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

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 elu- cidate 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 14-3-3 e as a NADE-binding protein in vivo . To examine whether 14-3-3 e affects the induction of p75NTR-mediated apoptosis, wild type or various deletion mutant forms of 14-3-3 e were co-expressed in HEK293, PC12nnr5, and oligodendrocytes. Interest-ingly, transient expression of the mutant form of 14-3-3 e lacking the 208–255 amino acid region blocked nerve growth factor-dependent p75NTR/NADE-mediated

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 14-3-3⑀ as a NADE-binding protein in vivo. To examine whether 14-3-3⑀ affects the induction of p75NTR-mediated apoptosis, wild type or various deletion mutant forms of 14-3-3⑀ were co-expressed in HEK293, PC12nnr5, and oligodendrocytes. Interestingly, transient expression of the mutant form of 14-3-3⑀ lacking the 208 -255 amino acid region blocked nerve growth factor-dependent p75NTR/NADE-mediated apoptosis, although this mutant form of 14-3-3⑀ continued to associate with NADE. These results suggest that 14-3-3⑀ plays an important role in the modulation of nerve growth factor-dependent p75NTR/NADE-mediated apoptosis.
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)(6)(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 mitogenactivated 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 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 14-3-3⑀ 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 14-3-3⑀ binds to NADE and that protein complexes consisting of p75NTR, NADE, and 14-3-3⑀ are formed in mammalian cells. Furthermore, the mutant form of 14-3-3⑀ 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 14-3-3⑀ is involved in the regulation of caspase-3 activity and in p75NTR/NADE-mediated apoptosis.

MATERIALS AND METHODS
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. ␤-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% CO 2 . 1.0 ϫ 10 6 HEK293 cells in 100-mm tissue culture dishes were transfected with 20 g 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% CO 2 . 2.5 ϫ 10 5 PC12nnr5 cells in 35-mm collagen-coated tissue culture dishes were transfected with 2 g 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% CO 2 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 g 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.
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 g/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 ( max ϭ 400 nm) and, upon cleavage of the substrate by caspase-3, free AFC emits a yellowish green fluorescence ( max ϭ 494 nm). The fluorometer (Hitachi) was used to measure fluorescence values as a means to quantify caspase-3 activity.

Association of NADE with 14-3-3⑀ 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 ␤-galactosidase activity tests were used to select candidate proteins associated with NADE. An estimated 8.0 ϫ 10 6 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 14-3-3⑀. These six positive clones contained the overlapping region encoding from Thr-91 to Leu-209 of 14-3-3⑀. This overlapping region contains a motif recognized by other 14-3-3-binding proteins (Fig. 1).
To confirm the interaction of NADE with 14-3-3⑀ in vitro, GST pull-down assays were performed. Lysates from HEK293 cells expressing Myc-tagged NADE was incubated with GST/ 14-3-3⑀ 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 Myctagged 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/14-3-3⑀ but not to GST alone ( Fig. 2A). The wild type 14-3-3⑀ 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, 14-3-3⑀ bound to GST/NADE fusion proteins but not to GST alone (Fig.  2B). These results further indicate that NADE interacts with 14-3-3⑀ in vitro.
A previous study showed that 14-3-3⑀ 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 14-3-3⑀. To map the region within NADE required for interaction with 14-3-3⑀, we utilized GST pull-down assays. Myctagged 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/ 14-3-3⑀ 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 (1-112)-NADE and (81-124)-NADE bound to GST/14-3-3⑀ but not to GST alone. However, NADE deletion mutants (1-70)-NADE and (1-80)-NADE bound neither to GST/14-3-3⑀ nor to GST alone ( Fig. 2A). The lysates from HEK293 cells expressing (1-112)-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 14-3-3⑀ required for interaction with NADE, we conducted a GST pull-down assay. FLAG-tagged deletion mutant forms of 14-3-3⑀ encoding amino FIG. 1. Location of 14-3-3⑀ and NADE binding region. A, schematic representation of the 14-3-3⑀ 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 14-3-3⑀ 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. 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 (1-207)-14-3-3⑀ and (121-207)-14-3-3⑀ bound to NADE/GST but not GST alone. However, 14-3-3⑀ deletion mutant (1-120)-14-3-3⑀ did not bind to NADE/ GST (Fig. 2B). The regions necessary for both bindings are summarized in Fig. 1.
NADE/14-3-3⑀ Complexes Were Detected in Mammalian Cells-To confirm the association of NADE with 14-3-3⑀ in vivo, both Myc-tagged NADE and FLAG-tagged 14-3-3⑀ 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 14-3-3⑀ 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 14-3-3⑀ in vivo (Fig. 3A, right).
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 14-3-3⑀ 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 14-3-3⑀ and the mutant (1-207)-14-3-3⑀, which lacks a NES motif, were localized in the cytoplasm of p75NTR/NADE/wt14-3-3⑀//HEK293 cells or p75NTR/NADE/(1-207)-14-3-3⑀//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 (1-207)-14-3-3⑀ is not due to changes in the subcellular localization of NADE and 14-3-3⑀ proteins.

DISCUSSION
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 14-3-3⑀ associates with NADE both in vitro and in vivo. To clarify whether 14-3-3⑀ is a key molecule in this signal transduction, we investigated the effect of 14-3-3⑀ on the induction of apoptosis.
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 ϭ 5.9) or post-translational modification of NADE. Wild type and (1-112)-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 (Cys 102 -Ser/NADE) was expressed in HEK293 cells. Expression of Cys 102 -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 Cys 102 , 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 14-3-3⑀.
To map the region of 14-3-3⑀ protein required for NADE binding, wild type and deletion mutant forms of 14-3-3⑀ 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 14-3-3⑀ 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 14-3-3⑀ in HEK293 cells that exogenously express NADE and 14-3-3⑀ (Fig. 3A). Since NADE directly interacts with p75NTR, we hypothesized that NADE acts an adaptor protein to bridge p75NTR with 14-3-3⑀. To test for the existence of this putative signaling protein complex, HEK293 cells were transfected with p75NTR, NADE, and a wild type 14-3-3⑀ and stimulated with NGF. The resulting cell lysates were used for various immunoprecipitation experiments. Complexes containing p75NTR, NADE, and 14-3-3⑀ 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/14-3-3⑀ was not detected (Fig. 3C).
We also examined association of endogenously expressed NADE with 14-3-3⑀ 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 ϫ 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 14-3-3⑀, 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/14-3-3⑀ interaction will be required to clarify these questions, our results suggested that NADE is a putative adapter protein that recruits 14-3-3⑀ to p75NTR in vivo, and these complex formations are required for signal transduction in apoptosis induced by NGF.
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 cycleregulating protein phosphatase Cdc25 and apoptosis-promoting protein BAD, and the mechanisms of the signal transduction have been reported (41)(42)(43)(44). NADE may play a role such as protein complexes in these signal transduction pathways.
In conclusion, we showed that 14-3-3⑀ binds to NADE and forms signaling complexes consisting of p75NTR, NADE, and 14-3-3⑀. A deletion mutant form of 14-3-3⑀ encoding amino acid residues 1-207 (i.e. lacking residues 208 -255 at the carboxylterminal 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 14-3-3⑀ is a key molecule in this signaling cascade.