Nur77, an orphan nuclear receptor, plays a key role in apoptosis in T cells. In cancer cell lines, Nur77 can induce apoptosis through the intrinsic apoptotic pathway, but the mechanism by which Nur77 kills T cells remains controversial. In this study, we provide biochemical, pharmacological, and genetic evidence demonstrating that Nur77 induces apoptosis through the activation of the intrinsic pathway in T cells. We also show that Nur77 is a physiological substrate of the MEK-ERK-RSK cascade. Specifically, we demonstrate that RSK phosphorylates Nur77 at serine 354 and this modulates Nur77 nuclear export and intracellular translocation during T cell death. Our data reveal that Nur77 phosphorylation and mitochondrial targeting, regulated by RSK, defines a role for the MEK1/2-ERK1/2 cascade in T cell apoptosis.
Nur77, a member of the NR4A group of nuclear orphan receptors, is identified as a transcription factor required both for TCR-induced apoptosis in T cell hybridomas (1, 2) and negative selection of CD4 and CD8 double positive (DP)3 thymocytes (3, 4). Although target genes regulated by Nur77 in T cells remain to be identified (5), it was assumed that induction of apoptosis by Nur77 in T cells occurred through activation of downstream genes. Consistent with this assumption, expression of a dominant negative form of Nur77 with no DNA binding activity delays TCR-induced apoptosis both in vitro and in vivo (2, 4, 6).
Unexpectedly, in cancer cell lines, it was shown that nuclear Nur77 could migrate to mitochondria, bind Bcl-2, and convert Bcl-2 from a cell death protector into a killer, thus implicating the intrinsic pathway in Nur77-mediated apoptosis in these cells (7). However, early studies in thymocytes from Nur77 transgenic mice suggested cytochrome c is not released from mitochondria during Nur77-induced death, thus casting doubt on the role of the canonical intrinsic apoptosis pathway in Nur77-mediated apoptosis in T cells (5). More recently, several studies suggest that Nur77 can translocate to the mitochondria following TCR activation in DP thymocytes (8, 9). Although these studies showed by biochemical or pharmacological methods that Nur77 mitochondrial translocation is coincident with apoptosis in T cells, direct genetic evidence linking specific intracellular translocation with onset of intrinsic apoptotic program in T cells has not yet been demonstrated. Therefore, the effector mechanism by which Nur77 induces apoptosis in T cells remains to be defined.
MAPKs, which include ERK1/2, JNK1–3, p38s (α, β, γ and δ), and ERK5, are a family of serine/threonine kinases that play essential roles in signal transduction and modulate various biological responses such as proliferation, differentiation, and apoptosis (10). Previous studies suggested all of these kinases may play roles in TCR-induced apoptosis of DP thymocytes (11, 12, 13, 14, 15, 16). Unfortunately, no substrates or targets of these kinases have been identified during T cells and it has been difficult to define the regulatory function of these kinases in T cell apoptosis. Nur77, a major contributor to TCR-induced apoptosis, is also a highly phosphorylated protein in T cells (17). Therefore, it possibly could be a candidate substrate for MAPKs. Consistent with this notion, it was reported recently that Nur77 is regulated by the ERK5 MAPK cascade either through transcriptional up-regulation or posttranslational modification mechanism in T cells (16, 18). However, conditional ablation of ERK5 in T cells affects neither transcription of Nur77 nor T cell development in thymus (19), suggesting the role of the MAPK cascades in regulation of Nur77-mediated T cell apoptosis should be re-evaluated.
Several lines of evidence suggest that ERK1/2 MAPK cascade, rather than the ERK5 cascade, regulates Nur77-mediated T cell apoptosis. Firstly, pretreatment of fetal thymic lobes with pharmacological inhibitors to prevent ERK1/2 activation can block apoptosis of thymocytes during negative selection triggered by high affinity TCR ligands (20). Secondly, negative selection signals provoke a transient but robust activation window of ERK1/2 (21, 22), which overlaps very well with the kinetics of Nur77 expression in TCR-activated T cells, providing a basis for regulation of Nur77 by ERK1/2 MAP kinase cascade. Thirdly, Nur77 can be phosphorylated by the ERK1/2 MAPK pathway. It has been reported that Nur77 can be phosphorylated by ERK2 in vitro and the phosphorylation site was mapped to threonine 142 (23). In addition, p90 ribosomal protein S6 kinase 2 (RSK2), a serine/threonine kinase acting as downstream effectors of the MEK1/2-ERK1/2 MAPK pathway, also can phosphorylate Nur77 in vitro or in 293T cells at serine 354 (24). These studies prompt us to hypothesize that the ERK1/2 MAPK pathway may phosphorylate Nur77 and modulate Nur77-mediated apoptosis in T cells.
In this study, we show that Nur77 is a physiological substrate of ERK1/2 MAPK pathway in T cells. For the first time, we provide genetic evidence to demonstrate that mitochondrial translocation of Nur77 is sufficient to activate the intrinsic apoptotic pathway. Using both pharmacological inhibitors and dominant negative mutant expression to block the ERK1/2 MAPK pathway, we demonstrate that the ERK1/2 MAPK cascade phosphorylates Nur77 during TCR-activation-induced and Nur77-mediated apoptosis in T cells. This phosphorylation event regulates Nur77 nuclear export and translocation to mitochondria, where Nur77 triggers the intrinsic apoptotic program. In addition, we define the specific site (serine 354) of in vivo RSK phosphorylation of Nur77. Mutation of this site into alanine or glutamic acid can impair or enhance Nur77 nuclear export and TCR activation-induced apoptosis in T cells. Our study elucidates a molecular mechanism linking the ERK1/2-MAPK pathway to Nur77 and provides insight into our understanding of how Nur77 induces apoptosis in T cells.
Materials and Methods
The Retro-X-Tet-off Advanced Inducible Expression System was purchased from Clontech. The Gateway cassette was cloned from the MSCV2.2-IRES-GFP Gateway destination vector and inserted into BamHI and EcoRI sites of pRetroX-tight-Pur (Clontech), generating pRetroX-tight-Pur-Gateway vector. Nur77-nuclear export sequence (NES) was generated in the MSCV 2.2-IRES-GFP vector by fusing the C terminus of Nur77 with NES (amino acids LALKLAGLDL) (25) using an in-fusion PCR cloning kit (Clontech). Nur77 and Nur77-NES were then PCR-cloned into EcoRI and NotI sites of pENTR1A (Invitrogen) followed by the insertion between SalI and EcoRI sites either of EGFP (from pEGFPC1, Clontech) or Flag tag at the N terminus of Nur77 coding sequence to generate pENTR1A-GFP-Nur77, pENTR1A-GFP-Nur77-NES, and pENTR 1A-Flag-Nur77-NES, respectively. Mutations were introduced by site-directed mutagenesis to create pENTA1A-GFP-Nur77S354A and pENTR1A- GFP-Nur77S354E. pENTR1A-GFP was generated by the insertion of EGFP PCR fragment between SalI and NotI sites of pENTR1A. Finally, all inserts in pENTR1A vectors were transferred into pRetroX-tight-Pur-Gateway vector using the Gateway LR Clonase II enzyme mix kit (Invitrogen) to obtain the corresponding pRetroX-tight vectors for the establishment of Tet-off inducible cell lines. pUSEamp-CA-HA-rat MEK1 was obtained from Millipore. pCMV5-Flag-Nur77 and pRK5-Myc-murine Rsk2 were obtained from Dr. Simon Arthur (University of Dundee, Dundee, Scotland) and Dr. Richard Huganir (Johns Hopkins University, Baltimore, MD), respectively. The Nur77S260A, Nur77S344A, Nur77S354A, and Nur77S536A mutations were introduced into CMV5-Flag-Nur77 by site-directed mutagenesis. Murine Bcl-2 and Bcl-XL cDNAs were purchased from Addgene and were PCR-cloned into pcDNA6/myc-HisA vector (Invitrogen) to generate pcDNA6-Bcl2-myc-His and pcDNA6-Bcl-XL-myc-His, respectively. MSCV-DN-MEK1-Flag-neo was generated by PCR in-fusion cloning of a cDNA encoding the murine DN-MEK1 (S218AS222A) from pBluescript-DN-MEK1 (a gift from Dr. Kunihiko Naito, Tokyo University, Tokyo, Japan) into MSCV-Neo (Clontech) with the carboxyl terminus of DN-MEK1 fused and in-frame with Flag tag.
The following reagents were obtained: Z-VAD-fmk, wortmannin, and LY-294002 (Biomol); PD 184352 (Axxora); SB203580, SP600125, and U0126 (Calbiochem); SL0101 (Toronto Research Chemicals); PMA, ionomycin, Lemptomycin B, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP), digitonin, trypsin, doxycycline (Sigma-Aldrich); active GST-RSK2 (Cell Signaling Technology); recombinant active mouse ERK2 (Calbiochem); myelin basic protein (Upstate Biotechnology); and Ni-NTA beads (Qiagen).
Cell culture, transfection, infection, and stable cell lines
DO11.10 and 293T cells were maintained in RPMI 1640/DME/Glutamine medium composed of 45% RPMI 1640, 45% DMEM, 10% FBS, and gentamicin. To transfect DO11.10 cells, 2.0–3.0 μg of DNA were electroporated into 2.5 × 106 cells using a mouse T cell kit and program S18 following the manufacturer’s instruction (Amaxa). The infection of DO11.10 cells was performed following a T cell spin infection protocol (26) by packaging retrovirus in 293T cells, which were transfected with 2.0 μg of pCL-Eco (Imgenex) and 2 μg of retroviral DNA containing the gene of interest using Fugene6 (Roche). Inducible Tet activator cell line (no. 21) and pRetroX-tet-off cell lines were established according to the manufacturer’s protocol (Clontech). Induction of all established Tet-off cell lines was performed by washing cells twice using PBS with 3-h interval.
Preparation of cell extracts and subcellular fractionation
Whole cell lysates were prepared by lysing cells using radioimmunoprecipitation assay buffer. Nuclear, cytosolic, and mitochondrial fractionation was performed as described previously (27, 28) and modified as follows. For nuclear and cytoplasmic fractionation, 1 × 106 cells were permeabilized with 150∼200 μl of isotonic lysis buffer (10 mM Tris (pH 7.5), 1.5 mM MgCl2, 20 mM KCl, 250 mM sucrose, 1.0 mM EDTA, and 1.0 mM EGTA; 10 μM cytochalasin B; 1.0 mM DTT, protease; and phosphatase inhibitors) containing 300 μg/ml digitonin before centrifugation at 1000 × g for 5 min to obtain a crude postnuclear supernatant (PNS) and crude nuclear fraction pellet. The crude PNS was centrifuged four to five times at 1000 × g for 5 min until no pellet was observed. One volume of CCB buffer (10 mM Tris (pH 7.5), 5.0 mM EDTA, 250 mM sucrose, 4.0% Trition X-100, 2.0% sodium deoxycholate, 0.4% SDS, 1.0 mM DTT, protease, and phosphatase inhibitors) was then added into three volumes of PNS, incubated on ice for 30 min and centrifuged at 14000 × g for 15 min to obtain cytoplasmic extracts. The crude nuclear pellet was washed once with nuclear wash buffer (NWB) (10 mM Tris (pH 7.5), 20 mM KCl, 1.5 mM MgCl2, 250 mM sucrose, 10 μM cytochalasin B, protease, and phosphatase inhibitors) containing 0.5% Triton X-100, followed by another wash with NWB buffer without Trition X-100. Nuclear pellets were resuspended in NLB (10 mM Tris (pH 7.5), 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 1.0% Triton X-100, 1.0 mM EDTA), incubated on ice for 30 min, and centrifuged at 14000 × g for 15 min to obtain nuclear extracts.
For nuclear, cytosolic, and mitochondrial fractionation, 5 × 107 cells were harvested and washed with cold PBS before resuspension into 400 μl of ice-cold hypotonic buffer (20 mM HEPES-KOH (pH 7.4), 10 mM KCl, 1.0 mM EGTA, 1.0 mM EDTA, 1.0 mM DTT, 10 μM cytochalasin B, and protease inhibitors) followed by incubation on ice for 20 min. The cell suspension was transferred into a 2 ml glass dounce homogenizer and homogenized by 20 strokes. Immediately, 125 μl of CIB (20 mM HEPES-KOH (pH 7.4), 7.5 mM MgCl2, 10 mM KCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.25 M sucrose, 10 μM cytochalasin B, and protease inhibitors) was added into cell homogenates followed by centrifugation at 1000 × g for 5 min three to five times to obtain supernatant as clear PNS. Nuclear and unbroken cell pellets were washed with NWB containing 0.5% Triton X-100 followed by their resuspension into 200 μl of NWB and loading on the top of 2.0 M over 1.6 M sucrose gradient (10 mM Tris (pH 7.5), 20 mM KCl, 1.5 mM MgCl2, 1.6 or 2.0 M sucrose, 10 μM cytochalasin B, and protease inhibitors). After centrifugation at 100,000 × g for 1.0 h, nuclear pellets were recovered, washed once with NWB, extracted with 100 μl of NLB for 30 min on ice, and centrifuged for 15 min at 14,000 × g to obtain supernatants as nuclear extracts. PNS was centrifuged at 10,000×g for 15 min to obtain mitochondrial pellets and supernatants (S-10). Mitochondrial pellets were extracted with MLB (10 mM Tris (pH 7.4), 5 mM EDTA, 150 mM NaCl, 1.0% Triton X-100, and protease inhibitors) for 30 min followed by centrifugation at 4°C at 10,000 × g for 15 min to obtain mitochondrial extracts. S-10 fractions were centrifuged at 4°C at 100,000 × g for 30 min to obtain supernatants (S100) as cytosolic extracts.
For trypsin digestion of mitochondria, mitochondria harvested from stimulated cells were resuspended into MSB (20 mM HEPES (pH 7.4), 10.0 mM KCl, 200 mM manitol, 68 mM sucrose, 1 mM EDTA, 1 mM EGTA, 0.1% BSA, 10 mM succinate, 2 mM ATP, 10 μM phosphocreatine, 10 μg/ml creatine kinase, and proteases inhibitors) with or without 200 μg/ml trypsin and incubated on ice for 30 min. Reaction was stopped by addition of SDS sample buffer. For alkali extraction of mitochondria, mitochondria harvested from stimulated cells were resuspended either into MSB or fresh prepared 0.1M Na2CO3 (pH 11.5), incubated on ice for 30 min and centrifuged at 13,000 × g for 30 min at 4°C to obtain pellets (alkali-resistant fractions) and supernatants (alkali-sensitive fractions). The pellets were extracted with radioimmunoprecipitation assay buffer.
To perform co-IP in 293T cells, nuclear and cytoplasmic extracts were prepared using a Nuclear Complex CO-IP Kit (Active Motif) and pooled together as cell lysates. Anti-Flag M2–agarose beads or control mouse IgG-agarose (Sigma-Aldrich) were blocked with 100 μg/ml BSA in PBS for 1.0 h, incubated with lysates overnight, washed five times with washing buffer (50 mM HEPES (pH 7.4), 1% Nonidet P-40, 250 mM NaCl) and incubated with 250 μg/ml Flag peptide in PBS for 30 min to elute Flag-Nur77-Rsk2 immune complexes from beads. To perform co-IP for endogenous Nur77 and Rsk2 in DO11.10 cells, cell lysates, prepared as above, were cleared by Protein A/G Plus beads (Santa Cruz Biotechnology), incubated with anti-Nur77 no. 357 mAb for 2.0 h and followed by Ag-capture for 2.0 h using Protein A/G Plus beads. Immunoprecipitates were then washed five times with washing buffer and boiled in SDS sample buffer.
To enrich exogenous Bcl-2 or Bcl-XL for immunoblot detection, 5 × 106 DO11.10 transduced and transfected with pcDNA6-EV/pRetroX-tight-GFP-Nur77-NES, pcDNA6-Bcl-2-myc-His/pRetroX-tight-GFP-Nur77-NES, or pcDNA6-Bcl-XL-myc-His/pRetroX-tight-GFP-Nur77-NES, were extracted with lysis buffer (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 5 mM immidazole, 1.0% Nonidet P-40 and protease inhibitors), incubated on a shaker for 30 min at 4°C, and centrifuged at 14,000 × g for 15 min to obtain supernatant as lysates. Twenty microliters of Ni-NTA agarose beads were incubated with lysates on a shaker for 2 h at 4°C, washed three times with lysis buffer, and eluted three times with 20 μl of elution buffer (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 250 mM imidazole, 1.0% Nonidet P-40) at room temperature for 10 min. Pooled elutes were added with 6× SDS sample buffer.
The following Abs were used for immunoblotting. Cytochrome c (A-8), Tom20 (FL-145), and Rsk2 (E-1) were from Santa Cruz Biotechnology. Caspase-3 (8G5), Caspase-9 (C9), Phospho-p44/p42 MAPK (no. 9101), p44/p42 MAPK (no. 9102), Bcl-XL (54H6), Bcl-2 (50E3), COX IV (no. 4844), and anti-RXRXXpS/T (Phopho-Ser/Thr-Akt substrate Ab) (no. 9611) were from Cell Signaling Technology. Anti-Flag M2 (no.3165) were from Sigma-Aldrich. Poly (ADP-ribose) polymerase (PARP) (C-2–10) was from eBioscience. GFP (3E6) was from Invitrogen. GAPDH was from Chemicon. Anti-Myc was tissue culture supernatant from hybridoma 9E10 (American Type Culture Collection). Nur77 (12, 13, 14) (BD Pharmingen) was used for immunoblot. Nur77 (no. 357) mAb, which was generated in our laboratory by the injection of purified Escherichia coli-derived Nur77 protein into mice, was used for immunoprecipitation and immunoblotting in this study.
Cell death assay
DO11.10 cells were stimulated to die with 10 nM PMA and 500 nM Ionomycin except as noted in Fig. 5, C and F. At 8–30 h poststimulation, cells were stained with Annexin V-FITC, PI (Calbiochem,) or Annexin V-Cy5 (Biovision) for flow cytometry analysis. Cell death was also quantified by measuring hypodiploid DNA levels in stimulated cells (Fig. 5C) according to Riccardi et al. (29). All flow cytometry data were acquired by BD FACSDiva (BD Biosciences) and analyzed by FlowJo (Tristar). Trypan blue exclusion was used to measure cell death in Figure S2D4 and 5A. Cytochrome c release was assessed by using InnoCyte flow cytometric cytochrome c release kit according to the manufacturer’s instruction (Calbiochem). Cells were stained either with JC-1 (Biotium) or tetramethylrhodamine, ethyl ester, perchlorate (TMRE) (50 nM) (Invitrogen) at 37°C for 15 min before flow cytomeric analysis of mitochondrial membrane potential. Cells that were pretreated with FCCP (10 μM) for 30 min at 37°C before TMRE staining were used as positive controls for mitochondrial membrane potential loss.
In vitro kinase assay and dephosphorylation in vitro
The Flag-Nur77 immunoprecipitates, purified from serum-starved 293T cells that have been transfected with CMV-Flag-Nur77 and were washed five times with washing buffer and once with kinase buffer (5 mM MOPS (pH 7.2), 2.5 mM β-glycerolphosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT) before the elution with kinase buffer containing 250 μg/ml Flag peptide. Kinase assays were performed at 30°C for 15 min with Flag-Nur77 eluates as a substrate either of active GST-human Rsk2 (Cell Signaling Technology) or active mouse ERK2 in a kinase buffer- based reaction containing 50 μM of ATP (containing 5μCi of γ-32P-ATP) in the presence or absence of 100 μM of SL0101. The reactions were stopped by adding 6× SDS-sample buffer and were subjected to SDS-PAGE. For each treatment, a mock kinase reaction was performed with the omission of ATP and RSK2/ERK2 and analyzed by immunoblotting to provide loading control for in vitro kinase assay. Densitometric analysis was performed using ImageJ software (National Institutes of Health).
To perform in vitro dephosphorylation, cells were lysed with lysis buffer (10 mM Tris, (pH 7.6), 150 mM NaCl, 5 mM EDTA, 1.0% Triton X-100, and protease inhibitors). With or without phosphatase inhibitors (10 mM sodium vanadate and 50 mM sodium fluoride), cell lysates containing 50 μg of protein were incubated with 400U Lamda phosphatase (New England Biolabs) following the manufacturer’s instruction. Incubation was performed at 30°C for 30 min followed by the addition of 6× sample buffer to stop the reaction.
Luciferase reporter assay
In brief, 2.45 μg of (NurRE)3-TK-Luc (30) and 50 ng of pRL-CMV (Promega) were transfected by nucleofector (Amaxa) into 2.5 × 106 DO11.10 stable cell lines, which were induced to express Nur77 or mutants 1 day in advance. Twenty hours after transfection, cells were lysed and luciferase assay was performed following the manufacturer’s protocol (Dual-luciferse Reporter Assay).
Immunofluorescence staining and confocal microscopy
DO11.10 cells were transferred onto slides by cytospin, fixed in 3.7% formaldehyde for 10 min, permeabilized with 0.1% Trition X-100 for 10 min, and blocked for 1.0 h in 5.0% BSA in PBS, before being incubated sequentially with primary Ab and secondary Ab (anti-Mouse IgG-Alexa 488 and anti-Rabbit IgG-Alexa-568, Invitrogen) in 5.0% BSA in PBS for 1.0 h each. Cells were stained with 4′,6-diamidino-2-phenylindole and mounted onto slides with Prolong Gold Anti-fade Reagent (Invitrogen). Photographs were taken through a Zeiss Axiovert 200M inverted microscope equipped with a Hamamatsu Orca camera.
Statistical significance was tested in an unpaired Student’s t test using the Graph Pad Prism software. Compared data with p < 0.05 were denoted to be statistically significant (p < 0.05).
Translocation to mitochondria is sufficient for Nur77 to activate intrinsic mitochondrial apoptotic program
Recently, several groups observed that in T cells, following TCR activation, Nur77 can migrate to mitochondria. Thompson et al. (9) reported that Nur77 translocates to mitochondria and binds Bcl-2 in TCR-stimulated DP thymocytes isolated from Bcl-2 transgenic mice, suggesting that Nur77 follows the same paradigm in cancer cells to kill thymocytes. However, Cunningham et al. (31) found that mitochondrial location of Nur77 is found in TCR-activated peripheral T cells, but not in activated thymocytes. Given that expression of Nur77 in peripheral T cells does not kill T cells (31), it is necessary to determine whether Nur77 mitochondrial translocation occurs in Nur77-mediated T cell apoptosis and physiological relevance of Nur77 mitochondrial translocation under this context should be carefully investigated.
To address these questions, we used the CD4+CD8+ DO11.10 T cell hybridoma, a cell line widely used to study T cell apoptosis. TCR activation by α-CD3/CD28 or treatment with PMA plus ionomycin (P+I) induce apoptosis in DO11.10 cells. It has been shown by previous studies that this is dependent on Nur77 expression (1, 2). Consistent with these studies, Nur77 was rapidly induced by P+I stimulation in DO11.10 as early as 1 h (Figure S2A). In contrast to the findings of Rajpal et al. (5) using thymocytes from Nur77 transgenic mice, induction of apoptosis in DO11.10 cells resulted in release of cytochrome c from mitochondria (Figure S1A), loss of mitochondrial member potential (Figure S1B), and cleavage of caspase 9 (Figure S1C). In our preliminary studies, we found that that during P+I-induced apoptosis in thymocytes, Nur77 is found in mitochondrial fraction as shown by Western blot (Figure S7). However, primary thymocytes can neither be transfected nor infected with exogenous DNA. Therefore to address the question of how Nur77 modifications influence translocation to the cytosol and induction of apoptosis, we eomployed the use of a CD4+, CD8+ T cell line. As previously reported, using subcellular fractionation techniques, we found in DO11.10 cells, that Nur77 migrates to mitochondria during T cell apoptosis (Figure S2B) and this translocation is blocked by Leptomycin B pretreatment before stimulation by P+I (Figure S2, C and D). Mitochondrially associated Nur77 is sensitive both to trypsin digestion and alkaline buffer extraction, suggesting that Nur77 loosely associates with outer membrane of mitochondria in dying DO11.10 cells (Figure S2, E and F). Our results confirm that Nur77 can translocate to mitochondria during apoptosis in T cells.
To determine whether Nur77 mitochondrial translocation is a cause or effect of TCR-activation-induced apoptosis in T cells, we used a gain-of-function approach to overexpress Nur77 in the cytosol of T cells. We generated Nur77 fusion proteins by fusing the C terminus of either GFP-Nur77 or Flag-Nur77 with a cyclic AMP-dependent protein kinase inhibitor-like NES (amino acids LALKLAGLDL) (25) which delivers GFP-Nur77 or Flag-Nur77 into the cytosol. Using a Tet-regulated gene expression system whereby Nur77 expression is turned on in the absence and turned off in the presence of doxycycline (Fig. 1,A), we showed that clones robustly expressing either GFP-Nur77 or Flag-Nur77 undergo apoptosis (Fig. 1,B) and display similar kinetics, suggesting the observed phenotype is Nur77 specific, and neither overexpression nor positional effects of Nur77 transgene integration into the genome. However, progression of apoptosis induced by the delivery of Nur77 into the cytosol is slower than that induced by P+I stimulation in DO11.10 cells (Fig. 1,B and data not shown; see Discussion). Confocal fluorescence microscopy data showed that cytosolically localized Nur77-NES partially overlapped with Tom20, a specific mitochodondrial membrane protein, suggesting that the expressed Nur77-NES localizes to mitochondria (Fig. 1 C).
Next, we investigated whether mitochondrial localization of Nur77 is sufficient to activate the intrinsic apoptotic pathway. Indeed, even in the absence of TCR signals, more than 20% of DO11.10 cells were found to release cytochrome c from mitochondria 2 days after inducing Nur77-NES expression (Fig. 1,D). Targeting Nur77 into the cytosol also led to significant loss of mitochondrial membrane potential in DO11.10 cells (Fig. 1,E). Additionally, both caspase 9 and PARP were cleaved, mirroring the activation of the mitochondrial apoptotic pathway (Fig. 1,F). Furthermore, Nur77-NES-induced apoptosis can be abrogated by pretreating these cells with the broad-spectrum caspase inhibtior zVAD-fmk before the induction of Nur77 protein expression (Fig. 1,G). Finally, we found that overexpressing Bcl-2 or Bcl-XL delays a Nur77-NES-initiated apoptotic program (Fig. 1,H) by preserving mitochondrial membrane potential (Fig. 1 I).
To rule out that Nur77-NES induces apoptosis primarily through transcriptional activation, driving the expression of other proapoptotic genes, we compared the transcriptional activity of wild-type (WT) GFP-Nur77 and GFP-Nur77-NES mutant in bulk populations of transduced cells. WT GFP-Nur77 was constitutively retained in the nucleus while GFP-Nur77-NES completely localized to the cytosol (Figure S3A). Consistent with their intracellular localization, WT GFP-Nur77 exhibited significantly higher transcriptional activity of Nur77 than that of GFP-Nur77-NES (Figure S3B). Interestingly, cells expressing GFP-Nur77-NES underwent more apoptosis than cells expressing WT GFP-Nur77 when these cell lines are stimulated with P+I (Figure S3C), implying that transcriptional activation of downstream genes by Nur77-NES is not the key effector mechanism contributing to the induction of apoptosis in T cells.
We conclude that the delivery of Nur77 into the cytosol, even in the absence of TCR signals, can induce apoptosis in T cells by triggering activation of the intrinsic apoptotic pathway.
Nur77 can be phosphorylated by activation of the MEK1-ERK1/2 MAPK pathway
Nur77 is phosphorylated in DO11.10 T cells undergoing apoptosis (17). Although phosphorylation of Nur77 regulating apoptosis or differentiation in cancer or neuronal cell lines has been well established (32, 33, 34, 35), during T cell apoptosis, the pathway and corresponding kinases that phosphorylate Nur77 remains to be defined. We found that treatment of lysates from apoptotic DO11.10 cells with lambda phosphatase results in the collapse of high molecular weight (mw) species of Nur77 (lane 3 in Fig. 2,A) and this is prevented by treating cell lysates with lambda phosphatase in the presence of phosphatase inhibitors (lane 5 in Fig. 2 A), demonstrating that Nur77 is phosphorylated in dying DO11.10 T cells.
Nur77 has been reported to be a substrate for several serine/threonine kinases in a variety of different cell types. To examine which kinases might phosphorylate Nur77 in apoptotic T cells, we used a variety of kinase inhibitors to ask which ones both blocked phosphorylation of Nur77 and induction of apoptosis. As shown in Fig. 2, B and C, pretreatment of DO11.10 with the MEK1 inhibitor U0126 resulted in the collapse of higher MW species of Nur77, while pretreatment of DO11.10 with the p38 inhibitor SB203580, the JNK inhibitor SP600125 and two compounds (Wortmannin or LY294002) that inhibit AKT activation, failed to affect the migration of Nur77 bands in the gel. These data suggest that the MEK1/2-ERK1/2 MAPK pathway plays a role in Nur77 phosphorylation.
The MEK1 inhibitor U0126 can inhibit both MEK1/2-ERK1/2 and MEK5-ERK5 pathways (36). To further explore the contribution of the ERK1/2 MAPK pathway to Nur77 phosphorylation, we used another MEK1 inhibitor PD184352. At low concentration (≤2 μM), this inhibitor will prevent the activation of MEK1/2-ERK1/2 MAPK pathway leaving the MEK5-ERK5 MAPK pathway unaffected (36). Indeed, pretreatment of DO11.10 with 2 μM PD184352 inhibited Nur77 phosphorylation (Fig. 2 D) supporting the conclusion that Nur77 is phosphorylated by the MEK1/2-ERK1/2 pathway.
The data described above are pharmacological. In a more direct approach, we coexpressed Nur77 and a constitutively active MEK1 mutant in 293T cells and this resulted in an upward shift of Nur77 MW (Fig. 2,E), whereas expressing a dominant negative MEK1 mutant in DO11.10 cells led to the predominance of hypophosphorylated lower bands (Fig. 2 F). Collectively, our pharmacological and genetic evidence demonstrates that the MEK1/2-ERK1/2 MAP kinase pathway is involved in the phosphoylation of Nur77.
MEK1/2-ERK1/2 MAPK pathway regulates Nur77 intracellular translocation and Nur77-mediated apoptosis in DO11.10 T cells
Given that MEK1/2-ERK1/2 MAPK cascade can phosphorylate Nur77 and Nur77 induces apoptosis through intracellular translocation in T cells, we wanted to explore whether phosphorylation of Nur77 by the MEK1/2-ERK1/2 pathway regulates Nur77 translocation during T cell apoptosis. To this end, we inhibited the activation of MEK1-ERK1/2 pathway by several strategies and examined the effects on Nur77 intracellular localization and induction of apoptosis.
Pretreatment of DO11.10 with the MEK1 inhibitor U0126 both impaired mitochondrial translocation of Nur77 as well as induction of apoptosis (Fig. 3, A and B). This was corroborated by pretreatment of DO11.10 cells with 2 μM PD184352 which both inhibited apoptosis and decreased the Nur77 abundance in mitochondrial fractions (Fig. 3, C and D). Furthermore, inhibiting the ERK1/2 MAPK pathway by expressing dominant negative MEK1 reduced induction of apoptosis and nuclear export of Nur77 in stimulated DO11.10 cells (Fig. 3, E and F). Thus, ERK1/2 MAPK pathway both regulates Nur77 nuclear export and mitochondrial translocation, as well as Nur77-mediated apoptosis in T cells.
RSK2 can phosphorylate and physically interact with Nur77
ERK2 previously was shown to be able to phosphorylate Nur77 at Thr142 in vitro (23). Consistent with this, we also found that active recombinant ERK2 can phosphorylate Nur77 immunoprecipitates in an in vitro kinase assay (Figure S4). However, we were unable to reproducibly assign radioactivity labeling activity by ERK2 in vitro to any of twelve potential ERK2 phosphorylation sites (XpS/TP) in Nur77 (data not shown). In addition, in 293T cells or DO11.10 cells, we found that overexpressed or endogenous Nur77 never bound ERK2 (data not shown). Therefore, we were unable to demonstrate that Nur77 is a physiological substrate of ERK2 in vivo.
The RSK family of kinases are downstream effectors of the MEK1/2-ERK1/2 pathway. Relevant to this study, RSK2 is known to phosphorylate Nur77 in vitro and in 293T cells at serine 354 (24). However, in this study E. coli-derived recombinant Nur77 was used as a substrate for RSK2 in an in vitro kinase. In our hands, degradation of Nur77 in bacteria prevented purification of full-length intact Nur77 for functional analysis (data not shown). To rule out artifacts caused by any truncation of Nur77, we took a different approach to obtain Nur77 protein and perform an in vitro kinase assay to determine whether RSK can directly phosphorylate Nur77. Purified Flag-Nur77 from 293T cells was incubated with recombinant active GST-RSK2 in the presence of 32P-ATP. As shown in Fig. 4, Nur77 was directly phosphorylated by RSK2 in vitro. This phosphorylation is inhibited in the presence of SL0101, an inhibitor of all RSK isoforms. The canonical RSK phosphorylation motif is RXRXXpS/T (37), however, arginine (R) at −6 position is not always required (37, 38). In Nur77, there are four potential serine residues (serine 260/344/354/536) that satisfy the minimum phosphorylation motif (RXXpS/T) of RSK2. To identify the exact phosphorylation site(s) of Nur77 by RSK2, we mutated all four serines in Nur77 separately and expressed these mutants in 293T cells to produce purified proteins for use in an in vitro kinase assay. Our data demonstrate that mutation of serine 354 abolishes the phosphorylation of Nur77 by recombinant RSK2. Densitometric analysis indicated that this mutant incorporated only 30% of radioactivity compared with WT Nur77 (Fig. 4,C). Other Nur77 mutants showed mild decreases in phosphorylation when compared with WT Nur77 (Fig. 4 C). Thus, serine 354 is the predominant site of phosphorylation of Nur77 by RSK2.
To investigate whether RSK phosphorylates Nur77 in vivo, we pretreated DO11.10 cells with SL0101 before P+I stimulation. We found that SL0101 blocks the appearance of higher MW species of Nur77 (Fig. 4,A), suggesting that RSK is involved in Nur77 phosphorylation in DO11.10 cells. To further demonstrate that Nur77 is a physiological substrate of RSK2, we investigated the physical interaction between Nur77 and RSK2. We serum-starved 293T cells that had been cotransfected with Nur77 and RSK2. In this way, de novo-synthesized RSK2 was inactive and was activated only by brief PMA treatment before harvest. As shown in Fig. 4,D, activation and phosphorylation of ERK1/2, which is required for RSK2 activation, is PMA stimulation-dependent. We also measured phosphorylation levels of Nur77 by RSK2 using anti-RXRXXpS/T, a polyclonal Ab recognizing the RSK2 phosphorylation motif. In the absence of RSK2, Nur77 was not phosphorylated by endogenous RSK2 (Fig. 4,D, lane 5) but was phosphorylated by endogenous RSK2 induced by PMA stimulation (Fig. 4,D, lane 6). Overexpression of RSK2 is sufficient to phosphorylate coexpressed Nur77 (Fig. 4,D, lane 7) and PMA stimulation further increased Nur77 phosphorylation (Fig. 4,D, lanes 7 and 8), suggesting that RSK2 can phosphorylate Nur77 in vivo. Furthermore, RSK2 was immunopreciptated by anti-Flag (Nur77) independent of whether it was activated by PMA through ERK1/2 (Fig. 4,D, lanes 7 and 8). Finally, we looked at the interaction between Nur77 and RSK2 in DO11.10 cells. RSK2 was immunoprecipitated by an Ab directed against Nur77, but not by isotype control IgG, in lysates from stimulated DO11.10 cells (Fig. 4 E). Overall, our data strongly suggest that Nur77 is a substrate of RSK2 in T cells.
Nur77 phosphorylation by RSK is required for Nur77 nuclear export and TCR activation-induced apoptosis in T cell
To explore the physiological relevance of Nur77 phosphorylation by RSK2 in T cells, we examined the effects of the inhibition of RSK activity on P+I-induced apoptosis in DO11.10 cells. We observed a significant decrease in apoptosis in cells pretreated with RSK inhibitor SL0101, compared with cells pretreated with methanol vehicle, before stimulation (Fig. 5,A). To demonstrate that decreased apoptosis in DO11.10 cells by the RSK inhibitor was attributed to reduced Nur77 phosphorylation, we overexpressed WT Nur77, the Nur77 phosphorylation-deficient mutant (Nur77S354A), or the Nur77 phosphomimetic mutant (Nur77S354E) proteins, respectively, in DO11.10 cells. Protein expression levels of Nur77 and its mutants were identical in stable cell lines upon induction (Fig. 5,B). However, we found that cells overexpressing Nur77S354A mutant exhibited significantly less apoptosis than cells overexpressing WT Nur77 following stimulation by P+I whereas cells overexpressing phosphomimetic mutant Nur77S354E restored killing activity to almost the same extent as cells overexpressing WT Nur77 (Fig. 5 C). Our data suggest that phosphorylation of Nur77 by RSK is required for Nur77-mediated apoptosis in DO11.10 cells.
Previously, others suggested that phosphorylation of Nur77 serine 354 results in impaired DNA binding and transcriptional activity and this blocks apoptosis in T cells (39, 40). However, a recent study showed that phosphorylation of this site had no effect on Nur77 transcriptional activity in MEFs (24). To further clarify whether transcriptional activity of Nur77, or the mutants we constructed, plays a role in modulating their apoptotic function, we used a luciferase assay to measure transcriptional activity of WT Nur77 and mutant proteins in DO11.10 cells. Expressed WT Nur77, Nur77S354A, and Nur77S354E localized mainly in nucleus (data not shown) and this resulted in robust Nur77 transcriptional activity (Fig. 5,D). However, we found that cells expressing Nur77S354A showed significantly higher transcriptional activity than cells expressing Nur77S354E (Fig. 5,D). Because cells expressing the Nur77S354A mutant exhibit less apoptosis than cells overexpressing WT Nur77 or Nur77S354E (Fig. 5 C), the transcriptional activity of Nur77S354A mutant apparently was not responsible for the defect in its proapoptotic function in T cells.
Finally, we investigated whether the inhibition of RSK activity in DO11.10 could recapitulate the Nur77 nuclear export defect imposed by inhibiting MEK1 activity (Fig. 3, A and C). Indeed, pretreating DO11.10 cells with the RSK inhibitor SL0101 significantly crippled Nur77 nuclear export (Fig. 5,E). Disrupting RSK phosphorylation of Nur77 by mutating serine 354 to alanine led to impaired nuclear export of Nur77 in DO11.10 cells stimulated with P+I, whereas mimicking RSK phosphorylation of Nur77 by mutating serine 354 to glutamic acid resulted in enhanced Nur77 nuclear export (Fig. 5 F). Our data demonstrate that intracellular translocation of Nur77, modulated by RSK through phosphorylation, regulates Nur77-mediated apoptosis in T cells.
In this study, we present evidence that the ERK1/2 MAPK pathway regulates T cell apoptosis. Furthermore, we demonstrate that the ERK1/2 MAPK cascade modifies Nur77, a key component of TCR-activation-induced apoptosis in T cells. Our data demonstrate that this pathway regulates Nur77 through phosphorylation by RSK, a downstream component of ERK1/2 MAPK cascade.
The association between ERK1/2 MAP cascade and Nur77 proapoptotic function in T cells is one of novel findings demonstrated in the present study. Although Cunningham et al. (31) suggested that the ERK1/2 MAPK pathway phosphorylates Nur77 and modulates its intracellular translocation in peripheral T cells, these authors did not investigate physiological consequences of this phosphorylation. Similarly, Fujii et al. (18) found that the ERK1/2 MAPK pathway can phosphorylate Nur77 both in vitro and in COS7 cells. Our studies extend these observations and demonstrate that phosphorylation of Nur77 by ERK1/2 MAPK cascade contributes to its killing activity in T cells (Figs. 2 and 3). Most investigations of ERK1/2 MAPK in T cells used the MEK1 inhibitors PD98059 or U0126. Recent data question the specificity of these inhibitors and attributed their ability to inhibit apoptosis to inactivation of ERK5 which, in turn, was suggested to regulate Nur77 by either transcriptional or posttranslation modifications (16, 18). However, these conclusions are contradicted by convincing genetic evidence. Arthur and colleagues (19) demonstrated that Nur77 transcription and T cell development both are normal in the absence of ERK5 in T cells. In addition, studies of the MEK5-ERK5 cascade did not address the role of the MEK1-ERK1/2 pathway in Nur77-mediated T cell apoptosis. In our study, we present both pharmacological and genetic evidence to demonstrate that the ERK1/2 MAPK pathway phosphorylates Nur77, regulates its intracellular localization and modulates its proapoptotic function in T cells. These data are supported further by evidence of direct phosphorylation of Nur77 by RSK2, a downstream member of the ERK1/2 MAPK pathway. We show that phosphorylation of serine 354 in Nur77 by RSK has a dramatic effect on both the intracellular localization and proapoptotic function of Nur77 in T cells. Both positive and negative selection signals can activate ERK1/2 cascade in DP thymocytes. However, only negative selection signals trigger transiently robust activation of ERK1/2 (15), which rapidly induces the translocation of ERK1/2 to the plasma membrane (41). Activated ERK1/2 may phosphorylate and carry RSK to the plasma membrane, where RSK receives additional activation signals from PDK1 (42). Subsequently activated RSK can translocate into the nucleus to phosphorylate Nur77 (43). Therefore modulation of Nur77 phosphorylation by MEK1/2-ERK1/2 cascade is very compatible with the intracellular behavior of ERK1/2 MAPK pathway components in T cells.
Although several studies have shown that RSK2 can phosphorylate Nur77 in vitro (24, 44, 45, 46), the present study is the first to show that RSK is involved in Nur77-mediated apoptosis in T cells. We used a specific RSK inhibitor to demonstrate Nur77 phosphorylation by RSK in vivo and linked RSK phosphorylation with Nur77 intracellular translocation during T cell apoptosis. In addition, we show that RSK2 can directly bind Nur77 in stimulated T cells. We mapped the phosphorylation site of Nur77 to serine 354, which is consistent with another recent study (24). The same site has been reported previously to be phosphorylated by AKT (39, 40). However, phosphorylation of Nur77 by AKT was demonstrated only in vitro or in 293T cells (39, 40). In addition, there is no cellular basis to support the notion that nuclear protein Nur77 in T cells can interact with AKT, a cytosolic protein (39). Importantly, the insensitivity of Nur77 phosphorylation to two inhibitors that block AKT activation in T cells (Fig. 2 C, this study) and the inability of recombinant active AKT to modify Nur7 in vitro demonstrated in another study (24), argue against the physiological role of AKT in modifying Nur77 in T cells. Instead, our data collectively demonstrate that Nur77 is a physiological substrate of RSK in T cells. There are four isoforms of RSK. Our data, however, do not identify which RSK isoform is specifically responsible for Nur77 phosphorylation in T cells. Future work is needed to address this issue.
Although previous studies using inhibitors or biochemical fractionation provided evidence of Nur77 mitochondrial translocation during T cell apoptosis, the in vivo physiological relevance of this translocation was not addressed. In this study, we present conclusive genetic evidence demonstrating that Nur77 is capable of directly triggering the mitochondrial apoptotic pathway. Using the Tet-off inducible expression system, we found that the delivery of Nur77 into the cytoplasm activates the intrinsic apoptotic pathways in DO11.10 cells (Fig. 1). This approach has several advantages. Firstly, it isolates Nur77 expression from other signaling events allowing us to specifically investigate Nur77-induced apoptosis in T cells. Secondly, it isolates Nur77 cytoplasmic killing activity from any nuclear proapoptotic functions. This gave us an opportunity to investigate whether targeting of Nur77 to the cytoplasm is sufficient to kill T cells in the absence of presumable Nur77 nuclear killing activity. Our data demonstrate, for the first time, that Nur77 translocates to mitochondria and this is sufficient to initiate the mitochondrial pathway during activation of apoptosis in DO11.10 T cells.
Apoptosis induced by the Nur77-triggered mitochondrial pathway proceeds very slowly compared with apoptosis induced by P+I treatment (Fig. 1 B). There are at least two explanations for this. Firstly, treatment of DO11.10 with P+I will activate both the mitochondrial apoptotic pathway (Nur77 and Bim-mediated) (47) and the death receptor pathway (FasL-mediated) (48). We found that Nur77 expression does not alter FasL expression (data not shown). Thus, it is possible that these two death pathways may synergize to kill TCR-stimulated DO11.10 cells. WT Nur77, which is retained in the nucleus in the absence of activation, can still kill DO11.10 cells although less efficiently than cytoplasmic-localized Nur77 (data not shown). This implies that Nur77 expressed in activated DO11.10 cells may possess both nuclear and mitochondrial killing activity. Secondly, Nur77 is a highly modified protein in activated DO11.10 cells. These posttranslational modifications, which are induced by P+I but absent in our inducible expression system, could modulate Nur77 proapoptotic function at the mitochondrial interface.
Finally, our work also indicates that effector mechanism of Nur77 to activate mitochondrial apoptotic pathway is different from that found in cancer cell lines. Seminal work from the Zhang laboratory demonstrates that in cancer cells Nur77 can bind and convert Bcl-2 from a cell protector into a killer to exert its proapoptotitic function at mitochondria (7, 28, 49). However, our data showed that in DO11.10 T cells, Bcl-2, and Bcl-XL preserved mitochondrial membrane potential and reduced the percentage of cells undergoing apoptosis induced specifically by mitochondrial-targeted Nur77 (Fig. 1, H and I). This is consistent with a recent finding in cutaneous T cell lymphoma whereby expression levels of Bcl-2 and Bcl-XL are highly correlated with resistance to Nur77-mediated cell death (50). Furthermore, we were unable to detect any physical interaction between Nur77 and Bcl2 or Bcl-XL in WT DO11.10 cell lines or DO11.10 overexpressing Nur77 (Figure S5 and S6). We argue that in DO11.10 T cells, Nur77 transduces the death signal at the mitochondria or in the cytosol independently of Bcl-2 family members. In the future, identifying Nur77-interacting proteins will provide insight to better understand the Nur77-mitochondrial pathway in T cells.
We thank Dr. Richard Hugnir at John Hopkins University School of Medicine for PRK5-myc-murine RSK2, Dr. Kunihiko Naito at University of Tokyo for mouse DN-MEK1-cDNA, Dr. Jing Chen at Emory University School of Medicine for MSCV2.2-IRES-GFP Gateway destination vector, and Dr. Simon Arthur at University of Dundee for pCMV5-Flag-Nur77. We thank Dr. John Blenis for many helpful conversations and Dr. Lisa Minter for critical review of the manuscript. Thanks are also given to Rebecca Lawlor and Kathie Curnick for their excellent technical support. We appreciate Dr. Dominique Alfandari’s generosity in allowing us to use his microscopy facility.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This study was supported by a grant from NIH AI049361 (to B.A.O.).
Abbreviations used in this paper: DP, double positive; RSK, ribosomal protein S6 kinase; PNS, postnuclear supernatant; P+I, PMA plus ionomycin; NES, nuclear export sequence; WT, wild type; FCCP, p-trifluoromethoxy carbonyl cyanide phenyl hydrazone; NWB, nuclear wash buffer; PARP, poly (ADP-ribose) polymerase; TMRE, tetramethylrhodamine, ethyl ester, perchlorate; MW, molecular weight.
The online version of this article contains supplemental material.