 |
INTRODUCTION |
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
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% 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 14-3-3
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 14-3-3
and NADE cDNAs were subcloned into pGEX5X-1
(Amersham Pharmacia Biotech). These GST fusion proteins, GST/14-3-3
and GST/NADE, were then expressed in DH5
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 14-3-3
, (1)-14-3-3
,
(1)-14-3-3
, or (121)-14-3-3
. GST/14-3-3
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 (
max = 400 nm)
and, upon cleavage of the substrate by caspase-3, free AFC emits a
yellowish green fluorescence (
max = 494 nm). The
fluorometer (Hitachi) was used to measure fluorescence values as a
means to quantify caspase-3 activity.
 |
RESULTS |
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 × 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 14-3-3
. 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).

View larger version (6K):
[in this window]
[in a new window]
|
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.
|
|
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 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/14-3-3
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.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 2.
Interaction of 14-3-3
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 14-3-3 GST
fusion protein (GST/14-3-3 ). Constructs that interacted with
GST/14-3-3 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/14-3-3 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 14-3-3 ((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
14-3-3 ; 2nd, 6th, and 10th lanes
are from cells expressing (1)-14-3-3 ; and 3rd, 7th,
and 11th lanes are from cells expressing
(1)-14-3-3 ; 4th, 8th, and 12th
lanes are from cells expressing (121)-14-3-3 . 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 14-3-3
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. 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/14-3-3
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)-NADE and (81)-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)-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 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)-14-3-3
and (121)-14-3-3
bound to NADE/GST but
not GST alone. However, 14-3-3
deletion mutant (1)-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).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
Interaction of 14-3-3
with the NADE/p75NTR complex in vivo.
A, HEK293 cells were co-transfected with Myc-tagged NADE and
FLAG-tagged 14-3-3 . 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-14-3-3 ,
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 14-3-3 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 14-3-3
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 14-3-3
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 14-3-3
, HEK293
cells were transiently transfected with p75NTR, Myc-tagged NADE, and
FLAG-tagged 14-3-3
(p75NTR/mycNADE/FLAG14-3-3
//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 14-3-3
associates with p75NTR in
p75NTR/mycNADE/FLAG14-3-3
//HEK293 cells (Fig. 3C). To
examine whether NADE is required for the association of p75NTR with
14-3-3
, HEK293 cells were co-expressed with p75NTR and FLAG-tagged
14-3-3
in the absence of NADE. FLAG-tagged 14-3-3
was
immunoprecipitated from lysates of p75NTR/FLAG14-3-3
//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, 14-3-3
did not associate with p75NTR (Fig.
3C).
14-3-3
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 14-3-3
protein on the
p75NTR/NADE-mediated apoptosis, wild type or deletion mutant forms of
14-3-3
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 14-3-3
(n = 5), and 46.9% in cells
transfected with p75NTR, NADE, and (1)-14-3-3
(n = 5) (Fig.
4A). In striking contrast, the
percentage of apoptotic cells transfected with p75NTR, NADE, and
(1)-14-3-3
was only 11.6% (Fig. 4A). To study
further the effect of 14-3-3
protein on the physiological
p75NTR-mediated apoptosis, these 14-3-3
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 14-3-3
(n = 5), and 31.3% in cells transfected with NADE and (1)-14-3-3
(n = 5) (Fig.
4B). In contrast, the percentage of apoptotic cells
transfected with NADE and (1)-14-3-3
was 18.9% (Fig.
4B).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Mutant forms of 14-3-3
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 14-3-3 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 14-3-3
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)-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)-14-3-3
is not due to changes in the subcellular localization of NADE and 14-3-3
proteins.
We have reported previously (26) that NGF induces 75NTR/NADE-mediated
apoptosis with DNA fragmentation. To examine whether (1)-14-3-3
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 14-3-3
(n = 4), and 43.3% in cells
transfected with p75NTR, NADE, and (1)-14-3-3
(n = 4) (Fig. 5B). Again, in contrast to the
other constructs, only 15.5% of the cells transfected with p75NTR,
NADE, and (1)-14-3-3
were positive in the TUNEL assays
(n = 4) (Fig. 5B).

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
Mutation of 14-3-3
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/wt14-3-3
//HEK293, and
p75NTR/NADE/ (1)-14-3-3
//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)-14-3-3
//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/wt14-3-3
//PC12nnr5, and
p75NTR/NADE/(1-120)-14-3-3
//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)-14-3-3
//PC12nnr5 was increased by 1.5-fold
(Fig. 6B). These data suggest that expression of mutant
(1)-14-3-3
attenuates apoptosis.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 6.
Mutant forms of 14-3-3
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)-14-3-3
inhibits NGF-induced apoptosis
under more physiological conditions, FLAG-tagged wild type or mutant
forms of 14-3-3
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 14-3-3
transfectants, 43.2% in (1)-14-3-3
transfectants (n = 4), 74.8% in (1)-14-3-3
transfectants (n = 4), and 72.1% in vector (without
insert) transfectants (n = 4) (Fig. 7B).
These results showed that (1)-14-3-3
also inhibits NGF-induced
apoptosis in oligodendrocyte.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 7.
(1-207)-14-3-3
inhibits NGF-induced apoptosis in primary oligodendrocytes.
FLAG-tagged wild type 14-3-3 and indicated mutant forms of 14-3-3
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 |
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.
To examine whether 14-3-3
directly interacts with NADE in
vitro, a GST pull-down assay was performed. The results indicated that NADE directly binds to 14-3-3
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 14-3-3
, 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 14-3-3
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 14-3-3
.
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
14-3-3
.
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 × 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.
To examine the effects of 14-3-3
on NGF-induced apoptosis, wild type
14-3-3
, the (1)-14-3-3
mutant, or the (1)-14-3-3
mutant was co-transfected with both p75NTR and NADE into HEK293 cells.
We found that co-expression of p75NTR, NADE, and (1)-14-3-3
inhibited NGF-dependent apoptosis (by 75%), as compared
with HEK293 cells co-expressing p75NTR, NADE, and wild type 14-3-3
.
In contrast, the percentage of apoptotic cells singly transfected with
wild type 14-3-3
, (1)-14-3-3
, and (1)-14-3-3
was
10-20% (data not shown), and there was no significant difference in
the percentage among these three single transfectants. In addition,
wild type 14-3-3
, (1)-14-3-3
mutant, or (1)-14-3-3
mutant was also co-transfected with NADE into PC12nnr5 cells to examine
the effect of (1)-14-3-3
under more physiological conditions.
PC12nnr5 cells endogenously express p75NTR but not TrkA (37). We found
that co-expression of NADE and (1)-14-3-3
inhibited
NGF-dependent apoptosis (by 59%), as compared with
PC12nnr5 cells co-expressing NADE and wild type 14-3-3
. Furthermore,
expression levels of p75NTR, NADE, and 14-3-3
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 14-3-3
protein. Furthermore, subcellular localization of these
proteins was similar in all transfectants. Hence, NADE, wild type
14-3-3
mutant (1)-14-3-3
, and mutant (1)-14-3-3
were
localized in the cytoplasm. These results suggest that inhibition of
cell death by (1)-14-3-3
is not due to changes in the
subcellular localization of NADE and 14-3-3
proteins. The wild type
14-3-3
and (1)-14-3-3
bound to NADE, but (1)-14-3-3
did not. More important, only (1)-14-3-3
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)-14-3-3
inhibit the activation of caspase-3 in
HEK293 and PC12nnr5 cells, as shown in Fig. 6, A and
B. These results suggest that 14-3-3
regulates caspase-3 activity, and the carboxyl-terminal region (residues 208-255) of
14-3-3
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)-14-3-3
also inhibited
NGF-induced apoptosis in oligodendrocytes as shown in Fig. 7,
A and B. From these results, we conclude that
(1)-14-3-3
has a dominant negative effect on p75/NADE-mediated
apoptosis. Thus, the carboxyl-terminal region of 14-3-3
, 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 14-3-3
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
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 14-3-3
is a key molecule in this signaling cascade.