14-3-3 Is Involved in p75 Neurotrophin Receptor-mediated Signal Transduction*

Makoto T. KimuraDagger §, Shinji IrieDagger , Shisako Shoji-HoshinoDagger , Jun Mukai, Daita NadanoDagger , Mitsuo Oshimura§, and Taka-Aki Sato||

From the Dagger  Molecular Oncology Laboratory, Tsukuba Institute, RIKEN (Institute of Physical and Chemical Research), Ibaraki 305-0074, the § Department of Molecular and Cell Genetics, School of Life Science, Faculty of Medicine, Tottori University, Tottori 683-8503, Japan, and the  Division of Molecular Oncology, Department of Otolaryngology/Head and Neck Surgery and Pathology, College of Physicians and Surgeons, Columbia University, New York, New York 10032

Received for publication, June 22, 2000, and in revised form, February 20, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The low affinity neurotrophin receptor (p75NTR) has been shown to mediate the apoptosis signaling to neural cells. However, the specific mechanisms of intracellular signal transduction of this process are largely unknown. To understand p75NTR-mediated signal transduction, we previously identified a protein that interacts with the intracellular domain of p75NTR, and we named it p75NTR-associated cell death executor (NADE). To elucidate further the signaling mechanisms utilized by p75NTR and NADE, we screened for NADE-binding protein(s) with the yeast two-hybrid method, and we identified 14-3-3epsilon as a NADE-binding protein in vivo. To examine whether 14-3-3epsilon affects the induction of p75NTR-mediated apoptosis, wild type or various deletion mutant forms of 14-3-3epsilon were co-expressed in HEK293, PC12nnr5, and oligodendrocytes. Interestingly, transient expression of the mutant form of 14-3-3epsilon lacking the 208-255 amino acid region blocked nerve growth factor-dependent p75NTR/NADE-mediated apoptosis, although this mutant form of 14-3-3epsilon continued to associate with NADE. These results suggest that 14-3-3epsilon plays an important role in the modulation of nerve growth factor-dependent p75NTR/NADE-mediated apoptosis.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell growth, cell differentiation, and genetically controlled programmed cell death are required for development of the neural system and for plasticity in the adult nervous system of vertebrates. Abnormal cell growth, differentiation, or apoptosis results in teratogenesis or degeneration of the neural system. To understand neural system development and plasticity, many researchers have tried to identify the molecule(s) that regulate those cellular responses (1). Nerve growth factor (NGF)1 was first identified as a growth factor required for survival of specific neuronal cells during normal development (2). However, some reports have indicated that NGF has diverse effects on the nervous system, including differentiation and apoptosis (2, 3). To reveal the mechanisms by which NGF induces various cellular responses, such as cell growth, differentiation, or apoptosis, many researchers have studied the mechanisms of NGF signal transduction (4).

NGF recognizes at least two cell surface receptors, the high affinity tyrosine kinase receptor (TrkA) and the low affinity non-tyrosine kinase type receptor p75 neurotrophin receptor (p75NTR) (5-7). The TrkA contains a tyrosine kinase motif within its intracellular region. Binding of NGF to TrkA activates the kinase and subsequently induces phosphorylation of multiple substrates that lead to the activation of mitogen-activated protein (MAP) kinase and phosphatidylinositol 3-kinase (8, 9). The TrkA receptor initiates cell survival and differentiation signals in neuronal cells (10, 11). In contrast, the role of p75NTR was mostly discussed as that of an accessory receptor modulating the survival signals through the TrkA receptor (12, 13). However, recent evidence suggests that NGF/p75NTR signaling actually induce apoptosis in some types of neuronal cells. In embryonic chick retinal cells that express p75NTR but not TrkA, NGF causes the death of retinal neurons (14). Furthermore, NGF treatment induces apoptosis in terminally differentiated primary oligodendrocytes expressing p75NTR but not TrkA (15). These findings indicate that p75NTR is involved in NGF-induced cell death. There have been reports of NGF/p75NTR-mediated cellular responses including nuclear factor kappa B activation in Schwann cells and stress-activated protein kinase or c-Jun amino-terminal kinase activation in oligodendrocytes (16-18). The only known consensus motif within the intracellular domain of p75NTR is a death domain, similar to that found in the p55 tumor necrosis factor receptor and in Fas. However, the precise mechanisms of apoptosis induced by p75NTR have remained elusive (19).

To identify regulatory proteins that control p75NTR-mediated signaling pathway, several groups have performed molecular cloning of p75NTR-binding proteins, such as zinc finger proteins (SC-1 and NRIF), tumor necrosis factor receptor-associated factors, protein tyrosine phosphatase (Fas-associated phosphatase-1), and GTP-binding protein (RhoA) (20-25). However, the mechanisms of p75NTR-mediated signal transduction are still not fully understood. Recently, we identified a p75NTR-binding protein named p75NTR-associated cell death executor (NADE) (26). Another group (27) reported this gene as brain expressed X-linked gene 3 (BEX3) but its function remained unclear. As we reported previously, NADE consists of 124 amino acids and does not contain any known biochemical motifs other than the nuclear export signal (NES) sequence. NADE binds to the intracellular domain of p75NTR in an NGF-dependent manner. HEK293 cells co-expressing both NADE and p75NTR showed NGF-dependent apoptotic cell death, whereas cells expressing NADE alone did not. It should be noted that HEK293 cells do not express the TrkA receptor. In cells that underwent apoptosis, the apoptosis executor protease, caspase-3, was activated (26). Furthermore, we also observed pheochromocytoma PC12nnr5, which expresses p75NTR but not TrkA, undergo NGF-dependent apoptosis when NADE was transiently expressed (26). These results suggest that NADE is an essential protein for p75NTR-mediated apoptosis; however, the molecular mechanisms by which NADE regulates apoptosis are not fully clarified.

To understand better the function of NADE, we performed extensive yeast two-hybrid screenings to identify NADE-associated protein(s). We identified 14-3-3epsilon as a candidate molecule that binds to NADE. 14-3-3 proteins were originally isolated as highly abundant acidic proteins in brain extracts (28). 14-3-3 proteins associate with a number of signaling molecules and are thought to play important roles in signal transduction pathways involved in cell cycle regulation and the induction of apoptotic cell death. Here, we show that 14-3-3epsilon binds to NADE and that protein complexes consisting of p75NTR, NADE, and 14-3-3epsilon are formed in mammalian cells. Furthermore, the mutant form of 14-3-3epsilon encoding 1-207 amino acids was found to suppress both caspase-3 activation and NGF-dependent-p75NTR/NADE-mediated apoptosis in HEK293, PC12nnr5, and oligodendrocytes. Taken together, these data suggest that 14-3-3epsilon is involved in the regulation of caspase-3 activity and in p75NTR/NADE-mediated apoptosis.

    MATERIALS AND METHODS
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ABSTRACT
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MATERIALS AND METHODS
RESULTS
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Yeast Two-hybrid Analysis-- Analysis of protein-protein interactions by yeast two-hybrid system was performed essentially as described by Vojtek et al. (29). The cDNA encoding full-length NADE was subcloned into pBTM116 (pBTM116-NADE), and the sequence was confirmed using an Applied Biosystems model 310 automated DNA sequencer. pBTM116-NADE was then transformed into the L40 yeast strain, and the yeast cells were propagated with appropriate selection. The expression of the fusion protein (LexA-NADE) was determined in protein extract by Western blotting with both an anti-LexA antibody (Santa Cruz Biotechnology) and an anti-NADE antibody (26). The L40 yeast cells containing pBTM116-NADE were transformed with a murine day 9.5 embryonic cDNA library in pVP16 (kindly provided by Dr. Stanley M. Hollenberg). Histidine prototrophy was determined on plates containing 5 mM 3-aminotriazole to screen for proteins that bind to NADE. beta -Galactosidase activity was utilized as a secondary screen. Clones that were positive in both interaction tests were sequenced, and their nucleotide sequences were subjected to a BLAST search.

Cell Culture and Transfection Procedures-- HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (Cell Culture Technologies) and cultured at 37 °C in 5% CO2. 1.0 × 106 HEK293 cells in 100-mm tissue culture dishes were transfected with 20 µg of total plasmid DNA using the calcium phosphate method as described previously (30). PC12nnr5 cells were maintained in RPMI 1640 medium (Sigma) supplemented with 5% fetal bovine serum (Cell Culture Technologies) and with 10% horse serum (JRH Biosciences) and were cultured at 37 °C in 10% CO2. 2.5 × 105 PC12nnr5 cells in 35-mm collagen-coated tissue culture dishes were transfected with 2 µg of total plasmid DNA using Effectene Transfection Reagent (Qiagen). Primary cortical cultures of oligodendrocytes were obtained from post-natal (P1-2) Wister rat and were kept in M15 media (DMEM containing 15% fetal bovine serum, 6 mg/ml glucose, 100 units/ml penicillin, and 100 mg/ml streptomycin) for 7 days. After shaking, precursor cells were plated on poly-D-lysine-coated dishes with M15 medium at 37 °C in 5% CO2 for 15 days. Then the cells were cultured in differentiation medium (DMEM supplemented with 6 mg/ml glucose, 100 units/ml penicillin, 100 mg/ml streptomycin, 25 mg/ml insulin, 30 ng/ml sodium selenite, 100 mg/ml transferrin, 20 nM progesterone, 60 mM putrescine, 50 mM thyroxine, and 20 mg/ml triiodothyronine) for 7 days. Those differentiated oligodendrocytes in 6-well plates were transiently transfected with 2 µg of total plasmid DNA using Effectene Transfection Reagent (Qiagen).

NGF Treatment-- HEK293 transfectants were cultured in growth medium for 24 h before any further treatments. 7 S NGF (Sigma) was then added at a final concentration of 100 ng/ml. During NGF treatment, transfected cells were grown in serum-free DMEM for 24 h. PC12nnr5 transfectants were cultured in growth medium for 24 h before any further treatments. 7 S NGF (Sigma) was then added at a final concentration of 100 ng/ml. During NGF treatment, transfected cells were grown in serum-free RPMI 1640 for 24 h. Oligodendrocytes transfected with each plasmid were cultured for 24 h and were treated with 7 S NGF (Sigma) at a final concentration of 100 ng/ml for 12 h.

Plasmid Constructs-- Murine 14-3-3epsilon cDNAs encoding the full-length, amino acid residues 1-207, 1-120, or 121-207 were subcloned into pCMV Tag2 (Stratagene). These FLAG epitope (MDYKDDDK amino acid sequence)-tagged constructs were then transfected into HEK293 or into PC12nnr5 for the binding assays and apoptosis assays and transfected into primary oligodendrocytes for apoptosis assays. cDNAs encoding either full-length amino acid residues 1-112, 1-80, 1-70 or 81-124 of murine NADE were subcloned into pcDNA3.1(-)myc-His vector (Invitrogen). The Myc epitope (EQKLISEEDL amino acid sequence)-tagged constructs were transfected into HEK293 cells for binding assays. For apoptosis assays, murine full-length NADE cDNA subcloned into pcDNA3 (Invitrogen) was transfected into HEK293 and PC12nnr5 cells. Human p75NTR subcloned into pcDNA3 (a gift from Dr. Moses V. Chao) was used for apoptosis assays. The coding regions of these constructs were fully sequenced and verified to be correct.

In Vitro Binding Assay-- To generate GST fusion proteins, the murine 14-3-3epsilon and NADE cDNAs were subcloned into pGEX5X-1 (Amersham Pharmacia Biotech). These GST fusion proteins, GST/14-3-3epsilon and GST/NADE, were then expressed in DH5alpha bacteria and purified onto glutathione-Sepharose beads using standard techniques (31). The beads containing immobilized fusion proteins were blocked with PBS containing 2% bovine serum albumin at 4 °C for 2 h and were washed with NETN buffer (0.5% Nonidet P-40, 1 mM EDTA, 20 mM Tris-HCl (pH 7.2), 150 mM NaCl, 1.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin and leupeptin). The beads were then incubated with cell lysates extracted from HEK293 transfectant expressing wild type 14-3-3epsilon , (1)-14-3-3epsilon , (1)-14-3-3epsilon , or (121)-14-3-3epsilon . GST/14-3-3epsilon beads were incubated with the cell lysates extracted from HEK293 transfectants expressing wild type NADE, (1)-NADE, (1-80)-NADE, (1-70)-NADE, or (81)-NADE. Lysates were prepared as described previously in NETN buffer (26). The incubations were carried out at 4 °C for 12-16 h, and the beads were washed three times with NETN buffer. Bound proteins were eluted from the beads by boiling in Laemmli sample buffer for 5 min and were subjected to SDS-polyacrylamide gel electrophoresis on gels containing 12.5% polyacrylamide. The proteins were then transferred to polyvinylidene difluoride membranes, and Western blotting analysis was performed.

Western Blotting Procedures-- Samples were diluted in Laemmli sample buffer, boiled for 5 min, subjected to SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. The membranes were incubated with 10% skim milk (Difco) at 25 °C for 1 h, were washed with PBS for 30 min, and then incubated with primary antibody. The primary antibodies used included anti-Myc 9E10 (Biomol) at 1:1000 in PBS, anti-FLAG M2 (Sigma) at 1:1000 in PBS, anti-14-3-3 (Santa Cruz Biotechnology) at 1:2000 in PBS, anti-p75NTR (Promega) at 1:10000 in PBS, and anti-NADE at 0.5 µg/ml in PBS. Immunoreactive bands were detected with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibodies (Bio-Rad) and visualized by using the enhanced chemiluminescence (ECL) procedure (Amersham Pharmacia Biotech).

Immunoprecipitation Procedures-- Cells were washed with ice-cold PBS and lysed in NETN buffer on ice for 20 min. Cell lysates were cleared by centrifugation at 15,000 rpm for 20 min at 4 °C, normalized for protein content, and subjected to immunoprecipitation. Lysates were incubated with anti-Myc (Biomol), anti-FLAG (Sigma), anti-14-3-3 (Santa Cruz Biotechnology), and anti-NADE antibodies (number 5), which were coupled to CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech), at 4 °C for 8 h. Anti-NADE polyclonal antibody (number 5) was raised against synthetic peptide including 112-124 amino acid residues (CHHDHHDEFCLMP) of murine NADE. As a negative control, pre-immune mouse or rabbit IgG coupled to CNBr-activated Sepharose 4B was used. Immunocomplexes were collected by centrifugation, washed with NETN buffer, and subjected to Western blotting, as described above.

Trypan Blue Staining-- At selected time points after NGF treatments, HEK293 cells were harvested and washed in PBS. Trypan blue (Sigma) was added to suspended cells at a concentration of 0.4% w/v. After 10 min, cells were transferred to a hemocytometer, and the number of dead (blue-stained) cells was determined using a light microscope.

Apoptosis Assays-- HEK293 or PC12nnr5 transfectants, which were treated with or without NGF, were harvested and used for TUNEL assay by MEBSTAIN Apoptosis Kit Direct (Medical and Biological Laboratories), according to the manufacturer's recommended conditions (32). After TUNEL assay, samples were analyzed on a FACScan system using the CELLQuest software (Becton Dickinson). For detection of apoptotic oligodendrocytes, NGF-treated oligodendrocyte transfectants were fixed with 4% paraformaldehyde at room temperature for 30 min, permeabilized with 0.1% sodium citrate containing 0.1% Triton X-100 for 2 min on ice, and stained with anti-FLAG monoclonal antibody (M2) (Sigma). After incubation with anti-FLAG antibody, samples were processed for TUNEL assay. Then, cells were incubated with Cy-5-conjugated anti-mouse IgG (Jackson ImmunoResearch). Stained cells were visualized by fluorescence microscopy. The numbers of TUNEL-positive or Cy-5-positive cells were counted.

Caspase Assays-- The activity of caspase-3 in the transfected cells were assessed with a CPP32/caspase-3 Fluorometric Protease Assay Kit (Medical and Biological Laboratories). Caspase-3 recognizes and cleaves the consensus peptide sequence DEVD. CPP32/caspase-3 Fluorometric Protease Assay is based on detection of cleaved substrate DEVD-AFC. DEVD-AFC emits a blue light (lambda max = 400 nm) and, upon cleavage of the substrate by caspase-3, free AFC emits a yellowish green fluorescence (lambda max = 494 nm). The fluorometer (Hitachi) was used to measure fluorescence values as a means to quantify caspase-3 activity.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Association of NADE with 14-3-3epsilon in Yeast and in Vitro-- A yeast expression library derived from 9.5-day embryonic cDNA (cDNAs were subcloned into pVP16) was screened for proteins that associate with NADE. The full-length NADE was subcloned into pBTM116 in frame with the DNA binding domain of LexA as a target. Expression of the NADE-LexA fusion protein in yeast L40 was confirmed by Western blotting using anti-LexA and anti-NADE antibody (data not shown). Histidine prototrophy and beta -galactosidase activity tests were used to select candidate proteins associated with NADE. An estimated 8.0 × 106 colonies were screened. One hundred positive clones were selected for sequencing, and the nucleotide sequences of these positive clones were then subjected to a BLAST search. Among these clones, six clones were found to encode a partial sequence of protein termed 14-3-3epsilon . These six positive clones contained the overlapping region encoding from Thr-91 to Leu-209 of 14-3-3epsilon . This overlapping region contains a motif recognized by other 14-3-3-binding proteins (Fig. 1).


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Fig. 1.   Location of 14-3-3epsilon and NADE binding region. A, schematic representation of the 14-3-3epsilon amino acid sequence. The NES is located at the carboxyl terminus and is indicated by a black box. All positive yeast clones that were selected by yeast two-hybrid screening containing the 14-3-3epsilon sequence shared the common region (amino acid residues 91-209). The thick line (amino acid residues 121-207) indicates the putative NADE binding domain. B, schematic representation of the NADE amino acid sequence. The NES is located at the carboxyl terminus and is indicated as a black box. A thin line indicates the p75NTR binding domain at amino acid residues 81-106 (26). The region indicated by a thick line contains the putative 14-3-3 binding domain (see the text). N and C indicate N terminus and C terminus, respectively.

To confirm the interaction of NADE with 14-3-3epsilon in vitro, GST pull-down assays were performed. Lysates from HEK293 cells expressing Myc-tagged NADE was incubated with GST/14-3-3epsilon or GST proteins conjugated with glutathione beads, as described under "Materials and Methods." The beads were then washed and subjected to Western blotting with an anti-Myc antibody. The lysates from HEK293 cells expressing Myc-tagged wild type NADE exhibited two immunoreactive bands, 22 and 44 kDa, on anti-Myc Western blotting (Fig. 2A). Both 22- and 44-kDa products bound to GST/14-3-3epsilon but not to GST alone (Fig. 2A). The wild type 14-3-3epsilon with a FLAG epitope tag was also transfected into HEK293 cells, and the resulting cell lysates were incubated with GST/NADE fusion proteins conjugated with glutathione beads. In these experiments, 14-3-3epsilon bound to GST/NADE fusion proteins but not to GST alone (Fig. 2B). These results further indicate that NADE interacts with 14-3-3epsilon in vitro.


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Fig. 2.   Interaction of 14-3-3epsilon with NADE. A, top panel, summary of results from GST pull-down assays. Wild type (wt) or various mutant deletion constructs of Myc-tagged NADE ((1-112), (1-80), (1-70), and (81)) were subjected to GST pull-down assay, using a 14-3-3epsilon GST fusion protein (GST/14-3-3epsilon ). Constructs that interacted with GST/14-3-3epsilon are indicated by +, and those that did not are indicated by -. Bottom panel, immunoreactive levels in the various samples against an anti-Myc antibody. 1st, 6th, and 11th lanes are from HEK293 cells expressing Myc tagged wild type NADE; 2nd, 7th, and 12th lanes are from cells expressing (1)-NADE; 3rd, 8th, and 13th lanes are from cells expressing (1-80)-NADE; and 4th, 9th, and 14th lanes are from cells expressing (1-70)-NADE; 5th, 10th, and 15th lanes are from cells expressing (81)-NADE. 1st to 5th lanes, immunoreactivity of total cell lysates against an anti-Myc antibody. 6th to 10th lanes, immunoreactivity in pull-down complexes incubated with GST/14-3-3epsilon against an anti-Myc antibody. 11th to 15th lanes, immunoreactivity in pull-down complexes incubated with GST alone. B, top panel, summary of results from GST pull-down assays. Wild type or three mutant deletion constructs of FLAG-tagged 14-3-3epsilon ((1-207), (1), and (121)) were subjected to GST pull-down assay, using a NADE GST fusion protein (GST/NADE). Constructs that interacted with GST/NADE are indicated by +, and those that did not are indicated by -. Bottom panel, immunoreactive levels in the various samples against an anti-FLAG antibody. 1st, 5th, and 9th lanes are from HEK293 cells expressing FLAG-tagged wild type 14-3-3epsilon ; 2nd, 6th, and 10th lanes are from cells expressing (1)-14-3-3epsilon ; and 3rd, 7th, and 11th lanes are from cells expressing (1)-14-3-3epsilon ; 4th, 8th, and 12th lanes are from cells expressing (121)-14-3-3epsilon 1st to 4th lanes, immunoreactivity of total cell lysates against an anti-FLAG antibody. 5th to 8th lanes, immunoreactivity in pull-down complexes incubated with GST/NADE against an anti-FLAG antibody. 9th to 12th lanes, immunoreactivity in pull-down complexes incubated with GST alone.

A previous study showed that 14-3-3epsilon binds to phosphorylated serine residues within the consensus amino acid sequence RSXpSXP (where X is any amino acid and pS is phosphorylated serine residue). NADE does not contain this motif, although the motif has been shown to be present in many proteins bound to 14-3-3epsilon . To map the region within NADE required for interaction with 14-3-3epsilon , we utilized GST pull-down assays. Myc-tagged NADE deletion mutants encoding amino acid residues 1-112, 1-80, 1-70, or 81-124 were transfected into HEK293 cells, and the resulting cell lysates were incubated with GST/14-3-3epsilon fusion proteins conjugated with glutathione beads. The incubated beads were then washed and subjected to Western blotting with anti-Myc antibody. The results showed that NADE mutants encoding (1)-NADE and (81)-NADE bound to GST/14-3-3epsilon but not to GST alone. However, NADE deletion mutants (1-70)-NADE and (1-80)-NADE bound neither to GST/14-3-3epsilon nor to GST alone (Fig. 2A). The lysates from HEK293 cells expressing (1)-NADE exhibited two immunoreactive bands estimated at 20 and 40 kDa on anti-Myc Western blotting. However, the lysates from (1-70)-NADE and (1-80)-NADE exhibited only one immunoreactive band estimated to be the same as their putative molecular weight (Fig. 2A). To map further the region within the 14-3-3epsilon required for interaction with NADE, we conducted a GST pull-down assay. FLAG-tagged deletion mutant forms of 14-3-3epsilon encoding amino acid residues 1-120, 1-207, or 121-207 were transfected into HEK293 cells, and the resulting cell lysates were incubated with GST/NADE fusion proteins conjugated with glutathione beads. The results showed that (1)-14-3-3epsilon and (121)-14-3-3epsilon bound to NADE/GST but not GST alone. However, 14-3-3epsilon deletion mutant (1)-14-3-3epsilon did not bind to NADE/GST (Fig. 2B). The regions necessary for both bindings are summarized in Fig. 1.

NADE/14-3-3epsilon Complexes Were Detected in Mammalian Cells-- To confirm the association of NADE with 14-3-3epsilon in vivo, both Myc-tagged NADE and FLAG-tagged 14-3-3epsilon were transiently transfected into HEK293 cells. The resulting cell lysates were subjected to immunoprecipitation with either CNBr-activated Sepharose 4B-conjugated anti-FLAG antibody, anti-Myc antibody, or murine IgG. We confirmed the association of 14-3-3epsilon with NADE in vivo by Western blotting with an anti-Myc antibody (Fig. 3A, left). The same immunoprecipitated samples were also subjected to Western blotting with an anti-FLAG antibody. These experiments also clearly showed that NADE associates with 14-3-3epsilon in vivo (Fig. 3A, right).


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Fig. 3.   Interaction of 14-3-3epsilon with the NADE/p75NTR complex in vivo. A, HEK293 cells were co-transfected with Myc-tagged NADE and FLAG-tagged 14-3-3epsilon . Cells were lysed, and the resulting lysates were used for immunoprecipitation (IP) experiments with an anti-FLAG, anti-Myc, or mouse IgG. The immune complexes were collected with either CNBr-activated Sepharose 4B-conjugated anti-Myc antibody, anti-FLAG antibody, or murine IgG. The immune complexes were washed and subjected to Western blotting analysis with anti-Myc (left panel) and anti-FLAG (right panel) antibodies. B, PC12nnr5 cells were lysed, and the resulting lysates were used for immunoprecipitation experiments with an anti-14-3-3epsilon , anti-NADE, or mouse IgG. The immune complexes were collected with either CNBr-activated Sepharose 4B-conjugated anti-Myc antibody, anti-FLAG antibody, or murine IgG. The immune complexes were washed and subjected to Western blotting analysis with anti-NADE (left panel) and anti-14-3-3 (right panel) antibodies. C, HEK293 cells were transfected with plasmids expressing either p75NTR, Myc-tagged NADE, or FLAG-tagged 14-3-3epsilon as indicated. The transfectants were cultured in the presence of 100 ng/ml NGF for 12 h, and the resulting lysates were subjected to immunoprecipitation with the indicated antibodies. An anti-p75NTR antibody (Promega) was used to detect by Western blotting.

In addition, co-immunoprecipitation assays under more physiological conditions were performed using the cell lysate of pheochromocytoma PC12nnr5. Cell lysates of PC12nnr5 cells were subjected to immunoprecipitation with either CNBr-activated Sepharose 4B-conjugated anti-14-3-3 polyclonal antibody, anti-NADE polyclonal antibody (number 5), or rabbit IgG. We confirmed the association of 14-3-3epsilon with NADE in PC12nnr5 cells by Western blotting with an anti-NADE antibody (Fig. 3B, left). We detected only 20-kDa NADE products in both immunoprecipitants with an anti-NADE antibody and with an anti-14-3-3 antibody. However, we did not detect any NADE products in immunoprecipitants with rabbit IgG. The same immunoprecipitated samples were subjected to Western blotting with an anti-14-3-3 antibody. The experiments clearly showed that NADE associates with 14-3-3epsilon also in vivo (Fig. 3B, right).

We previously reported that NADE interacts with p75NTR (26). To confirm that the protein complexes contain p75NTR, NADE, and 14-3-3epsilon , HEK293 cells were transiently transfected with p75NTR, Myc-tagged NADE, and FLAG-tagged 14-3-3epsilon (p75NTR/mycNADE/FLAG14-3-3epsilon //HEK293). The resulting lysates were subjected to immunoprecipitation with an anti-FLAG antibody, and the immune complexes were washed and analyzed by anti-p75NTR Western blotting. The results showed that exogenously expressed 14-3-3epsilon associates with p75NTR in p75NTR/mycNADE/FLAG14-3-3epsilon //HEK293 cells (Fig. 3C). To examine whether NADE is required for the association of p75NTR with 14-3-3epsilon , HEK293 cells were co-expressed with p75NTR and FLAG-tagged 14-3-3epsilon in the absence of NADE. FLAG-tagged 14-3-3epsilon was immunoprecipitated from lysates of p75NTR/FLAG14-3-3epsilon //HEK293 cells with an anti-FLAG antibody, and the resulting immune complexes were subjected to Western blotting with an anti-p75NTR antibody. In the absence of NADE, 14-3-3epsilon did not associate with p75NTR (Fig. 3C).

14-3-3epsilon Mutant Lacking a Carboxyl-terminal Region Inhibits p75/NADE-mediated Apoptosis-- Co-expression of NADE and p75NTR-induced apoptosis followed by caspase-3 activation in HEK293 cell (26). To examine the effect of 14-3-3epsilon protein on the p75NTR/NADE-mediated apoptosis, wild type or deletion mutant forms of 14-3-3epsilon were co-transfected into HEK293 cells expressing both p75NTR and NADE. At 24 h after transfection, cells were treated with 100 ng/ml NGF for 24 h. The transfectants were then harvested, and apoptotic cells were enumerated by trypan blue staining. The percentage of apoptotic cells was 45.2% in cells transfected with p75NTR and NADE (n = 7), 48.1% in cells transfected with p75NTR, NADE, and wild type 14-3-3epsilon (n = 5), and 46.9% in cells transfected with p75NTR, NADE, and (1)-14-3-3epsilon (n = 5) (Fig. 4A). In striking contrast, the percentage of apoptotic cells transfected with p75NTR, NADE, and (1)-14-3-3epsilon was only 11.6% (Fig. 4A). To study further the effect of 14-3-3epsilon protein on the physiological p75NTR-mediated apoptosis, these 14-3-3epsilon constructs were transfected into PC12nnr5 cells with NADE. At 24 h after transfection, cells were treated with 100 ng/ml NGF for 24 h. The percentage of apoptotic cells was 32.9% in cells transfected with NADE (n = 5), 31.9% in cells transfected with NADE and wild type 14-3-3epsilon (n = 5), and 31.3% in cells transfected with NADE and (1)-14-3-3epsilon (n = 5) (Fig. 4B). In contrast, the percentage of apoptotic cells transfected with NADE and (1)-14-3-3epsilon was 18.9% (Fig. 4B).


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Fig. 4.   Mutant forms of 14-3-3epsilon differentially affect p75NTR/NADE-mediated apoptosis. Various expression constructs, as indicated, were transfected into HEK293 cells (A) and into PC12nnr5 cells (B). The transfectants were cultured in the presence (black bars) or absence (open bars) of 100 ng/ml NGF for 24 h. A minimum of 100 cells was analyzed by the trypan blue exclusion method. Data shown indicates average percentage of apoptotic cells ± S.E. C, immunoreactivity levels of p75NTR, NADE, and wild type or mutant forms of 14-3-3epsilon in HEK293 cells transfected with indicated plasmids. Cleared whole cell lysates were subjected to Western blotting with anti-p75NTR (top), anti-NADE (middle), or anti-FLAG (bottom) antibodies, as described under "Materials and Methods."

To investigate whether differences in the rate of apoptosis were caused by different expression levels of p75NTR, NADE, and type or deletion mutant forms of FLAG-tagged 14-3-3epsilon in transfectants, Western blotting analyses were performed. The expression levels of these proteins were relatively equal across transfectants (Fig. 4C). Furthermore, subcellular localizations of these proteins were examined using fluorescence microscopy. Both NADE and 14-3-3 contain the nuclear exporting signal (NES) sequence that is necessary for mediating nuclear export of large carrier proteins (33). Both wild type 14-3-3epsilon and the mutant (1)-14-3-3epsilon , which lacks a NES motif, were localized in the cytoplasm of p75NTR/NADE/wt14-3-3epsilon //HEK293 cells or p75NTR/NADE/(1-207)-14-3-3epsilon //HEK293 cells (data not shown). NADE was also found to be localized in the cytoplasm of these cells (data not shown). These results suggest that inhibition of cell death by (1)-14-3-3epsilon is not due to changes in the subcellular localization of NADE and 14-3-3epsilon proteins.

We have reported previously (26) that NGF induces 75NTR/NADE-mediated apoptosis with DNA fragmentation. To examine whether (1)-14-3-3epsilon could block DNA fragmentation, TUNEL assays (31) followed by flow cytometry were performed on the transfectants (Fig. 5A). Representative histogram data of HEK293 transfectants are shown. The percentage of TUNEL positives in HEK293 cells transfected with p75NTR and NADE was 39.2% (n = 4), 40.8% in cells transfected with p75NTR, NADE, and wild type 14-3-3epsilon (n = 4), and 43.3% in cells transfected with p75NTR, NADE, and (1)-14-3-3epsilon (n = 4) (Fig. 5B). Again, in contrast to the other constructs, only 15.5% of the cells transfected with p75NTR, NADE, and (1)-14-3-3epsilon were positive in the TUNEL assays (n = 4) (Fig. 5B).


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Fig. 5.   Mutation of 14-3-3epsilon affects p75/NADE-mediated apoptosis. A, HEK293 cells were transfected with various expression plasmids as indicated and cultured in the presence of 100 ng/ml NGF for 24 h. The percentages of apoptotic cells were determined by the TUNEL assay. Representative histograms from these transfectants are shown. Dotted lines represent the data from cells transfected with vector, and black lines represent cells transfected with the indicated constructs. B, the percentages of TUNEL-positive cells in response to 100 ng/ml NGF for 24 h are shown. Data are expressed as average ± S.E. from four separate experiments.

We reported previously (26) that NGF-dependent p75NTR/NADE-mediated apoptosis induces caspase-3 activation. To examine caspase-3 activities in each transfectant, a CPP32/caspase-3 fluorometric protease assay was performed. Caspase-3 activity was evaluated in the various transfected cell lines, as compared with the activity of control vector transfected cell. As shown in Fig. 6A, the caspase-3 activity ratio of the p75NTR/NADE//HEK293, p75NTR/NADE/wt14-3-3epsilon //HEK293, and p75NTR/NADE/ (1)-14-3-3epsilon //HEK293 cell lines was increased by 2.1-2.4-fold, as compared with control vector//HEK293. However, the caspase-3 activity ratio of p75NTR/NADE/(1-207)-14-3-3epsilon //HEK293 was increased by only 1.2-fold (Fig. 6A). In addition, caspase-3 activity in PC12nnr5-transfected cell lines was also examined. The caspase-3 activity ratio of the p75NTR/NADE//PC12nnr5, p75NTR/NADE/wt14-3-3epsilon //PC12nnr5, and p75NTR/NADE/(1-120)-14-3-3epsilon //PC12nnr5 was increased by 2.3-2.7-fold, as compared with control vector//PC12nnr5 (Fig. 6B). However, the caspase-3 activity ratio of p75NTR/NADE/(1-207)-14-3-3epsilon //PC12nnr5 was increased by 1.5-fold (Fig. 6B). These data suggest that expression of mutant (1)-14-3-3epsilon attenuates apoptosis.


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Fig. 6.   Mutant forms of 14-3-3epsilon differentially affect caspase-3 activity. Caspase-3 activity in cells was evaluated with a CPP32/caspase-3 fluorometric protease kit (Medical and Biological Laboratories). These representative histograms show the relative activity of caspase-3 in the cells expressing the indicated constructs. After treatment with 100 ng/ml NGF for 24 h, activity of caspase-3 in cells expressing indicated constructs was determined and expressed as a ratio of each transfectant with vector transfectant. Data are expressed as average ± S.E., from three separate experiments. The data from HEK293 and PC12nnr5 transfectants are shown in A and B, respectively.

To examine whether (1)-14-3-3epsilon inhibits NGF-induced apoptosis under more physiological conditions, FLAG-tagged wild type or mutant forms of 14-3-3epsilon were expressed in primary oligodendrocytes from post-natal (P1-2) Wistar rats. The numbers of TUNEL-positive and of FLAG-tagged fusion proteins expressing oligodendrocytes were counted. TUNEL-positive oligodendrocytes showed green signals and transfectants expressing FLAG-tagged protein showed red signals (Fig. 7A). One hundred oligodendrocytes that expressed FLAG-tagged protein were examined per each experiment, and we performed four independent experiments. The percentages of TUNEL-positive cells were 69.4% (n = 4) in wild type 14-3-3epsilon transfectants, 43.2% in (1)-14-3-3epsilon transfectants (n = 4), 74.8% in (1)-14-3-3epsilon transfectants (n = 4), and 72.1% in vector (without insert) transfectants (n = 4) (Fig. 7B). These results showed that (1)-14-3-3epsilon also inhibits NGF-induced apoptosis in oligodendrocyte.


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Fig. 7.   (1-207)-14-3-3epsilon inhibits NGF-induced apoptosis in primary oligodendrocytes. FLAG-tagged wild type 14-3-3epsilon and indicated mutant forms of 14-3-3epsilon were transfected into oligodendrocytes, and those transfectants were treated with 100 ng/ml NGF for 12 h. A, immunostaining for FLAG-tagged protein and TUNEL assay were performed. Red signals indicate FLAG-tagged protein-positive and green signals indicate TUNEL-positive. B, percentage of TUNEL-positive transfectants expressing indicated constructs are represented.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The goal of our research was to identify the molecules involved in NGF-induced apoptosis mediated by p75NTR and to characterize the functions of these proteins in the signal transduction pathway. Previously, we have identified (26) a p75NTR-associated protein called NADE, which is essential for NGF-induced apoptosis through p75NTR. In this report, we found that 14-3-3epsilon associates with NADE both in vitro and in vivo. To clarify whether 14-3-3epsilon is a key molecule in this signal transduction, we investigated the effect of 14-3-3epsilon on the induction of apoptosis.

To examine whether 14-3-3epsilon directly interacts with NADE in vitro, a GST pull-down assay was performed. The results indicated that NADE directly binds to 14-3-3epsilon in vitro (Fig. 2, A and B). Although most 14-3-3-binding proteins contain the 14-3-3-binding consensus motif, RSXpSXP, NADE does not. ADP-ribosyltransferase, exoenzyme S (ExoS) from Pseudomonas aeruginosa, also binds to 14-3-3 in the absence of this consensus motif (34). However, there is no similarity in amino acid sequence between NADE and ExoS. To map the region required for interaction of NADE with 14-3-3epsilon , GST pull-down assays were performed using the lysates of HEK293 cells transfected with wild type and deletion mutant forms of NADE. The results clearly showed that wild type NADE and (1)-NADE interacted with 14-3-3epsilon but (1-90)- and (1-70)-NADE did not (Fig. 2A), suggesting that amino acids 90-112 of NADE are necessary for the interaction of NADE with 14-3-3epsilon .

Two immunoreactive bands, a monomer of 22 kDa and a dimer of 44 kDa, were contained in the cell lysate of HEK293 transfected with Myc-tagged NADE on Western blotting with an anti-Myc antibody (Fig. 2A). These two immunoreactive bands were exhibited also in cell lysates that contain NADE without tag, by Western blotting with an anti-NADE antibody. The molecular size of the smaller immunoreactive band, estimated at 22 kDa by Western blotting, seems to be slightly larger than the molecular weight predicted from its nucleotide sequence of Myc-tagged NADE. This difference might be caused by the low pI value (pI = 5.9) or post-translational modification of NADE. Wild type and (1)-NADE exhibited both two immunoreactive bands. However, (1-80)- and (1-70)-NADE showed only the lower (monomer) band (Fig. 2A). These findings indicate that amino acids 90-112 are required for dimerization of NADE protein. To confirm this, the NADE point mutant (Cys102-Ser/NADE) was expressed in HEK293 cells. Expression of Cys102-Ser/NADE resulted in only the 22-kDa immunoreactive band on anti-Myc Western blotting (data not shown). This result confirmed that NADE is homodimerized by a disulfide bound at Cys102, resulting in the 44-kDa band. This dimerization form could not be separated by exposure to chelating reagents (data not shown). These findings imply that a tightly dimerized form of NADE may be more efficient for association with 14-3-3epsilon .

To map the region of 14-3-3epsilon protein required for NADE binding, wild type and deletion mutant forms of 14-3-3epsilon tagged with the FLAG epitope were expressed in HEK293 cells, and cell lysates were subjected to in vitro binding assay. These experiments suggested that amino acids within 121-207 in 14-3-3epsilon are required for the binding to NADE (Fig. 2B). This region has been also found to be required for the binding to other 14-3-3-interacting proteins such as Raf-1 (35, 36).

We showed that NADE associated with 14-3-3epsilon in HEK293 cells that exogenously express NADE and 14-3-3epsilon (Fig. 3A). Since NADE directly interacts with p75NTR, we hypothesized that NADE acts an adaptor protein to bridge p75NTR with 14-3-3epsilon . To test for the existence of this putative signaling protein complex, HEK293 cells were transfected with p75NTR, NADE, and a wild type 14-3-3epsilon and stimulated with NGF. The resulting cell lysates were used for various immunoprecipitation experiments. Complexes containing p75NTR, NADE, and 14-3-3epsilon proteins were co-immunoprecipitated. The bands detected by anti-p75NTR antibody were same patterns as reported previously. However, in the absence of NADE, the protein complex p75NTR/NADE/14-3-3epsilon was not detected (Fig. 3C).

We also examined association of endogenously expressed NADE with 14-3-3epsilon in PC12nnr5 cells (Fig. 3B). On anti-NADE Western blotting, we detected only a monomer of 20-kDa NADE in immunoprecipitated samples with either an anti-NADE antibody or with an anti-14-3-3 antibody (Fig. 3B, left). This result might be explained by degradation of native NADE protein or by difference of subcellular localization between a dimer NADE and a monomer NADE under physiological conditions. In fact, native NADE protein in cell lysates can be degraded rapidly in the absence of proteasome inhibitors (26), and a dimer form of native NADE can be separated from monomer NADE by centrifugation at 100,000 × g (data not shown). In immunoprecipitation experiment using PC12nnr5 cells (Fig. 3B), we detected two immunoreactive bands (Fig. 3B, right). This result might be explained because NADE associates with other 14-3-3 isoforms in addition to 14-3-3epsilon , and because we used antibody that recognizes all murine 14-3-3 proteins in these Western blottings. Interestingly, other 14-3-3 isoforms were also isolated by our initial yeast two-hybrid screening (data not shown). Although further biochemical studies on native NADE/14-3-3epsilon interaction will be required to clarify these questions, our results suggested that NADE is a putative adapter protein that recruits 14-3-3epsilon to p75NTR in vivo, and these complex formations are required for signal transduction in apoptosis induced by NGF.

To examine the effects of 14-3-3epsilon on NGF-induced apoptosis, wild type 14-3-3epsilon , the (1)-14-3-3epsilon mutant, or the (1)-14-3-3epsilon mutant was co-transfected with both p75NTR and NADE into HEK293 cells. We found that co-expression of p75NTR, NADE, and (1)-14-3-3epsilon inhibited NGF-dependent apoptosis (by 75%), as compared with HEK293 cells co-expressing p75NTR, NADE, and wild type 14-3-3epsilon . In contrast, the percentage of apoptotic cells singly transfected with wild type 14-3-3epsilon , (1)-14-3-3epsilon , and (1)-14-3-3epsilon was 10-20% (data not shown), and there was no significant difference in the percentage among these three single transfectants. In addition, wild type 14-3-3epsilon , (1)-14-3-3epsilon mutant, or (1)-14-3-3epsilon mutant was also co-transfected with NADE into PC12nnr5 cells to examine the effect of (1)-14-3-3epsilon under more physiological conditions. PC12nnr5 cells endogenously express p75NTR but not TrkA (37). We found that co-expression of NADE and (1)-14-3-3epsilon inhibited NGF-dependent apoptosis (by 59%), as compared with PC12nnr5 cells co-expressing NADE and wild type 14-3-3epsilon . Furthermore, expression levels of p75NTR, NADE, and 14-3-3epsilon were relatively equal in each transfectant (data not shown). Therefore, this inhibitory effect was not due to differences in expression levels of p75NTR, NADE, or 14-3-3epsilon protein. Furthermore, subcellular localization of these proteins was similar in all transfectants. Hence, NADE, wild type 14-3-3epsilon mutant (1)-14-3-3epsilon , and mutant (1)-14-3-3epsilon were localized in the cytoplasm. These results suggest that inhibition of cell death by (1)-14-3-3epsilon is not due to changes in the subcellular localization of NADE and 14-3-3epsilon proteins. The wild type 14-3-3epsilon and (1)-14-3-3epsilon bound to NADE, but (1)-14-3-3epsilon did not. More important, only (1)-14-3-3epsilon had an inhibitory effect on p75NTR/NADE-mediated apoptosis. As reported previously, caspase-3 is activated in p75NTR/NADE-mediated apoptotic cells. Expression of (1)-14-3-3epsilon inhibit the activation of caspase-3 in HEK293 and PC12nnr5 cells, as shown in Fig. 6, A and B. These results suggest that 14-3-3epsilon regulates caspase-3 activity, and the carboxyl-terminal region (residues 208-255) of 14-3-3epsilon is related to regulation of caspase-3 activity.

NGF-induced apoptosis has been shown in primary cultured oligodendrocytes, and NADE is thought to relate to this signaling pathway (26). In this report, (1)-14-3-3epsilon also inhibited NGF-induced apoptosis in oligodendrocytes as shown in Fig. 7, A and B. From these results, we conclude that (1)-14-3-3epsilon has a dominant negative effect on p75/NADE-mediated apoptosis. Thus, the carboxyl-terminal region of 14-3-3epsilon , within amino acid residues 208-255, may contain a functional motif that regulates this apoptosis signal.

It has been reported that 14-3-3 regulates UVC irradiation-induced apoptosis mediated by p38 MAP kinase activation (38). We also examined effects of a specific inhibitor of p38 MAP kinase on p75NTR/NADE-mediated apoptosis. However, apoptosis was not blocked completely by treatment with the SB202190 (data not shown). Other signaling pathways may be involved in p75NTR/NADE-mediated apoptosis.

More than 30 proteins have been found to bind to 14-3-3, and the biological functions of 14-3-3 have been studied. 14-3-3 regulates GTPase Ras signaling in eye development of Drosophila (39, 40). 14-3-3 proteins associate with the cell cycle-regulating protein phosphatase Cdc25 and apoptosis-promoting protein BAD, and the mechanisms of the signal transduction have been reported (41-44). NADE may play a role such as protein complexes in these signal transduction pathways.

In conclusion, we showed that 14-3-3epsilon binds to NADE and forms signaling complexes consisting of p75NTR, NADE, and 14-3-3epsilon . A deletion mutant form of 14-3-3epsilon encoding amino acid residues 1-207 (i.e. lacking residues 208-255 at the carboxyl-terminal end) had a dominant negative effect on p75NTR/NADE-mediated apoptosis and blocked caspase-3 activation. Further study will be required for a better understanding of the specific mechanisms of p75NTR/NADE-mediated apoptosis. This study clearly demonstrated that 14-3-3epsilon is a key molecule in this signaling cascade.

    ACKNOWLEDGEMENTS

We thank Makiko Iwai and Lydia Reveron for excellent secretarial assistance. We are grateful to Mariko Nagayoshi and Setsuko Kobayashi for assistance with maintenance of cell lines.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant R01 GM55147 (to T. S.) and by the Ribosome Engineering Project (The Organized Research Combination) of the Japanese Science and Technology Agency.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Division of Molecular Oncology, Dept. of Otolaryngology/Head and Neck Surgery and Pathology, College of Physicians and Surgeons, Columbia University, 630 West 168th St., P&S 11-451, New York, NY 10032. Tel.: 212-305-1701; Fax: 212-305-1736; E-mail: ts174@columbia.edu.

Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M005453200

    ABBREVIATIONS

The abbreviations used are: NGF, nerve growth factor; p75NTR, p75 neurotrophin receptor; NADE, p75NTR-associated cell death executor; HEK293, human embryonic kidney derived 293; MAP kinase, mitogen-activated protein kinase; TRAF, tumor necrosis factor receptor-associated factor; FAP-1, Fas-associated phosphatase-1; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP-biotin end labeling; AFC, 7-amino-4-trifluoromethylcoumarin; NES, nuclear export signal.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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