{'fraction_non_alphanumeric': 0.03588990575058052, 'fraction_numerical': 0.02882119928971452, 'mean_word_length': 4.983857785042909, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Akt Inhibits the Orphan Nuclear Receptor Nur77 and T-cell Apoptosis*

Akt is a common mediator of cell survival in a variety of circumstances. Although some candidate Akt targets have been described, the function of Akt is not fully understood, particularly because of the cell type- and context-dependent apoptosis regulation. In this study, we demonstrate that one of the mechanisms by which Akt antagonizes apoptosis involves the inhibition of Nur77, a transcription factor implicated in T-cell recep-tor-mediated apoptosis. It has been suggested that Akt phosphorylates Nur77 directly, but whether Akt suppresses biological functions of Nur77 remains unknown. We found that Akt inhibited the DNA binding activity of Nur77 and stimulated its association with 14-3-3 in a phosphorylation site-dependent manner. Moreover, we found that expression of Akt suppressed Nur77-induced apoptosis in fibroblasts and activation-induced cell death of T-cell hybridomas. The inhibition of Nur77 by Akt suggests a mechanism that explains how T-cell receptor activation can promote survival in some in-stances even when Nur77 is induced. Collectively, these results may suggest that Akt is a negative regulator of Nur77 in T-cell apoptosis. The regulation of survival versus apoptosis is a central issue during T-cell development and activation. The survival/death regulation of thymocytes through T-cell receptor (TCR) 1 activation

The regulation of survival versus apoptosis is a central issue during T-cell development and activation. The survival/death regulation of thymocytes through T-cell receptor (TCR) 1 activation plays a key role in establishing a functional T-cell repertoire. In the thymus, immature T cells that moderately recognize self-peptide-MHC complexes survive and are induced to differentiate (positive selection), whereas self-reactive T cells that recognize self-peptide-MHC complexes with high affinity/ avidity are induced toward cell death (negative selection), thus eliminating potentially toxic T cells (1,2). Although both forms of thymic selection, as well as activation-induced cell death (AICD) of mature T cells, are mediated by TCR, the intracellular signaling mechanisms by which TCR regulates T-cell survival and apoptosis are not fully understood and have been subject to intense investigation.
Among the molecules implicated in T-cell apoptosis is Nur77 (also known as NGFI-B or TR3), a member of the orphan nuclear receptor superfamily. Nur77 was originally identified as an immediate early gene transiently induced by serum, growth factors and nerve growth factor (3, 4). It has also been shown that Nur77 and Nor-1, a related member of the Nur77 family, are induced during TCR-mediated apoptosis (5,6). Expression of a dominant-negative Nur77 blocks activation-induced cell death in T-cell hybridomas as well as negative selection in transgenic mice (7,8). Conversely, transgenic mice that express wild type Nur77 or Nor-1 exhibit massive apoptosis and a reduction in thymocyte numbers and the proportion of double-positive (DP) thymocytes (7,9). Therefore, it seems likely that Nur77 plays an important role in T-cell apoptosis. Nur77 transcription activity correlates well with its apoptotic function in T cells (10), although this might not be the case in other cell types (11).
When Nur77 is induced by growth factors or TCR stimulation, it often becomes transcriptionally inactive due to posttranslational modification (12). This is consistent with the observation that expression of Nur77 does not necessarily correlate with induction of apoptosis. In particular, we found that TCR activation resulted in Nur77 expression even under a condition mimicking positive selection, in which TCR promotes survival (this study). This and other studies suggest the existence of a mechanism to inhibit the proapoptotic function of Nur77 (13). It has been reported that phosphorylation of Nur77 on Ser-350 negatively regulates its function. Ser-350 resides within the domain required for specific binding of Nur77 to DNA, and its phosphorylation results in reduction of both the DNA binding and transcriptional activities of Nur77 in PC12 cells (12,14). In vivo phosphorylation of Ser-350 takes place in T cells as well as in other cells (15)(16)(17). Therefore, it appeared likely that a kinase involved in survival signaling would phosphorylate Nur77 and thus antagonize the proapoptotic function of Nur77.
The phosphatidylinositol 3-kinase (PI3-K)-Akt pathway is activated in response to TCR activation and is implicated in mediating survival signals in T cells. For instance, thymocytes derived from mice deficient in p110␥, a catalytic subunit of PI3-K, exhibited enhanced apoptosis (18), whereas those derived from pten Ϫ/Ϫ mice, in which the PI3-K-Akt pathway is constitutively active, were more resistant to TCR-mediated cell death and resulted in a defect in thymic negative selection (19,20). Moreover, both DP thymocytes and mature T cells derived from transgenic mice expressing gag-akt, a constitutively active Akt, showed enhanced viability in culture and resistance to various apoptosis-inducing stimuli such as ␥-irradiation, dexamethasone, and Fas ligand, further supporting the survivalpromoting function of the PI3-K-Akt pathway in T cells (21).
Recently, it has been shown that Akt phosphorylates Nur77 directly in vitro and in vivo (22). However, whether Akt inhibits the proapoptotic functions of Nur77 and how Akt-mediated phosphorylation inhibits Nur77 remain to be determined. In this study, we show that phosphorylation of Nur77 by Akt results in reduction of its DNA binding activity and stimulation of its association with 14-3-3 in a phosphorylation site-dependent manner. We also show that Akt suppresses Nur77-mediated cell death and thus propose an antagonistic interaction between apoptosis and survival signaling downstream of TCR.
MHC-deficient mice had been generated by crossing the I-A␤ Ϫ/Ϫ line of MHC class II-deficient mice with ␤ 2 -microglobulin-deficient mice (23). These mice fail to develop mature T cells, and most of the thymocytes derived from MHC knockout mice are immature, CD8ϩ/CD4ϩ double-positive cells. A previous report (24) suggested that engagement of T cells with anti-TCR-␤ monoclonal antibody mimicked positive selection and differentiation, as indicated by an increase in cell surface expression of TCR-␣ chain and CD5 and a decrease in RAG-1 and CD4/CD8 expression. On the other hand, stimulation with anti-CD3⑀ antibody resulted in induction of apoptosis, mimicking negative selection (25).
Immunoprecipitation and Immunoblotting-Cells were washed twice with phosphate-buffered saline and lysed with a cell lysis buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM dithiothreitol, and protease inhibitors, phenylmethylsulfonyl fluoride, leupeptin, aprotinin, and pepstatin. Proteins were immunoprecipitated from cell lysates after incubation with 2 g of appropriate antibodies and protein A-Sepharose beads (Amersham Pharmacia Biotech) for 2 h at 4°C. Cell lysates were subjected to SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore). Immunoreactive bands were detected by an ECL system (Amersham Pharmacia Biotech).
Electrophoretic Mobility Shift Assay-Cells were lysed in a buffer containing 50 mM Hepes-KOH, pH 7.8, 420 mM KCl, 0.1 mM EDTA, 5 mM MgCl 2 , 20% glycerol, and protease inhibitors. Oligonucleotide probes were end-labeled with T4 polynucleotide kinase. Cell lysates and labeled probes were incubated for 1 h in a buffer containing 10 mM Hepes-KOH, pH 7.8, 50 mM KCl, 1 mM EDTA, 5 mM MgCl 2 , 5 mM dithiothreitol, 10% glycerol, and protease inhibitors and subjected to polyacrylamide gel electrophoresis. The electrophoretically shifted bands including the complex of Nur77 and DNA were detected after autoradiography.
Retroviral Infection and Cell Sorting-Human CD8 sequence lacking cytoplasmic domain was subcloned into pMX-IRES retroviral vector to generate pMX-IRES-CD8. Akt mutants were inserted into this vector to generate pMX-Akt-IRES-CD8. Recombinant retroviruses were obtained by transfection of retroviral vector plasmids into ecotropic virus packaging cells (PLAT-E cells) as described elsewhere (27). Conditioned media from these cells were collected and added to T-cell cultures and incubated for 16 h in the presence of 5 g/ml Polybrene (Sigma). Infected (CD8-positive) cells or noninfected (CD8-negative) cells were separated by the use of microbead-conjugated anti-CD8 antibody and automagnetic-activated cell sorting separation units (Miltenyi Biotec) according to the manufacturer's instructions.
Luciferase Assay-After stimulation, the luciferase activity in the cell lysates was measured according to the manufacturer's instructions (Promega). Relative luciferase activity was normalized with co-expressed ␤-galactosidase activity.
Fetal Thymus Organ Cultures (FTOCs)-FTOCs were prepared essentially as described previously (28). Briefly, fetal thymi were dissected from C57BL/6 mouse embryos (embryonic day 15) and incubated for 8 days at 37°C in RPMI 1640 medium containing 10% fetal bovine serum. The cells were then harvested and dissociated to analyze cell surface expression of CD8 and CD4 using a FACScan flow cytometer (Becton Dickinson).

Nur77 Induction and Akt Activation in Thymocytes-A major question in T-cell development is how TCR activation in imma-
ture CD4ϩ/CD8ϩ DP thymocytes is translated into either survival (positive selection) or apoptotic (negative selection) signals (1, 2). One possible mechanism to discriminate between these outcomes might be that TCR activation in negative selection, but not in positive selection, induces expression of proapoptotic proteins such as Nur77. Therefore we compared expression levels of Nur77 under conditions mimicking either positive or negative selection. Immature DP thymocytes isolated from the MHC knockout mice were treated with anti-TCR-␤ antibody or with anti-CD3⑀/CD28 antibodies to mimic positive or negative selection, respectively (Ref. 24; see details under "Experimental Procedures"). Consistent with previous reports, CD3⑀/CD28 engagement in thymocytes derived from MHC knockout mice resulted in sustained expression of Nur77 protein (Fig. 1). Importantly, the stimulation of thymocytes with anti-TCR-␤ antibody also induced expression of Nur77 protein, although the induction was transient and not robust Double-positive thymocytes prepared from MHC knockout mice were stimulated with plate-bound anti-CD3⑀ plus anti-CD28 antibodies or anti-TCR-␤ antibody for the indicated times to mimic negative or positive selection, respectively. The level of Nur77 protein was detected by Western blotting with anti-Nur77 antibody. Phosphorylation state at Ser-473 and the protein level of Akt were also examined by immunoblotting with anti-phospho-Akt and anti-Akt antibodies.
( Fig. 1). This result suggests that Nur77 is induced to a higher level in the context of negative selection (apoptosis) compared with positive selection (survival) and also suggests that a threshold level should exist for the apoptosis-inducing function of Nur77. One mechanism for determination of such a threshold might be a survival signal that would counteract this proapoptotic function of Nur77.
Although it has been shown that Akt is activated in response to TCR activation in mature T cells and contributes to their survival, it was not known whether this is also the case in immature DP thymocytes. We found that Akt became phosphorylated in response to the cross-linking of both anti-TCR-␤ and anti-CD3⑀/CD28 to a similar extent (Fig. 1). We then asked whether the PI3-K-Akt pathway might serve as a survival promoter to counteract the proapoptotic function of Nur77 in thymocytes.
PI3-K Activity Is Necessary for Thymocyte Survival-To determine whether the PI3-K-Akt pathway is indeed essential for survival of immature thymocytes, we examined the effect of LY294002, a PI3-K inhibitor, on FTOCs. FTOCs taken from embryonic day 15 mouse embryos were incubated for 2 days in the presence or absence of LY294002, and then the numbers of double-negative (CD4Ϫ/CD8Ϫ), single-positive (CD4ϩ/CD8Ϫ or CD4Ϫ/CD8ϩ), and DP cells were determined by fluorescence-activated cell-sorting analysis. Treatment of FTOCs with LY294002 markedly reduced all types of immature thymocytes in a dose-dependent manner (Fig. 2). A low dose (5 M) of LY294002 resulted in reduction of primarily DP cells (Fig. 2), suggesting that DP cells are most sensitive to PI3-K inhibition. This result is consistent with the recent finding that PI3-K␥ knockout mice shows moderate reduction of immature thymocytes (18).
Akt Suppresses Transcriptional Activation of Nur77 Induced by TCR Stimulation-Because the PI3-K-Akt pathway and Nur77 appear to have opposite effects on death/survival regulation of T cells, and because it has recently been reported that Akt is capable of phosphorylating Nur77 directly (22), we asked whether Akt has an inhibitory effect on the functions of Nur77 in T lymphocytes. We first examined the transcriptional activity of endogenous Nur77 in T lymphocytes, as monitored by a luciferase gene reporter construct under the control of Nur77 binding elements. We obtained DO11.10 T-cell hybridomas stably expressing either a wild type, constitutively active, or kinase-negative form of Akt by infecting cells with a retrovirus encoding both Akt and human CD8, an expression marker, linked with a bicistronic IRES sequence (see Fig. 3A). After infection, Akt-expressing cells were separated by cell surface expression of human CD8. The profile of human CD8 expression levels was not affected by the types of Akt (Fig. 3B). In noninfected cells or in cells expressing only human CD8, the transcriptional activity of Nur77 increased 12-14-fold after TCR stimulation for 4 h and was accompanied by an increase in the level of endogenous Nur77 protein (Fig. 3C). This increase in transcriptional activity was blocked by expression of N-terminaltruncated dominant-negative Nur77 (Fig. 3C), confirming that this reporter assay indeed reflects the transcriptional activity of Nur77. Expression of either a wild type or constitutively active Akt, but not of a kinase-negative Akt, inhibited activation of Nur77 transcriptional activity in response to TCR stimulation (Fig. 3C). Importantly, the level of Nur77 protein induced by TCR stimulation was not altered by expression of any of the Akt constructs (Fig. 3C). Induction of Nur77 binding element-driven luciferase by ectopic expression of Nur77 was also suppressed when active Akt was co-expressed, whereas the level of Nur77 protein was not suppressed (Fig. 3D). Therefore, it is likely that Akt suppresses the transcriptional activity of Nur77 in T lymphocytes through posttranslational modification, presumably through phosphorylation.
Akt Suppresses DNA Binding Activity of Nur77-How does Akt-mediated phosphorylation inhibit the transcriptional activity of Nur77? The most likely possibility may be that phosphorylation on Ser-350 reduces DNA binding activity as reported previously using recombinant Nur77 protein (29). We therefore examined whether the DNA binding activity of endogenous Nur77 induced by T-cell activation could indeed be inhibited by expression of an active Akt. A shifted band (Nur77-DNA complex) was detected in electrophoretic mobility shift assay when control cells or cells expressing a kinasenegative Akt were stimulated with a phorbol ester (PMA) plus CaI (Fig. 4). The identity of the shifted band (Nur77-DNA complex) was confirmed by supershift analysis with anti-Nur77 antibody (data not shown) and a chase experiment with cold probes (Fig. 4). Expression of an active Akt resulted in reduction of the Nur77-DNA complex (Fig. 4), although the protein levels of Nur77 were essentially unchanged between the control cell extract and the Akt-expressing cell extract used in these experiments (Fig. 3C), suggesting that Akt inhibits the DNA binding activity of Nur77.
Association of Nur77 with 14-3-3 in a Phosphorylation-dependent Manner-Another mechanism by which Akt-mediated phosphorylation inhibits the function of Nur77 might be phosphorylation-dependent binding of 14-3-3 to Nur77. There are a large number of proteins whose functions are regulated by phosphorylation-dependent binding of 14-3-3. For example, some of the targets of Akt, such as Bad and the Forkhead family of transcription factors, bind to 14-3-3 and are functionally modulated (30,31). Interestingly, the sequence around Ser-350 of Nur77 corresponds to the consensus sequence for 14-3-3 binding, R-X-X-phospho-S/T-X-P when it is phosphorylated and is highly conserved among the Nur77 family (Nor-1 and Nurr1) and between species (Fig. 5A). Therefore, the possibility that Nur77 forms a complex with 14-3-3 upon phosphorylation of this site is intriguing. We observed Akt phosphorylation of Nur77 on Ser-350 in vitro (Fig. 5B) and in vivo (Fig. 5C) by use of a phosphorylation site mutant (S350A) and phospho-Ser-350-specific antibody, which confirmed the recent finding of Pekarsky et al. (22). We then tested whether recombinant (His-tagged) Nur77 binds to recombinant (GST-tagged) 14-3-3 in vitro when it is phosphorylated by Akt using a GST pulldown assay. We found that the wild type phosphorylated His-Nur77 could be bound to GST-14-3-3 (Fig. 5D). Neither the S350A mutant nor nonphosphorylated Nur77 was able to bind to GST-14-3-3 (Fig. 5D). We also tested their in vivo association. Myc-tagged Nur77 and FLAG-tagged 14-3-3 were cotransfected into 293T cells with or without various Akts, and co-precipitation of FLAG-tagged 14-3-3 with Myc-tagged Nur77 was observed. 14-3-3 was found to associate with wild type Nur77, but not with S350A Nur77, and did so only when active Akt was co-expressed (Fig. 5E). These data strongly suggest that 14-3-3 binds to Nur77 phosphorylated on Ser-350 both in vitro and in vivo. Because it has been shown that some of the 14-3-3-binding proteins were excluded from the nucleus after complex formation via a mechanism utlizing a nuclear export signal of 14-3-3 protein, we examined the subcellular localization of green fluorescent protein-fused Nur77 in DO11.10 cells. However, Nur77 protein remained in the nucleus even after cells were stimulated with PMA/CaI or when active Akt was co-expressed (data not shown).
Akt Inhibits Activation-induced Cell Death in T-cell Hybridomas-Nur77 is thought to play a key role in AICD of mature T cells as well as in negative selection of immature DP thymocytes. We examined whether the activation of Akt is capable of suppressing AICD in the T-cell hybridoma DO11.10. AICD was induced by treating cells with PMA/CaI for 7 h, and dead cells FIG. 4. Effects of Akt on the DNA binding activity of Nur77. Either control or Akt-expressing DO11.10 cells were stimulated with 10 nM PMA plus 1 M CaI A23187 for 7 h to induce Nur77 expression. Cell lysates were analyzed by electrophoretic mobility shift assay with a probe specific for Nur77 binding. Shifted bands of Nur77-DNA complexes were confirmed after chasing with a 10-fold excess of unlabeled cold probe but not with mutated cold probe.

FIG. 3. Effects of Akt on the transcriptional activity of Nur77.
A, schematic structure of retroviral vectors harboring both Akt and human CD8 linked with a bicistronic IRES sequence to ensure simultaneous expression of both genes. B, CD8 expression on the cell surface was confirmed after retrovirus infection of DO11.10 cells. After magnetic-activated cell sorting, the CD8Ϫ cells (dashed line) and CD8ϩ cells (solid line) were detected by FACScan using fluorescein isothiocyanate-conjugated anti-CD8 antibody. C, transcriptional activity of Nur77 after TCR stimulation. Nur77-responsive reporter plasmid (NBRE-luc) was electroporated into DO11.10 cells that stably expressed each Akt mutant or dominant-negative Nur77. After cells were stimulated with plate-bound anti-CD3⑀ monoclonal antibody for 4 h, the luciferase activity in the cell lysates was measured. The level of Nur77 protein in each subpopulation after TCR stimulation was examined by immunoblotting with anti-Nur77 antibody. D, Rat1a cells were transfected with Nur77 expression plasmid and reporter plasmid with or without Akt, and then the luciferase activity and expression level of Nur77 protein were measured after 18 h.
were detected by use of a membrane-impermeable dye, 7-amino-actinomycin D. Cells expressing only human CD8 or a kinase-negative Akt underwent AICD to a similar extent as the control (noninfected) cells (Fig. 6, A and B). In contrast, expression of a wild type or constitutively active Akt markedly inhibited the cell death induced by PMA/CaI treatment (Fig. 6B), suggesting that Akt mediates survival signals to prevent AICD. This result further supports the idea that Akt might counteract the effect of Nur77 in death/survival regulation of T cells.
Akt Inhibits Nur77-induced Cell Death in a Ser-350-dependent Manner-Finally, we asked whether Akt directly inhibits the proapoptotic function of Nur77. To address this, we attempted to examine the proapoptotic function of Nur77 in T-cell hybridomas, but expression of Nur77 per se appeared insufficient for inducing apoptosis in these cells (data not shown). Therefore, we utilized Rat1a fibroblasts, in which expression of Nur77 was sufficient to induce apoptosis. Expression of active Akt suppressed apoptosis induced by Nur77 almost to the levels seen in the absence of Nur77 (Fig. 7). In contrast, active Akt was unable to inhibit apoptosis induced by the mutant Nur77 containing the Ser-350 to Ala substitution (Fig. 7). These results suggest that Akt inhibits Nur77-induced FIG. 5. Association of Nur77 protein with 14-3-3 in a phosphorylation-dependent manner. A, schematic structure of Nur77. The boxed region indicates amino acid sequences of the Nur77 family that correspond to the consensus sequence for 14-3-3 binding (R-X-X-S/T-X-P). The consensus sequence for Akt phosphorylation is underlined. B, in vitro phosphorylation of Nur77 by Akt. Bacterially expressed GST-fused recombinant Nur77 proteins (wild type or S350A mutant) were incubated with or without active Akt immunoprecipitates in a kinase reaction buffer and subjected to immunoblotting with anti-phospho-Ser-350 Nur77 antibody. C, in vivo phosphorylation of Nur77. Myc-tagged Nur77 (wild type or S350A) and each Akt were co-expressed in 293T cells. Anti-Myc immunoprecipitates (anti-Myc) were blotted with either anti-phospho-Ser-350 Nur77 antibody or with anti-Myc antibody. Total cell extracts were also subjected to immunoblotting with anti-Akt antibody. An active Akt that lacks pleckstrin homology domain migrates faster than full-length Akt. D, in vitro binding of Nur77 with 14-3-3 protein. Bacterially expressed His-tagged Nur77 proteins (wild type or S350A) were preincubated with or without active Akt immunoprecipitate in a kinase reaction buffer. Nur77 proteins were subsequently mixed with either GST or GST-14-3-3 protein and glutathione-Sepharose for 1 h at 4°C, allowing the complexes to bind to the resin. The Nur77 proteins that bound to GST or GST-14-3-3 were detected by immunoblotting with anti-His antibody. E, in vivo binding of Nur77 with 14-3-3 protein. 293T cells were transfected with or without Myc-tagged Nur77 (wild type or S350A), FLAG-tagged 14-3-3, and each Akt. Complex formation was analyzed by immunoprecipitation with anti-Myc antibody followed by immunoblotting with anti-FLAG antibody to detect bound 14-3-3 proteins. apoptosis directly through phosphorylation of Ser-350. DISCUSSION Akt, a downstream target of PI3-K, is emerging as a common mediator of cell survival in a variety of models. Recent data suggest that Akt antagonizes the proapoptotic functions of Bad, caspase-9, the Forkhead family members, and glycogen synthase kinase-3 or activates the nuclear factor B pathway, which may participate in the survival-promoting effects of Akt (32,33). However, the function of Akt in T cells was still largely unknown. Our studies demonstrate that one of the mechanisms by which Akt antagonizes apoptosis involves the inhibition of Nur77. We show here that expression of an active Akt suppresses activation-induced cell death of T-cell hybridoma DO11.10, in which Nur77 plays a pivotal role. Moreover, Akt also suppresses apoptosis induced by expression of Nur77 in a phosphorylation site-dependent manner, suggesting that Akt directly inhibits the proapoptotic function of Nur77. This role in inhibition may account for the observation that Nur77 is expressed in the absence of induction of apoptosis under conditions in which TCR activation promotes survival in thymocytes, as well as in other cells.
The fate of a developing thymocyte is determined by the interaction between TCR and its ligands (MHC-peptide complexes). Studies suggest that both the qualitative and quantitative aspects of TCR activation guide a T cell to either survive and mature or undergo apoptosis. In the quantitative model, strong activation signals result in cell death (negative selection), whereas moderate signals result in survival (positive selection) (34). Although the signaling requirement for regulating strong versus weak signals from the TCR remains largely unknown, the Nur77 family proteins appear to play a qualitatively distinct role, that is, an important role only in apoptosis (negative selection), and not in survival (positive selection). In our study, the levels of Nur77 expressed in response to TCR activation under conditions mimicking either positive or negative selection were low and high, respectively (Fig. 1). Therefore, we would predict the existence of a threshold that determines whether Nur77 induces apoptosis or not. We propose that a survival signal via the PI3-K-Akt pathway might play a part in this threshold based on several reasons: first, the proapoptotic function of Nur77 can be antagonized by Akt-mediated phosphorylation (this study). Second, the inhibition of PI3-K induces robust apoptosis of DP thymocytes (this study and Ref. 18), and third, the activation of the PI3-K-Akt pathway inhibits negative selection (20,21) or activation-induced cell death (this study).
The phosphorylation of Ser-350 has been shown to inhibit the transcriptional activity of Nur77 (12). Expression of an active Akt consistently suppressed the transcriptional activity of both exogenous and endogenous Nur77 induced by TCR activation (this study). In this study, we have suggested possible mechanisms by which Akt inhibits the transcriptional activity of Nur77. One mechanism would be Akt inhibition of the DNA binding activity of Nur77 because we observed reduced DNA binding activity of Nur77 upon expression of Akt in T-cell hybridomas (this study) and because a previous study has shown that the phosphorylation of the recombinant DNA-binding domain of Nur77 results in reduction of the DNA binding activity in vitro (29). Another mechanism (although these mechanisms are not mutually exclusive) might be the 14-3-3 binding of Nur77 induced by the phosphorylation of Ser-350. The binding of 14-3-3 often leads to inactivation of the protein or sequestration of the protein from its functional targets (30,31). Therefore, the binding of Nur77 with 14-3-3 might result in inactivation of its transcriptional activity or sequestration from its targets or functional compartments such as the nucleus (or the mitochondria; see below). This sequestration model is supported by the previous observations that the phosphorylated form of Nur77 is predominantly cytoplasmic (22,35).
It is not clear how Akt suppresses the proapoptotic function of Nur77 in T cells and fibroblasts. We show that expression of Akt suppresses both the transcriptional and proapoptotic activities of Nur77, but this does not necessarily mean that Akt inhibits Nur77-induced apoptosis through inhibition of its transcriptional activity. A recent study has shown that expression of Nur77 induces apoptosis in prostate cancer LNCaP cells through targeting to the mitochondria even in the absence of the DNA-binding domain (11), which apparently contradicts the findings of other reports in which the transcriptional activity correlates well with the apoptosis-inducing activity of Nur77 (7,8,10). In our hands, Nur77 was localized diffusely in the nucleus and not in the mitochondria when Nur77 was ectopically expressed with either a Myc tag or a green fluorescent protein tag in T-cell hybridomas and Rat-1 fibroblasts (data not shown), although this does not exclude the possibility that a trace amount of mitochondrially targeted Nur77 is responsible for the induction of apoptosis. In that case, the 14-3-3 binding of Nur77 induced by Akt-mediated phosphorylation might play an essential role in inactivation of the proapoptotic function of Nur77.
In summary, we have shown that Akt antagonizes the proapoptotic function of Nur77 through its direct phosphorylation. This finding reveals a novel nexus between apoptosis and survival signaling, which might play a critical role in determination of cell death/survival decision of T cells and other cells.