the catalytically inactive form of JNK1-AF, instead of JNK2-AF, strongly inhibited JNK activator-induced repression of TR3 transcriptional activity (Fig. 5DGo). Collectively, these data demonstrate that JNK1-induced TR3 phosphorylation down-regulates the TR3 transactivational activity.


Figure 5
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  		FIG. 5. JNK-regulated transactivation activity and DNA binding of TR3. A–D, Effect of JNK activator (JA) on TR3-dependent transcriptional activity (A and C) regulated by dominant-negative MKK4/7 (B) and dominant-negative JNK1/2 (D). TR3 (or different TR3 mutants) and TR3-Luc reporter was transfected into 293T cells with or without MKK4/7-DN or JNK1/2-AF as indicated. After treatment of JNK activator for 1 h, luciferase activity was measured and normalized by ß-gal activity. All transfections were performed in three independent assays, and the data represented the means ± SD. E, Effect of JNK on TR3 DNA binding. GFP-TR3 or its point mutant TR3/S95A was transfected into 293T cells, respectively, and then treated with JNK activator or JNK inhibitor for 1 h. Nuclear proteins were prepared, and homodimerization of TR3 was analyzed by EMSA and probed with IRDye800-labeled TR3 oligonucleotides. To determine the formation of TR3 homodimer, antibody specific for GFP (aGFP) was preincubated with nuclear proteins for 30 min at room temperature before the EMSA. As control, preimmune serum (PI) was also incubated with nuclear proteins for 30 min at room temperature. To protect TR3 from degradation, cells were pretreated with MG132 for 30 min. F, Phosphorylation of TR3 by JNK in vitro. Bacterially expressed and purified GST-TR3 or GST-TR3/S95A was incubated with JNK and then subjected to in vitro phosphorylation assay as described in Materials and Methods. GST and GST-c-Jun were used as negative and positive control, respectively. Coomassie blue staining was used to indicate the GST fusion protein expression levels (lower panel). G, Effect of phosphorylation on TR3 DNA binding. To get the phosphorylated protein, bacterially expressed GST-TR3 or GST-TR3/S95A was preincubated with JNK and then subjected to the EMSA as described in E.

 
One possible mechanism by which JNK-induced phosphorylation represses the transcriptional activity of TR3 is to inhibit TR3 DNA binding activity. We therefore examined whether the DNA binding activity of TR3 is impaired in the presence of JNK activator. Gel retardation assay was performed to determine the binding of TR3 protein to NurRE, a TR3 homodimer-binding site. As shown in Fig. 5EGo, nuclear proteins of GFP-TR3-transfected 293T cells formed a strong complex that bound to NurRE, and this complex band was completely up-shifted only when the nuclear proteins were preincubated with anti-GFP antibody rather than preimmune serum, indicating that the complex is composed of homodimeric TR3. We found that this complex formation could be inhibited by JNK activator but not JNK inhibitor, suggesting that JNK phosphorylation inhibits the DNA binding activity of TR3. We further analyzed the DNA binding activity of TR3 point mutant TR3/S95A that was not phosphorylated by JNK activation. As expected, JNK activator did not showed any effect on TR3 homodimerization. In addition, to exclude the possibility that retardation of TR3 to DNA is due to decreased amount of TR3 by JNK activator, MG132 was added to protect TR3 from degradation. However, TR3 complex remained to be inhibited by JNK activator as effectively as the non-MG132 group, further confirming a relationship between phosphorylation and DNA binding of TR3 by JNK activation.

To further understand the relationship between TR3 phosphorylation and its DNA binding in response to JNK activation, we studied how JNK phosphorylates TR3 in bacterially expressed GST-TR3 fusion protein system. Using an in vitro phosphorylation assay, the purified GST-TR3, but not GST-TR3/S95A, was found to be phosphorylated when incubated with JNK (Fig. 5FGo). In control experiments, the positive control GST-c-Jun also showed strong phosphorylation by JNK, whereas no phosphorylation could be observed on the negative control GST. The bacterially expressed GST-TR3 could form a complex with NurRE in gel retardation assay. However, when phosphorylated by JNK, binding of GST-TR3 to NurRE was largely diminished (Fig. 5GGo). On the other hand, the binding activity of TR3 point mutant TR3/S95A was not changed after incubation with JNK. Taken together, these data demonstrate that JNK phosphorylation suppresses the transactivational function of TR3 by inhibiting its DNA binding activity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
JNK is a crucial regulator of nuclear receptor function. It has the ability to phosphorylate a wide range of nuclear receptors, including glucocorticoid receptor, estrogen receptor, peroxisomal proliferators-activated receptor, RAR, and RXR (39, 40, 41, 42, 43). Similarly, JNK also phosphorylates TR3 (24), a nuclear orphan receptor that belongs to the superfamily of steroid/thyroid/retinoid receptor. In the present study, we found that JNK activator anisomycin induced TR3 to be phosphorylated by JNK but not p38 and ERK. We further showed that JNK phosphorylates TR3 on the serine 95 that matches the JNK phosphorylation consensus sequence. Such phosphorylation causes the degradation of TR3 through ubiquitination pathway and finally results in the loss of TR3 mitogenic activity on cell proliferation. Our results highlight the regulatory mechanism of JNK signaling on TR3 and offer a novel insight into the repression of TR3 DNA binding and transcriptional activity by JNK phosphorylation.

Although JNK pathway has been shown to regulate activities of several nuclear receptors (44, 45, 46, 47), the molecular basis for regulation of these receptors by JNK is different and appears to be complicated. TR3 is a cellular target of JNK phosphorylation (Fig. 1AGo). Anisomycin, a potent activator of JNK and p38 (31) but not for ERK (16, 27), could induce TR3 phosphorylation in a JNK-dependent and p38- and ERK-independent manner (Fig. 1BGo). Among JNK isoforms, JNK1 played a causal role in TR3 phosphorylation (Fig. 1DGo). JNK-induced TR3 phosphorylation is specifically mediated through MKK pathway because the dominant-negative MKK4/7, upstream activators of JNK (48, 49, 50), could effectively suppress the TR3 phosphorylation by JNK (Fig. 1CGo). Therefore, TR3 is one of the critical effecters of JNK activation. Further deletion analysis showed that JNK phosphorylated TR3{Delta}N92 but not TR3{Delta}N98 (Fig. 2BGo), suggesting that the N-terminal a.a. 93–98 region of TR3 is critical for JNK phosphorylating action. Indeed, this sequence (93-ATSPAS-98) contains a typical phosphorylation consensus of JNK, S/P (37). In further point mutant analysis, replacement of serine with Alanine in TR3 abolished the phosphorylation of TR3 by JNK. Therefore, serine 95 is the only residue responsible for TR3 phosphorylation by JNK. The phosphorylation consensus in other structural domain, such as kinase-docking site, has been shown to be required for JNK-specific phosphorylation. For example, JNK activates c-Jun by phosphorylating its Ser63 and Ser73 (51). However, the mutant of v-Jun, which deletes docking site but retains phosphorylation site Ser63 and ser73, is not phosphorylated by JNK (52), indicating that docking site is also required for JNK to phosphorylate v-Jun. The substrates of JNK normally contain a conserved docking site LXL (also referred to as D site) (53). Interestingly, there are three potential D sites in TR3. Whether these sites are also involved in the phosphorylation of TR3 by JNK needs to be further addressed.

We found that phosphorylation of TR3 by JNK was closely linked to its degradation through the ubiquitination/proteasome pathway (Fig. 3Go, A, B, and D) because JNK activator did not result in TR3 degradation in the presence of inhibitors for 26S proteasome, MG132 and ALLN (Fig. 3CGo). Furthermore, JNK did not induce the degradation of TR3 mutants lacking JNK phosphorylation sites (Fig. 3EGo), suggesting that ubiquitin targeting to TR3 is phosphorylation dependent (Fig. 3FGo). Phosphorylation might profoundly change the properties and functions of TR3, such as altering the conformation, DNA binding, and transcriptional activation of TR3. Phosphorylation-mediated conformational changes are expected to regulate TR3 dimerization and DNA binding (54).

Multiple kinase signaling pathways, including protein kinase A, protein kinase C, and MAPK, are involved in the gene expression as well as the transactivation function of TR3 (55, 56). In addition, modulation of transcriptional activity by JNK has been demonstrated for other nuclear receptors, including RXR (43, 57), RAR (39), and glucocorticoid receptor (42). It has been reported that MEKK1 inhibits TR3 transcriptional activity and TR3-mediated cellular proliferation through the activation of JNK (24). In contrast, Slagsvold et al. (58) showed that ERK2 mediates the phosphorylation of TR3 in vitro when a survival signal is received from growth factors such as epithelial growth factor. Our data showed that JNK could effectively phosphorylate TR3 at serine 95 in N terminus, indicating that this process might be crucial for TR3 transactivation because its N terminus is responsible for TR3 transactivation function. Indeed, we observed that phosphorylation of TR3 by JNK inhibited TR3 transcriptional activity (Fig. 5Go, A–C), which was closely related to the blockage of TR3 binding to DNA (Fig. 5EGo). The inhibition on DNA binding of TR3 is presumably via the modulation of its dimerization because no TR3 homodimers could be detected in the presence of JNK activator (Fig. 5EGo). More recently it has been shown that activation of JNK by 4-[3-Cl-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid plays a role in translocation of TR3 from the nucleus to the cytoplasm, in which TR3 was hyperphosphorylated (16). However, induction of TR3 nuclear export by JNK required other signaling pathway or protein factors, such as inhibition of Akt phosphorylation on TR3 (16). These evidences inspire us to ponder over the possible strategy to turn TR3 protein to good account in cancer cell inhibition through different signaling pathway because TR3 is constitutively expressed in cancer cells.

JNK cascade is important in regulating cell death decisions. Cytokines, such as TNF-{alpha} and IL-1, usually transiently activate JNK and promote cell survival (59, 60). However, genotoxic stresses, such as UV and {gamma}-irradiation, induce sustained JNK activation and trigger cell death (61, 62). Collectively, these studies demonstrated that JNK can either promote or inhibit cell proliferation, depending on the nature of cell type and stimulus. TR3 is an immediate-early response gene and rapidly reacts to several mitogenic inducers, including serum growth factor, epidermal growth factor, and fibroblast growth factor (1, 2, 63, 64). TR3 is overexpressed in most cancer cells due to the uncontrolled expression of growth factors (6, 65). TR3 overexpression might confer a proliferative advantage to cancer cells (66, 67). For example, ectopic expression of TR3 in lung cancer cells stimulates their cell cycle progression and proliferation, whereas inhibition of endogenous TR3 expression suppresses proliferation induced by growth factors (24). Moreover, overexpression of TR3 prevents ceramide-induced cell death in neuronal cells and protects cells from TNF-induced apoptosis in mouse embryonic fibroblasts (6, 68, 69). Similarly, we found that TR3 was directly involved in promoting 293T cell proliferation, and such TR3-mediated cell proliferation could be repressed by JNK signal through inhibition of DNA synthesis (Fig. 4Go, A and B). In fact, the mitogenic effect of TR3 required its DNA binding and transactivation functions (Fig. 5EGo) (24). Consistent with the notion that the N terminus of TR3 is associated with the major transactivation activity (70), TR3 lacking the N terminus failed to promote the 293T cell growth (Fig. 4CGo). Furthermore, in the presence of JNK, phosphorylated TR3 lost its mitogenic role in cell proliferation (Fig. 4Go), showing a good agreement with the loss of TR3 DNA binding and transactivation activity (Figs. 4Go and 5Go, E and G). Our in vitro phosphorylation assay (Fig. F) further confirmed this negative relationship between TR3 phosphorylation by JNK and its DNA binding activity. Clearly the mitogenic effect of TR3 on cell proliferation depends on its DNA binding and transcription activities, and JNK phosphorylation represses these activities.

In summary, this study demonstrates that TR3 ubiquitination and degradation are triggered by JNK activation, which is closely associated with TR3 phosphorylation by JNK. JNK appears to be one of the principal regulators of TR3 functions. It is likely that JNK phosphorylates and consequently interferes with the metabolism and signaling of TR3 in cells, resulting in the repression of its DNA binding and transcriptional activation and finally contributing to the dysfunction of TR3 in cell proliferation.


   Acknowledgments
 
We thank Dr. Jia-huai Han and Dr. Sheng-cai Lin (Xiamen University, Xiamen, Fujian, China) for plasmids of JNK1-AF, JNK2-AF, MKK4-DN, and MKK7-DN.


   Footnotes
 
This work was supported by the National Outstanding Young Science Foundation (30425014); Grant 2004CCA02100 from the Ministry of Science and Technology of China); The National Natural Science Foundation of China (30370715, 30570936); The National Natural Science Foundation of Fujian Province (C0410003); and Grant 2005105 from Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering.

Disclosure Statement: The authors have nothing to disclose.

First Published Online October 5, 2006

Abbreviations: a.a., Amino acids; BrdU, 5-bromo-2'-deoxyuridine; CIAP, calf intestinal alkaline phosphatase; ß-gal, ß-galactosidase; GFP, green fluorescent protein; GST, glutathione-S-transferase; HEK, human embryonic kidney; JNK, c-Jun N-terminal kinase; MEKK, mitogen-activated protein/ERK kinase; MKK, MAPK kinase; OA, okadaic acid; RA, retinoic acid; RAR, retinoic acid receptor; RARE, RA response element; RXR, retinoid X receptor.

Received June 14, 2006.

Accepted for publication September 25, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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