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J Biol Chem, Vol. 275, Issue 20, 15336-15342, May 19, 2000
From the We have isolated a novel member of the TNFR
family, designated TAJ, that is highly expressed during embryonic
development. TAJ possesses a unique cytoplasmic domain with no sequence
homology to the previously characterized members of the TNFR family.
TAJ interacts with the TRAF family members and activates the JNK
pathway when overexpressed in mammalian cells. Although it lacks a
death domain, TAJ is capable of inducing apoptosis by a
caspase-independent mechanism. Based on its unique expression profile
and signaling properties, TAJ may play an essential role in embryonic development.
Members of the
TNFR1 superfamily
play an important role in regulating diverse biological activities,
such as cell proliferation, differentiation, and programmed cell death
or apoptosis. The majority of TNFR family members are type I membrane
proteins that share significant sequence homology in their
extracellular domains (1). This homology is due to the presence of
highly conserved cysteine residues in so-called cysteine-rich
pseudo-repeats, a hallmark of this family. The cytoplasmic domains of
the various TNFR family members, on the other hand, differ greatly not
only in their sequence but also in their length. Some of the
apoptosis-inducing members of this family, such as TNFR1,
CD95/Fas/APO-1, DR3/TRAMP/APO-3, DR4/TRAIL/APO-2, and DR5/TRAIL-R2,
contain a conserved domain of approximately 80 amino acids, called a
death domain, which is essential for mediation of cell death (2-4).
Although death domains are absent in other members of the TNFR family,
some of these non-death domain-containing receptors, such as TNFR2,
CD30, and LT- In the present report, we describe the isolation and characterization
of a novel member of the TNFR family, designated TAJ (for
Toxicity And JNK inducer). We
present evidence that, based on its unique expression profile and
signaling activities, TAJ may be a key regulator of cell activation and
death during embryonic development.
Cell Lines and Reagents--
293T cells were obtained from Dr.
David Han (University of Washington, Seattle). 293EBNA cells were
obtained from Invitrogen. Rabbit polyclonal antibodies against FLAG and
HA tags were obtained from Santa Cruz Laboratories. FLAG beads were
obtained from Sigma. The pull-down kinase assay kit for JNK was
obtained from New England Biolabs, and the constructs for the
Pathdetect luciferase reporter assay were purchased from Stratagene.
YOPRO-1 was obtained from Molecular Probes.
Cloning of TAJ cDNA--
Two murine EST clones (IMAGE
consortium clones 650744 and 664665) encoding the extracellular domain
of a new member of the TNFR superfamily were identified by searching
the data base of expressed sequence tags (dbEST) for sequences sharing
homology to the extracellular domain of human DR3. The sequence
corresponding to the cytoplasmic domain of this receptor was obtained
by using 3'-rapid amplification of cDNA ends (RACE) on murine
spleen Marathon Ready cDNA (CLONTECH) using the
Marathon cDNA amplification kit (CLONTECH) and
following the manufacturer's instructions. A repeat search of the EST
data base for sequences with homology to the cytoplasmic tail of
murine TAJ revealed the presence of a human EST clone (IMAGE consortium
clone 340844). The sequence corresponding to the 5' end of human TAJ
(hTAJ) was obtained by performing 5'-RACE on human fetal spleen
Marathon-ready cDNA (CLONTECH).
Northern Blot Analysis--
Northern blot analysis was performed
using mouse embryo and human multiple tissue Northern blots
(CLONTECH). Blots were hybridized under high
stringency conditions with 32P-labeled probes corresponding
to the protein-coding regions of mTAJ and hTAJ and following the
instructions of the manufacturer.
Expression Vectors--
To construct FLAG or Myc epitope-tagged
receptors, the amino acids 23-424 of hTAJ were amplified using
Pfu polymerase (Stratagene) with a primer containing a
BglII site at the 5' end and a SalI site at the
3' end and then were ligated to a modified pSectag A vector
(Invitrogen) containing a FLAG or a Myc epitope downstream of a murine
Ig Reporter Assays--
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-cJun) (50 ng), a
reporter plasmid encoding the luciferase gene downstream of the GAL4
upstream activating sequence (pFR-luc) (500 ng), as well as a RSV/LacZ
( Caspase Activation Assay--
293T cells (2 × 105) were transfected with 2 µg of an empty vector or
expression vectors encoding TNFR1 or hTAJ along with a GFP-encoding
plasmid. Cells were examined under a fluorescent microscope 32 hours
later to ensure equal transfection efficiency. Cells were subsequently
lysed in 100 µl of lysis buffer (0.1% Triton X-100, 20 mM sodium phosphate, pH 7.4, 150 mM NaCl). For assaying caspase 3 activation, 10 µl of cell extract was mixed with
80 µl of assay buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 10 mM dithiothreitol, 1 mM EDTA, 10% glycerol) in a 96-well microtiter plate in
triplicate, and the was reaction started by the addition of 10 µl of
Ac-DEVD-pNA substrate. Caspase 3-mediated cleavage of
Ac-DEVD-pNA into p-nitroanilide was measured using a plate reader at 405 nm.
DNA Content Analysis--
293T cells (2 × 106)
were transfected with the various test plasmids along with a
GFP-encoding plasmid. Approximately 32 h post-transfection, cells
were examined under a fluorescent microscope to ensure equal
transfection efficiency. Cells were subsequently trypsinized, washed
once with PBS, and fixed in 50% ethanol at 4 °C. After washing with
PBS, cells were treated with 500 µg of RNase A (Sigma) in 100 µl of
PBS for 30 min at 37 °C and resuspended in 0.5 ml of PBS containing
50 µg/ml propidium iodide. After further incubation at 4 °C for 15 min, cells were analyzed by flow cytometry.
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 along with 1 µg of a
hemagglutinin-tagged GFP-encoding plasmid (HA-GFP) by calcium phosphate
coprecipitation. Eighteen to thirty-six 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 1 EDTA-free mini-protease inhibitor tablet per 10 ml (Roche Molecular
Biochemicals). For immunoprecipitation, cell lysates (500 µl) were
incubated for 1 h at 4 °C with 10 µl of FLAG or control mouse
Ig beads precoated with 2% bovine serum albumin. Beads were washed
twice with lysis buffer, twice with a wash buffer containing 0.1%
Triton X-100, 20 mM sodium phosphate, pH 7.4, 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 blot.
Cloning of Murine and Human TAJ cDNAs--
In order to
identify new members of the TNFR family, we searched the EST data base
(dbEST) for sequences with homology to the extracellular domain of
Death Receptor 3 (DR3) and identified two mouse cDNA clones. Both
clones originated from cDNA libraries made from 13.5- to 14.5-day
mouse embryos and, upon sequencing, were found to represent the
alternatively spliced forms of the same gene. One of the clones, named
mTAJ-
A repeat search of the EST data base for clones homologous to the
cytoplasmic tail of mTAJ led to the identification of a human EST clone
derived from an embryonic heart library constructed from a 19-week-old
embryo. Based on the sequence of this clone, primers were designed, and
5'-RACE was used to clone the full-length human TAJ (hTAJ) clone. The
full-length hTAJ clone encoded a protein of 423 amino acids having
68.4% amino acid identity and 79.2% amino acid similarity with
mTAJ- Expression of TAJ--
As discussed above, almost all the EST
clones encoding TAJ are derived from cDNA libraries originating
from embryonic tissues. To test further the expression of TAJ during
embryonic development, Northern analysis was performed on a multiple
tissue Northern blot containing poly(A) RNA from 7-, 11-, 15-, and
17-day mouse embryos. The protein-coding region of mTAJ-
Expression of TAJ in adult human tissues was studied by Northern
analysis using the protein-coding region of hTAJ cDNA as a probe.
Major expression was seen only in the prostate gland with only very low
expression seen in other tissues such as spleen, thymus, testis,
uterus, small intestine, colon, and peripheral blood leukocytes (Fig.
2). Expression of TAJ was also detected in 293 (human embryonic kidney)
and LNCaP (prostate cancer) cell lines (data not shown).
TAJ Activates the JNK Pathway--
Activation of the JNK and the
NF-
TAJ-induced JNK activation was successfully blocked by the JNK-binding
domain of JIP1 (15), a recently isolated specific inhibitor of the JNK
pathway (Fig. 3C). TRAF2 and its homologs have been shown to
play an essential role in the JNK activation by TNFR family members by
activating the protein kinase ASK1 (16). However, as shown in Fig.
3D, dominant-negative mutants of TRAF2 and ASK1 failed to
block JNK activation via TAJ, while successfully blocking TRAF2-induced
JNK activation.
We also investigated the ability of TAJ to activate the NF- TAJ Induces Caspase-independent Cell Death--
During the
course of investigating the above signaling functions of TAJ, we
noticed that it could also induce cell death. This result was somewhat
unexpected since TAJ does not possess a death domain. To characterize
further the death inducing property of TAJ, 293T cells were transfected
with a control plasmid, or plasmids containing TNFR1 or TAJ, along with
a reporter plasmid encoding
A comparison with TNFR1-transfected cells revealed several unique
features of TAJ-induced apoptosis. First, cell death induced by TAJ was
slightly delayed as compared with that induced by TNFR1 (32 versus 24 h). Second, whereas TAJ-transfected cells
showed cytoplasmic swelling and nuclear condensation, they demonstrated relatively little membrane budding as compared with the
TNFR1-transfected cells (Fig. 4A). Third, based on nuclear
staining with YOPRO-1, a cell-impermeant DNA-intercalating dye, we have
recognized differences in the nuclear morphology of cells undergoing
apoptosis in response to TAJ or TNFR1. As shown in Fig. 4C,
whereas the majority of the vector-transfected cells did not stain with
YOPRO-1, a large number of TAJ or TNFR1-transfected cells showed
nuclear staining with this dye reflecting the loss of membrane
integrity. However, unlike the TNFR1-transfected cells, those dying in
response to TAJ did not demonstrate nuclear fragmentation, a key
feature of caspase-induced cell death (Fig. 4C). Finally, as
compared with TNFR1-transfected cells, TAJ-transfected cells
demonstrated a complete absence of oligonucleosomal DNA fragmentation,
one of the essential features of caspase-induced apoptosis (Fig.
4D).
Absence of nuclear fragmentation in the TAJ transfected cells was
confirmed by DNA content analysis using flow cytometry. As shown in
Fig. 4E, transfection of TNFR1 in 293T cells is accompanied by the appearance of a hypodiploid population of cells, which is absent
in cells transfected with an empty vector or TAJ. Furthermore, there
was no significant difference in the cell cycle distribution of cells
transfected with TAJ or control vector, making it unlikely that TAJ
induces cell death via a block in cell cycle transition.
To characterize further TAJ-induced cell death, we tested the ability
of several known inhibitors of apoptosis to block cell death induced by
it. TAJ-induced cell death was not blocked by two virally encoded
caspase inhibitors, CrmA and p35, and a synthetic caspase inhibitor,
benzyloxycarbonyl-VAD-fmk, all of which successfully blocked
TNFR1-induced apoptosis (Fig.
5A and data not shown). Similarly, MRIT/cFLIP, Orf-K13, and dominant-negative inhibitors of
FADD and caspase 8 (caspase 8 C360S) had no effect on TAJ-induced cell
death (Fig. 5B). Finally, JBD-JIP1 failed to effectively block TAJ-induced cell death, ruling out a major role of the JNK pathway in TAJ-induced cell death (Fig. 5C).
Lack of activation of the caspase cascade during TAJ-induced cell death
was further supported by a chromogenic assay based on caspase
3-mediated cleavage of the chromogenic substrate, pNA-DEVD. Caspase 3 is one of the executioner caspases of the caspase cascade and
is activated during death receptors-induced apoptosis (17). As shown in
Fig. 5D, cell lysates from cells transfected with TNFR1
demonstrated caspase 3 activation, whereas TAJ-transfected cells failed
to do so.
TAJ Interacts with TRAF Family Members--
TRAF family
members have been previously implicated in the signal transduction by
various TNFR family members (18). To test the involvement of TRAFs in
the signaling via TAJ, we tested their ability to interact with each
other using a coimmunoprecipitation assay in 293T cells. As shown in
Fig. 6, TAJ successfully
coimmunoprecipitated with TRAF1, TRAF2, TRAF3, and TRAF5 in the above
assay. However, consistent with the absence of a death domain in its
cytoplasmic tail, TAJ failed to interact with FADD (Fig. 6).
Programmed cell death or apoptosis plays an important role
in the development and morphogenesis of multicellular organisms by controlling cell number and removing mutated or defective cells (19). Despite the recent progress in the identification of downstream effector molecules involved in developmental cell death (20), the
cell surface receptors regulating this process remain to be identified.
Members of the TNFR family are well known for their role in the
mediation of cell death in adult tissues. Based on its unique
expression profile during development, TAJ may play a similar role
during embryogenesis.
In addition to its full-length isoform, we have isolated two
alternative spliced forms of murine TAJ that are likely to act as decoy
and soluble receptors, respectively. Such receptors have been
previously identified for other members of the TNFR family and shown to
block signaling via the full-length receptors (14, 21, 22). It remains
to be seen whether the mTAJ- Despite sharing significant sequence homology with TNFR family members
in the extracellular ligand-binding domain, TAJ possesses a unique
cytoplasmic domain, which suggests that it utilizes novel signal
transduction pathways for the activation of JNK and cell death
pathways. Consistent with this hypothesis, TAJ-induced JNK activation
was not blocked by dominant-negative inhibitors of TRAF2, TRAF5, or
ASK1, which have been previously implicated in JNK activation via TNFR1
and CD40. However, coimmunoprecipitation assays revealed that TAJ is
capable of binding a number of different TRAF family members, and it is
possible that TAJ-induced JNK activation is mediated by an as yet
untested TRAF homolog, such as TRAF6. Similarly, in addition to ASK1,
alternative pathways for JNK activation via TNFR family members have
been described (23-25), and it remains to be seen whether TAJ utilizes
one of these alternative pathways.
A surprising result of this study was the ability of TAJ to induce cell
death since it does not possess a death domain. Our results indicate
that TAJ induces cell death by a mechanism independent of the classical
death domain receptor-FADD-caspase 8/10 pathway. Although,
caspase-mediated cell death is the best characterized form of
apoptosis, several caspase-independent forms of death are also known in
the literature. For example, FADD, in addition to its role in the
activation of caspases, can also induce caspase-independent cell death
(26). This death is resistant to caspase inhibitors and is accompanied
by cellular swelling without apoptotic bodies or fragmentation of
chromatin (26). Similar forms of caspase-independent death have been
previously reported for both Fas and TNFR1 as well (27, 28). Several
non-death domain-containing members of the TNFR family have been shown
to induce cell death under certain conditions (5-7, 29, 30), and it
remains to be seen whether TAJ shares a common caspase-independent
mechanism of cell death induction with these receptors. Several
additional examples of caspase-independent cell death have been
recently reported as well (31-37).
There are several potential mediators of caspase-independent cell death
via TAJ. First, Bax and Bax-like proteins have been known to kill
mammalian cells even in the presence of caspase inhibitors, provoking
chromatin condensation and membrane alterations but without caspase
activation or DNA degradation (38-40). It has been proposed that Bax
and Bax-like proteins might mediate caspase-independent death via their
channel forming ability that could promote mitochondrial permeability
transition or puncture the outer mitochondrial membrane (41). Second,
nitric oxide, like Bax, also induces cell death accompanied by
chromatin condensation, nuclear compaction, and mitochondrial swelling
but without caspase activation or DNA fragmentation (42). The recent
isolation of apoptosis-inducing factor may provide an additional
mechanism for caspase-independent cell death induction by TAJ (43, 44).
Apoptosis-inducing factor is a flavoprotein of approximately 57 kDa
that is normally confined to mitochondrial intermembrane space but
translocates to the nucleus when apoptosis is induced.
Apoptosis-inducing factor can induce chromatin condensation in isolated
nuclei, dissipation of mitochondrial The ligand for TAJ has not been identified yet. Recently, two novel
ligands belonging to the TNF family, designated APRIL (45) and
THANK/BlyS (46, 47), respectively, have been identified. Besides
Blys/THANK, TAJ could also be a receptor for VEGI, another member of
the tumor necrosis factor family for which receptor has not been
identified. Studies are in progress to test whether one of these
represents the ligand for TAJ.
We thank Drs. Vishva Dixit, Roger Davis,
Audrey Minden, Richard Gaynor, Melanie Cobb, Yukiko Gotoh, Michael
Wright, and David Han for various plasmids.
*
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF167552-AF167555.
¶
To whom correspondence and reprint requests should be
addressed: The Hamon Center for Therapeutic Oncology Research,
University of Texas 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.
The abbreviations used are:
TNFR, tumor necrosis
factor receptor;
DR3, death receptor 3;
EST, expressed sequence tag;
MRIT, Mach-related inducer of toxicity;
TRAF, tumor necrosis
factor-associated factor;
JNK, c-Jun N-terminal kinase;
NF-
TAJ, a Novel Member of the Tumor Necrosis Factor Receptor Family,
Activates the c-Jun N-terminal Kinase Pathway and Mediates
Caspase-independent Cell Death*
,,,¶
Hamon Center for Therapeutic Oncology
Research, University of Texas Southwestern Medical Center,
Dallas, Texas 75390-8593 and the § University of
Washington, Seattle, Washington 98195
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
R, are nevertheless capable of inducing cell death
under certain circumstances (5-7).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
chain signal peptide. Expression constructs for dominant-negative FADD, caspase 8 C360S, CrmA, p35, MRIT, Orf-K13, and
TRAFs have been described previously (8-10).
-galactosidase) reporter construct (75 ng). Transfection was
performed using calcium phosphate coprecipitation method. Forty hours
later cell extracts were prepared using the Luciferase Cell Culture
Lysis Reagent (Promega, Madison, WI), and luciferase assays were
performed using 20 µl of cell extract. The cell lysate was diluted
1:20 with phosphate-buffered saline, pH 7.4, and used for the
measurement of -galactosidase activity. Luciferase activity was
normalized relative to the -galactosidase activity to control for
the difference in the transfection efficiency. Western blot analysis on
cell extracts prepared from similarly transfected cells was used to
confirm that the various expression constructs lead to the production
of the desired proteins. The NF-B reporter assay was performed
essentially as described above using a luciferase reporter plasmid
containing four copies of a consensus NF-B binding site (11).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S, was found to encode an open reading frame of 214 amino acids
(Fig. 1A). Sequence analysis of this clone revealed the presence of an N-terminal signal peptide (amino acids 1-23), cysteine-rich pseudo-repeats with significant sequence homology to the extracellular domain of other members of the
TNFR family (20-25% sequence identity and 35-46% sequence similarity), a hydrophobic stretch of amino acids representing the
transmembrane region (amino acids 171-193), and a short cytoplasmic tail (Fig. 1A). Based on the presence of the short
cytoplasmic tail, this clone may encode for a decoy receptor. The
sequence of the second murine clone, named mTAJ-, was identical to
mTAJ-S in the N-terminal 149 amino acids. This region contains its
signal peptide, and the cysteine-rich pseudo-repeats representing the majority of the ligand-binding domain (Fig. 1A). However,
mTAJ- has a stop codon after amino acid 150 and, therefore, may
represent a soluble receptor lacking a transmembrane domain (Fig.
1A). Based on the sequence of mTAJ-S, primers were
designed and used in 3'-RACE to clone the sequence representing the
cytoplasmic tail of TAJ. The full-length cDNA clone, named
mTAJ-L, was found to contain an open reading frame of 416 amino
acids with a unique cytoplasmic domain having no significant homology
to any other member of the TNFR family (Fig. 1A).
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Fig. 1.
A, amino acid sequence of murine and
human TAJ isoforms. The signal peptides and transmembrane domains are
shown in bold and italics, respectively.
B, sequence alignment of the extracellular domains of TNFR
family members. Identical amino acids are shaded dark and
homologous residues shaded gray.
L (Fig. 1A). In addition, hTAJ was found to have
significant sequence similarity to other members of the TNFR family in
its extracellular ligand-binding domain (Fig. 1B).
L cDNA
was used as a probe. A strong signal of 4.4 kilobase pairs was detected
in lanes containing RNA from day 11, 15, and 17 embryos indicating the
expression of TAJ during embryonic development (Fig.
2).
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Fig. 2.
Northern blot analysis of TAJ expression
during murine embryonic development and human adult tissues.
kb, kilobase pairs; PBL, peripheral blood
leukocytes.
B pathways is a common feature of the TNFR family members.
Previous studies have also shown that members of this family can be
activated in a ligand-independent fashion upon transient
transfection-based overexpression (8, 12-14). Therefore, the ability
of TAJ to activate the JNK pathway upon transient overexpression in
293EBNA cells was tested using a luciferase-based c-Jun transcriptional
assay. In this assay, luciferase expression is driven by JNK-mediated
phosphorylation of the activation domain of the transcription factor
c-Jun that is fused to the GAL4 DNA-binding domain. TAJ could strongly
activate the JNK pathway in these cells which was comparable in
magnitude to that observed with CD40 (Fig.
3A). The ability of TAJ to
activate the JNK pathway was also confirmed by a "pull-down" kinase
assay based on in vitro phosphorylation of GST-cJun (Fig.
3B).
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Fig. 3.
A, TAJ mediates c-Jun transcriptional
activation. 293 EBNA cells were transfected with an empty vector or
expression vectors encoding human TAJ or CD40 receptors along with
pFA-cJun, pFR-luc, and RSV/LacZ encoding plasmids in duplicate. The
experiment was performed as described under "Experimental
Procedures." Values shown are mean ± S.E. of a representative
of two independent experiments performed in duplicate. B,
TAJ activates JNK pathway. 293EBNA cells (3 × 106)
were transfected with the indicated plasmid and JNK activation measured
by a "pull-down" JNK assay kit (New England Biolabs). GST-c-Jun
coupled to agarose beads was used to both pulling down the endogenously
expressed JNK and as a substrate for activated JNK-induced
phosphorylation. A representative of two independent experiments.
C, JBD-JIP1 blocks TAJ-induced JNK activation. 293EBNA cells
(1.2 × 105) were transfected with an empty vector or
an hTAJ expression vector (250 ng/well) with and without JBD-JIP1 (500 ng/well) in each well of a 24-well plate in duplicate. The total amount
of transfected DNA was kept constant by adding empty vector. The
experiment was performed as described for A. Values shown
are mean ± S.E. of a representative of two independent
experiments performed in duplicate. D, dominant-negative
mutants of TRAF2 and ASK1 fail to block TAJ-induced JNK activation. The
experiment was performed essentially as described for C.
Values shown are mean ± S.E. of a representative of two
independent experiments performed in duplicate.
B
pathway. However, transient transfection of TAJ led to only a weak
activation of NF-B in 293EBNA cells and completely failed to
activate this pathway in 293T or MCF7 cells (not shown).
-galactosidase or the green fluorescent
protein (GFP). Approximately 32 h post-transfection,
TAJ-transfected cells became rounded and started to detach from the
plate (Fig. 4, A and
B). In addition to 293T cells, TAJ also induced cell death
in 293EBNA cells. However, we have so far failed to observe significant
TAJ-induced apoptosis in COS cells, which might reflect the tissue or
species specificity of this response.
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Fig. 4.
A, TAJ induces cell death. 293T cells
(2 × 106) were transfected with the indicated
plasmids (5 µg) along with a -galactosidase-encoding plasmid (2 µg). Cells were fixed and stained with
5-bromo-4-chloro-3-indolyl--D-galactopyranoside 36 h post-transfection as described previously (8). Dying cells have a
dark rounded appearance and are becoming detached from the plate.
B, 293T cells (2 × 106) were transfected
with a control vector or a TAJ expression vector along with a
GFP-encoding vector. Thirty six hours after transfection, cells were
examined under a fluorescent microscope and photographed.
TAJ-transfected cells have a rounded appearance and are becoming
detached from the plate. C, absence of nuclear fragmentation
during TAJ-induced cell death. 293T cells were transfected with an
empty vector or vectors encoding TNFR1 or TAJ. Approximately
40 h later cells were stained with YOPRO-1, which stains only
those cells that have lost membrane integrity. Fragmented nuclei of
TNFR1-transfected cells are shown with arrowheads.
Insets show nuclear morphology of TNFR1- and TAJ-transfected
cells under higher magnification. D, cell death induced by
TAJ is not accompanied by oligonucleosomal DNA fragmentation. 293T
cells (2 × 106) were transfected with the indicated
plasmids (5 µg), and DNA fragmentation assay was performed after
38 h essentially as described (48). E, DNA content
frequency distribution. Cell death induced by TNFR1 is accompanied by
an increase in the hypodiploid cell population (<2n) that
is absent in the TAJ-transfected cells. The percentage of cells in
various stages of cell cycle is also shown. DNA content analysis was
performed using flow cytometry on ethanol-permeabilized cells stained
with propidium iodide. The transfection efficiency of the tested
plasmids, as determined by the expression of cotransfected GFP, ranged
from 40 to 50%.
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Fig. 5.
A, 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. 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.
Cells were fixed, stained, and percentage of apoptotic cells determined based on criteria described in Fig.
4A. Values shown are mean ± S.E. of a representative
of two independent experiments performed in duplicate. B,
lack of inhibitory effect of dominant-negative FADD, caspase 8 C360S,
MRIT/cFLIP, and Orf-K13 on TAJ-induced cell death. The experiment was
performed essentially as described for A. C,
JBD-JIP1 fails to block TAJ-induced cell death. The experiment was
performed essentially as described for A. Values shown are
mean ± S.E. of a representative of two independent experiments
performed in duplicate. D, TAJ fails to activate caspase 3. 293T (2 × 106) cells were transfected with the
indicated plasmids (5 µg each). Cells were lysed 36 h
post-transfection and 10 µl of cellular extracts used for the
measurement of caspase 3 activation as described under "Experimental
Procedures." Values shown are mean ± S.E. of a representative
of two independent experiments performed in duplicate or
triplicate.
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Fig. 6.
TAJ coimmunoprecipitates TRAFs but fails to
coimmunoprecipitate FADD. 293T cells were transfected with the
indicated plasmids and cell lysates (L) immunoprecipitated
with FLAG (F) beads or control beads (C).
Coimmunoprecipitated proteins were detected by Western analysis with
the indicated antibodies. Lack of immunoprecipitation of cotransfected
GFP-HA demonstrates the specificity of the interactions. The
lower band in the right-most panel corresponds to
the mouse Ig light chain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S and mTAJ- isoforms play a similar
role in regulating signaling via the full-length TAJ receptor.
m, and exposure of
phosphatidylserine in the plasma membrane. None of these effects are
blocked by treatment with broad-spectrum caspase inhibitor
benzyloxycarbonyl-VAD-fluoromethyl ketone (43, 44). We are currently
testing the contribution of the above mediators to TAJ-induced cell death.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
B, nuclear factor kappa B;
JIP1, JNK interacting protein 1;
JBD, JNK
binding domain;
GFP, green fluorescent protein;
, ASK1, apoptosis
signal-regulating kinase 1;
HA, hemagglutinin;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
PBS, phosphate-buffered saline;
RACE, rapid amplification of cDNA ends;
hTAJ, human TAJ;
pNA, p-nitroanilide;
GST, glutathione S-transferase;
FADD, Fas-associated death
domain.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Gruss, H. J.,
and Dower, S. K.
(1995)
Blood
85,
3378-3404 2.
Tartaglia, L. A.,
Ayres, T. M.,
Wong, G. H.,
and Goeddel, D. V.
(1993)
Cell
74,
845-853[CrossRef][Medline]
[Order article via Infotrieve]
3.
Itoh, N.,
and Nagata, S.
(1993)
J. Biol. Chem.
268,
10932-10937 4.
Ashkenazi, A.,
and Dixit, V. M.
(1998)
Science
281,
1305-1308 5.
Force, W. R.,
Cheung, T. C.,
and Ware, C. F.
(1997)
J. Biol. Chem.
272,
30835-30840 6.
Vandenabeele, P.,
Declercq, W.,
Vanhaesebroeck, B.,
Grooten, J.,
and Fiers, W.
(1995)
J. Immunol.
154,
2904-2913[Abstract]
7.
Lee, S. Y.,
Park, C. G.,
and Choi, Y.
(1996)
J. Exp. Med.
183,
669-674 8.
Chaudhary, P. M.,
Eby, M.,
Jasmin, A.,
Bookwalter, A.,
Murray, J.,
and Hood, L.
(1997)
Immunity
7,
821-830[CrossRef][Medline]
[Order article via Infotrieve]
9.
Chaudhary, P. M.,
Eby, M. T.,
Jasmin, A.,
and Hood, L.
(1999)
J. Biol. Chem.
274,
19211-19219 10.
Chaudhary, P. M.,
Eby, M. T.,
Jasmin, A.,
and Hood, L.
(1999)
Oncogene
18,
5738-5746[CrossRef][Medline]
[Order article via Infotrieve]
11.
Berberich, I.,
Shu, G. L.,
and Clark, E. A.
(1994)
J. Immunol.
153,
4357-4366[Abstract]
12.
Chinnaiyan, A. M.,
O'Rourke, K., Yu, G. L.,
Lyons, R. H.,
Garg, M.,
Duan, D. R.,
Xing, L.,
Gentz, R.,
Ni, J.,
and Dixit, V. M.
(1996)
Science
274,
990-992 13.
Pan, G.,
O'Rourke, K.,
Chinnaiyan, A. M.,
Gentz, R.,
Ebner, R.,
Ni, J.,
and Dixit, V. M.
(1997)
Science
276,
111-113 14.
Pan, G.,
Ni, J.,
Wei, Y. F., Yu, G.,
Gentz, R.,
and Dixit, V. M.
(1997)
Science
277,
815-818 15.
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 16.
Nishitoh, H.,
Saitoh, M.,
Mochida, Y.,
Takeda, K.,
Nakano, H.,
Rothe, M.,
Miyazono, K.,
and Ichijo, H.
(1998)
Mol. Cell
2,
389-395[CrossRef][Medline]
[Order article via Infotrieve]
17.
Salvesen, G. S.,
and Dixit, V. M.
(1997)
Cell
91,
443-446[CrossRef][Medline]
[Order article via Infotrieve]
18.
Darnay, B. G.,
and Aggarwal, B. B.
(1997)
J. Leukocyte Biol.
61,
559-566[Abstract]
19.
Vaux, D. L.,
and Korsmeyer, S. J.
(1999)
Cell
96,
245-254[CrossRef][Medline]
[Order article via Infotrieve]
20.
Los, M.,
Wesselborg, S.,
and Schulze-Osthoff, K.
(1999)
Immunity
10,
629-639[CrossRef][Medline]
[Order article via Infotrieve]
21.
Sheridan, J. P.,
Marsters, S. A.,
Pitti, R. M.,
Gurney, A.,
Skubatch, M.,
Baldwin, D.,
Ramakrishnan, L.,
Gray, C. L.,
Baker, K.,
Wood, W. I.,
Goddard, A. D.,
Godowski, P.,
and Ashkenazi, A.
(1997)
Science
277,
818-821 22.
Pitti, R. M.,
Marsters, S. A.,
Lawrence, D. A.,
Roy, M.,
Kischkel, F. C.,
Dowd, P.,
Huang, A.,
Donahue, C. J.,
Sherwood, S. W.,
Baldwin, D. T.,
Godowski, P. J.,
Wood, W. I.,
Gurney, A. L.,
Hillan, K. J.,
Cohen, R. L.,
Goddard, A. D.,
Botstein, D.,
and Ashkenazi, A.
(1998)
Nature
396,
699-703[CrossRef][Medline]
[Order article via Infotrieve]
23.
Yuasa, T.,
Ohno, S.,
Kehrl, J. H.,
and Kyriakis, J. M.
(1998)
J. Biol. Chem.
273,
22681-22692 24.
Liu, Z. G.,
Hsu, H.,
Goeddel, D. V.,
and Karin, M.
(1996)
Cell
87,
565-576[CrossRef][Medline]
[Order article via Infotrieve]
25.
Mathias, S.,
Pena, L. A.,
and Kolesnick, R. N.
(1998)
Biochem. J.
335,
465-480
26.
Kawahara, A.,
Ohsawa, Y.,
Matsumura, H.,
Uchiyama, Y.,
and Nagata, S.
(1998)
J. Cell Biol.
143,
1353-1360 27.
Vercammen, D.,
Brouckaert, G.,
Denecker, G.,
Van de Craen, M.,
Declercq, W.,
Fiers, W.,
and Vandenabeele, P.
(1998)
J. Exp. Med.
188,
919-930 28.
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 29.
Frade, J. M.,
Rodriguez-Tebar, A.,
and Barde, Y. A.
(1996)
Nature
383,
166-168[CrossRef][Medline]
[Order article via Infotrieve]
30.
Rabizadeh, S.,
Oh, J.,
Zhong, L. T.,
Yang, J.,
Bitler, C. M.,
Butcher, L. L.,
and Bredesen, D. E.
(1993)
Science
261,
345-348 31.
Pettersen, R. D.,
Gaudernack, G.,
Olafsen, M. K.,
Lie, S. O.,
and Hestdal, K.
(1998)
J. Immunol.
160,
4343-4352 32.
Drenou, B.,
Blancheteau, V.,
Burgess, D. H.,
Fauchet, R.,
Charron, D. J.,
and Mooney, N. A.
(1999)
J. Immunol.
163,
4115-4124 33.
Deas, O.,
Dumont, C.,
MacFarlane, M.,
Rouleau, M.,
Hebib, C.,
Harper, F.,
Hirsch, F.,
Charpentier, B.,
Cohen, G. M.,
and Senik, A.
(1998)
J. Immunol.
161,
3375-3383 34.
Maroney, A. C.,
Finn, J. P.,
Bozyczko-Coyne, D., TM, O. K.,
Neff, N. T.,
Tolkovsky, A. M.,
Park, D. S.,
Yan, C. Y.,
Troy, C. M.,
and Greene, L. A.
(1999)
J. Neurochem.
73,
1901-1912[Medline]
[Order article via Infotrieve]
35.
Stefanis, L.,
Park, D. S.,
Friedman, W. J.,
and Greene, L. A.
(1999)
J. Neurosci.
19,
6235-6247 36.
Belaud-Rotureau, M. A.,
Lacombe, F.,
Durrieu, F.,
Vial, J. P.,
Lacoste, L.,
Bernard, P.,
and Belloc, F.
(1999)
Cell Death Differ.
6,
788-795[CrossRef][Medline]
[Order article via Infotrieve]
37.
Heibein, J. A.,
Barry, M.,
Motyka, B.,
and Bleackley, R. C.
(1999)
J. Immunol.
163,
4683-4693 38.
Xiang, J.,
Chao, D. T.,
and Korsmeyer, S. J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
14559-14563 39.
McCarthy, N. J.,
Whyte, M. K.,
Gilbert, C. S.,
and Evan, G. I.
(1997)
J. Cell Biol.
136,
215-227 40.
Gross, A.,
Jockel, J.,
Wei, M. C.,
and Korsmeyer, S. J.
(1998)
EMBO J.
17,
3878-3885[CrossRef][Medline]
[Order article via Infotrieve]
41.
Adams, J. M.,
and Cory, S.
(1998)
Science
281,
1322-1326 42.
Okuno, S.,
Shimizu, S.,
Ito, T.,
Nomura, M.,
Hamada, E.,
Tsujimoto, Y.,
and Matsuda, H.
(1998)
J. Biol. Chem.
273,
34272-34277 43.
Lorenzo, H. K.,
Susin, S. A.,
Penninger, J.,
and Kroemer, G.
(1999)
Cell Death Differ.
6,
516-524[CrossRef][Medline]
[Order article via Infotrieve]
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.
Hahne, M.,
Kataoka, T.,
Schroter, M.,
Hofmann, K.,
Irmler, M.,
Bodmer, J. L.,
Schneider, P.,
Bornand, T.,
Holler, N.,
French, L. E.,
Sordat, B.,
Rimoldi, D.,
and Tschopp, J.
(1998)
J. Exp. Med.
188,
1185-1190 46.
Mukhopadhyay, A.,
Ni, J.,
Zhai, Y., Yu, G. L.,
and Aggarwal, B. B.
(1999)
J. Biol. Chem.
274,
15978-15981 47.
Moore, P. A.,
Belvedere, O.,
Orr, A.,
Pieri, K.,
LaFleur, D. W.,
Feng, P.,
Soppet, D.,
Charters, M.,
Gentz, R.,
Parmelee, D.,
Li, Y.,
Galperina, O.,
Giri, J.,
Roschke, V.,
Nardelli, B.,
Carrell, J.,
Sosnovtseva, S.,
Greenfield, W.,
Ruben, S. M.,
Olsen, H. S.,
Fikes, J.,
and Hilbert, D. M.
(1999)
Science
285,
260-263 48.
Hsu, H.,
Xiong, J.,
and Goeddel, D. V.
(1995)
Cell
81,
495-504[CrossRef][Medline]
[Order article via Infotrieve]
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