From the Hamon Center for Therapeutic Oncology Research and the Division of Hematology-Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8593
Received for publication, September 12, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ectodermal dysplasia receptor (EDAR) is a
recently isolated member of the tumor necrosis factor receptor family
that has been shown to play a key role in the process of ectodermal
differentiation. We present evidence that EDAR is capable of activating
the nuclear factor- Anhidrotic (or hypohidrotic) ectodermal dysplasia is a disorder of
ectodermal differentiation characterized by a triad of signs consisting
of sparse hair, abnormal or missing teeth, and an inability to sweat
(1). A similar phenotype is seen in mice with mutations involving the
downless locus, suggesting the existence of a common
underlying genetic defect. Recently, mutations in EDAR,1 a novel receptor of
the TNFR family, were found in several families with autosomal dominant
and recessive forms of anhidrotic ectodermal dysplasia and in
downless mice (2, 3). Although the above studies established
the role of EDAR in ectodermal differentiation, the signaling pathways
activated by this receptor remain to be elucidated.
Death domain-containing receptors of the TNFR family are believed to
activate two main signaling cascades: a kinase cascade leading to
NF- Cell Lines and Reagents--
293T and MCF7 cells were obtained
from Dr. David Han (University of Washington, Seattle, WA). 293 EBNA cells were obtained from Invitrogen. Rabbit polyclonal
antibodies against FLAG, Myc, and hemagglutinin tags were obtained from
Santa Cruz Biotechnology. FLAG beads and control beads were obtained
from Sigma. The pull-down kinase assay kit for JNK was obtained from
New England Biolabs Inc., and the constructs for the Pathdetect
luciferase reporter assay were purchased from Stratagene. YOPRO-1 was
obtained from Molecular Probes, Inc. Synthetic caspase inhibitors
(Z-VAD-fmk and t-butoxycarbonyl-Asp-fmk) were purchased from Calbiochem.
Expression Constructs--
EDAR cDNA encoding amino
acids 22-448 was amplified by reverse transcription-polymerase chain
reaction using normal prostate RNA as a template. The primers used for
amplification also carried restriction enzyme sites at their 5'
termini. The amplified product was subsequently cloned in a modified
pSecTagA vector carrying a FLAG epitope tag downstream of the murine
Ig Electrophoretic Mobility Shift Assay--
293T cells (3 × 105) were transfected with 2 µg of various constructs in
each well of a six-well plate. After 36 h, nuclear extracts
were prepared, and electrophoretic mobility shift assay was
performed essentially as described previously (9).
Luciferase Reporter Assays--
The NF-
For the c-Jun transcriptional activation assay, 293 EBNA cells
(1.2 × 105) were transfected in duplicate with
various expression constructs (500 ng) along with a fusion
transactivator plasmid containing the yeast Gal4 DNA-binding
domain fused to transcription factor c-Jun (pFA-c-Jun) (50 ng), a
reporter plasmid encoding the luciferase gene downstream of the Gal4
upstream activating sequence (pFR-luc) (500 ng), as well as a Rous
sarcoma virus/LacZ ( Coimmunoprecipitation Assays--
For studying in
vivo interaction, 2 × 106 293T cells were plated
in a 100-mm plate and cotransfected 18-24 h later with 5 µg/plate of
each epitope-tagged construct by calcium phosphate coprecipitation. A
hemagglutinin-tagged green fluorescent protein-encoding plasmid was
also included in some experiments. Twenty-four hours post-transfection, cells were lysed in 1 ml of lysis buffer containing 0.1% Triton X-100,
20 mM sodium phosphate (pH 7.4), 150 mM NaCl,
and one EDTA-free mini-protease inhibitor tablet (Roche Molecular
Biochemicals)/10 ml. Cell lysates (500 µl) were incubated for 1 h at 4 °C with 10 µl of FLAG or control mouse Ig beads precoated
with a supersaturated casein solution. Beads were washed twice with
lysis buffer; twice with wash buffer containing 0.1% Triton X-100, 20 mM sodium phosphate (pH 7.4), and 500 mM NaCl;
and again with lysis buffer. Bound proteins were eluted by boiling,
separated by SDS-polyacrylamide gel electrophoresis, transferred to a
nitrocellulose membrane, and analyzed by Western blotting. Essentially
a similar procedure was used for experiments involving
coimmunoprecipitation of TRADD and TRAFs, except cells were lysed in a
modified radioimmune precipitation assay buffer containing 150 mM NaCl, 50 mM Tris (pH 7.4), 1% Nonidet P-40,
0.1% sodium deoxycholate, and 1 mM EDTA, and the beads
were washed extensively in the above buffer containing 1 M NaCl.
Receptor-Ligand Interaction Assays--
293T cells were
transfected with expression plasmids encoding EDAR and TAJ
immunoadhesins, and 12 h post-transfection, the medium was changed
to 293SFM (Life Technologies, Inc.). Supernatants containing the
secreted immunoadhesin were collected 48 h later and stored in
aliquots at
For coimmunoprecipitation assay, equal volumes (500 µl) of the
immunoadhesin and Myc-EDA supernatants were mixed in a buffer containing 50 mM Tris (pH 7), 150 mM NaCl, and
0.1% Triton X-100 and incubated at 4 °C overnight with gentle
shaking. Supernatants were subsequently divided into two halves and
immunoprecipitated with goat anti-mouse IgG1 beads (Sigma) or control
antibody beads precoated with supersaturated casein solution. After
extensive washing, the bound proteins were eluted by boiling, separated by SDS-polyacrylamide gel electrophoresis, transferred to a
nitrocellulose membrane, and analyzed by Western blotting.
For enzyme-linked immunosorbent assay, 5 µl of a control supernatant
or the supernatant containing Myc-EDA I were immobilized overnight at
4 °C in the wells of a microwell plate in
Na2CO3/NaHCO3 buffer (pH 9.6).
After washing with Tris-buffered saline containing 0.05% Tween (TBST),
5 µl of supernatants containing FLAG-tagged immunoadhesins (or a
control supernatant) diluted in Tris-buffered saline were added to the
wells and incubated for 4 h at room temperature. After washing,
goat anti-mouse peroxidase (1:2000 in TBST) was added for 1 h.
Color was developed using o-phenylenediamine dihydrochloride (Sigma), and the absorbance was measured at 490 nm.
EDAR Activates the NF- Mechanism of EDAR-induced NF-
The NIK and IKK serine/threonine kinases have been shown to be involved
in the activation of the NF-
As IKKs function by mediating inducible phosphorylation and degradation
of I Mutagenesis Analysis of EDAR-induced NF-
We next tested whether two EDAR mutations seen in association with
anhidrotic ectodermal dysplasia could affect the ability of EDAR to
activate the NF- EDAR Activates the JNK Pathway--
In addition to NF-
We used deletion and point mutagenesis to map the region of the EDAR
cytoplasmic domain responsible for JNK activation. These studies
revealed that, although the EDAR
Finally, we tested the ability of the two EDAR mutants known to be
associated with anhidrotic ectodermal dysplasia to activate the JNK
pathway. As in the situation with NF-
TRAF2 has been shown to play an essential role in JNK activation via
various members of the TNFR family (17, 18). However, as shown in Fig.
4D, a dominant-negative mutant of TRAF2 that could
effectively block CD40-induced JNK activation failed to block JNK
activation via EDAR. These results suggest either that TRAF2 is not
involved in JNK activation via EDAR or that it plays a functionally
redundant role in this process. Finally, EDAR-induced JNK activation
was effectively blocked by the JNK-binding domain of JIP1 (Fig.
4E), a recently described inhibitor of the JNK pathway (19).
EDAR Induces Caspase-independent Cell Death--
As discussed
above, EDAR is known to possess a region in its cytoplasmic domain with
partial sequence homology to the "death domain" present in the
apoptosis-inducing members of the TNFR family. Previous studies have
demonstrated that transient transfection of EDA, the putative ligand
for EDAR, in MCF7 cells leads to cellular rounding and detachment,
which are not inhibited by the caspase inhibitor Z-VAD-fmk (20, 21).
Consistent with these results, transient transfection of EDAR in 293T,
293 EBNA, or MCF7 cells led to cellular rounding and detachment, two
features suggestive of cell death (Fig.
5, A-C). However, unlike
TNFR1-transfected cells, cellular rounding and detachment were
relatively delayed features of EDAR-transfected cells (24 versus 30 h). Finally, unlike TNFR1-transfected cells,
those cells transfected with EDAR failed to demonstrate membrane
budding, a feature associated with caspase-dependent cell
death (Fig. 5A).
We were next interested in testing whether EDAR-expressing cells
actually undergo cell death. To test this hypothesis, we used nuclear
staining with YOPRO-1, a cell-impermeable DNA-intercalating dye. As
shown in Fig. 5D, although the majority of
vector-transfected cells failed to show nuclear staining with this dye,
a large number of EDAR-transfected cells stained positively, suggesting
a lack of membrane integrity indicative of cell death. However, unlike the TNFR1-transfected cells, those cells dying in response to EDAR
failed to show nuclear fragmentation, another key feature of
caspase-dependent cell death (Fig. 5D).
The lack of caspase activation during EDAR-induced cell death was
characterized further by using several known inhibitors of this
pathway. EDAR-induced cell death was not blocked by Z-VAD-fmk and
t-butoxycarbonyl-Asp-fmk, two synthetic cell-permeable
caspase inhibitors, and by CrmA and p35, two virally encoded caspase
inhibitors. In contrast, all these caspase inhibitors effectively
blocked cell death induced by TNFR1 (Fig.
6, A and B).
Similarly, EDAR-induced cell death was not blocked by MRIT/cFLIP,
MC159L, and dominant-negative mutants of caspase-8 (caspase-8-C360S)
and FADD, suggesting that EDAR uses a FADD- and caspase-8-independent
pathway for inducing cell death (Fig. 6C). Finally,
EDAR-induced cell death was not blocked by a dominant-negative I
Caspase-3 is one of the executioner caspases of the caspase cascade and
is activated during apoptosis induced by death receptors belonging to
the TNFR1 family (22). We used a chromogenic assay, based on
caspase-3-mediated cleavage of the chromogenic substrate Z-DEVD-p-nitroanilide, to test the activation of
caspase-3 during EDAR-induced cell death. As shown in Fig.
6D, cell lysates from TNFR1- or DR4-transfected cells
demonstrated caspase-3 activation, whereas EDAR-transfected cells
failed to do so. Collectively, the above results suggest that cellular
rounding and eventual cell death induced by EDAR overexpression are not
mediated by a caspase-dependent mechanism.
We used deletion and point mutagenesis to map the region in the EDAR
cytoplasmic domain responsible for induction of cell death. As shown in
Fig. 6E, the EDAR EDAR Interacts with TRAFs and NIK, but Fails to Interact with FADD
or TRADD--
The death domain-containing adaptor proteins TRADD and
FADD have been shown to play an essential role in signaling via various death domain-containing receptors of the TNFR family (4). Therefore, we
tested the ability of these proteins to interact with EDAR using a
coimmunoprecipitation assay. EDAR failed to coimmunoprecipitate FADD
and TRADD when overexpressed with them in 293T cells (Fig. 7, A and B).
Control experiments, performed in parallel, confirmed successful
coimmunoprecipitation of FADD with Fas and TRADD with TNFR1, thereby
demonstrating the validity of the assay.
In addition to the death domain-containing adaptor proteins, different
members of TRAF family have been shown to interact with various members
of the TNFR family (13). Therefore, we tested the ability of these
proteins to interact with EDAR using coimmunoprecipitation assay. EDAR
successfully coimmunoprecipitated murine TRAF1, murine TRAF2, and TRAF3
when these proteins were coexpressed in 293T cells (Fig. 7,
C-F). Deletion mutagenesis revealed that the C-terminal 94 amino acids, encoding the death domain, were not essential for
interaction of EDAR with murine TRAF1. Consistent with this hypothesis,
the death domain point mutants E79K and R420Q were as effective as the
wild-type protein in coimmunoprecipitating murine TRAF1. Finally, EDAR
successfully coimmunoprecipitated with NIK, a protein known to be
involved in NF- EDA Is the Ligand for EDAR--
EDA is believed to be the ligand
for EDAR based on the similarity in the clinical features of genetic
disorders resulting from the mutations in these genes (2, 23, 24).
However, EDA has never been shown to bind to EDAR. To test the ability of EDA to bind to EDAR, we generated a baculovirus construct containing the extracellular receptor-binding domain of EDA fused to an N-terminal Myc epitope tag. As shown in Fig.
8A, the Myc-EDA I construct contained the second and third collagenous repeats of the EDA-A1 isoform, in addition to its TNF homology domain. Myc-tagged soluble EDA
proteins was collected from the supernatant of baculovirus-infected insect cells and tested for its ability to bind to EDAR-Fc or mTAJ-Fc
in a coimmunoprecipitation assay. As shown in Fig. 8B, EDAR-Fc successfully coimmunoprecipitated the Myc-EDA I protein, whereas mTAJ-Fc failed to do so. The ability of Myc-EDA I to bind to
EDAR was further confirmed using enzyme-linked immunosorbent assay
(Fig. 8C).
During the process of skin differentiation, the mitotically active
cells of the basal epithelium cease proliferating and then migrate
outwards and undergo terminal differentiation (25). The NF- We have observed that EDAR-induced NF- Our results further suggest that, like the other death domain receptors
of the TNFR family, the death domain of EDAR plays a key role in the
activation of the NF- Previous studies had demonstrated that transfection of an EDA
expression construct in MCF7 cells led to cell rounding and detachment,
resembling the morphology of cells undergoing cell death (20, 21).
However, these morphological changes could not be blocked by the
cell-permeable caspase inhibitor Z-VAD-fmk, suggesting the lack of a
role for caspase activation in this process. In the present study, we
have similarly demonstrated that transfection of EDAR in 293T, 293 EBNA, and MCF7 cells also leads to cell rounding and detachment,
followed by cell death. Cells dying in response to EDAR overexpression
do not show any morphological or biochemical features of caspase
activation, suggesting that EDAR induces cell death by using a
caspase-independent mechanism. Such a caspase-independent form of cell
death has been described previously for several death domain- and
non-death domain-containing members of the TNFR family, and it remains
to be seen whether EDAR shares a common mechanism of cell death
induction with them (11, 32-39). Several potential mediators of
caspase-independent cell death have been recently described, such as
Bax, nitric oxide, and apoptosis-inducing factor (40-44). It will be
interesting to test the involvement of these proteins in EDAR-induced
cell death. It is also conceivable that cell death induced by EDAR is a
consequence of cellular detachment from the plate.
Finally, in this report, we demonstrate for the first time that
extracellular domains of EDAR and EDA can physically interact with each
other. EDA is unique among the ligands of the TNF family in possessing
three collagenous repeat domains, in addition to a TNF homology domain
(20, 21, 24, 45, 46). Like other ligands of the TNF family, EDA is
expressed in a trimeric form, and it is conceivable that the
collagenous repeats of EDA help in this process (45). Several
alternatively spliced isoforms of EDA that lack one or more of its
subdomains have been described recently (20, 24, 46). Future studies
should address the role of each of these subdomains of EDA in its
interaction with EDAR.
B, JNK, and caspase-independent cell death
pathways and that these activities are impaired in mutants lacking its
death domain or those associated with anhidrotic ectodermal dysplasia and the downless phenotype. Although EDAR possesses a death
domain, it did not interact with the death domain-containing adaptor
proteins TRADD and FADD. EDAR successfully interacted with various TRAF family members; however, a dominant-negative mutant of TRAF2 was incapable of blocking EDAR-induced nuclear factor-
B or JNK
activation. Collectively, the above results suggest that EDAR utilizes
a novel signal transduction pathway. Finally, ectodysplasin A can
physically interact with the extracellular domain of EDAR and thus
represents its biological ligand.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B and JNK activation and a caspase cascade leading to cell death
(4). As the cytoplasmic domain of EDAR was reported to contain a region
with partial homology to the death domain, we decided to test its
abilities to activate the above signaling cascades (2, 3). In this
report, we present evidence that, like the classical death
domain-containing receptors, EDAR is capable of activating the NF-
B,
JNK, and cell death pathways. However, EDAR does not interact with the
death domain-containing adaptor proteins TRADD or FADD and does not
activate the caspase cascade. Our results suggest the existence of a
novel signaling pathway utilized by EDAR.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
signal peptide. The above FLAG-EDAR construct was used to
generate deletion mutants EDAR
C223, EDAR
C164, and EDAR
C94 by
taking advantage of the naturally occurring SmaI,
SalI, and XhoI sites in the EDAR cytoplasmic domain, whereas the deletion mutant EDAR
C38 was generated using a
custom primer containing an XbaI site. Site-directed
mutagenesis was carried out using the Quick-Change mutagenesis kit
(Stratagene) and using the FLAG-EDAR plasmid as a template. To generate
the EDAR immunoadhesin (EDAR Fc), the 5'-fragment of the FLAG-EDAR construct containing the signal peptide, the FLAG epitope, and nucleotides encoding the extracellular domain of EDAR (amino acids 21-177) was amplified using custom primers. The resulting product was
cloned upstream of the Fc portion (amino acids 236-462) of the murine
Ig heavy chain. This construct also carried a C-terminal FLAG tag. A
murine TAJ immunoadhesin construct (mTAJ-Fc) was generated similarly by
fusing the extracellular ligand-binding domain of murine TAJ protein
(amino acids 1-172) to the Fc portion of the murine Ig heavy chain. To
construct Myc-EDA I the nucleotide sequence corresponding to amino
acids 134-391 of the EDA-A1 isoform was amplified by reverse
transcription-polymerase chain reaction using total RNA derived
from human prostate gland as a template. The upstream
primer used for amplification was 5'-CGCGGGATCCCGCCCTATTGAATTTCTTC-3', and the downstream primer used was
5'-CGCGGTCGACCTAGGATGCAGGGGCTTCAC-3'. The amplified product was
digested with BamHI and SalI enzymes and
subsequently cloned into a modified pFastBAC1 vector (Life Technologies, Inc.), which contained a Myc epitope tag downstream of a
baculovirus gp67 signal peptide. The sequences of all constructs were
confirmed by automated fluorescent sequencing. Constructs encoding NIK
and its mutants (5), the IKK2 mutant (6); I
B
-S32A/S36A (7); and
murine TRAF1, murine TRAF2 and its mutant, TRAF3, I-TRAF, FADD, DR5,
CD40, and NF-
B/luciferase reporter constructs have been
described previously (8-10) and were obtained from the indicated
sources. The dominant-negative IKK1 mutant was kindly provided by Dr.
Richard Gaynor.
B reporter assay was
performed essentially as described previously (9). Briefly, 293T cells
were transfected in duplicate in a 24-well plate with the various test
plasmids along with an NF-
B/luciferase reporter construct (75 ng/well) and a Rous sarcoma virus promoter-driven
-galactosidase
reporter construct (pRcRSV/LacZ; 75 ng). Twenty-four to thirty hours
later, cells were lysed, and extracts were used for the measurement of
luciferase and
-galactosidase activities, respectively. Luciferase
activity was normalized relative to
-galactosidase activity to
control for the difference in the transfection efficiency.
-galactosidase) reporter construct (75 ng).
Cells were lysed 24 h later, and luciferase assay was performed as
described previously (10).
70 °C until use. Myc-EDA I protein was produced by
infection of Sf9 insect cells with the corresponding baculovirus
construct following the manufacturer's instructions (Life
Technologies, Inc.). Supernatant containing the secreted protein was
collected 48 h post-infection, filtered, and stored at
70 °C
until use.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B Pathway--
We began by testing the
ability of EDAR to activate an NF-
B-driven luciferase reporter
construct upon transient transfection in 293T cells. As shown in Fig.
1A, transfection of EDAR in
these cells led to significant activation of the NF-
B pathway that was comparable in magnitude to that induced by TNFR1. In comparison, TAJ/TROY, another TNFR family member that is highly expressed in skin
during embryonic development (11, 12), failed to activate the NF-KB
pathway. NF-
B activation by EDAR was further confirmed using an
electrophoretic mobility shift assay (Fig. 1B).
View larger version (33K):
[in a new window]
Fig. 1.
EDAR activates the
NF- B pathway. A, NF-
B
reporter assay. 293T cells were transfected with the indicated
constructs (500 ng/well) along with an NF-
B/luciferase reporter
construct (75 ng/well) and a Rous sarcoma virus/LacZ
(
-galactosidase) reporter construct (75 ng/well), and the experiment
was performed as described under "Materials and Methods." The
values shown are averages (mean ± S.E.) of one representative
experiment out of three, in which each transfection was performed in
duplicate. B, electrophoretic mobility shift assay. 293T
cells were transfected with the indicated vectors. Approximately
36 h post-transfection, cells were lysed, and nuclear extracts
were used for the gel shift assay. The position of the induced NF-
B
complex is marked by an arrowhead, and the position of a
constitutive NF-
B complex is indicated by an
asterisk.
B Activation--
TRAF2 has been
known to mediate NF-
B activation by various TNFR family members, and
TANK/I-TRAF has been known to regulate this process (13). Therefore, we
investigated the roles of TRAF2 and TANK/I-TRAF in the activation of
the NF-
B pathway by EDAR. An N-terminal deletion mutant of TRAF2
(14) that could effectively block NF-
B activation by CD40 was
ineffective in blocking NF-
B induction by EDAR (Fig.
2A). Similarly, EDAR-induced
NF-
B activation was not affected by coexpression of TANK/I-TRAF
(Fig. 2A). Collectively, these results suggest either that
TRAF2 and TANK/I-TRAF are not involved in NF-
B activation via EDAR
or that they play a functionally redundant role in this process. We
have also tested the ability of dominant-negative mutants of the
receptor-interacting protein and TRADD to block EDAR-induced NF-
B,
but have failed to observe any significant inhibitory effect (data not
shown).
View larger version (30K):
[in a new window]
Fig. 2.
Mechanism of EDAR-induced
NF- B activation. 293T cells were
transfected with the indicated plasmids, and the experiment was
performed as described under "Materials and Methods." The amount of
inhibitor plasmids (450 ng/well) was three times the amount of receptor
plasmids (150 ng/well), and the total amount of DNA transfected was
kept constant by adding empty vector. The values shown are averages
(mean ± S.E.) of a representative of at least two independent
experiments, in which each transfection was performed in duplicate.
A, dominant-negative TRAF2 (DN-TRAF2) and
TANK/I-TRAF fail to block EDAR-induced NF-
B activation;
B, dominant-negative mutants of NIK (NIK-K429R
(NIK-KR) and NIK-2101) effectively block EDAR-induced
NF-
B activation, whereas a similar mutant of MEKK1 (MEKK1-D1369A
(MEKK1-D-A)) fails to do so; C, kinase-inactive
mutants of IKK1 and IKK2 (IKK1-K44M (IKK1-KM) and IKK2-K44M
(IKK2-KM), respectively) block EDAR-induced NF-
B
activation; D, a phosphorylation-resistant mutant of
I
B
(I
B
-S32A/S36A
(I
B
S32/36A)) effectively blocks
EDAR-induced NF-
B activation.
B pathway by the members of the TNFR and
interleukin-1 receptor families (15). To determine the role of these
proteins in EDAR-induced NF-
B activation, we took advantage of the
dominant-negative inhibitors of these kinases. As shown in Fig.
2B, a C-terminal deletion mutant (NIK-2101) and a catalytic
site mutant (NIK-K429R) of NIK could effectively block NF-
B induced
by EDAR. However, a dominant-negative mutant of MEKK1 (MEKK1-D1369A), a
related MAPK, failed to block EDAR-, CD40-, or TNFR1-induced NF-
B,
suggesting the specificity of the observed effect. EDAR-induced NF-
B
activation was also effectively blocked by dominant-negative mutants of
IKK1 (IKK1-K44M) and IKK2 (IKK2-K44M), respectively (Fig.
2C).
B proteins, we tested the ability of a dominant-negative mutant
of I
B
(I
B
-S32A/S36A) to block NF-
B induction by EDAR. This mutant contains serine-to-alanine substitutions at amino acids 32 and 36, respectively, and is resistant to phosphorylation-induced degradation of I
B
(16). As shown in Fig. 2D, NF-
B
induction by EDAR was effectively blocked by I
B
-S32A/S36A. Taken
together, the above results suggest that EDAR activates NF-
B by NIK-
and IKK-induced phosphorylation and degradation of the I
B
protein.
B Activation--
We
used C-terminal deletion mutagenesis to map the domain of EDAR
responsible for NF-
B activation. Deletion mutants EDAR
C38 and
EDAR
C94, which are missing the C-terminal 38 and 94 amino acids,
respectively, were only minimally effective in NF-
B activation (Fig.
3, A and B). The
EDAR
C38 mutant possesses a partial death domain, whereas the
EDAR
C94 mutant is missing it entirely (Fig. 3A). These
results suggest that the death domain plays a crucial role in NF-
B
activation by EDAR. A complete lack of NF-
B activation was also
observed upon expression of mutants EDAR
C164 and EDAR
C223, which
are missing additional C-terminal sequences of the cytoplasmic domain
(Fig. 3, A and B).
View larger version (18K):
[in a new window]
Fig. 3.
Mutagenesis analysis of EDAR-induced
NF- B. A, a schematic
representation of the wild-type and mutant EDAR constructs. The
ligand-binding domain is shown in stripes, the transmembrane
domain in gray, and the death domain (DD) in
black. The approximate positions of the two point mutants
are shown by arrowheads. B, NF-
B activation by
various deletion and point mutants of EDAR. The experiment was
performed essentially as described for Fig. 1A. Western
analysis on the total cell lysate was used to confirm equivalent
expression of the various EDAR proteins. The values shown are averages
(mean ± S.E.) of a representative of two independent experiments,
in which each transfection was performed in duplicate. C,
dose response of NF-
B activation by wild-type EDAR
(EDAR.WT) and its two point mutants.
B pathway. The E379K mutation is an autosomal
recessive mutation in the death domain of murine EDAR and is
responsible for the spontaneous downlessJackson
phenotype, whereas the R420Q mutation has been detected in the death
domain of human EDAR in a family with autosomal dominant anhidrotic
ectodermal dysplasia (2, 3). We used site-directed mutagenesis to
generate the corresponding mutants of the human EDAR gene. As shown in
Fig. 3 (B and C), whereas the E379K mutant retained significant residual ability to activate the NF-
B pathway, the R420Q mutant demonstrated a more severe loss of this activity. These results suggest that the recessive phenotype of the E379K mutant
may be due to the need for two mutant alleles to significantly influence NF-
B signaling. We would like to further point out that,
in addition to the R420Q mutation, a nonsense mutation in the
cytoplasmic domain of EDAR has also been detected in a family with
autosomal dominant anhidrotic ectodermal dysplasia (2). This
mutation (R358ter) results in the production of a truncated protein
that is missing the C-terminal 90 amino acid residues and that closely
resembles the deletion mutant EDAR
C94, which failed to activate the
NF-
B pathway (Fig. 3, A and B). Taken together, the above results suggest that the impaired ability to
activate NF-
B may be a key determinant in the pathogenesis of
anhidrotic ectodermal dysplasia.
B
activation, different members of the TNFR family are also known to
activate the JNK pathway. Therefore, we tested the ability of EDAR to
activate this pathway using a luciferase-based c-Jun transcriptional
activation assay. In this assay, luciferase expression is driven by
JNK-mediated phosphorylation of the activation domain of transcription
factor c-Jun that is fused to the Gal4 DNA-binding domain. As shown in
Fig. 4A, expression of EDAR in
the 293 EBNA cells led to modest activation of the JNK pathway. In
contrast to the situation with NF-
B activation, the JNK-inducing
ability of EDAR was relatively weak as compared with that of TAJ/TROY.
Activation of the JNK pathway by EDAR was further confirmed using a
pull-down kinase assay based on in vitro phosphorylation of
GST-c-Jun (Fig. 4B).
View larger version (23K):
[in a new window]
Fig. 4.
JNK activation by EDAR. A,
EDAR mediates c-Jun transcriptional activation. 293 EBNA cells were
transfected with the indicated plasmids, and the c-Jun transcriptional
activation assay was performed as described under "Materials and
Methods." The values shown are representative of two independent
experiments, in which each transfection was performed in duplicate.
B, EDAR activates the JNK pathway. 293 EBNA cells (3 × 106) were transfected with the indicated plasmids, and JNK
activation was measured by a pull-down JNK assay kit. GST-c-Jun coupled
to agarose beads was used both to pull down the endogenously expressed
JNK and as a substrate for activated JNK-induced phosphorylation.
C, mutagenesis analysis of EDAR-induced c-Jun
transcriptional activation. The experiment was performed essentially as
described for A. D, dominant-negative TRAF2
(DN-TRAF2) fails to block EDAR-induced c-Jun transcriptional
activation. The amount of inhibitor plasmids (750 ng/well) was three
times the amount of test plasmids (250 ng/well), and the total amount
of transfected DNA was kept constant by adding empty vector. The values
shown are means ± S.E. of a representative of two independent
experiments performed in duplicate. E, JBD-JIP1 blocks
EDAR-induced c-Jun transcriptional activation. The experiment was
performed essentially as described for D.
C38 and EDAR
C94 deletion mutant
have some residual JNK activation ability, almost a complete lack of
this ability is present in deletion mutants EDAR
C164 and EDAR
C223
(Fig. 4C). Thus, the putative death domain of EDAR is
essential for both NF-
B and JNK activation.
B activation, the E379K mutant
was almost half as effective as the wild-type protein in JNK
activation, whereas a more severe impairment of JNK activation was seen
with the R420Q mutant.
View larger version (46K):
[in a new window]
Fig. 5.
EDAR induces cell death.
A, 293T cells (1.5 × 105) were
transfected with the indicated plasmids (500 ng) along with a
-galactosidase-encoding plasmid (75 ng). Cells were fixed and
stained with 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside 36 h post-transfection as
described previously (47). EDAR- and TNFR1-transfected cells have a
dark rounded appearance and condensed nuclei and are becoming detached
from the plate. However, unlike TNFR1-transfected cells,
EDAR-transfected cells lack membrane budding. B, MCF7 cells
were transfected with the indicated plasmids along with a
-galactosidase-encoding plasmid using Superfect (QIAGEN Inc.), and
the experiment was performed essentially as described for A. C, 293 EBNA cells (2 × 106) were
transfected with the indicated expression constructs (5 µg) along
with a green fluorescent protein-encoding plasmid (1 µg). Cells were
examined under a fluorescent microscope and photographed 36 h
after transfection. EDAR-transfected cells have a rounded appearance
and are detaching from the plate, whereas those cells transfected with
the EDAR
C164 mutant have a normal morphology. D, shown is
the absence of nuclear fragmentation during EDAR-induced cell death.
293T cells were transfected with an empty vector or vectors encoding
EDAR or TNFR1. Approximately 40 h later, cells were stained with
YOPRO-1, which stains dead cells that have lost membrane integrity.
TNFR1-transfected cells have fragmented nuclei, which are absent in
cells transfected with EDAR.
B
mutant or JBD-JIP1, suggesting the lack of involvement of the NF-
B
and JNK pathways in this process (data not shown).
View larger version (33K):
[in a new window]
Fig. 6.
Mechanism of EDAR-induced cell death.
A, synthetic caspase inhibitors fail to block TAJ-induced
cell death. 293T cells (2 × 105) were transfected
with an empty vector or the indicated receptor plasmids along with a
-galactosidase reporter plasmid in duplicate in each well of a
24-well plate. Twelve hours post-transfection, cells were treated with
Me2SO (DMSO), Z-VAD-fmk (20 µM),
or t-butoxycarbonyl-Asp-fmk (BOC-D-fmk; 50 µM). Approximately 36 h post-transfection, cells
were fixed and stained, and the percentage of apoptotic cells was
determined based on the criteria described for Fig. 5A. The
values shown are means ± S.E. of a representative of two
independent experiments performed in duplicate. B, virally
encoded caspase inhibitors fail to block EDAR-induced cell death. 293T
cells were transfected with the indicated plasmids, and the experiment
was performed essentially as described for A. The amount of
inhibitor plasmids (CrmA and p35) was three times the amount of
receptor plasmids, and the total amount of transfected DNA was kept
constant by adding empty vector. The values shown are means ± S.E. of a representative of two independent experiments performed in
duplicate. C, lack of inhibitory effect of dominant-negative
FADD (DN-FADD), caspase-8-C360S (CASP8 C360S),
MRIT/cFLIP, and MC159L on EDAR-induced cell death. The experiment was
performed essentially as described for B. D, EDAR
fails to activate caspase-3. 293T cells (2 × 106)
were transfected with the indicated plasmids (5 µg each). Cells were
lysed 36 h post-transfection, and 20 µl of the cellular extracts
were used for the measurement of caspase-3 activation as determined by
the cleavage of its chromogenic substrate
(Z-DEVD-p-nitroanilide) following the manufacturer's
instructions (Alexis). The values shown are means ± S.E. of a
representative of two independent experiments performed in duplicate.
E, Mutagenesis analysis of EDAR-induced cell death. The
experiment was performed essentially as described for
A.
C38 deletion mutant, which retains a
partial death domain, demonstrated significant residual ability to
induce cell death, whereas the EDAR
C94 mutant, which completely
lacks the death domain, possessed only a minor cell death-inducing
ability. However, a complete lack of this activity was present in the
deletion mutants lacking the death domain, such as EDAR
C164 and
EDAR
C223. Finally, the point mutants E379K and R420Q demonstrated
significant residual cell death-inducing ability.
View larger version (32K):
[in a new window]
Fig. 7.
EDAR coimmunoprecipitates TRAFs and NIK, but
fails to coimmunoprecipitate TRADD or FADD. 293T cells were
transfected with the indicated plasmids, and cell lysates
(L) were immunoprecipitated (I.P.) with FLAG
beads (F) or control mouse IgG beads (C).
Coimmunoprecipitated proteins were detected by Western analysis with
the indicated antibodies. A and B, lack of
interaction of EDAR with TRADD or FADD. C and D,
interactions of full-length EDAR (EDAR-FL) and its deletion
and point mutants with murine TRAF1 (mTRAF1). Western
analysis of total cell lysates was used to confirm equivalent
expression of various EDAR constructs. Lack of coimmunoprecipitation of
cotransfected hemagglutinin (HA)-tagged green fluorescent
protein (GFP) in C indicates the specificity of
the interaction. E-G, EDAR interacts with murine TRAF2,
TRAF3, and NIK. HVEM, herpes virus entry mediator.
B activation by various members of the TNFR family
(Fig. 7G). However, we have so far failed to detect an
interaction between a GST fusion protein containing the EDAR
cytoplasmic domain and in vitro transcribed and translated
TRAF2 or NIK (data not shown). Similarly, no interaction has been
detected between the cytoplasmic domain of EDAR and TRAF2 or NIK using
a yeast two-hybrid assay (data not shown). Collectively, the above
results suggest that the interaction between EDAR and TRAFs or NIK
might be facilitated by the presence of intermediate bridging proteins
present in the 293T cells.
View larger version (21K):
[in a new window]
Fig. 8.
EDA binds to EDAR. A, a
schematic representation of wild-type EDA-A1 and the Myc-tagged
baculovirus construct used in this study. TM, transmembrane
region; CR, collagenous repeat domain; Sig. Pep.,
signal peptide. B, EDAR-Fc coimmunoprecipitates Myc-EDA I. The experiment was performed as described under "Materials and
Methods." The blot was reprobed with a goat anti-mouse IgG
(G M) to demonstrate the expression and
immunoprecipitation of EDAR and murine TAJ immunoadhesin. mTAJ-Fc
comigrated with the heavy chain of the control antibody. S,
supernatant; C, control antibody beads;
M, goat anti-mouse IgG1 beads. C,
interaction of EDAR-Fc with Myc-EDA I using enzyme-linked immunosorbent
assay. The values shown are means ± S.E. of an experiment
performed in duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B
proteins are initially present in the cytoplasm of basal cells, but
later migrate to the nuclei of suprabasal cells, suggesting a role for
NF-
B activation in the switch from proliferation to growth arrest
and differentiation (25). This hypothesis is supported by the result of
a recent study involving a functional blockade of NF-
B via the
expression of a dominant-negative mutant of I
B
in transgenic
murine and human epidermis. This mutant resulted in the production of
hyperplastic epithelium due to increased thickness of the suprabasal
squamous layer (25). More recently, targeted disruption of the IKK1
gene has resulted in a similar phenotype (26-28). As the
IKK1-deficient keratinocytes exhibited near normal IKK activation in
response to a number of pro-inflammatory stimuli, such as TNF-
,
interleukin-1, and lipopolysaccharide, these results have led to the
suggestion that IKK1 is critical for I
B-dependent
activation of NF-
B in response to an as yet unidentified
developmental signal that triggers keratinocyte differentiation (26-28). In the present study, we demonstrate that EDAR-induced NF-
B activation is effectively blocked by a dominant-negative IKK1
mutant, suggesting that EDAR may be the missing developmental signal
required for keratinocyte differentiation. The key role of EDAR-induced
NF-
B activation in the process of ectodermal differentiation is also
supported by our results demonstrating the impaired ability of EDAR
mutants associated with anhidrotic ectodermal dysplasia to activate the
NF-
B pathway. However, these mutants also demonstrated impaired
ability to activate the JNK and cell death pathways. Thus, it is
conceivable that defects in EDAR-mediated JNK and cell death pathways
may also contribute to the clinical and pathological phenotype of
anhidrotic ectodermal dysplasia.
B activation could be blocked
by a dominant-negative mutant of IKK2. However, no obvious ectodermal
defect has been reported in IKK2-deficient animals (29, 30). These
results might be explained by the functionally redundant role of IKK2
in EDAR-induced NF-
B activation. Premature embryonic lethality of
the IKK2-deficient animals might have also prevented the manifestation
of the ectodermal defects in these animals (29, 30).
B, JNK, and cell death pathways. However, the
putative death domain of EDAR possesses only a weak sequence homology
to the classical death domains present in the known apoptosis-inducing
death receptors and does not interact with either TRADD or FADD.
Therefore, we tend to favor the hypothesis that the death domain of
EDAR may be involved in NF-
B, JNK, and cell death pathways by acting
as a more general protein recruitment domain, as has been suggested
recently (3, 31). Although EDAR could interact with various TRAF family
members and NIK in the coimmunoprecipitation assay in 293T cells, we
have so far failed to detect an interaction between these proteins in
mammalian cell-free systems, suggesting that the interaction between
EDAR and TRAFs or NIK might be facilitated by the presence of
intermediate bridging proteins. Future studies aimed at isolation of
the adaptor proteins that directly bind to the cytoplasmic domain of
EDAR will greatly enhance our understanding of EDAR signaling in the process of ectodermal differentiation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Richard Gaynor, David Han, Hiroyasu Nakano, Roger Davis, Gioacchino Natoli, Vishva Dixit, Edward Clark, and Melanie Cobb for various expression plasmids.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Grant P30 AR41940-09 from the National Institutes of Health and by a grant from the Howard Hughes Medical Institutes (to P. M. C.).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.
These authors contributed equally to this work.
§ To whom correspondence and reprint requests should be addressed: Hamon Center for Therapeutic Oncology Research, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8593. Tel.: 214-648-1837; Fax: 214-648-4940; E-mail: pchaud@mednet.swmed.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M008356200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
EDAR, ectodermal
dysplasia receptor;
TNF, tumor necrosis factor;
TNFR, TNF receptor;
NF-B, nuclear factor-
B;
JNK, c-Jun N-terminal kinase;
EDA, ectodysplasin A;
TRADD, TNFR1-associated death domain;
FADD, Fas-associated death domain;
Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone;
TAJ, toxicity
and JNK inducer;
NIK, NF-
B-inducing kinase;
IKK, I
B kinase;
TRAF, TNFR-associated factor;
I-TRAF, TRAF-interacting protein;
TANK, TRAF-associated NF-
B activator;
MEKK, mitogen-activated protein
kinase/extracellular signal-regulated kinase kinase kinase;
MAPK, mitogen-activated protein kinase;
GST, glutathione
S-transferase;
JIP1, JNK-interacting protein-1;
MRIT, Mach-related inducer of toxicity;
JBD, JNK-binding domain;
DR, death
receptor;
EBNA, Epstein-Barr nuclear antigen.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Freire-Maia, N., and Pinheiro, M. (1984) Ectodermal Dysplasias: A Clinical and Genetic Study , Alan R. Liss, Inc., New York |
2. | Monreal, A. W., Ferguson, B. M., Headon, D. J., Street, S. L., Overbeek, P. A., and Zonana, J. (1999) Nat. Genet. 22, 366-369[CrossRef][Medline] [Order article via Infotrieve] |
3. | Headon, D. J., and Overbeek, P. A. (1999) Nat. Genet. 22, 370-374[CrossRef][Medline] [Order article via Infotrieve] |
4. |
Ashkenazi, A.,
and Dixit, V. M.
(1998)
Science
281,
1305-1308 |
5. |
Natoli, G.,
Costanzo, A.,
Moretti, F.,
Fulco, M.,
Balsano, C.,
and Levrero, M.
(1997)
J. Biol. Chem.
272,
26079-26082 |
6. |
Nakano, H.,
Shindo, M.,
Sakon, S.,
Nishinaka, S.,
Mihara, M.,
Yagita, H.,
and Okumura, K.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3537-3542 |
7. | Brockman, J. A., Scherer, D. C., McKinsey, T. A., Hall, S. M., Qi, X., Lee, W. Y., and Ballard, D. W. (1995) Mol. Cell. Biol. 15, 2809-2818[Abstract] |
8. |
Berberich, I.,
Shu, G. L.,
and Clark, E. A.
(1994)
J. Immunol.
153,
4357-4366 |
9. | Chaudhary, P. M., Jasmin, A., Eby, M. T., and Hood, L. (1999) Oncogene 18, 5738-5746[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Chaudhary, P. M.,
Eby, M. T.,
Jasmin, A.,
and Hood, L.
(1999)
J. Biol. Chem.
274,
19211-19219 |
11. |
Eby, M. T.,
Jasmin, A.,
Kumar, A.,
Sharma, K.,
and Chaudhary, P. M.
(2000)
J. Biol. Chem.
275,
15336-15342 |
12. |
Kojima, T.,
Morikawa, Y.,
Copeland, N. G.,
Gilbert, D. J.,
Jenkins, N. A.,
Senba, E.,
and Kitamura, T.
(2000)
J. Biol. Chem.
275,
20742-20747 |
13. | Baker, S. J., and Reddy, E. P. (1996) Oncogene 12, 1-9[Medline] [Order article via Infotrieve] |
14. |
Takeuchi, M.,
Rothe, M.,
and Goeddel, D. V.
(1996)
J. Biol. Chem.
271,
19935-19942 |
15. |
Verma, I. M.,
and Stevenson, J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11758-11760 |
16. | Baldwin, A. S., Jr. (1996) Annu. Rev. Immunol. 14, 649-683[CrossRef][Medline] [Order article via Infotrieve] |
17. | Lee, S. Y., Reichlin, A., Santana, A., Sokol, K. A., Nussenzweig, M. C., and Choi, Y. (1997) Immunity 7, 703-713[Medline] [Order article via Infotrieve] |
18. | Yeh, W. C., Shahinian, A., Speiser, D., Kraunus, J., Billia, F., Wakeham, A., de la Pompa, J. L., Ferrick, D., Hum, B., Iscove, N., Ohashi, P., Rothe, M., Goeddel, D. V., and Mak, T. W. (1997) Immunity 7, 715-725[Medline] [Order article via Infotrieve] |
19. |
Dickens, M.,
Rogers, J. S.,
Cavanagh, J.,
Raitano, A.,
Xia, Z.,
Halpern, J. R.,
Greenberg, M. E.,
Sawyers, C. L.,
and Davis, R. J.
(1997)
Science
277,
693-696 |
20. |
Bayes, M.,
Hartung, A. J.,
Ezer, S.,
Pispa, J.,
Thesleff, I.,
Srivastava, A. K.,
and Kere, J.
(1998)
Hum. Mol. Genet.
7,
1661-1669 |
21. |
Ezer, S.,
Schlessinger, D.,
Srivastava, A.,
and Kere, J.
(1997)
Hum. Mol. Genet.
6,
1581-1587 |
22. | Salvesen, G. S., and Dixit, V. M. (1997) Cell 91, 443-446[Medline] [Order article via Infotrieve] |
23. | Kere, J., Srivastava, A. K., Montonen, O., Zonana, J., Thomas, N., Ferguson, B., Munoz, F., Morgan, D., Clarke, A., Baybayan, P., Chen, E. Y., Ezer, S., Saarialho-Kere, U., de la Chapelle, A., and Schlessinger, D. (1996) Nat. Genet. 13, 409-416[Medline] [Order article via Infotrieve] |
24. |
Srivastava, A. K.,
Pispa, J.,
Hartung, A. J.,
Du, Y.,
Ezer, S.,
Jenks, T.,
Shimada, T.,
Pekkanen, M.,
Mikkola, M. L.,
Ko, M. S.,
Thesleff, I.,
Kere, J.,
and Schlessinger, D.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13069-13074 |
25. |
Seitz, C. S.,
Lin, Q.,
Deng, H.,
and Khavari, P. A.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
2307-2312 |
26. |
Takeda, K.,
Takeuchi, O.,
Tsujimura, T.,
Itami, S.,
Adachi, O.,
Kawai, T.,
Sanjo, H.,
Yoshikawa, K.,
Terada, N.,
and Akira, S.
(1999)
Science
284,
313-316 |
27. |
Hu, Y.,
Baud, V.,
Delhase, M.,
Zhang, P.,
Deerinck, T.,
Ellisman, M.,
Johnson, R.,
and Karin, M.
(1999)
Science
284,
316-320 |
28. |
Li, Q.,
Lu, Q.,
Hwang, J. Y.,
Buscher, D.,
Lee, K. F.,
Izpisua-Belmonte, J. C.,
and Verma, I. M.
(1999)
Genes Dev.
13,
1322-1328 |
29. |
Li, Q.,
Van Antwerp, D.,
Mercurio, F.,
Lee, K. F.,
and Verma, I. M.
(1999)
Science
284,
321-325 |
30. |
Li, Z. W.,
Chu, W.,
Hu, Y.,
Delhase, M.,
Deerinck, T.,
Ellisman, M.,
Johnson, R.,
and Karin, M.
(1999)
J. Exp. Med.
189,
1839-1845 |
31. | Feinstein, E., Kimchi, A., Wallach, D., Boldin, M., and Varfolomeev, E. (1995) Trends Biochem. Sci. 20, 342-344[CrossRef][Medline] [Order article via Infotrieve] |
32. |
Kawahara, A.,
Ohsawa, Y.,
Matsumura, H.,
Uchiyama, Y.,
and Nagata, S.
(1998)
J. Cell Biol.
143,
1353-1360 |
33. |
Vercammen, D.,
Brouckaert, G.,
Denecker, G.,
Van de Craen, M.,
Declercq, W.,
Fiers, W.,
and Vandenabeele, P.
(1998)
J. Exp. Med.
188,
919-930 |
34. |
Vercammen, D.,
Beyaert, R.,
Denecker, G.,
Goossens, V.,
Van Loo, G.,
Declercq, W.,
Grooten, J.,
Fiers, W.,
and Vandenabeele, P.
(1998)
J. Exp. Med.
187,
1477-1485 |
35. | Lee, S. Y., Park, C. G., and Choi, Y. (1996) J. Exp. Med. 183, 669-674[Abstract] |
36. |
Force, W. R.,
Cheung, T. C.,
and Ware, C. F.
(1997)
J. Biol. Chem.
272,
30835-30840 |
37. | Frade, J. M., Rodriguez-Tebar, A., and Barde, Y. A. (1996) Nature 383, 166-168[CrossRef][Medline] [Order article via Infotrieve] |
38. | Rabizadeh, S., Oh, J., Zhong, L. T., Yang, J., Bitler, C. M., Butcher, L. L., and Bredesen, D. E. (1993) Science 261, 345-348[Medline] [Order article via Infotrieve] |
39. |
Vandenabeele, P.,
Declercq, W.,
Vanhaesebroeck, B.,
Grooten, J.,
and Fiers, W.
(1995)
J. Immunol.
154,
2904-2913 |
40. |
Xiang, J.,
Chao, D. T.,
and Korsmeyer, S. J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14559-14563 |
41. |
McCarthy, N. J.,
Whyte, M. K.,
Gilbert, C. S.,
and Evan, G. I.
(1997)
J. Cell Biol.
136,
215-227 |
42. |
Gross, A.,
Jockel, J.,
Wei, M. C.,
and Korsmeyer, S. J.
(1998)
EMBO J.
17,
3878-3885 |
43. |
Okuno, S.,
Shimizu, S.,
Ito, T.,
Nomura, M.,
Hamada, E.,
Tsujimoto, Y.,
and Matsuda, H.
(1998)
J. Biol. Chem.
273,
34272-34277 |
44. | Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Snow, B. E., Brothers, G. M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., Larochette, N., Goodlett, D. R., Aebersold, R., Siderovski, D. P., Penninger, J. M., and Kroemer, G. (1999) Nature 397, 441-446[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Ezer, S.,
Bayes, M.,
Elomaa, O.,
Schlessinger, D.,
and Kere, J.
(1999)
Hum. Mol. Genet.
8,
2079-2086 |
46. |
Ferguson, B. M.,
Brockdorff, N.,
Formstone, E.,
Ngyuen, T.,
Kronmiller, J. E.,
and Zonana, J.
(1997)
Hum. Mol. Genet.
6,
1589-1594 |
47. | Chaudhary, P. M., Eby, M., Jasmin, A., Bookwalter, A., Murray, J., and Hood, L. (1997) Immunity 7, 821-830[Medline] [Order article via Infotrieve] |