Skip Navigation


Carcinogenesis Advance Access originally published online on August 19, 2008
Carcinogenesis 2008 29(11):2078-2088; doi:10.1093/carcin/bgn197
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
29/11/2078    most recent
bgn197v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Chen, H.-Z.
Right arrow Articles by Wu, Q.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, H.-Z.
Right arrow Articles by Wu, Q.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Akt phosphorylates the TR3 orphan receptor and blocks its targeting to the mitochondria

Hang-Zi Chen, Bi-Xing Zhao, Wen-Xiu Zhao, Li Li, Bing 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

* To whom correspondence should be addressed. Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen 361005, Fujian Province, China. Tel: +86 592 2187959; Fax: +86 592 2086630; Email: qiaow{at}xmu.edu.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 
Acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) phosphorylates and regulates the function of many cellular proteins involved in processes such as metabolism, apoptosis and proliferation. However, the precise mechanisms by which Akt promotes cell survival and inhibits apoptosis have been characterized in part only. TR3, an orphan receptor, functions as a transcription factor that can both positively or negatively regulate gene expression. We have reported previously that the translocation of TR3 from the nucleus to the mitochondria can elicit a proapoptotic effect in gastric cancer cells. In our present study, we demonstrate that Akt phosphorylates cytoplasmic TR3 through its physical interaction with the N-terminus of TR3. When coexpressed with Akt, TR3 mitochondrial targeting was blocked and this protein adopted a diffuse expression pattern in the cytoplasm. Moreover, Akt displayed an ability to disrupt the interaction of TR3 with Bcl-2, which is thought to be a critical requirement for mitochondrial TR3 to elicit apoptosis. Consistently, insulin was also found to induce the phosphorylation of TR3 and abolish 12-O-tetradecanoylphorbol-13-acetate-induced mitochondrial localization, which was dependent upon the activation of the phophatidylinositol-3-OH-kinase–Akt signaling pathway. Taken together, our current data demonstrate a unique role for Akt in inhibiting TR3 functions that are not related to transcriptional activity but that correlate with the regulation of its mitochondrial association. This may represent a novel signal pathway by which Akt exerts its antiapoptotic effects in gastric cancer cells, i.e. by regulating the phosphorylation and redistribution of orphan receptors.

Abbreviations: AKT, acutely transforming retrovirus AKT8 in rodent T cell lymphoma; CIAP, calf intestine alkaline phosphatase; DBD, DNA-binding domain; DN-Akt, dominant-negative Akt; ERK, extracellular signal-regulated kinase; OA, okadaic acid; PBS, phosphate-buffered saline; PI3K, phophatidylinositol-3-OH-kinase; TM, transmembrane domain; TPA, 12-O-tetradecanoylphorbol-13-acetate


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
The immediate-early gene product TR3 (also known as Nur77 or nerve growth factor-induced gene-B) is an orphan nuclear receptor of the steroid/thyroid/retinoid receptor superfamily (1–3). The members of this family mainly act as transcription factors that can both positively or negatively regulate gene expression (4–6). TR3 is highly expressed in a variety of cell types (7–9) and has been implicated in the regulation of cell proliferation and differentiation. Recent studies strongly suggest that TR3 also acts as a potent proapoptotic molecule (4,5,10). Transgenic mice that express wild-type TR3 exhibit massive apoptosis (11,12). Conversely, expression of a dominant-negative TR3 blocks activation-induced cell death in T-cell hybridomas as well as negative selection in transgenic mice (11,13). A rapid induction of TR3 messenger RNA has also been found in other carcinoma cells after stimulation with a variety of apoptosis-inducing agents, such as phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), calcium ionophore A23187 [GenBank] and etoposide VP-16 (7,12,14). Recently, we and other groups have observed that in gastric and prostate cancer cells, TR3 mitochondrial targeting, rather than the activation of TR3 transcription activity, is critical in promoting the induction of apoptosis (8,14,15). This suggests a distinct mechanism for TR3 in the initiation of the apoptotic response.

The activity of TR3 is controlled in part by phosphorylation and this orphan receptor is heavily phosphorylated in vivo on multiple sites around its N-terminus, whereas its C-terminus is devoid of phosphorylation sites (16). The phosphorylation of TR3 at Ser351, which resides within its DNA-binding domain (DBD), has been shown to inhibit the DNA binding of TR3 in vitro (17). In PC12 cells, the nerve growth factor-induced phosphorylation of Ser351 results in a 60% decrease in TR3 transactivation activity (18). In addition, the phosphorylation of Ser105 of TR3 in PC12 cells, via the TrK/Ras/mitogen-activated protein kinase pathway upon treatment with nerve growth factor, seems to promote its translocation from the nucleus to the cytoplasm (19). Therefore, it is probably that the phosphorylation of TR3 within its DBD may contribute to the modulation of its transactivation function. In addition, the phosphorylation of TR3 within its N-terminus may be associated with its nucleocytoplasmic translocation. However, whether the phosphorylation of cytoplasmic TR3 molecules affects their mitochondrial targeting and the biological functions associated with this, such as the proapoptotic effects of this localization in cancer cells, has not yet been reported.

The protein kinase acutely transforming retrovirus AKT8 in rodent T cell lymphoma (Akt) is a critical molecule in the transduction of proliferative and antiapoptotic signals and can be activated by a variety of stimuli in a phophatidylinositol-3-OH-kinase (PI3K)-dependent manner (20–23). Akt promotes cell survival and represses apoptosis through diverse mechanisms in numerous cell types. One of the best-known examples of this is the promotion of cell survival by Akt through the direct phosphorylation of Bad, a key regulator of the apoptotic cascade and a member of the Bcl-2 family. Akt phosphorylates Bad upon residue Ser316, which promotes the sequestration of Bad by 14-3-3 proteins in the cytosol, thus preventing its interaction with Bcl-2 at the mitochondrial membrane (24,25). Additionally, Akt may also promote cell survival via other pathways. For instance, by inactivating GSK-3, Akt could stabilize cyclin D1, an important protein in cell cycle progression, by elevating the levels of β-catenin (26,27).

The number of identified physiological substrates of Akt is expanding rapidly and includes hormone receptors. The fact that Akt-mediated phosphorylation of the estrogen receptor correlates with cellular resistance to tamoxifen suggests that the upregulation of Akt may contribute to antiestrogen resistance (28). Akt has also been shown to exert a similar effect upon the androgen receptor (29). TR3 is also a target of Akt. In 293T and NIH3T3 cells, Akt interacts with TR3 and inactivates this receptor by phosphorylation at Ser351 in a PI3K-dependent manner (30). Similar observations in T-cells have also illustrated that the phosphorylation of TR3 by Akt at Ser351 results in reduction of its DNA-binding activity and stimulation of its association with 14-3-3 in a phosphorylation site-dependent manner (31). Moreover, the expression of Akt suppresses TR3-induced apoptosis in fibroblasts and activation-induced cell death of T-cell hybridomas (31). These findings strongly suggest that Akt suppresses TR3-mediated cell death and highlight an antagonistic interaction between apoptosis and survival signaling pathways. However, the precise mechanism of how Akt suppresses TR3-mediated cell death is still not well understood. In our present study, we find that Akt inhibits apoptosis by preventing TR3 mitochondrial targeting, and that this process is closely associated with the phosphorylation of cytoplasmic TR3 by Akt. Analysis of TR3 deletion mutants further revealed that the N-terminus of TR3 is responsible for phosphorylation by Akt in the cytoplasm. Moreover, phosphorylation by Akt led to the disassociation of TR3 and Bcl-2, resulting in a suppression of the apoptotic response function of TR3. Our current study therefore reveals novel aspects of the mechanism underlying the antagonistic interactions between the cellular protein kinase Akt and the orphan receptor TR3 in terms of their biological functions and signaling transduction.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Cell culture
Human gastric cancer cell line BGC-823 was purchased from the Institute of Cell Biology, Shanghai, China, and maintained in RPMI-1640 medium. Human embryonic kidney 293T cell line was obtained from American Type Culture Collection (ATCC, Maryland) and cultured in Dulbecco’s modified Eagle’s medium. The medium was supplemented with 10% fetal bovine serum, 100 IU penicillin and 100 mg/ml streptomycin.

Immunofluorescent staining and microscopic observation
Cells were seeded on cover glass overnight and then transfected with different expression plasmids as required. Twenty-four hours after transfection, cells were fixed in 4% paraformaldehyde. For staining exogenous Akt and dominant-negative Akt (DN-Akt) proteins, cells were incubated with anti-Myc (Santa Cruz Biotechnology, Santa Cruz, CA) antibody followed by Cy5-conjugated secondary antibody. For staining endogenous TR3 and cytochrome c proteins, cells were incubated with anti-TR3 (Santa Cruz or Cell Signaling Technology, Beverly, MA) or anti-cytochrome c (Santa Cruz) antibody, followed by fluorescein isothiocyanate- (Pharmingen, San Diego, CA) or Texas red-conjugated (Sigma, St. Louis, MO) secondary antibody. For mitochondrial staining, cells were incubated with anti-Hsp60 antibody (Santa Cruz) followed by Texas red-conjugated secondary antibody. To display the nuclei, cells were stained with propidium iodine (50 µg/ml) or 4,6-diamidino-2-phenylindole (Roche, Meylan, France, 50 µg/ml). Stained cells were visualized under a confocal microscope (Bio-Rad MRC-1024ES).

Coimmunoprecipitation
Cells were transfected with different expression plasmids as required, and the cell extracts were prepared as described previously (32). After centrifuge, the supernatant was mixed with protein-A beads (Santa Cruz), incubated for 30 min, centrifuged for 5 min at 3000g for preclearing and then incubated with the primary antibody for 2 h. Protein-A beads were added again, incubated for another 1 h and centrifuged for 3 min at 3000g. The immunoprecipitates were collected and washed three times with radioimmunoprecipitation assay buffer and finally subjected to western blot analysis.

Western blot analysis
Different cellular fractions, including nuclear, cytoplasmic and mitochondrial fractions, were prepared as described below. Protein was electrophoresed on 10% denaturing gel and electroblotted onto a polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK). The membrane was incubated with different antibodies as required at 4°C overnight, followed by corresponding secondary antibody at room temperature for 1 h. A Pierce enhanced chemiluminescence kit was used to detect the antibody reactivity according to the manufacturer’s instruction.

Subcellular fractionation was performed as described with minor modifications (33). Briefly, cells were suspended in mannitol-sucrose buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris–HCl at pH 7.5 and 1 mM ethylenediaminetetraacetic acid at pH 7.5) containing protease inhibitor and then homogenized using a Dounce homogenizer. The homogenate was spun at 1300g for 10 min at 4°C, then the pellet was washed in phosphate-buffered saline (PBS) and resuspended in mannitol-sucrose buffer. After centrifugation at 1300g, the supernatant was subjected to another centrifugation at 17 000g for 30 min at 4°C. The pellet was collected, resuspended in mannitol-sucrose buffer and layered on a sucrose gradient, i.e. 2 ml of 1 M and 5 ml of 1.5 M sucrose buffer (10 mM Tris–HCl, pH 7.5, and 1 mM ethylenediaminetetraacetic acid), and then centrifuged at 60 000g for 30 min at 4°C. Gradient-purified mitochondria were collected at the interface of the sucrose gradients and dissolved in dilution buffer (5 mM Tris–HCl at pH 7.5 and 1 mM ethylenediaminetetraacetic acid).

Apoptosis analysis
Cells were transfected with various expression plasmids and then harvested. After fixing in 4% paraformaldehyde, cells were stained with 4,6-diamidino-2-phenylindole (50 µg/ml, Roche). Apoptotic cells showing typical morphology of nuclear condensation and fragmentation were counted among 300 transfected cells randomly. The results represent the mean values of three independent experiments.

Detection of serine phosphorylation in vivo
To detect the phosphorylation on serine in cells, 293T cells that were transfected with GFP-TR3, TR3 mutants and other plasmids as indicated were lysated and immoprecipitated using an anti-green fluorescent protein antibody (Santa Cruz). The pellet was then boiled and subjected to western blotting and immunoblotted with anti-phospho-serine antibody (Zymed, San Francisco, CA).

Flow cytometry
BGC-823 cells were transfected with si-Akt or si-TR3 (5'-ACAGTCCAGCCATGCTCCT-3'); 24 h after transfection, cells were treated with TPA for another 24 h. Cells were then fixed with 4% paraformaldehyde. After washed with PBS, cells were incubated with Alexa Fluor 488-conjugated anti-phosphatidylserine antibodies (Upstate, Lake Placid, NY) on ice for 2 h. Add PBS to each tube to wash out unbound antibody and centrifuged at 2000 r.p.m. for 5 min at 4°C. Resuspend cells in PBS and subject to fluorescent activated cell sorting (Beckman Coulter, Fullerton, CA).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Akt partially inhibits TPA-induced nuclear TR3 export
It has been reported previously that Akt is involved in the inhibition of 4-[3-(1-adamantyl)-4-hydroxyphenyl]-3-chlorocinnamic acid-induced TR3 nuclear export in lung cancer cells H460 (34). Although a similar mechanism of inhibition by Akt, of TPA-induced nucleocytoplasmic translocation, was also observed in BGC-823 gastric cancer cells in our current experiments, we noted that Akt did not completely block TR3 trafficking (Figure 1A and B). Our observations by confocal microscopy indicated that the transfection of exogenous Akt that possesses phosphorylation activity (Figure 1C) could block TPA-induced TR3 nuclear export, whereas a DN-Akt with no phosphorylation function (Figure 1C) failed to do so (Figure 1A). However, this Akt inhibition of TR3 translocation was partial only; TR3 was detected in the cytoplasm of BGC-823 cells in response to TPA treatment (Figure 1B, left panel), and the amount of cytoplasmic TR3 in Akt transfected cells was 50% of the controls (Figure 1B, right panel).


Figure 1
View larger version (27K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1. Akt inhibits the TPA-induced nucleocytoplasmic translocation of TR3. (A) Akt blocks TPA-induced TR3 nuclear export. BGC-823 cells were transfected with Myc-Akt or Myc-DN-Akt and then treated with TPA (100 ng/ml) for 6 h. Cells were immunostained with anti-TR3 antibody followed by fluorescein isothiocyanate-conjugated antibody to detect endogenous TR3 protein and with anti-Myc antibody followed by Cy5-conjugated antibody to reveal exogenous Akt. Simultaneously, cells were stained with propidium iodine to display the nuclei. The fluorescent images were examined under a confocal microscope. Approximately 80% of the TPA-treated cells showed a cytosolic localization of TR3; about 50% of the cells transfected with Myc-Akt exhibited a nuclear localization of TR3 and Myc-DN-Akt almost had no effect upon TR3 nuclear export. (B) The redistribution of TR3 by Akt in response to TPA. 293T cells were transfected with GFP-TR3, Myc-Akt or Myc-DN-Akt. After transfection, cells were treated with TPA for 6 h, and nuclear and cytoplasmic fractions were prepared. The levels of TR3 were determined by western blotting using an anti-green fluorescent protein antibody. Lamin B and tubulin were used to quantify the levels of nuclear and cytoplasmic proteins. The amount of cytoplasmic TR3 was quantified and is shown by the histogram in the right panel. (C) The phosphorylation activity of Akt in BGC-823 cells. Myc-Akt and Myc-DN-Akt were introduced into these cells and lysates were immunoprecipitated with anti-Myc antibody. The precipitates were then immunoblotted with anti-phospho-Akt (Ser473) antibody. (D) Effects of insulin, wortmannin and PD98059 on the TPA-induced subcellular localization of TR3. BGC-823 cells were stained with anti-Akt antibody to reveal the localization of endogenous Akt as indicated in the upper panel. More than 85% of the cells displayed the Akt distribution pattern shown. To evaluate the effects of insulin on TR3, BGC-823 cells were serum starved overnight and then pretreated with or without wortmannin (100 nM) or PD98059 (40 µM) for 30 min and with insulin (100 nM) for 3 h. This was followed by exposure to TPA for another 6 h. More than 30% of cells displayed localization of TR3 as showed. (E) Effects of insulin, wortmannin or PD98059 on the activity of Akt. BGC-823 cells were cultured normally or serum starved, and this was followed by treatment with insulin, wortmannin or PD98059 as described above. The activity of Akt was then determined as described in (C). (F) Endogenous TR3 expression levels in BGC-823 cells were assayed by western blotting. Cells were treated with different reagents as described in (D).

 
Since endogenous Akt localized in both the nucleus and cytoplasm of BGC-823 cells (Figure 1D, upper panel), as detected by confocal microscopy, we used insulin to activate endogenous Akt phosphorylation activity (Figure 1E) (35) in BGC-823 cells and found that this treatment blocked TPA-induced TR3 nuclear export (Figure 1D, lower panel). However, pretreatment with wortmannin (Figure 1E), which inhibits the insulin-induced activation of Akt (35), attenuated the insulin effects upon TPA-induced TR3 shuttling and TPA-induced TR3 translocation from the nucleus to the cytoplasm was observed (Figure 1D, lower panel). The use of mitogen activated protein/extracellular signal regulated kinase kinase 1 inhibitor, PD98059, which does not affect insulin-activated Akt phosphorylation activity (Figure 1E), had no effect upon the block of TPA-induced TR3 translocation by insulin (Figure 1D, down panel), suggesting that the PI3K–Akt but not the mitogen activated protein/extracellular signal regulated kinase kinase 1–extracellular signal-regulated kinase (ERK) pathway is responsible for the insulin-induced repression of TR3 translocation, at least in the current experimental system. Further analysis of the TR3 expression levels in nuclear and cytoplasmic fractions by western blotting produced result that were consistent with our confocal observations i.e. the cytoplasmic TR3 was ~50% of the control levels (TPA treatment only) after treatment with insulin and TPA (Figure 1F). These results indicate that although the activation of Akt plays an important role in blocking TR3 nuclear export, it does so only partially.

Akt interacts with the N-terminus of TR3
In agreement with previous reports that TR3 interacts with Akt (30,31), we detected endogenous Akt in BGC-823 gastric cancer cell lysates that had been immunoprecipitated using anti-TR3 antibodies, examined by coimmunoprecipitation assays and vice versa (Figure 2A). Similar results were obtained in 293T cells that had been cotransfected with TR3 with Akt. As a control, we used green fluorescent protein alone that did not bind to Akt (Figure 2B). The domain of TR3 that interacted with Akt was not clear at this stage and to map this region, several TR3 deletion mutants were constructed as indicated in Figure 2C. When Akt was coexpressed with each of these deletion mutant products in 293T cells, the TR3{Delta}C1 mutant, harboring a 25 amino acid deletion in the C-terminus of TR3, interacted with Akt in the same manner as the full-length TR3. However, two other deletion mutants, TR3{Delta}N1 and TR3{Delta}CN, both of which lacked a 106 amino acid region of the N-terminus, did not interact with Akt (Figure 2D). This indicated that the N-terminal region of TR3 is required for Akt binding. Another of the TR3 deletion mutants in our series, TR3{Delta}N2, which lacks the first N-terminal 50 amino acid could also be coimmunoprecipitated with Akt (Figure 2D), further demonstrating that residues 51–105 in the N-terminus are responsible for the interaction of TR3 with Akt.


Figure 2
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2. Mapping of the interaction region between TR3 and Akt. (A) TR3 interacts with Akt at endogenous levels. Cell lysates from BGC-823 cells were immunoprecipitated with anti-TR3 or anti-Akt antibodies and subjected to western blotting. The input represents 5% of the cell lysates used in the coimmunoprecipitation assay. IgG was used as a control. (B) TR3 binding to Akt at exogenous levels. Lysates from 293T cells that were cotransfected with GFP-TR3 or with Myc-Akt were immunoprecipitated with an anti-green fluorescent protein antibody. These immunoprecipitates were then examined by western blotting using an anti-Myc antibody. As a control, green fluorescent protein alone was tested and found not to interact with Akt (right panel). (C) Schematic representation of full-length TR3 and a deletion mutant series. (D) Determination of the Akt interaction domain on TR3. The different GFP-TR3 deletion mutants indicated were cotransfected into 293T cells with Myc-Akt. Cell lysates were immunoprecipitated by green fluorescent protein antibody and the immunoprecipitates were examined by western blotting.

 
Akt blocks mitochondrial targeting by cytoplasmic TR3
TR3 localizes in the nucleus in gastric cancer BGC-823 cells but TPA induces the mitochondrial targeting of this orphan receptor (Figure 3A, upper panel), with ~60% of TPA-treated cells demonstrating colocalization of TR3 and Hsp60. We further found that upon insulin pretreatment of BGC-823 cells, TPA-induced TR3 mitochondrial localization is reduced (Figure 3B). However, the addition of wortmannin abolished this insulin effect, strongly suggesting the possibility that Akt blocks cytoplasmic TR3 mitochondria targeting in addition to its inhibitory role in TR3 nuclear export. To verify this possibility, we constructed a TR3 deletion mutant, TR3{Delta}DBD, which has been described previously (14) and which lacks DBD (Figure 3C). Confocal microscopic analysis showed that when transfected into BGC-823 cells alone, TR3{Delta}DBD displayed a mitochondrial localization as indicated by its overlapping expression with the mitochondrial marker Hsp60 (Figure 3A, lower panel). However, when coexpressed with Akt, TR3{Delta}DBD remained cytoplasmic with a diffuse expression pattern. Under this circumstance, the mitochondrial TR3 levels were far lower, as detected by western blotting, when compared with cells transfected using TR3{Delta}DBD alone (Figure 3D). In contrast to these findings, DN-Akt had no such inhibitory effects upon TR3{Delta}DBD mitochondrial targeting (Figure 3A and D). These results thus reveal that Akt is capable of blocking cytoplasmic TR3 mitochondrial targeting.


Figure 3
View larger version (24K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3. Akt abolishes the mitochondrial targeting of TR3. (A) TPA induces but Akt blocks TR3 mitochondrial targeting. BGC-823 cells were transfected with GFP-TR3, treated with TPA for 6 h and then stained with Hsp60. Approximately 60% of the TPA-treated cells demonstrated colocalization of TR3 and Hsp60 (upper panel). GFP-TR3{Delta}DBD together with Myc-Akt or Myc-DN-Akt was cotransfected into BGC-823 cells. Cells were then immunostained for Akt and Hsp60 with corresponding antibodies. About 40% of the TR3{Delta}DBD proteins colocalized with Hsp60. (B) Insulin inhibits the mitochondrial targeting of TR3. BGC-823 cells were starved overnight and then treated with insulin, wortmannin or TPA as described in Figure 1. The mitochondrial fractions were then purified and subjected to western blotting. (C) Schematic representation of a TR3 deletion mutant series. (D) Inhibitory effects of Akt upon the mitochondrial localization of TR3{Delta}DBD. GFP-TR3{Delta}DBD, together with Myc-Akt or Myc-DN-Akt, was transfected into BGC-823 cells from which mitochondrial fractions were prepared. The levels of TR3 protein were then determined by western blotting with anti-green fluorescent protein antibody. Hsp60 was used to quantify the amounts of mitochondrial proteins loaded in each lane.

 
Akt phosphorylates cytoplasmic TR3 at the N-terminus
The mechanism by which Akt inhibits TR3 mitochondrial targeting is currently unknown. Since the phosphorylation of nuclear TR3 at Ser351 by Akt contributes to a suppression of the nucleocytoplasmic translocation of TR3 (34), we wished to determine whether Akt also exerts a phosphorylating effect upon cytoplasmic TR3. We first assessed whether residue Ser351 of TR3 is the only Akt phosphorylation site. For this purpose, a point mutant was constructed in which Ser351 was replaced by alanine (S351A). When cotransfected into 293T cells, however, Akt could still phosphorylate TR3 S351A as effectively as wild-type TR3 (Figure 4A). In contrast, DN-Akt did not phosphorylate TR3 and/or the S351A mutant. This evidence strongly suggests that additional Akt phosphorylation sites are present on TR3. In addition, we found by western blotting that when TR3{Delta}DBD and Akt were coexpressed in 293T cells, the upshifted band for TR3{Delta}DBD became more evident when compared with TR3{Delta}DBD transfected alone (Figure 4B). To identify whether band shift represented a phosphorylated form of TR3, okadaic acid (OA), a serine/threonine phosphatase inhibitor, was used to treat intact cells, and calf intestine alkaline phosphatase (CIAP) was also incubated with lysates extracted from transfected cells. Consistent with our conjecture, the upshifted band disappeared after CIAP treatment. In contrast, OA treatment increased the intensity of this band shift (Figure 4B). Moreover, we also observed that the phosphoserine signal weakened in the presence of CIAP but not OA (Figure 4B). Since TR3{Delta}DBD lacks a Ser351 site and localize in the cytoplasm, this result not only supports the presence of additional Akt phosphorylation sites on the TR3 molecule but also it suggests that Akt indeed phosphorylates cytoplasmic TR3.


Figure 4
View larger version (49K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4. Akt phosphorylates the N-terminus of TR3. (A) Akt phosphorylates TR3 at sites other than the Ser351 residue. A GFP-TR3 S351A product, together with Myc-Akt or Myc-DN-Akt, was transfected into 293T cells. The cell lysates were then immunoprecipitated with anti-green fluorescent protein antibody, and the precipitates were subjected to western blotting with anti-phosphoserine antibodies. GFP-TR3 was used as a positive control. (B) Akt phosphorylates cytoplasmic TR3. GFP-TR3{Delta}DBD was cotransfected with Myc-Akt into 293T cells, which were then treated with OA (100 nM) for 2 h, or the cell lysates were incubated with CIAP (0.2 U/µl) for 1 h. Cell lysates were then immunoprecipitated with anti-green fluorescent protein antibody. The lysates were immunoblotted with anti-green fluorescent protein antibody, and the phosphorylation of TR3{Delta}DBD was determined by incubation with anti-phosphoserine antibody. (C) Effects of Akt phosphorylation of different TR3 mutants. Myc-Akt together with the GFP-TR3 mutants indicated was cotransfected into 293T cells, which were then treated with CIAP or OA as described above. Phosphorylation of the TR3 mutants was determined by western blotting with an anti-green fluorescent protein antibody (asterisks). (D) Akt phosphorylation occurs in the N-terminus of TR3. GFP-TR3/N1-194 together with Myc-Akt or Myc-DN-Akt was transfected into 293T cells. Cell lysates were then immunoprecipitated with anti-green fluorescent protein antibody. The phosphorylation levels were determined by western blotting with an anti-phosphoserine antibody as described above. (E) Effects of insulin and wortmannin on TR3 phosphorylation. GFP-TR3, GFP-TR3 S351A and GFP-TR3/N1-194 was transfected into BGC-823 cells; after overnight serum starvation, these cells were pretreated with wortmannin and then treated with insulin as described above. Cell lysates were then immunoprecipitated with anti-green fluorescent protein antibody. The phosphorylation status was again determined by anti-phosphoserine antibody blots. (F) Interaction of Akt with TR3 deletion mutants. The GFP-TR3 deletion mutants indicated were cotransfected with Myc-Akt into 293T cells. Cell lysates were generated and then immunoprecipitated using an anti-Myc antibody. The immunoprecipitates were next subjected to western blotting using an anti-green fluorescent protein antibody to assay for complex formation between Akt and these various TR3 deletion mutants. TR3 was used as a positive control.

 
We next performed deletion analyses to map the Akt phosphorylation region on cytoplasmic TR3, based on the structure of the TR3{Delta}DBD mutant. Two truncation mutants, TR3/N1-194 and TR3/C440-598 (Figure 3C) both of which show a cytoplasmic localization (data not shown) (36), were used in these experiments. The degree of phosphorylation of these truncation mutants by Akt was examined in a parallel experiment. As shown in Figure 4C, TR3/N1-194, but not TR3/C440-598, was clearly phosphorylated by Akt (band indicated with an asterisk). Consistently, CIAP treatment drastically weakened, whereas OA enhanced, the intensity of the band shift, indicating that the N-terminus of TR3 is required for phosphorylation by Akt.

We additionally found that Akt could phosphorylate TR3/N1-194 (Figure 4D), but not TR3/C440-598 (data not shown), as revealed using phosphoserine antibodies, further showing that the phosphorylation of TR3 by Akt occurs N-terminally. On the other hand, upon activation of endogenous Akt by insulin, the phosphorylation of TR3 wild-type, TR3 N-terminus (TR3/N1-194) and the TR3 S351A point mutant were all enhanced. In contrast, wortmannin abolished the insulin effects on TR3 phosphorylation (Figure 4E). Finally, by coimmunoprecipitation assay, we found that Akt interacts with the TR3 N-terminus but not with its C-terminus (Figure 4F). Hence, the N-terminus of TR3 is a key interaction and phosphorylation domain for Akt and is a prerequisite for Akt phosphorylation of this orphan receptor.

Phosphorylation by Akt disrupts the interaction of TR3 with Bcl-2
Since Bcl-2, acting as a carrier, is required for TR3 mitochondrial targeting (37), it was of interest to know whether Akt might impact upon this interaction, which would probably explain the diffuse distribution of TR3 in the cytoplasm upon Akt phosphorylation. To this end, we first tested whether Bcl-2 is required for TR3 export from the nucleus to the cytoplasm. When TR3 or its point mutant S351A together with Bcl-2 were cotransfected into 293T cells that lack detectable levels of endogenous Bcl-2 (37), the subcellular localization of TR3 and S351A remained nuclear (Figure 5A, left panel). However, treatment of these cells with TPA resulted in the efflux of both products from the nucleus to the mitochondria, as revealed by the white color when TR3 is overlaid with Bcl-2 and Hsp60 (Figure 5A, left panel). This indicated that Bcl-2 is not required for the nucleocytoplasmic translocation of TR3, and that TPA is responsible for this export. In addition, we found that the transfection of TR3{Delta}DBD or TR3/C440-598 alone in 293T cells resulted in a diffuse pattern of expression of the exogenous products in the cytoplasm, whereas the coexpression of Bcl-2 resulted in a mitochondrial localization for both even in the absence of TPA. This suggested that once localized in the cytoplasm, TR3 is potently recruited into mitochondria by Bcl-2 (Figure 5A, right panel).


Figure 5
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5. Akt disrupts the interaction between TR3 and Bcl-2. (A) TPA-induced and Bcl-2-mediated TR3 mitochondrial targeting. GFP-TR3 or TR3 S351A together with Flag-Bcl-2 was transfected into 293T cells. Cells were then treated with TPA for 6 h and immunostained for Bcl-2 using an anti-Flag antibody and for endogenous Hsp60 with an anti-Hsp60 antibody. Approximately 50% of the TPA-treated cells that were transfected with Bcl-2 showed a mitochondrial localization of TR3. About 65% of the TR3 S351A proteins colocalized with Bcl-2 when treated with TPA, and ~40% of cells showed colocalization of TR3{Delta}DBD and Bcl-2. More than 40% of the cells showed colocalization of TR3 C440-598 and Bcl-2. (B) Redistribution of TR3 and its mutants in the mitochondria in the presence of Akt. GFP-TR3 and the indicated mutants, together with Flag-Bcl-2 and Myc-Akt, were transfected into 293T cells that were then treated with TPA and fractionated. The levels of mitochondrial TR3 were determined by western blotting with an anti-green fluorescent protein antibody. (C) Inhibitory effects of Akt on Bcl-2-mediated TR3 mitochondrial targeting. Different GFP-TR3 deletion mutants, together with Flag-Bcl-2 and Myc-Akt, were transfected into 293T cells, respectively. Cells were simultaneously immunostained with anti-Flag and anti-Myc antibodies. TR3, Bcl-2 and Akt were visualized using confocal microscopy. Less than 20% of the cells that were transfected with TR3{Delta}DBD, Bcl-2 and Akt showed a colocalization of Bcl-2 and TR3{Delta}DBD. Almost 40% of the cells exhibited a colocalization of TR3 C440-598 with Bcl-2 regardless of the presence of exogenous Akt. (D) Bcl-2 is indispensable for TR3 mitochondrial targeting. TR3{Delta}DBD together with Bcl-2 or a Bcl-2{Delta}TM mutant that cannot localize in mitochondria was transfected into 293T cells. Mitochondrial fractions were then prepared and subjected to western blotting. (E) Akt disrupts the interaction between TR3 and Bcl-2. Different GFP-TR3 deletion mutants, together with Flag-Bcl-2 and Myc-Akt, were cotransfected into 293T cells. Interactions of the TR3 mutants with Akt or Bcl-2 were detected by coimmunoprecipitation assay as described above. Cell lysates were immunoprecipitated by green fluorescent protein antibody. The immunoprecipitates were then examined by western blotting using Flag or Myc antibodies to detect TR3–Bcl-2 or TR3–Akt complexes.

 
The requirement of Bcl-2 for the mitochondrial targeting of TR3 and the inhibitory effects of Akt upon this targeting were confirmed by preparations of mitochondrial fractions from 293T cells. When TR3 or its mutants including S351A, {Delta}DBD and C440-598 were coexpressed with Bcl-2, the levels of these exogenous TR3 products in the mitochondrial fractions were dramatically increased (Figure 5B). However, the transfection of Akt led to decrease in the mitochondrial levels of TR3, S351A and {Delta}DBD. In contrast to this, a large portion of the expressed TR3/C440-598 protein was still present in the mitochondrial fraction even in the presence of Akt (Figure 5B). Consistent with these western blotting results, confocal microscope examinations showed that {Delta}DBD and C440-598 were exclusively localized in mitochondria when coexpressed with Bcl-2 in 293T cells. However, upon cotransfection of Akt, TR3{Delta}DBD was no longer present in mitochondria and was diffusely cytoplasmic although the Akt non-reactive mutant TR3/C440-598 remained in the mitochondria (Figure 5C). In a further experiment, a mutant of Bcl-2, {Delta}TM, in which the transmembrane domain is deleted and hence no mitochondrial targeting of the truncated protein occurs (37), was cotransfected with TR3{Delta}DBD in 293T cells. In these transfectants, Bcl-2{Delta}TM did not promote the import of TR3{Delta}DBD into the mitochondria (Figure 5D), which provided further supporting evidence for a function of Bcl-2 as a carrier for TR3 into mitochondria.

We also examined the effects of Akt upon the interaction between TR3 and Bcl-2 by coimmunoprecipitation assay. When TR3{Delta}DBD or TR3/C440-598, together with Bcl-2, were transfected into 293T cells, a significant portion of the mutant TR3 proteins were immunoprecipitated with Bcl-2. However, upon Akt transfection, the TR3{Delta}DBD–Bcl-2 complexes were abolished and TR3{Delta}DBD–Akt complexes could be detected (Figure 5E). In contrast to these results, the interaction between TR3/C440-598 and Bcl-2 was unaffected by Akt, and no TR3/C440-598/Akt complexes were detectable (Figure 5E). Taken together, these findings suggest that Akt phosphorylates TR3 and elicits a conformational change in this orphan receptor, which disrupts its interaction with Bcl-2, prevents its mitochondrial targeting and results in a cytoplasmic distribution.

Akt inhibits TR3-mediated apoptosis and cytochrome c release
We investigated the role of Akt on TR3-mediated biological functions. Given that TR3 mitochondrial targeting is closely associated with the apoptotic response induced by TPA in gastric, lung and prostate cancer cells (8,14,36), we speculated that the Akt-mediated inhibition of TR3 mitochondrial targeting might prevent cells from TR3-induced apoptosis. This would represent a novel way for Akt to inhibit apoptosis. To further assess the biological activities of Akt in this process, we tested its effects on TR3-induced apoptosis in BGC-823 gastric cancer cells. TPA treatment was found to induce nuclear TR3 to translocate into the mitochondria, thereby increasing the number of apoptotic cells from 5 to 27% (Figure 6A). However, the transfection of Akt, but not DN-Akt, potently reduced the number of TPA-induced apoptotic cells from 27 to 14% (Figure 6A). In addition, when Ser351 of TR3 was mutated to alanine, Akt could still inhibit TPA-induced apoptosis from 31 to 20%, which further indicated that Akt can induce phosphorylation of TR3 other than S351.


Figure 6
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6. Effects of Akt on TR3-mediated apoptosis. (A) Effects of Akt on TPA-induced apoptosis. BGC-823 cells that were cotransfected with TR3 or TR3 S351A and Akt or DN-Akt were treated with TPA for 24 h and then stained with 4,6-diamidino-2-phenylindole. Apoptotic cells were scored by examining 500 cells based on their nuclear fragmentation and chromatin condensation. (B) Akt inhibits TR3-mediated apoptosis in BGC-823 cells. Different TR3 mutants were cotransfected with Myc-Akt and then stained with 4,6-diamidino-2-phenylindole. Apoptotic cells were scored by examining 500 cells. (C) The knockdown of Akt enhances the apoptotic response induced by TPA. BGC-823 cells were transfected with si-TR3 or si-Akt. After transfection, cells were treated with TPA for 24 h. After staining with Alexa Fluor 488-conjugated anti-phosphatidylserine antibodies, cells were subjected to fluorescent activated cell sorting analyses. The effect of si-TR3 and si-Akt were also presented by western blotting. (D and E) Akt blocks TR3-triggered cytochrome c release. Myc-Akt was coexpressed with GFP-TR3{Delta}DBD or GFP-TR3/C440-598 in BGC-823 cells. Cells were then simultaneously immunostained with anti-Myc, -Hsp60 and -cytochrome c antibodies. Approximately 60% of the TR3{Delta}DBD and Hsp60 colocalized cells displayed diffuse cytochrome c staining. Less than 20% of the TR3{Delta}DBD and Akt cotransfected cells showed a diffuse cytochrome c expression pattern, whereas >50% of the TR3 C440-598 and Akt cotransfected cells showed a cytoplasmic localization of cytochrome c. The expression levels of cytochrome c localized in the cytoplasm or mitochondria were determined by western blotting, as shown in Figure 6E.

 
To further elucidate the antiapoptotic effects of Akt upon TR3-mediated apoptosis, not only via the inhibition of nuclear export but also through a TR3 mitochondrial localization block, various TR3 deletion mutants (TR3{Delta}DBD and TR3/C440-598) that localized in the cytoplasm were analyzed for their abilities to induce apoptosis in the absence or presence of Akt. When TR3{Delta}DBD alone was transfected into BGC-823 cells, more apoptotic cells (25%) were detected. However, upon the cotransfection of Akt, a significantly decrease in the proapoptotic effects of TR3{Delta}DBD was observed in BGC-823 cells (Figure 6B), consistent with the fact that the mitochondrial targeting of TR3{Delta}DBD would be blocked by Akt. We further found that Akt only marginally downregulated the apoptotic response induced by TR3/C440-598 (Figure 6B), concordant with its inability to interact with and phosphorylate this TR3 mutant. Moreover, when endogenous TR3 was inhibited by small interfering RNA, TPA-induced apoptosis was decreased from 26.3 to 17.1%. Conversely, small interfering RNA silencing of Akt in BGC-823 cells further enhanced TR3-mediated cell apoptosis with the levels reaching 41.2% (Figure 6C). These results together indicate strongly that TR3 mediates TPA-induced apoptosis and that Akt exerts a negative effect upon this pathway in gastric cancer cells.

TR3-mediated apoptosis leads to a release of cytochrome c from the mitochondria in cancer cells (8,37–39). In our current analyses, we also observed in BGC-823 cells that cytochrome c was released when TR3{Delta}DBD was localized in the mitochondria. However, upon coexpression of Akt, a clear block of cytochrome c release was evident, whereas even in the presence of Akt, the TR3/C440-598 mutant still induced cytochrome c release after it had targeted to the mitochondria (Figure 6D). Further analysis of both cytoplasmic and mitochondrial fractions of the same cells produced consistent results (Figure 6E). Taken together, these data convincingly demonstrate that the inhibitory role of Akt in TR3-mediated apoptosis in gastric cancer cells is through the suppression of TR3 mitochondrial targeting and cytochrome c release.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
Akt signaling has often been detected in a variety of human cancers (40). The exact mechanisms underlying the role of Akt in promoting cell survival and inhibiting apoptosis have been partly characterized but have yet to be fully elucidated. One mechanism through which Akt functions to promote cell survival is through the phosphorylation and inactivation of proapoptotic proteins such as Bad, caspase-9 and Apaf-1 (24,25,41–43). TR3 is another demonstrated target of Akt (30,31). In our present study, we provide the first evidence to date that Akt inhibits cytoplasmic TR3 mitochondrial targeting through phosphorylation and thereby affects the apoptotic function of TR3. This process may thus represent a novel antiapoptotic signaling pathway mediated by Akt in cancer cells.

In gastric cancer cells, TPA treatment caused the translocation of the TR3 nuclear orphan receptor from the nucleus to the cytoplasm, after which it localizes in the mitochondria to initiate apoptosis (8,14). Akt on the other hand blocks TPA-induced TR3 nucleocytoplasmic translocation and mitochondrial targeting via an interaction with the TR3 N-terminus. Upon deletion of this domain of TR3, Akt loses its ability to bind and cytoplasmic TR3 is successfully targeted to the mitochondria. Accordingly, the interaction with Akt seems to negatively control TR3 trafficking to the mitochondria.

Akt phosphorylates nuclear TR3 and negatively regulates TR3 nuclear export. In this process, the Akt phosphorylation site on TR3, Ser351, which is located in the DBD, is critical (34). It is noteworthy that the phosphorylation of TR3 on Ser351 has been shown to influence diverse activities of TR3, including DNA binding and transactivation (30,31). Although we observe in our present study that Akt blocks TPA-induced TR3 nucleocytoplasmic translocation in gastric cancer cells, the precise regulation of TR3 mitochondrial targeting by Akt remains largely unknown. A recent report has indicated that the intracellular localization of TR3 from the nucleus to the cytoplasm in PC12 cells is regulated in part by the phosphorylation of Ser105 (19). The phosphorylation of this residue induced by nerve growth factor causes a conformational change that results in exposure of the nuclear export signal on the surface of the protein and subsequent export of TR3 to the cytoplasm through Trk/ERK/mitogen-activated protein kinase-signaling pathways (19). This finding raises the possibility that the export of nuclear TR3 to the cytoplasm may render it available for phosphorylation by cytoplasmic Akt.

The phosphorylation of nuclear TR3 by Akt leads to a suppression of its nuclear export (34). Whether the phosphorylation of cytoplasmic TR3 by Akt affects its mitochondrial targeting is therefore a salient question. Although TR3 lacks a classical mitochondrial-targeting sequence (44), Bcl-2, with its ability to interact with TR3, acts as a carrier for TR3 mitochondrial localization, which triggers cytochrome c release and induces apoptosis (37). IGFBP-3, which induces the intrinsic (mitochondria dependent) pathway of apoptosis by causing TR3 to target mitochondria and induce cytoplasmic cytochrome c release, was reported to inhibit the phosphorylation and activity of Akt, indicating that the activity of Akt may be important in impairing TR3-induced apoptosis (45). To further elucidate the mechanism whereby Akt phosphorylation of TR3 causes its diffuse distribution in the cytoplasm by preventing its mitochondrial localization, we tested whether the TR3–Bcl-2 interaction could be disrupted also by Akt. It was evident from our analyses that TR3{Delta}DBD, which can be phosphorylated by Akt, remained localized in the cytoplasm and formed a complex with Akt rather than with Bcl-2. In contrast, the TR3/C440-598 mutant that cannot be phosphorylated by Akt was translocated to the mitochondria even in the presence of Akt. Under this circumstance, Akt could not disrupt the TR3–Bcl-2 interaction. Thus, it is probably that Akt phosphorylates TR3 and elicits a conformational change that prevents its interaction with Bcl-2 and leads to its cytoplasmic localization.

Although the transcriptional activity of TR3 is well correlated with its apoptosis-inducing activity in a variety of cancer cell lines (11,13,31,46), this may not be the only mechanism for Akt to target and inhibit TR3-induced apoptosis. Our results in BGC-823 cells show that TR3 induces cytochrome c release upon localization in the mitochondria, resulting in the initiation of apoptosis. This strongly suggests that TR3 indeed exerts its proapoptotic role at the post-transcriptional level. Akt directly inhibits TR3-induced apoptosis, and this suppression correlates well with the inhibition of TR3 mitochondrial targeting and cytochrome c release and relies on the interaction between Akt and TR3. Hence, the involvement of Akt in survival signaling, at least in the gastric cancer cells, is through its interaction with and further phosphorylation of TR3, which antagonizes the proapoptotic function of TR3, rather than by directly inhibiting the activation of individual proapoptotic Bcl-2 family members. We therefore speculate that this may represent a novel signaling transduction pathway for Akt in its antiapoptotic role in carcinoma cells.

The activation of Akt by various survival and growth factors, such as insulin and platelet-derived growth factor, involves the PI3K-dependent phosphorylation of the Thr308 and Ser473 residues of Akt (23,47). Wortmannin, a PI3K inhibitor, inhibits this activation of Akt (48). The activation of Akt by treatment of cells with insulin is a model system that is often used to assay the phosphorylation of various Akt targets (23). In our current experiments, we found that Akt phosphorylates TR3 in a PI3K-dependent manner. Moreover, although insulin also triggers the activity of ERK, the ERK inhibitor PD98059 (49) had no effect upon TR3 function. This further demonstrated that the PI3K–Akt but not ERK pathway is responsible for regulating the phosphorylation and relocalization of TR3.

The consensus sequences for Akt phosphorylation are reported to be RxRxxS or RxRxxT, where x represents any amino acid (35). However, there may be other unknown sequences or macromolecular interactions within cells that allow Akt to phosphorylate motifs other than the minimally required RxRxxS/T. For example, the proposed Akt phosphorylation sequence for adenosine triphosphate citrate lyase is PSRTApS (serine is located at residue 454) (50), which differs from the classical consensus motif that has been reported. In our current study, the sequence RGRLPS (incorporating the Ser351 residue) matches well with the consensus Akt phosphorylation motif. In addition to this sequence, no RxRxxS/T consensus is present in TR3. However, Akt was found to phosphorylate TR3 even when Ser351 was deleted or mutated. Clearly, further work will be necessary to determine other potential Akt phosphorylation sites in the TR3 molecule.

In summary, the mitochondrial localization of TR3 is sufficient to induce apoptosis in gastric cancer cells, but such apoptotic activity can be inhibited by Akt phosphorylation of TR3. Our data clearly demonstrate a cross talk between the protein kinase Akt and this orphan receptor and strongly suggest that TR3 is a potential molecular target for developing new anticancer agents.


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
National Outstanding Young Science Foundation (30425014); Ministry of Science and Technology of China (2007CB914402); National Natural Science Foundation of China (30570936); Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering (2004111).


   Acknowledgments
 
We thank Dr Shengcai Lin for providing the Myc-Akt and Myc-DN-Akt constructs and Dr Binhua P.Zhou for providing the si-Akt1 construct.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

  1. Hazel TG, et al. A gene inducible by serum growth factors encodes a member of the steroid and thyroid hormone receptor superfamily. Proc. Natl Acad. Sci. USA (1988) 85:8444–8448.[Abstract/Free Full Text]
  2. Milbrandt J. Nerve growth factor induces a gene homologous to the glucocorticoid receptor gene. Neuron (1988) 1:183–188.[CrossRef][Web of Science][Medline]
  3. Chang C, et al. Identification of a new member of the steroid receptor super-family by cloning and sequence analysis. Biochem. Biophys. Res. Commun. (1988) 155:971–977.[CrossRef][Web of Science][Medline]
  4. Zhang XK. Vitamin A and apoptosis in prostate cancer. Endocr. Relat. Cancer (2002) 9:87–102.[Abstract]
  5. Kastner P, et al. Nonsteroid nuclear receptors: what are genetic studies telling us about their role in real life? Cell (1995) 83:859–869.[CrossRef][Web of Science][Medline]
  6. Winoto A, et al. Nuclear hormone receptors in T lymphocytes. Cell (2002) 109(suppl.):S57–S66.[CrossRef][Web of Science][Medline]
  7. Uemura H, et al. Antisense TR3 orphan receptor can increase prostate cancer cell viability with etoposide treatment. Endocrinology (1998) 139:2329–2334.[Abstract/Free Full Text]
  8. Wu Q, et al. Dual roles of Nur77 in selective regulation of apoptosis and cell cycle by TPA and ATRA in gastric cancer cells. Carcinogenesis (2002) 23:1583–1592.[Abstract/Free Full Text]
  9. Wu Q, et al. Modulation of retinoic acid sensitivity in lung cancer cells through dynamic balance of orphan receptors nur77 and COUP-TF and their heterodimerization. EMBO J. (1997) 16:1656–1669.[CrossRef][Web of Science][Medline]
  10. Maruyama K, et al. The NGFI-B subfamily of the nuclear receptor superfamily (review). Int. J. Oncol. (1998) 12:1237–1243.[Web of Science][Medline]
  11. Calnan BJ, et al. A role for the orphan steroid receptor Nur77 in apoptosis accompanying antigen-induced negative selection. Immunity (1995) 3:273–282.[CrossRef][Web of Science][Medline]
  12. Weih F, et al. Apoptosis of nur77/N10-transgenic thymocytes involves the Fas/Fas ligand pathway. Proc. Natl Acad. Sci. USA (1996) 93:5533–5538.[Abstract/Free Full Text]
  13. Zhou T, et al. Inhibition of Nur77/Nurr1 leads to inefficient clonal deletion of self-reactive T cells. J. Exp. Med. (1996) 183:1879–1892.[Abstract/Free Full Text]
  14. Li H, et al. Cytochrome c release and apoptosis induced by mitochondrial targeting of nuclear orphan receptor TR3. Science (2000) 289:1159–1164.[Abstract/Free Full Text]
  15. Ye X, et al. Distinct role and functional mode of TR3 and RARalpha in mediating ATRA-induced signalling pathway in breast and gastric cancer cells. Int. J. Biochem. Cell Biol. (2004) 36:98–113.[CrossRef][Web of Science][Medline]
  16. Davis IJ, et al. Functional domains and phosphorylation of the orphan receptor Nur77. Mol. Endocrinol. (1993) 7:953–964.[Abstract/Free Full Text]
  17. Hirata Y, et al. The phosphorylation and DNA binding of the DNA-binding domain of the orphan nuclear receptor NGFI-B. J. Biol. Chem. (1993) 268:24808–24812.[Abstract/Free Full Text]
  18. Katagiri Y, et al. Differential regulation of the transcriptional activity of the orphan nuclear receptor NGFI-B by membrane depolarization and nerve growth factor. J. Biol. Chem. (1997) 272:31278–31284.[Abstract/Free Full Text]
  19. Katagiri Y, et al. Modulation of retinoid signalling through NGF-induced nuclear export of NGFI-B. Nat. Cell Biol. (2000) 2:435–440.[CrossRef][Web of Science][Medline]
  20. Burgering BM, et al. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature (1995) 376:599–602.[CrossRef][Web of Science][Medline]
  21. Franke TF, et al. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell (1995) 81:727–736.[CrossRef][Web of Science][Medline]
  22. Meier R, et al. Mitogenic activation, phosphorylation, and nuclear translocation of protein kinase Bbeta. J. Biol. Chem. (1997) 272:30491–30497.[Abstract/Free Full Text]
  23. Chan TO, et al. AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu. Rev. Biochem. (1999) 68:965–1014.[CrossRef][Web of Science][Medline]
  24. Datta SR, et al. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell (1997) 91:231–241.[CrossRef][Web of Science][Medline]
  25. del PL, et al. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science (1997) 278:687–689.[Abstract/Free Full Text]
  26. Diehl JA, et al. Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev. (1998) 12:3499–3511.[Abstract/Free Full Text]
  27. Shtutman M, et al. The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway. Proc. Natl Acad. Sci. USA (1999) 96:5522–5527.[Abstract/Free Full Text]
  28. Campbell RA, et al. Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance. J. Biol. Chem. (2001) 276:9817–9824.[Abstract/Free Full Text]
  29. Lin HK, et al. Akt suppresses androgen-induced apoptosis by phosphorylating and inhibiting androgen receptor. Proc. Natl Acad. Sci. USA (2001) 98:7200–7205.[Abstract/Free Full Text]
  30. Pekarsky Y, et al. Akt phosphorylates and regulates the orphan nuclear receptor Nur77. Proc. Natl Acad. Sci. USA (2001) 98:3690–3694.[Abstract/Free Full Text]
  31. Masuyama N, et al. Akt inhibits the orphan nuclear receptor Nur77 and T-cell apoptosis. J. Biol. Chem. (2001) 276:32799–32805.[Abstract/Free Full Text]
  32. Wu Q, et al. Role of tumor metastasis suppressor gene KAI1 in digestive tract carcinomas and cancer cells. Cell Tissue Res. (2003) 314:237–249.[CrossRef][Web of Science][Medline]
  33. Lin XF, et al. RXRalpha acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells. J. Cell Sci. (2004) 117:5609–5621.[Abstract/Free Full Text]
  34. Han YH, et al. Regulation of Nur77 nuclear export by c-Jun N-terminal kinase and Akt. Oncogene (2006) 25:2974–2986.[CrossRef][Web of Science][Medline]
  35. Manning BD, et al. AKT/PKB signaling: navigating downstream. Cell (2007) 129:1261–1274.[CrossRef][Web of Science][Medline]
  36. Cao X, et al. Retinoid X receptor regulates Nur77/TR3-dependent apoptosis [corrected] by modulating its nuclear export and mitochondrial targeting. Mol. Cell. Biol. (2004) 24:9705–9725.[Abstract/Free Full Text]
  37. Lin B, et al. Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3. Cell (2004) 116:527–540.[CrossRef][Web of Science][Medline]
  38. Liu S, et al. Induction of apoptosis by TPA and VP-16 is through translocation of TR3. World J. Gastroenterol. (2002) 8:446–450.[Web of Science][Medline]
  39. Kolluri SK, et al. Mitogenic effect of orphan receptor TR3 and its regulation by MEKK1 in lung cancer cells. Mol. Cell. Biol. (2003) 23:8651–8667.[Abstract/Free Full Text]
  40. Nicholson KM, et al. The protein kinase B/Akt signalling pathway in human malignancy. Cell. Signal. (2002) 14:381–395.[CrossRef][Web of Science][Medline]
  41. Zhou H, et al. Akt regulates cell survival and apoptosis at a postmitochondrial level. J. Cell Biol. (2000) 151:483–494.[Abstract/Free Full Text]
  42. Yamaguchi H, et al. The protein kinase PKB/Akt regulates cell survival and apoptosis by inhibiting Bax conformational change. Oncogene (2001) 20:7779–7786.[CrossRef][Web of Science][Medline]
  43. Cardone MH, et al. Regulation of cell death protease caspase-9 by phosphorylation. Science (1998) 282:1318–1321.[Abstract/Free Full Text]
  44. Moll UM, et al. p53 and Nur77/TR3 - transcription factors that directly target mitochondria for cell death induction. Oncogene (2006) 25:4725–4743.[CrossRef][Web of Science][Medline]
  45. Lee KW, et al. Contribution of the orphan nuclear receptor Nur77 to the apoptotic action of IGFBP-3. Carcinogenesis (2007) 28:1653–1658.[Abstract/Free Full Text]
  46. Kuang AA, et al. Nur77 transcription activity correlates with its apoptotic function in vivo. Eur. J. Immunol. (1999) 29:3722–3728.[CrossRef][Web of Science][Medline]
  47. Bellacosa A, et al. Akt activation by growth factors is a multiple-step process: the role of the PH domain. Oncogene (1998) 17:313–325.[CrossRef][Web of Science][Medline]
  48. Ahmed NN, et al. The proteins encoded by c-akt and v-akt differ in post-translational modification, subcellular localization and oncogenic potential. Oncogene (1993) 8:1957–1963.[Web of Science][Medline]
  49. Favata MF, et al. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J. Biol. Chem. (1998) 273:18623–18632.[Abstract/Free Full Text]
  50. Berwick DC, et al. The identification of ATP-citrate lyase as a protein kinase B (Akt) substrate in primary adipocytes. J. Biol. Chem. (2002) 277:33895–33900.[Abstract/Free Full Text]
Received February 26, 2008; revised August 11, 2008; accepted August 16, 2008.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
29/11/2078    most recent
bgn197v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Chen, H.-Z.
Right arrow Articles by Wu, Q.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chen, H.-Z.
Right arrow Articles by Wu, Q.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?