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J. Biol. Chem., Vol. 277, Issue 16, 13973-13982, April 19, 2002

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Structure-Function Analysis of NADE

IDENTIFICATION OF REGIONS THAT MEDIATE NERVE GROWTH FACTOR-INDUCED APOPTOSIS^*

Jun Mukai, Shisako Shoji^§, Makoto T. Kimura^§, Shuichi Okubo, Hajime Sano^¶, Petro Suvanto, Yin Li, Shinji Irie^§, and Taka-Aki Sato

From the  Division of Molecular Oncology, Department of Otolaryngology/Head & Neck Surgery and Pathology, College of Physicians & Surgeons, Columbia University, New York, New York 10032 and the ^§ Molecular Oncology Laboratory, RIKEN (Institute of Physical and Chemical Research), Ibaraki 305-0074, Japan

Received for publication, September 16, 2001, and in revised form, December 20, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Nerve growth factor (NGF) can induce apoptosis in neural cells via activation of the low affinity neurotrophin receptor p75NTR. NADE (p75NTR-associated cell death executor) is a p75NTR-associated protein that mediates apoptosis in response to NGF by interacting with the death domain of p75NTR in 293T, PC12, and nnr5 cells (Mukai, J., Hachiya, T., Shoji-Hoshino, S., Kimura, M. T., Nadano, D., Suvanto, P., Hanaoka, T., Li, Y., Irie, S., Greene, L. A., and Sato, T. A. (2000) J. Biol. Chem. 275, 17566-17570). We performed extensive mutational analysis on NADE, to better characterize its structural and functional features. Truncation of a minimal region, including amino acid residues 41-71 of NADE, was found to be sufficient to induce apoptosis. The designated regulatory region includes the C-terminal amino acid residues (72-112) and is essential for NGF-dependent regulation of NADE-induced apoptosis. Furthermore, the mutants with amino acid substitutions in the leucine-rich nuclear export signal (NES) sequence (residues 90-100) abolished the export of NADE from the nucleus to the cytoplasm. Mutation of the NES also abolished self-association of NADE, its interaction with p75NTR, and NGF-dependent apoptosis. Expression of a fragment of NADE (amino acid residues 81-124) blocked NGF-induced apoptosis in oligodendrocytes, suggesting that this region has a dominant negative effect on NGF/p75NTR-induced apoptosis. These studies identify distinct regions of NADE that are involved in regulating specific functions involved in p75NTR signal transduction.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Many mammalian cells undergo apoptosis in response to various stimuli, including DNA damage, growth factor deprivation, and abnormal expression of oncogenes or tumor suppressor genes (1-3). Apoptosis induced by these various signals appears to be mediated by a common set of downstream elements that act as regulators and effectors of programmed cell death. Apoptosis also occurs during the course of normal development. In neural cells, neurotrophins have been shown to promote apoptosis during development (4). Nerve growth factor (NGF)¹ was first identified as a growth factor that is required for the survival of specific neuronal cells during normal development (5). However, more recently, it has been shown that NGF has other diverse effects on the nervous system, including differentiation and apoptosis (4-6).

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) (7-9). 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, phosphatidylinositol 3-kinase, and other intracellular signaling cascades (10, 11). The TrkA receptor promotes cell survival and initiates differentiation signals in neuronal cells (12, 13). In contrast, p75NTR, which is a member of the tumor necrosis factor (TNF) receptor superfamily, mediates neurotrophin-induced apoptosis (14). Several lines of evidence from various systems, including cultured cells as well as knockout and transgenic mice, suggest that p75NTR has a pro-apoptotic role (15-19). Furthermore, NGF induces apoptosis in terminally differentiated primary oligodendrocytes expressing p75NTR (20). These studies suggested that p75NTR is involved in NGF-induced apoptosis. 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 (p55TNFR) and in Fas. However, the molecular mechanisms by which p75NTR mediates pro-apoptotic signaling have not been well characterized.

Recently, members of the TNF receptor-associated factor (TRAF) family, FAP-1, SC-1, NRIF, NRAGE, and RhoA, have been reported to interact with the intracellular domain (ICD) of p75NTR (21-27). Of these, TRAF2, NRIF, and NRAGE have been reported to function in the p75NTR-mediated apoptotic pathway. Co-expression of TRAF2 with p75NTR enhanced cell death (22), whereas co-expression of TRAF6 was cytoprotective (21). NRIF is known to block cell division. The retinae of nrif ^/ mice show reduced cell death, and this reduction is quantitatively similar to that seen in p75^/ and ngf^/ mice (25). NRAGE binds with p75NTR both in vitro and in vivo and blocks the physical association of p75NTR with TrkA. NRAGE overexpression facilitates cell cycle arrest and permits NGF-dependent apoptosis within sympathetic neuron precursor cells (26). However, the mechanisms of p75NTR-mediated signal transduction are still not fully understood. Recently, we identified a novel protein that we termed p75NTR-associated cell death executor (NADE), which associated with the p75NTR(ICD) in a yeast two-hybrid screen (28). A data base search using the putative NADE amino acid sequence identified a homologous, partially characterized human cDNA clone termed HGR74/Bex3 (29). Co-expression of NADE and p75NTR induced cell death in 293T cells. NGF induced a dose-dependent association of NADE with the death domain of p75NTR, and p75NTR·NADE-induced cell death required NGF but not BDNF, NT-3, or NT-4/5. Similar results were also obtained in PC12 and PC12 nnr5 cells (28). After forebrain ischemia, p75NTR and NADE gene expression were induced in degenerating rat hippocampal CA1 neurons. NADE contributes to p75NTR-induced cortical neuronal death (30). Furthermore, 14-3-3 proteins associate with NADE and play a role in p75NTR·NADE-mediated apoptosis in HEK293 cells, PC12 nnr5 cells, and oligodendrocytes (31).

NADE has a nuclear export signal (NES) consensus motif, which is both necessary and sufficient to mediate nuclear export of large carrier proteins (32). Recent reports have demonstrated that the cellular localization of many proteins is tightly regulated by this motif, including that of HIV-Rev (33), PKI (34), and MAPKK (35). Thus, the NES-mediated intracellular transport system is a widely conserved mechanism that controls the subcellular localization of proteins in cells.

In this study, we have analyzed three issues relating to the functional and structural properties of mouse NADE. First, by mutational analysis, we have defined the subregions of NADE that are required for NGF-induced cell death. Second, we have shown that NADE contains a functional NES domain and that this sequence is responsible for self-association, interaction with p75NTR and induction of cell death. Finally, we have identified a dominant negative form of NADE, comprised of amino acids 81-124, which inhibited NGF-induced apoptosis in oligodendrocytes.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Constructs-- NADE (WT) (pcDNA3.1/Myc-His()A/mNADE WT) was constructed as described previously (28). Expression vectors for mouse NADE deletion mutants were constructed by PCR amplification of NADE coding sequences using oligonucleotide pairs as shown, digesting the resulting fragments with XhoI/BamHI, and ligating the resulting fragments into XhoI/BamHI-digested pcDNA3.1/Myc-His() A: for N-(1-120), FX29 (5'-ATCCTCGAGCGATCATGGCCAATGTCCAC-3') and RB360 (5'-ATCGGATCCGAATTCATCATGGTGATC-3'); for N-(1-112), FX29 and RB336 (5'-ATCGGATCCGTTAGACAGCTCCCCCAT-3'); for N-(1-100), FX29 and RB300 (5'-ATCGGATCCTCTCAGCTGTAGCTCCCT-3'); for N-(1-71), FX29 and RB213 (5'-ATCGGATCCGTCATTCATCTGCCTGTT-3'); for N-(1-20), FX29 and RB60 (5'-ATCGGATCCTTCCTGTCCATTCTGCAG-3'); for N-(41-124), FX121 (5'-ATCCTCGAGACCATGCACAACCATAACCACAAC-3') and RB27 (5'-ATCGGATCCAGGCATAAGGCAGAATTC-3'); for N-(81-124), FX241 (5'-ATCCTCGAGACCATGGAAATGTTCATGGAGGAG-3') and RB27; for N-(101-124), FX301 (5'-ATCCTCGAGACCATGAATTGTCTACGCATCCTT-3') and RB27; for N-(41-71), FX121 and RB213; for FLAG-tagged N(WT), FX29, and RBFLAG (5'-CGCGGATCCTCACTTATCGTCGTCATCCTTGTAATCAGGCATAAGGCAGAATTC-3').

Point mutants for NADE were constructed by PCR amplification of NADE coding sequences using oligonucleotide pairs as described below, followed by digesting the resulting PCR product with DpnI: for N(L99A) and GFP-N(L99A), in which Leu-99 is replaced with Ala, F-L99A (5'-AGGGAGCTACAGGCGAGAAATTGTCTA-3'), and R-L99A (5'-TAGACAATTTCTCGCCTGTAGCTCCCT-3'); for N(L94A/L97A/L99A) and GFP-N(L94A/L97A/L99A), in which Leu-94, Leu-97, and Leu-99 are replaced with Ala, F-L97A (5'-AAAGCTTAGGGAGGCACAGCTGAGAAA-3'), R-L97A (5'-TTTCTCAGCTGTGCCTCCCTAAGCTTT-3') and F-L97A/L99A (5'-AGGGAGGCACAGGCGAGAAATTGTCTA-3'), R-L97A/L99A (5'-TAGACAATTTCTCGCCTGTGCCTCCCT-3') and F-L94A/L97A (5'-ATCCGGAGAAAGGCTAGGGAGGCACA-3'), R-L94A/L97A (5'-TGTGCCTCCCTAGCCTTTCTCCGGAT-3').

Expression plasmids encoding fusion proteins of green fluorescence protein (GFP) and NADE proteins were constructed as follows: GFP cDNA was PCR-amplified from pEGFP-N2 (CLONTECH, Palo Alto, CA) by using the primer pair 5'-CTAGCTAGCATCATGGTGAGCAAGGGCGAG-3' and 5'-CCGCTCGAGTCTTGTACAGCTCGTCCAT-3'. The product was cloned into the NheI and XhoI sites of pcDNA3.1/Myc-His()A/mNADE. Expression plasmids for glutathione S-transferase (GST)-fused p75NTR proteins have been described previously (28).

pVP16/N(WT) and pVP16/N(L94A/L97A/L99A) were constructed by PCR amplification of N(WT) and N(L94A/L97A/L99A), respectively, using oligonucleotide pairs as shown, digesting the resulting fragments with BamHI/NotI, and ligating the fragments into BamHI/NotI-digested pVP16: forward primer (5'-GGGATCCTAATGGCCAATGTCCACCAGGAA-3') and reverse primer (5'-ATAGTTTAGCGGCCGCAATCAAGGCATAAGGCA-3'). pBTM116/N(WT) was constructed by PCR amplification of N(WT) using oligonucleotide pairs as shown, digesting the resulting fragments with BamHI/SalI, and ligating the fragments into BamHI/SalI-digested BTM116: forward primer (5'-GGGATCCTAATGGCCAATGTCCACCAGGAA-3') and reverse primer (5'-TGCGGTCGACGCTAAGGCATAAGGCAGAA-3'). pBTM116/p75DD was constructed by PCR amplification of pcDNA3/p75NTR using oligonucleotide pairs as shown, digesting the resulting fragments with EcoRI/SalI and ligating the fragments into EcoRI/SalI-digested BTM116: forward primer (5'-CGGAATTCAAGGGTGATGGCAACCTC-3') and reverse primer (5'-GCGTCGACTCAGTAACCCAGCTCGCCTGC-3').

Recombinant Adenovirus Construction-- Adenoviral vectors were constructed using the Adeno-X Expression system (CLONTECH) according to the protocol recommended by the manufacturer. pShuttle/Myc-GFP, pShuttle/Myc-N(WT), and pShuttle/Myc-N-(81-124) were constructed by digesting the pcDNA3.1/Myc-His()A/GFP, pcDNA3.1/Myc-His()A/N(WT), and pcDNA3.1/Myc-His()A/N-(81-124) with NheI/AflII, and ligating the resulting fragments into the NheI/AflII sites of the pShuttle vector. pAdeno-X/Myc-GFP, pAdeno-X/Myc-N(WT), and pAdeno-X/Myc-N-(81-124) were constructed by digesting pShuttle constructs with PI-SceI/I-CeuI and ligating the resulting fragments into the PI-SceI/I-CeuI sites of the pAdeno-X adenoviral vector. Recombinant adenoviral plasmids were packaged into infectious adenoviral particles by transfecting human embryonic kidney (HEK) 293 cells. The adenoviral particles were propagated in HEK293 cells and purified on cesium chloride gradients. Recombinant adenoviruses were screened for expression of the introduced genes by fluorescent microscopy and/or Western blot analysis.

Adenoviral-mediated Gene Transfer to Oligodendrocytes-- Adenoviral infection was performed on oligodendrocytes that were cultured for 7 days in Basal Medium Eagle (BME), which is described under "Cell Culture and Transfection." Cells were infected with 4-10 plaque-forming units/cell of adenovirus in BME. After incubation for 6 h at 37 °C, cultures were replaced with fresh BME for 18 h. For the apoptosis assay, infected oligodendrocytes were treated with 100 ng/ml NGF.

Reagents and Antibodies-- N-Acetyl-Leu-Leu-norleucinal (ALLN), NGF, leptomycin B (LMB), and the anti-FLAG monoclonal antibody were obtained from Sigma Chemical Co. (St. Louis, MO). Anti-rabbit IgG-Alexa Fluor 568, and anti-mouse IgG-Alexa Fluor 350 were obtained from Molecular Probes (Eugene, OR). The anti--NADE polyclonal antibody was prepared as described previously (28). The anti-O1 mouse monoclonal antibody was a kind gift from Dr. S. Pfeiffer. The anti-myc polyclonal antibody was obtained from MBL International Corp. (Watertown, MA). The anti-myc monoclonal antibody was obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA).

Cell Culture and Transfection-- 293T cells were obtained from the American Type Culture Collection; 293T cells were maintained in DMEM supplemented with 10% fetal bovine serum. For transfection, 293T cells (1.5 × 10⁶ per 100-mm dish) were transiently transfected with 25 µg of DNA using the calcium phosphate method in DMEM supplemented with 10% fetal bovine serum and cultured for 10 h. After withdrawing the serum, the cells were treated with 100 ng/ml NGF for 36 h. Oligodendrocytes were prepared as described previously (36). Mixed glial cultures were prepared from P2-rat cortex. After plating on poly-D-lysine-coated 75-cm² flasks, cultures were grown at 37 °C in a humidified incubator with 5% CO₂ for 7 days. Oligodendrocytic cultures were typically preplated and grown for 24 h in NM15 media (minimal essential medium supplemented with 15% FCS, 6 mg/ml glucose, 10 units/ml penicillin, and 10 mg/ml streptomycin). Cultures were then switched to oligodendrocyte differentiation media, containing BME:Ham's F-12 (1:1 v/v) supplemented with 6 mg/ml D-glucose, 100 units/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml transferrin, 25 µg/ml insulin, 20 nM progesterone, 60 µM putrescine, 30 nM selenium, 6.6 mM glutamine, and 0.5 µM thyroxine.

Subcellular Localization Analysis-- 293T cells were plated onto glass coverslips and transfected with GFP-containing constructs. At 24 h after transfection, cells were fixed with 3.7% paraformaldehyde, washed with phosphate-buffered saline (PBS), and stained with Hoechst 33342 to visualize the nucleus. The images of representative fields were captured on a fluorescence microscopy EFD-3 (Nikon) and recorded with a SPOT-RT digital charge-coupled device camera (Diagnostic Instruments).

Subcellular Fractionation-- Subcellular fractionation was performed as described previously (37). Transfected 293T cells were washed in PBS. Half of the cells were used for preparation of cytoplasmic and nuclear extracts, respectively. For preparation of cytoplasmic extracts, the cells were resuspended in 300 µl of buffer A (50 mM HEPES, pH 7.4, 50 mM KCl, 5 mM EDTA, 2 mM MgCl₂, 0.1 mM dithiothreitol, 20 µM leupeptin, 10 µg/ml pepstatin A, 1 mM PMSF, and 1 mM benzamidine), and allowed to swell by incubation for 10 min on ice. The cells were gently homogenized with a Dounce homogenizer. Small aliquots of the lysate were taken and stained with trypan blue to determine the progression of cell lysis. Homogenization was continued until more than 95% of the cells were broken. After centrifugation at 1500 × g, the resultant supernatant was carefully removed and supplemented with 60 µl of 6-fold concentrated standard reducing sample buffer. For preparation of nuclei, cells were incubated on ice for 10 min in buffer B (10 mM HEPES, pH 7.4, 10 mM KCl, 2 mM MgCl₂, 0.1 mM dithiothreitol, 20 µM leupeptin, 10 µg/ml pepstatin A, 1 mM PMSF, and 1 mM benzamidine) and homogenized as described for the preparation of cytoplasmic extracts. When more than 95% of the cells were lysed, the salt concentration was adjusted to 150 mM, and the suspension was layered over 20% (w/v) sucrose in buffer B and centrifuged at 800 × g. The nuclei were washed, resuspended in 300 µl of buffer B, and supplemented with 60 µl of 6-fold concentrated standard reducing sample buffer.

In Vitro Binding Assay-- In vitro translated [³⁵S]methionine-labeled proteins were generated with the TnT-coupled reticulocyte lysate system (Promega, Madison, WI). Binding assays were performed as described previously (28).

Co-immunoprecipitation and Western Blot-- The transfected 293T cells were lysed in a buffer containing 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.2% Nonidet P-40, 1 mM PMSF, 1 mM benzamidine, 50 µg/ml leupeptin, 7 µg/ml pepstatin A, and 25 µM ALLN). The lysates were then centrifuged at 15,000 × g for 15 min at 4 °C. Myc was then immunoprecipitated from the resulting supernatants with 0.8 µg of an anti-Myc monoclonal antibody coupled to CNBr-activated Sepharose 4B (Amersham Biosciences, Inc., Uppsala, Sweden). The immune complexes were then subjected to SDS-PAGE on 12.5% polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). The membrane was blocked for 5 h at room temperature in blocking buffer containing 10% skim milk and 0.1% NaN₃ in PBS. FLAG-tagged constructs were detected by Western blotting with an anti-FLAG monoclonal antibody and visualized with the ENHANCE chemiluminescence system (Amersham Biosciences, Inc.).

Immunocytochemistry-- The O1 mouse monoclonal antibody was used to identify oligodendrocytes. Cells were gently washed with PBS, and viable cells were incubated with the O1 antibody for 30 min at room temperature (1:100 dilution in Hanks' balanced salt solution buffer containing 3% bovine serum albumin and 3% fetal calf serum). For double-staining, cells were then fixed with 3.7% paraformaldehyde for 30 min at room temperature, permeabilized with 0.1% sodium citrate containing 0.1% Triton X-100 for 2 min on ice, and processed for TUNEL as described below. Cells were incubated with the anti-Myc polyclonal antibody in PBS containing 1% bovine serum albumin and 1% normal goat serum (Sigma) for 1 h at room temperature. After several rinses with PBS, cells were incubated with the anti-rabbit IgG-Alexa Fluor 568 and anti-mouse IgG-Alexa Fluor 350 at room temperature for 30 min. These antibodies were used at 1:100 dilutions in PBS supplemented with 0.5 M NaCl and 1% normal goat serum.

TUNEL Assay and DAPI Staining-- In situ detection of apoptotic oligodendrocytes was performed by using a TUNEL method (38). After incubation with the O1 antibody, oligodendrocytes were fixed and permeabilized, as described above. After several rinses, samples were processed for TUNEL using the in situ cell death detection assay according to the protocol suggested by the manufacturer (MBL International Corp.). In some experiments, Alexa 568-dUTP (Molecular Probes) was used instead of FITC·dUTP as a substrate for the TUNEL reaction. Double-stained cells were visualized by fluorescence microscopy EFD-3 (Nikon) and recorded with a SPOT-RT digital charge-coupled device camera (Diagnostic Instruments). Apoptotic cells were determined by counting the percentage of TUNEL-positive cells among O1-positive cells in four fields across the coverslip. At least 400 cells were counted for each condition.

The transfected 293T cells were washed with PBS, fixed in 3.7% paraformaldehyde, and stained with 50 µg/ml DAPI. A minimum of 400 cells was analyzed by fluorescence microscopy in each sample. Within this group, the number of cells that had the nuclear morphology typical of apoptosis was determined in each sample.

Yeast Two-hybrid Analysis-- Analysis of protein·protein interactions by yeast two-hybrid system was performed essentially as described by Vojtek et al. (39). The cDNAs encoding a full-length NADE and a death domain of p75NTR were subcloned into pBTM116 (pBTM116/N(WT) and pBTM116/p75DD). Each plasmid was then transformed into the L40 yeast strain (Mata trp1 leu2 his3 ade2 LYS2::(LexAop)₄-HIS3 URA::(LexAop)₈-LacZ), and the yeast cells were propagated with appropriate selection. The L40 yeast cells containing pBTM116/N(WT) or pBTM116/p75DD were transformed with pVP16/N(WT) or pVP16/N(L94A/L97A/L99A). Histidine prototrophy was determined on plates containing 5 mM 3-aminotriazole to detect the association of NADE or p75DD.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of NADE Deletion Mutants for Apoptosis-- To determine the structural elements of NADE that are required for apoptosis, we generated a series of NADE deletion mutants and point mutations of NES sequence, as shown in Fig. 1 (A and B). To analyze p75NTR·NADE-mediated apoptosis, we assessed nuclear morphology by DAPI staining. 293T cells were transiently transfected with pcDNA3.1/Myc-His/mNADE (N(WT)) or/and pcDNA3p75NTR, then incubated in the presence or absence of 100 ng/ml NGF. Cells co-transfected with N(WT) and p75NTR displayed the typical morphological characteristics of apoptosis, including nuclear condensation and fragmentation, in response to NGF treatment (Fig. 2A). In contrast, cells singly transfected with either N(WT) or p75NTR displayed a normal nuclear morphology, similar to that in cells transfected with the control vector alone. When apoptotic cells were scored based on morphological criteria, ~45% of 293T cells that were co-transfected with both N(WT) and p75NTR displayed apoptotic morphology in response to NGF (Fig. 2B). In contrast, only about 6% of cells transfected with the empty vector, N(WT) alone, or p75NTR alone exhibited apoptotic morphology.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Schematic representation of NADE mutants. A, deletion mutants of NADE. The domain structure of full-length mouse NADE (N(WT)) is shown at the top, and amino acid numbers are listed above. The nuclear export signal (NES) (90-100) and ubiquitin sequence (US) (91-112) domains are indicated by black and cross-hatching, respectively. B, point mutations in the NES of NADE. The NES sequences for wild-type and NES mutants are shown.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   NGF-dependent regulation of p75NTR·NADE-induced apoptosis in 293T cells. A, morphological analysis in 293T cells. Cells were transiently transfected with pcDNA3/rat-p75NTR and/or pcDNA3.1/Myc-His()A/N(WT) and cultured for 10 h. After withdrawing the serum, cells were treated in the presence (+) or absence () of 100 ng/ml NGF for 36 h. Cells were fixed with 3.7% paraformaldehyde and nuclear morphology was analyzed by DAPI staining, as described under "Experimental Procedures." B, overall percentage of apoptotic cells as determined by DAPI staining. Fluorescence microscopy was used to determine the number of cells with nuclear morphology typical of apoptosis from a total of at least 400 cells in each sample. The data are expressed as the average percentage of apoptotic cells ± S. D. from four separate experiments.

Various truncation mutants of NADE were co-transfected into 293T cells with or without p75NTR. The cells were then treated with NGF, and the percentage of apoptotic cells were evaluated. As shown in Fig. 3 and Table I, we found that two N-terminal deletion mutants, N-(81-124) and N-(101-124), failed to induce apoptosis, whereas deletion of the N-terminal 40 amino acids (N-(41-124)) did not affect apoptosis. The ability to induce apoptosis was retained in the C-terminal deletion mutant, N-(1-71) but not in N-(1-20), a shorter C-terminal deletion mutant. Furthermore, expression of a minimal region of NADE containing only residues 41-71 (N-(41-71)) was sufficient to induce apoptosis. In cells expressing wild-type NADE (N(WT)), the presence of p75NTR was required to induce apoptosis. In contrast, expression of N-(1-71), which lacks the p75NTR-binding domain (residues 81-106), retained its pro-apoptotic function even in the absence of p75NTR expression. Similar results were obtained with the N-(41-71) and N-(1-100) mutants, which failed to associate with p75NTR but still induced apoptosis. On the other hand, expression of N-(1-120), N-(1-112), and N-(41-124), each of which can associate with p75NTR, induced apoptosis in an NGF-dependent manner. Thus, the C-terminal amino acid residues (72-112), designated as the regulatory domain of NADE, are essential for NGF-dependent regulation of apoptosis.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Mapping analysis of NADE for apoptosis. The indicated constructs were transiently transfected into 293T cells with (open bars) or without (black bars) p75NTR, as described under "Experimental Procedures." Cells were fixed with 3.7% paraformaldehyde and nuclear morphology was analyzed by DAPI staining. The data are expressed as the average percentage of apoptotic cells among the total number of cells counted ± S.D. (n = 4).

View this table: [in this window] [in a new window]   Table I Summary of cell death assay and the association with p75NTR for NADE and its mutants In each cell death assay, + represents apoptotic cells that are scored more than 30% of 293T cells;  represents apoptotic cells that are scored more than 10% of 293T cells.

NADE NES Is Necessary for Self-association, Interaction with p75NTR, and Apoptosis-- In our previous report, we observed that the C-terminal residues of NADE between amino acids 90-100 conform to the consensus sequence for a functional NES motif, as indicated by the similarity of this region to other known NESs, shown in Fig. 4A. We demonstrated that wild-type NADE, which has an intact NES, was localized in the cytoplasm, but that the GFP-NES mutants (L94A and L97A) were retained in the nucleus (28). Because the NES is localized within the regulatory domain of NADE and partially includes the p75NTR-binding domain at residues 81-106, we hypothesized that the NES may play a role in the regulation of p75NTR·NADE-induced apoptosis. To more specifically analyze the function of this motif, we constructed two NADE point mutations. In N(L99A), a single leucine was mutated to alanine, and in N(L94A/L97A/L99A) three leucines were mutated to alanines at residues 94, 97, and 99 within the NES (Fig. 1B). Expression and subcellular distribution of GFP-NADE fusion proteins derived from these mutants were visualized in 293T cells by fluorescence microscopy. GFP-N(L94A/L97A/L99A) was observed both in the nucleus and in the cytoplasm (Fig. 4B), whereas wild-type NADE and N(L99A) were localized exclusively in the cytoplasm.

View larger version (35K): [in this window] [in a new window]   Fig. 4.   Effect of NADE NES function. A, NADE contains a conserved Rev-like NES motif. Mouse NADE is aligned with homologous NADE sequences from rat and human, and with the NES of PKI, HIV, Rev, MDM2 and MAPKK. B, subcellular localization of NES mutants in 293T cells. Cells were transfected with GFP-vector, GFP-N(WT), GFP-N(L99A), or GFP-N(L94A/L97A/L99A), as indicated. Hoechst 33342 was used to visualize the nucleus, and subcellular localization was determined as described under "Experimental Procedures." C, effect of leptomycin B (LMB). 293T cells transiently expressing GFP-N(WT) or GFP-N(L94A/L97A/L99A) were treated with or without 5 ng/ml LMB. After 3 or 12 h, cells were analyzed by fluorescent microscopy. D, subcellular distribution of NADE. 293T cells transiently expressing N(WT) or N(L94A/L97A/L99A) were treated with or without 5 ng/ml LMB. After 12 h, cells were homogenized and separated into cytoplasmic (C) and nuclear (N) fractions. The distribution of N(WT) or N(L94A/L97A/L99A) was analyzed by Western blotting with an anti-Myc antibody.

To determine whether the cytoplasmic localization of wild-type NADE is predominantly caused by the presence of a leucine-rich NES, GFP-N(WT)-transfected 293T cells were treated with 5 ng/ml leptomycin B (LMB). LMB has been shown to bind directly and irreversibly to CRM1 and inhibits NES-mediated active nuclear export (40). Within 3 h of LMB treatment, GFP-N(WT) was observed both in the nucleus and the cytoplasm, but GFP-N(WT) did not accumulate in the nucleus (Fig. 4C). After long term treatment with LMB (12 h), GFP-N(WT) was still distributed diffusely all over the cell. In contrast, the distribution of N(L94A/L97A/L99A) was not affected with LMB treatment (Fig. 4C). To verify these results, we performed subcellular fractionation and detected the distribution of N(WT) and N(L94A/L97A/L99A) using Western blotting. Consistent with the subcellular localization results of GFP constructs, Myc-N(WT) was detected in the cytoplasmic fraction but not in the nuclear fraction without LMB treatment and partially moved to the nuclear fraction from the cytoplasmic fraction after 12 h of LMB treatment (Fig. 4D, left panel). In contrast, the distribution of N(L94A/L97A/L99A), which was detected both in the nuclear and cytoplasmic fractions without LMB treatment, did not change following LMB treatment (Fig. 4D, right panel).

We previously reported that in 293T, PC12, and nnr5 cells transfected with wild-type NADE, two bands (22 and 44 kDa) were detected by Western blotting when SDS-PAGE was performed under reducing conditions (28). These bands correspond to the monomeric and dimeric forms of NADE, respectively. To confirm the self-association of NADE and to identify the region required for self-association, both FLAG-tagged NADE (N(WT)) and myc-tagged NADE mutants were transiently transfected into 293T cells. Immunoprecipitation of myc-tagged NADE mutants from cell extracts with an anti-myc antibody co-immunoprecipitated FLAG-tagged N(WT), as determined by immunoblotting with an anti-FLAG antibody. This experiment showed that N-(1-112) and N-(81-124) retained their ability to interact with N(WT) (Fig. 5A). In contrast, N-(1-100) failed to associate with N(WT), indicating that the region between residues 81-112, which includes the NES, is critical for self-association. We next transiently co-transfected 293T cells with FLAG-tagged N(WT) and myc-tagged N(L99A) or N(L94A/L97A/L99A) (Fig. 5B). These experiments showed that N(L94A/L97A/L99A) does not dimerize with wild-type NADE, although N(L99A) dimerized with wild-type NADE under reducing conditions. Three of the key hydrophobic residues of NES, Leu-94, Leu-97, and Leu-99, reside within the p75NTR binding domain of NADE, suggesting that these NES mutations may also be affecting the interaction of p75NTR with NADE. We therefore performed in vitro binding assays using GST fusion proteins to determine whether proteins containing these point mutations are able to interact with p75NTR(ICD). As shown in Fig. 5C, the N(L99A) single mutant associated with the p75NTR(ICD), however, the N(L94A/L97A/L99A) triple mutant did not. In addition, a N(L94A/L97A) double mutant associated with p75NTR(ICD), albeit much less efficiently than did wild-type NADE (data not shown). To verify these results, we performed interaction analysis of N(L94A/L97A/L99A) with N(WT) or the death domain of p75NTR (p75DD) in the yeast two-hybrid system. Consistent with the immunoprecipitation or the in vitro binding assay results, N(WT) self-associated with N(WT) or associated with p75DD, but N(L94A/L97A/L99A) did not (Fig. 5D). These results suggest that the NES is necessary for self-association of NADE and that the association or dissociation of NADE monomers may be linked to the regulation of its nuclear export.

View larger version (49K): [in this window] [in a new window]   Fig. 5.   Self-association of NADE. A, FLAG- and myc-tagged N(WT) or various NADE mutants were expressed in 293T cells as indicated in the top panel. 24 h after transfection, cell lysates myc-tagged proteins were immunoprecipitated from cell lysates, as described under "Experimental Procedures." Samples were then subjected to immunoblotting with an anti-FLAG antibody (upper panel). Cleared whole cell lysates were also immunoblotted with anti-FLAG (middle panel) and anti-myc (lower panel) antibodies. Asterisks indicate IgG bands. B, mutation of leucines in NES disrupts NADE self-association. 293T cells were transfected with plasmids producing FLAG-tagged N(WT) and Myc-tagged N(WT) or indicated NES mutants shown at top panel. Asterisks indicate IgG bands. C, interaction of wild-type and NES mutants with p75NTR. A GST fusion protein containing the cytoplasmic region of p75NTR was incubated with either wild-type or mutant NADE that had been translated in vitro and labeled with [35S]methionine. 35S-Labeled bound complexes were precipitated as described previously (28). D, self-association of NADE in yeast. L40 yeast cells were transformed with pBTM116/N(WT), or pBTM116/p75DD, and indicated NADE constructs in pVP16. Yeast transformants were streaked on solid growth media with (+His) or without (His) histidine. Growth of colonies on His-free plates is an indication that two proteins interact in yeast. p75DD, a death domain of p75NTR.

We next evaluated apoptosis in cells expressing the various NES point mutants of NADE (Fig. 6). This analysis showed that cells expressing the N(L94A/L97A/L99A) triple mutant failed to undergo apoptosis, whereas cells expressing the N(L99A) single mutant did undergo apoptosis. Thus, the NES motif of NADE is crucial not only for nuclear export, self-association, and interaction of NADE with p75NTR but is also required for NGF-dependent p75NTR·NADE-induced apoptosis.

View larger version (11K): [in this window] [in a new window]   Fig. 6.   Effect of NES mutations on apoptosis. Various NADE constructs were co-transfected with p75NTR into 293T cells, as indicated. Cells were fixed with 3.7% paraformaldehyde, and nuclear morphology was analyzed by DAPI staining. Data shown are expressed as the average percentage of apoptotic cells among the total number of cells counted ± S. D. (n = 4).

Dominant Negative Effect of N-(81-124) on NGF-induced Apoptosis in Oligodendrocytes-- NGF treatment induces apoptosis in terminally differentiated primary oligodendrocytes expressing high levels of p75NTR. For this reason, we used oligodendrocytes to characterize the mechanism by which NADE promotes a death signal through p75NTR. Interestingly, after NGF treatment, the expression of NADE mRNA and protein was increased in oligodendrocytes (28). To evaluate the NADE-mediated events that induce apoptosis in oligodendrocytes, we introduced the N-(81-124) NADE mutant into mature oligodendrocytes. The protein encoded by this construct does associate with p75NTR, but lacks a pro-apoptotic region (Table I). Oligodendrocytes were infected with a recombinant adenovirus carrying the myc-tagged NADE cDNA. As a control, other oligodendrocytes were infected with an adenoviral vector containing myc-tagged GFP. Typically, more than 90% of oligodendrocytes were infected with adenovirus, as assessed by immunofluorescence microscopy. As shown in Fig. 7 (A and B), oligodendrocytes were identified as blue signals by staining with an anti-O1 monoclonal antibody and an Alexa Fluor 350 anti-mouse IgG secondary antibody. Green signals were detected in control cells expressing GFP, and TUNEL-positive oligodendrocytes were visualized with red signals using Alexa 568·dUTP as a substrate (Fig. 7, A and B). In oligodendrocytes expressing GFP, the percentage of TUNEL-positive cells was 68.1% (n = 4) and 10.5% (n = 4), in the presence and absence of NGF, respectively (Fig. 7C). The percentage of TUNEL-positive cells among those that were not infected with adenovirus was not significantly affected by NGF treatment (Fig. 7C). Transfectants expressing myc-tagged N-(81-124) and N(WT) exhibited red fluorescence, and TUNEL-positive oligodendrocytes exhibited green fluorescence with FITC·dUTP used as a substrate (Fig. 7B). In the presence of NGF, 25.6% of cells expressing N-(81-124) were TUNEL-positive, whereas 72.1% of cells expressing N(WT) were TUNEL-positive (n = 4 in each group). Expression of N-(81-124) inhibited NGF-induced apoptosis in oligodendrocytes (Fig. 7C). Thus, N-(81-124) appears to have a dominant negative effect on NGF-induced apoptosis in oligodendrocytes.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Dominant negative effect of N-(81-124) on NGF-induced apoptosis in oligodendrocytes. Differentiated oligodendrocytes were infected with an adenovirus carrying GFP, N-(81-124), and N(WT). Infected cells were treated with 100 ng/ml NGF for 18 h. Oligodendrocytes were visualized with an anti-O1 monoclonal antibody and an Alexa 350 anti-mouse IgG second antibody, resulting in a blue signal. A, GFP expression was visualized as a green signal. TUNEL-positive oligodendrocytes were identified by red fluorescence, using Alexa 568-dUTP as a substrate of TUNEL reaction. The superimposed arrows indicate positive signals of GFP and TUNEL. B, cells expressing myc-tagged N-(81-124) and N(WT) are visualized as red fluorescence with an anti-myc polyclonal antibody and an Alexa 568-anti-rabbit IgG second antibody. TUNEL-positive oligodendrocytes are visualized as green fluorescence using FITC·dUTP as a substrate of TUNEL reaction. The superimposed arrows indicate positive signals of myc-tagged proteins and TUNEL. C, effect of N(WT) versus N-(81-124) NADE on NGF-induced apoptosis in oligodendrocytes. Data shown are expressed as the average percentage of TUNEL-positive cells among the total number of cells counted ± S.D. (n = 4).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The recent identification of distinct classes of receptor-associated signal transducers has provided insight into how members of the TNF receptor superfamily initiate downstream signaling events (41). The death domain is essential for the transduction of death signals elicited by ligands in the TNFR/Fas family, which belong to a superfamily of receptors that includes p75NTR. Several downstream targets of the TNFR and Fas death domains (subtype 1) have been identified (42). These proteins also contain death domain sequences, suggesting that there is a signaling mechanism triggered by the association of death domain-containing proteins, including self-association of death domains such as MORT1/FADD (43). However, the functional role of the downstream targets of the p75NTR death domain (subtype 2), including death-associated protein kinase, ankyrin, and NF-B p100 and p105 is still unclear. Structural analysis by NMR suggested that there is a potential site within the death domain of p75NTR that may interact with downstream targets in a ligand-dependent manner (44). We have reported that NADE associates with the death domain of p75NTR in an NGF-dependent manner and that this association induces apoptosis (28). To better understand the mechanism by which NADE mediates cell death, we have used mutational analysis to define the regions that correlate with the cellular functions of NADE. These studies revealed that NADE has a structure composed of modular regions that mediate specific functions.

Deletion mapping was the first approach we used to delineate the regions involved in NADE function. A schematic overview of the structural regions of NADE that were correlated with specific cellular functions is shown in Fig. 8. Expression of a minimal region of NADE-(41-71) was termed the pro-apoptotic region, because this fragment was sufficient to induce apoptosis even in the absence of NGF. In previous studies, we demonstrated that p75NTR·NADE-induced apoptosis was NGF-dependent in 293T cells after the interaction of NADE with p75NTR (28). Further analysis showed that apoptosis occurred in an NGF-dependent manner in cells expressing mutants in which both the pro-apoptotic and the p75NTR-binding regions were conserved. On the other hand, cells expressing mutants containing the pro-apoptotic region but not the p75NTR-binding region underwent apoptosis even in the absence of p75NTR. These results indicate that the C-terminal region regulates the pro-apoptotic region in response to NGF. This region (72-112) is termed the regulatory region, and includes the p75NTR-binding region.

View larger version (10K): [in this window] [in a new window]   Fig. 8.   Summary of the structure-function relationship of various regions of NADE. The structure of full-length mouse NADE (N(WT)) is shown at the top, and amino acid numbers are indicated above. The nuclear export signal (NES) is localized at amino acids 90-100, and the ubiquitin sequence (US) is localized at 91-112. The pro-apoptotic region resides within 41-71. The regulatory region resides between 72 and 112 and contains the p75NTR-binding region (81-106) and the self-association region (81-112). The 14-3-3 binding region is located between 81 and 124.

We also generated a series of point mutations within the NES motif of NADE, because this subregion (90-100) is located within the regulatory region of NADE (Figs. 4A and 8). We constructed several point mutants, including N(L99A) and N(L94A/L97A/L99A), because analogous mutations in other NES-containing proteins have been reported to prevent nuclear export (45, 46). Expression of N(L94A/L97A/L99A) decreased the efficiency of nuclear export (Fig. 4C). Moreover, LMB inhibited export of N(WT) from nucleus (Fig. 4C), suggesting that NADE is exported from the nucleus by a mechanism involving an LMB-sensitive NES between amino acids 90 and 100. These NES-mutational studies also showed that expression of N(L94A/L97A/L99A) decreased the interaction of NADE with p75NTR and self-association, suggesting that the NES motif of NADE plays a crucial role in each of these functions.

Interestingly, the regulatory region of NADE also associates with 14-3-3 proteins (32). Some 14-3-3 binding proteins have a functional nuclear localization signal (NLS)/NES that lies adjacent to the site of 14-3-3 binding (47-49). The N(L94A/L97A/L99A) triple mutant failed to associate with 14-3-3, suggesting that the NES motif of NADE might be important for 14-3-3 binding (data not shown). Thus, the NADE NES triple mutant lost p75NTR- and 14-3-3-binding activity as well as self-associating activity. Taken together, these findings suggest that the NADE NES might be an important platform for switching the conformation of NADE to regulate pro-apoptotic activity.

NADE has at least two homologues, Bex1 and Bex2, that are highly homologous to each other at amino acid level (29). In the sublocalization analysis using immunocytochemistry, human Bex1 is diffusely localized in both the nucleus and the cytoplasm, suggesting that Bex1 does not have a functional NES (data not shown). Indeed, in the region corresponding to NADE NES in human Bex1 and Bex2 (RQLMEKLREKQLS), the third leucine is replaced with lysine in the NES motif. Moreover, human Bex1 failed to dimerize, associate with p75NTR and NADE, and to induce p75NTR-dependent apoptosis in 293T cells (data not shown), suggesting that Bex1 is not involved in p75NTR signaling. Although the regions, corresponding to the NADE-regulatory region in human Bex1 and Bex2, are identical to each other, further investigation is necessary to analyze hBex2 on nuclear export, p75NTR binding, oligomerization, and p75NTR-dependent apoptosis.

NGF-induced apoptosis has been shown to occur in primary cultured oligodendrocytes (20, 36). In a previous study, we proposed that NADE is involved in this signaling pathway, because the expression of NADE mRNA and protein were increased by NGF in oligodendrocytes (28). NADE expression was also increased in response to zinc-induced apoptosis in rat cortical neurons (30). Because zinc-induced apoptosis was blocked by an antisense oligonucleotide of NADE, NADE appears to be involved in this type apoptosis in neurons (30). In the present report, we analyzed the effects of expressing various NADE mutants on NGF-induced apoptosis in oligodendrocytes. Expression of N-(81-124) inhibited NGF-induced apoptosis in oligodendrocytes, suggesting that N-(81-124) has a dominant negative effect on NGF/p75NTR-mediated apoptosis. Thus, the N-terminal region of NADE, which contains the pro-apoptotic region, may have a functional motif that transduces the apoptotic signal.

In our previous report, we showed that 14-3-3 is involved in NGF-induced apoptosis in oligodendrocytes (31). We also observed that 14-3-3 can associate with N-(81-124). However, co-expression of N-(81-124) and 14-3-3 did not induce NGF/p75NTR-dependent apoptosis in HEK293 cells (data not shown). These findings suggest that the recruitment of 14-3-3 to the p75NTR·NADE complex is necessary but not sufficient to mediate apoptosis through p75NTR. We hypothesize that, after NADE is recruited to the p75NTR(ICD) in response to NGF, the NADE protein undergoes a conformational change that exposes the pro-apoptotic region to as yet unknown signaling elements that mediate the downstream apoptosis-signaling pathway. In future studies, it will be of interest to identify the specific downstream molecules targeted by this pro-apoptotic region.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant R01-GM55147 (to T. S.).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.

^¶ Present address: Dept. of Otolaryngology, School of Medicine, Kitasato University, Kanagawa 228-8555, Japan.

To whom correspondence should be addressed: Division of Molecular Oncology, Dept. of Otolaryngology/Head & Neck Surgery and Pathology, College of Physicians & Surgeons, Columbia University, 630 W. 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 5, 2002, DOI 10.1074/jbc.M106342200

	ABBREVIATIONS

The abbreviations used are: NGF, nerve growth factor; ICD, intracellular domain; p75NTR, p75 neurotrophin receptor; GFP, green fluorescence protein; PBS, phosphate-buffered saline; GST, glutathione S-transferase; FITC, fluorescein isothiocyanate; DMEM, Dulbecco's modified Eagle's medium; DAPI, 4,6-diamidino-2-phenylindole; TrkA, tyrosine kinase receptor; MAP, mitogen-activated protein; MAPKK, MAP kinase kinase; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; ICD, intracellular domain; NADE, p75NTR-associated cell death executor; NES, nuclear export signal; WT, wild-type; HEK, human embryonic kidney; BME, Basal Medium Eagle; ALLN, N-acetyl-Leu-Leu-norleucinal; LMB, leptomycin B; PMSF, phenylmethylsulfonyl fluoride; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; MORT1, mediator of receptor-induced toxicity; FADD, Fas-associating death domain protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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