From the Department of Life Science, Kwangju Institute of Science and Technology, Puk-gu, Kwangju 500-712 and the § Division of Molecular Life Science, Ewha University, Seoul 120-750, Korea
Received for publication, April 3, 2001, and in revised form, May 4, 2001
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ABSTRACT |
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FLASH is a protein recently shown to interact
with the death effector domain of caspase-8 and is likely to be a
component of the death-inducing signaling complex in receptor-mediated
apoptosis. Here we show that antisense oligonucleotide-induced
inhibition of FLASH expression abolished TNF- TNF- In contrast, with respect to stress signaling and immune response,
TNF-R interacts with TRAFs and RIP, leading to the activation of
NF- It has recently been reported that FLASH is likely to be a component of
DISC involved in Fas- and TNF-mediated apoptosis (26). FLASH
contains a death-effector-domain-recruiting domain (DRD) in the
C-terminal region, which interacts with the death effector domain (DED)
of caspase-8 or FADD (26). Still, transient overexpression of FLASH
marginally affects apoptosis (26, 27), making its precise function with
respect to receptor-mediated signaling (e.g. via TNF-R)
unclear. In addition, the finding that caspase-8 and FADD may be
involved in the signaling to NF- Reagents--
Anti-I Construction of Recombinant Expression Plasmids--
pME18S-FLAG
and pME18S-FLAG-FLASH were kindly provided by Dr. Yonehara (University
of Kyoto, Japan). pHA-FLASH was generated by subcloning the FLASH
cDNA into the EcoRI/XbaI sites of
pcDNA-HA plasmid. FLASH deletion constructs were assembled by
polymerase chain reaction (PCR) using the following synthetic
oligonucleotides as primers: 5'-CCGGAATTCATGGCAGATGATGACAATGGT-3' and
5'-ATAAGAATGCGGCCGCCTAGCTCTCCATGCTAACAACT-3' for
pME18S-FLAG- Cell Culture, Stable Cells, and DNA Transfection--
HEK293 and
Jurkat cells were cultured in Dulbecco's modified Eagle's medium and
RPMI 1640, respectively, supplemented with 10% fetal bovine serum
(BIOFLUIDS). Cells were subcultured to a density of 2 × 105 cells/well in 6-well dishes and allowed to stabilize
for 1 day. Cells were then typically transfected with 600 ng of
NF- Antisense Oligonucleotide Treatment--
AS-2
(5'-ATTCAGCAACTTACTTGC-3') is an antisense oligonucleotide
complementary to human FLASH mRNA and corresponds to a location around the stop codon, 5942-5959 bp downstream of the translation initiation site. Comparison of this oligonucleotide sequence with the
database detected the only homology to the FLASH sequence. The
following scrambled sequence was used as a control:
(5'-GCTACTAGTAGCAGCTAC-3'). Cells (3 × 106 per well)
were continuously treated with 5 µM FLASH antisense or
the scrambled oligonucleotide for 48 h in culture medium
containing LipofectAMINE reagent.
RNA Isolation and RT-PCR--
Total RNA was isolated from HEK293
cells using TRIzol reagent (Life Technologies, Inc.). RT-PCR was
performed for quantification of FLASH mRNA using Luciferase and Generation of Anti-FLASH Antibody and Western Blot
Analysis--
GST-DRD-FLASH fusion proteins were expressed in
BL21(DE3) by addition of 0.2 mM
isopropyl- In Vitro Binding Assay--
The expression of GST fusion
proteins in BL21 (DE3) harboring pGEX-4T, pGEX-DRD-FLASH, or
pGEX-NAD-FLASH was induced with 0.2 mM
isopropyl- Immunoprecipitation--
HEK293 cells were transfected with
pHA-FLASH, pHA- Suppression of TNF-
Because we could detect exogenous FLASH expression (Fig.
2B) but failed to detect
expression of endogenous FLASH with Western blot analysis using
anti-FLASH antibody, the effects of AS-2 or the scrambled
oligonucleotide on FLASH expression were assessed by RT-PCR (Fig.
1C). RT-PCR and Northern blot analysis have both been used
to examine the effects of antisense on gene expression (32). FLASH
mRNA was undetectable in HEK293 cells treated with AS-2, whereas
tubulin expression was unaffected, and readily detectable in cells
treated with the scrambled oligonucleotide, indicating that FLASH
expression was reduced by AS-2 treatment. These results suggest that
FLASH is involved in TNF- Activation of NF-
FLASH contains DRD at its C terminus and a putative oligomerization
domain at its N terminus (Fig.
3A) (26). To ascertain which
of these mediates induction of NF-
Because it is known that TNF- FLASH Signals NF- FLASH Interacts with TRAF2 in Vitro and in Vivo through
NAD--
That FLASH interacts with TRAF2 was then examined with
in vitro binding assay and immunoprecipitation. GST
pull-down assay showed that GST-NAD fusion protein could bind to TRAF2,
whereas GST-DRD interacted only with caspase-8, indicating that NAD of FLASH specifically interacts with TRAF2 in vitro (Fig.
5). Immunoprecipitation assays were then
performed to examine cellular interaction of FLASH and TRAF2 in Jurkat,
HEK 293, and HeLa cells. Immunoprecipitation with anti-FLASH antibody
and subsequent Western blot analysis using anti-TRAF2 antibody revealed
that endogenous TRAF2 was co-precipitated with endogenous FLASH, but
not by preimmune serum (Fig.
6A). Endogenous FLASH could
not be detected with Western blot analysis using anti-FLASH antibody in
the immunoprecipitates probably because of its low expression level in
Jurkat cells. We have then generated HeLa cells stably expressing
exogenous FLASH tagged with HA (HeLa/HA-FLASH) and further examined the
intracellular interactions. Immunoprecipitation with anti-HA antibody
followed by Western blotting using anti-TRAF2 antibody or anti-HA
antibody showed the presence of TRAF2 and HA-FLASH in the
immunoprecipitates (Fig. 6B). These results indicate that
FLASH interacts with TRAF2 in the cells.
We have then examined cellular interaction of NAD of FLASH with TRAF2.
HEK293 cells were transfected with pHA-FLASH (Fig. 6C) or
pHA- Previous studies have suggested that TNF-R-mediated
apoptosis and NF- FLASH as a component of apoptotic signaling complexes is likely
to mediate apoptosis signals probably triggered by cell surface receptors. However, the fact that FLASH activates NF- FLASH seems to be an upstream component of various
receptor-mediated signals including TNF--induced activation of
NF-
B in HEK293 cells, as determined by luciferase reporter gene
expression driven by a NF-
B responsive promoter. Conversely,
overexpression of FLASH dose-dependently activated NF-
B,
an effect suppressed by dominant negative mutants of TRAF2, NIK,
and IKK
, and partially by those of TRAF5 and TRAF6. TRAF2 was
co-immunoprecipitated with FLASH from the cell extracts of HEK293 cells
or HeLa cells stably expressing exogenous FLASH (HeLa/HA-FLASH).
Furthermore, serial deletion mapping demonstrated that a domain
spanning the residues 856-1191 of FLASH activated NF-
B as
efficiently as the full-length and could directly bind to TRAF2
in vitro and in the transfected cells. Taken
together, these results suggest that FLASH coordinates downstream
NF-
B activity via a TRAF2-dependent pathway in the TNF-
signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 is a
pleiotropic cytokine associated with various cellular defense
responses, with lethal effects such as septic shock with inflammation,
and with apoptosis in susceptible cells (1, 2). TNF-
signaling is
transduced through its receptor, TNF-R, to simultaneously elicit two
opposing effects: apoptosis and activation of an anti-apoptotic
transcription factor NF-
B (3-5). During initiation of apoptosis,
FADD is complexed with activated TNF-R1 and TRADD via the death domain
(4, 6, 7) and recruits caspase-8 to the resultant death-inducing
signaling complex (DISC), which leads to apoptosis via activation of a
caspase cascade (4, 8-10). Similarly, Fas (CD95/APO-1) recruits FADD
to its activated receptor to induce apoptosis (11).
B. Whereas overexpression of the wild type TRAF2, -5, or -6 activates NF-
B, their truncated versions lacking zinc-binding domains inhibit NF-
B activation induced by various stimuli (12-17). Whereas TRAF2 transduces TNF-
-mediated activation of NF-
B, TRAF6 is associated with interleukin-1 and CARD4 signaling (17, 18), indicating that TRAFs are common mediators for NF-
B activation and
display an ability to stimulate signal-specific NF-
B activation. Subsequent activation of NIK, a member of the mitogen-activated protein
kinase family (4, 19, 20), and the downstream kinases, IKK
and
IKK
, leads to the phosphorylation of I
Bs for degradation and the
activation of NF-
B (20-25).
B activation as well as to apoptosis
(28, 29) suggests that FLASH may function to coordinate stress
responses, a possibility that prompted us to investigate the role of
FLASH in NF-
B activation. In this report, we used an antisense
oligonucleotide (AS) and overexpression analysis to show that FLASH
transduces the TNF-
signal, leading to the activation of
NF-
B via a TRAF2-NIK-IKK-dependent pathway.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
and anti-tubulin antibodies were
purchased from Santa Cruz Biotechnology (Santa Crutz, CA) and Sigma,
respectively. Anti-HA antibody and anti-TRAF2 antibody (SC-7346) were
from Roche Molecular Biochemicals (Mannheim, Germany) and Santa Cruz
Biotechnology, respectively. TNF-
and all other molecular biology
grade materials were from Sigma or New England Biolabs (Hertfordshire, UK).
A-(1-858) (pFL-
A-FLASH);
5'-CCGGAATTCATGGAGAGCTCATGTGCAATT-3' and
5'-ACCGGGCCCCTATCCAGTTCTAGGCAAAGA-3' for pcDNA-HA-
B-(856-1552) (pHA-
B-FLASH); 5'-CCGGAATTCATGGCAGATGATGACAATGGT-3' and
5'-ATAAGAATGCGGCCGCCTACAGTGAAGATTTAAAATTC-3' for
pME18S-FLAG-
C-(1-1191) (pFL-
C-FLASH);
5'-CCGGAATTCATGGAGAGCTGCTCATGTGCAATT-3' and 5'-CATTTAGGTGACACTA-3' for
pME18S-FLAG-
D-(1553-1962) (pFL-
D-FLASH); 5'-CCGGAATTCATGGAGAGCTCATGTGCAATT-3' and 5'-CATTTAGGTGACACTA-3' for
pME18S-FLAG-
E-(856-1962) (pFL-
E-FLASH). The PCR products were
then inserted into the EcoRI/NotI sites of
pME18S-FLAG (
A,
C,
D, and
E), the
EcoRI/ApaI sites of pcDNA3-HA
(pHA-
B-FLASH), or the EcoRI/NotI sites of
pcDNA3-HA (pHA-
D-FLASH). GST-NAD-FLASH and GST-DRD-FLASH fusion
proteins were generated by subcloning PCR products amplified by
5'-CGCGGATCCCCTAGAGTTTCTGCTGAA-3' and 5'-CCGCTCGAGTTACAGTGAAGATTTAAATT-3' for pGST-NAD-FLASH and
5'-CGCGGATCCGATAAGAGTAAACTAACTC-3' and
5'-CCGCTCGAGTTATTCACAGGAGCCAGGAGA-3' for pGST-DRD-FLASH into the
BamHI/XhoI sites of pGEX4T-3 (Amersham
Pharmacia Biotech.). All PCR products were confirmed by
DNA sequencing. pTRAF2, pTRAF5, pTRAF6, pFL-NIK, pCR3.1-IKK
,
dominant negative forms of TRAF2, TRAF5, TRAF6, NIK, and IKK
,
pNF-
B-luc, and pCasp8 were described previously (30, 37).
B-luciferase reporter plasmid (pNF-
B-luc), 200 ng of
pCMV-
-gal, and 1 µg of vector or the indicated expression plasmid
using LipofectAMINE according to the manufacturer's instructions (Life
Technologies, Inc.). The total amount of transfected plasmid DNA was
kept constant within individual experiments by adding appropriate
amounts of pcDNA or pME18S. HeLa cells stably expressing HA-FLASH
(HeLa/HA-FLASH) were generated as described by Chung et al.
(38).
-actin mRNA
as a control. Two sets of oligonucleotides were designed;
5'-TAGGTGCTTTTATTGACTTGACACAA-3' (sense) and
5'-CAGGAATTCAGCAACTTATCTGCAT-3' (antisense) (predicted product length:
725 bp) for FLASH primer 1 and 5'-GAAGGTAATCATCCTGCATTAGCTGT-3' (sense)
and 5'-GAGCTTCATTAGCTGCTGGAATCTT-3' (antisense) (predicted product
length: 714 bp) for FLASH primer 2. The nucleotide sequences of the
-actin primers were 5'-CAACCGCGAGAAGATGACCC-3' (sense) and
5'-GAAGGAAGGCTGGAAGAGTG-3' (antisense) (predicted product length: 457 base pairs). The PCR products were confirmed by DNA sequencing.
-Galactosidase Assays--
Cells were
harvested 24 h after transfection, and luciferase activities in
the cell extracts were determined using a luciferase assay system
(Promega). To measure
-galactosidase activity, the cell extracts
were mixed with equal amounts of
-galactosidase assay buffer (2×)
containing 200 mM sodium phosphate (pH 7.3), 2 mM MgCl2, 100 mM
-mercaptoethanol, and 1.33 mg/ml
O-nitrophenyl-
-D-galactopyranoside and
incubated at 37 °C for 30 min. The absorbance at 420 nm was then
measured using an ELISA reader (Molecular Device, Sunnyvale, CA).
-D-thiogalactoside, purified using glutathione-Sepharose 4B (Amersham Pharmacia Biotech) and administered into a rabbit in a series of four injections. Anti-FLASH antibody was
purified from the serum by antigen-affinity chromatography. For Western
blot analysis, cell lysates were prepared, and protein concentrations
were determined using a DC protein assay kit (Bio-Rad). Western
blotting was then carried out as previously described (31); proteins
were visualized using an enhanced chemiluminescence system (ECL,
Amersham Pharmacia Biotech).
-D-thiogalactoside during exponential growth. Harvested cells were resuspended and lysed by sonication in 50 mM Tris-HCl buffer (pH 7.4) containing 1 mM
dithiothreitol, 0.5 mM EDTA, and 10% (v/v) glycerol. The
supernatant lysates were incubated with glutathione-Sepharose 4B.
TRAF2, or caspase-8 labeled with [35S]methionine using
the TNT system (Promega) were then added to GST fusion proteins (20 µg each) coupled to glutathione-Sepharose 4B in a final volume of 500 µl of binding buffer (50 mM Tris-HCl, pH 7.4, 1 mM dithiothreitol, 0.5 mM EDTA, 0.01% Triton
X-100, 0.5 mg/ml bovine serum albumin, and 10% (v/v) glycerol). After being incubated at 4 °C for 2 h with gentle mixing, the beads were washed three times with the binding buffer, separated by 12%
SDS-PAGE, and detected by autoradiography.
B-FLASH, and pTRAF2 plasmids and lysed in radioimmune
precipitation buffer (50 mM Tris-HCl, pH 7.4, 1% Nonidet
P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml each of aprotinin, leupeptin, and pepstatin, 1 mM
Na3VO4, and 1 mM NaF). FLASH was
immunoprecipitated from cell lysates after incubation with anti-FLASH
and anti-HA antibodies and protein-A-coupled-Sepharose CL-4B (Amersham
Pharmacia Biotech.) at 4 °C for 2 h. TRAF2, HA-FLASH, and
HA-
B-FLASH were then detected by Western blot analysis using anti-TRAF2 and anti-HA monoclonal antibodies, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-induced NF-
B Activation by a FLASH
Antisense Oligonucleotide--
To identify a role for FLASH in TNF-
signaling, we initially examined its contribution to NF-
B activation
by directly targeting FLASH expression using an AS. Four different ASs
were synthesized based on the nucleotide sequence of human FLASH and
when examined, they showed essentially similar effects on
TNF-
-signaling (data not shown). Treating HEK293 cells with AS-2,
but not with a scrambled oligonucleotide (as a negative control),
abolished TNF-
-induced activation of NF-
B, as assessed by
luciferase reporter gene expression driven by a NF-
B responsive
promoter (Fig. 1A), and also
suppressed TNF-
-induced degradation of I
B
(Fig.
1B).
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Fig. 1.
Suppression of
TNF- -induced activation of
NF-
B using a FLASH antisense
oligonucleotide. HEK293 cells were co-transfected for 36 h
with NF-
B-luciferase reporter plasmid (pNF-
B-luc), pCMV-
-gal,
and either 5 µM FLASH AS-2 or a scrambled oligonucleotide
(random), and were then incubated with TNF-
(30 ng/ml) for an
additional 26 h. A, activity of luciferase reporter
genes was normalized to that of
-galactosidase, which served as an
internal control. Bars represent mean ± S.D. from at
least four independent experiments. B, HEK293 cells were
lysed, and Western blotting was performed with anti-I
B
antibody.
For an internal control, the same extracts were probed with antibody to
-tubulin. C, reverse transcription of RNA isolated from
cells treated with scrambled (Ran) or AS-2 (AS)
oligonucleotides. The PCR reaction was carried out with two different
sets of FLASH primers. PCR of
-actin was performed to normalize
FLASH expression.
-induced activation of NF-
B.
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Fig. 2.
NF- B activation
induced by FLASH expression. HEK293 cells were transfected for
36 h with pNF-
B-luc, pCMV-
-gal and the indicated amounts of
pFLASH, pTRAF2, or pHA-
D-FLASH. A, relative
NF-
B-driven luciferase activities in transfectants expressing FLASH,
TRAF2, or
D-FLASH; activity in the control cells was arbitrarily set
to a value of 1. B, Western blots showing the expression
levels of exogenous FLASH (anti-FLASH antibody),
D-FLASH (anti-HA
antibody), and TRAF2 (anti-TRAF2 antibody).
B by FLASH Expression and Domain Mapping for
NF-
B Activation--
To more directly assess the role of FLASH in
the activation of NF-
B signaling, we examined an effect of its
overexpression on NF-
B activity in HEK293 cells. We found that,
indeed, NF-
B activity was dose-dependently related to
FLASH expression (Fig. 2A). The relative levels of
expression of FLASH and TRAF2 were confirmed by Western blot analysis
(Fig. 2B).
B activity, effects of several
FLASH deletion mutants (Fig. 3A,
A-
E) were examined. Expression
of these deletions was confirmed in the transfected HEK293 cells by
Western blot analysis using anti-HA or anti-FLAG antibodies (data not
shown). Determination of NF-
B activity following the respective
expression of each of these constructs showed that the truncation in
the
B-,
C-, and
E-FLASH constructs had no effect on the
ability of FLASH to activate NF-
B (Fig. 3B). The
D-FLASH, by contrast, completely abolished NF-
B activation, whereas the
A-FLASH partially induced NF-
B activity (Fig.
3B). Because the
B-,
C-, and
E-FLASH contain a
common region including a putative oligomerization domain, the region
responsible for the activation of NF-
B apparently spans most of the
oligomerization domain and part of the
A-FLASH domain (Fig.
3A). We designate the common region in the
B-,
C-, and
E-FLASH spanning residues 856-1191 as the NF-
B activation domain
(NAD) of FLASH.
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Fig. 3.
Mapping of the FLASH domain responsible for
NF- B activation. A, schematic
diagrams of full-length FLASH and its deletion (
) constructs. The
death-effector domain-recruiting domain (DRD) and a putative
NF-
B-activating domain (NAD) are indicated. B,
HEK293 cells were co-transfected with pNF-
B-luc and either pcDNA
(control), full-length FLASH, or deletion constructs. One
day later, luciferase reporter gene assays were performed and for each
construct, luciferase activities were adjusted so that the control was
1. Bars indicate mean ± S.D. of the induction of
NF-
B activity relative to the control from at least four independent
experiments. C, HEK293 cells were left untreated, treated
with TNF-
for 1 h, or transfected with pcDNA-HA, pHA-FLASH,
pHA-
B-FLASH, pFL-
D-FLASH, or pTRAF2. After 1 day, cell extracts
were prepared and analyzed by Western blotting with anti-I
B
antibody.
treatment of cells leads to the
activation of NF-
B through the phosphorylation and degradation of
I
B
(33, 34), exposure of HEK293 cells to TNF-
resulted in the
degradation of I
B
(Fig. 3C, left panel). Transient
expression of FLASH, TRAF2, or
B-FLASH led to the degradation of
I
B
, whereas overexpression of the
D-FLASH did not affect the
degradation of I
B
(Fig. 3C, right panel), consistent
with the results of NF-
B activity assays in Fig. 3B.
These results demonstrate and confirm that FLASH expression through NAD
in
B-FLASH induces NF-
B activation by the degradation of
I
B
.
B Activation through
TRAF2-NIK-IKKs--
It has previously been shown that various
signaling proteins downstream of TNF-R, including TRADD, TRAFs, RIP,
NIK, and IKKs, are involved in TNF-
-induced activation of NF-
B.
We therefore examined the respective roles of these proteins in
FLASH-induced activation of NF-
B. Expression of dominant negative
mutants of TRAF2-(87-501), NIK (K429A,K430A), or IKK
(K44A)
inhibited NF-
B activation induced by FLASH (Fig.
4A) or TNF-
(Fig.
4B). On the other hand, although dominant negative mutants
of TRAF5-(205-558) and TRAF6-(289-530) partially suppressed TNF-
-
or FLASH-induced activation of NF-
B (Fig. 4B), they
showed little effect on both TNF-
- and FLASH-induced activation of
NF-
B (Fig. 4, A and B), which thus appears to
be mainly mediated via a TRAF2-NIK-IKK-pathway.
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Fig. 4.
Effects of various dominant negative mutants
on FLASH-induced activation of NF- B.
A, HEK293 cells were left untreated or treated with TNF-
after co-transfection with pNF-
B-luc and either pcDNA
(control) or expression plasmids encoding the wild type or
dominant negative mutants (D/N) of the indicated mediators
in the presence or absence of pHA-FLASH. One day later, luciferase
activities were measured and normalized to that of
-galactosidase.
B, HEK293 cells were co-transfected with pNF-
B-luc and
the indicated dominant negative mutants. The cells were then exposed to
TNF-
(30 ng/ml) for 6 h, after which luciferase activities were
measured.
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Fig. 5.
In vitro binding of NAD to
TRAF2. GST, GST-NAD, and GST-DRD fusion proteins were purified
from Escherichia coli. Proteins bound to their affinity
resins (each equivalent to 20 µg of protein) were incubated with
TRAF2 or caspase-8 labeled with [35S]methionine as
described under "Experimental Procedures." After separation by 12%
SDS-PAGE, the bound proteins were detected by autoradiography
(upper panels), and resin-coupled proteins were visualized
by Western blotting with anti-GST polyclonal antibody (lower
panels).
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Fig. 6.
Cellular interaction of FLASH with
TRAF2. A, proteins from Jurkat cell extracts were
immunoprecipitated (IP), separated by SDS-PAGE, and
immunoblotted with preimmune (pre) or anti-TRAF2 monoclonal
antibody. B, proteins from HeLa cell permanently expressing
HA-FLASH (HeLa/HA-FLASH) were immunoprecipitated with anti-HA antibody
and probed with anti-TRAF2 antibody (upper panel) or anti-HA
antibody (lower panel). C, HEK293 cells were
transiently transfected with pHA-FLASH in the presence or absence of
pTRAF2. One day later, proteins were immunoprecipitated with anti-FLASH
antibody or anti-HA antibody and immunoblotted with anti-TRAF2
antibody. D, HEK293 cells were co-transfected with
pHA- B-FLASH and pTRAF2, immunoprecipitated with anti-HA antibody,
and immunoblotted with anti-TRAF2 (upper panels) or anti-HA
antibody (lower panels).
B-FLASH (Fig. 6D) in the presence or absence of
TRAF2. Immunoprecipitation and Western blot analysis showed that
endogenous (Fig. 6C, middle panel) or exogenous
TRAF2 (Fig. 6C, right panel) was co-precipitated
with FLASH by anti-FLASH antibody or anti-HA antibody. As expected from
in vitro binding assays,
B-FLASH was co-immunoprecipitated with TRAF2 by anti-HA antibody from lysates of
HEK293 cells transfected with both pHA-
B-FLASH and pTRAF2 (Fig.
6D). These results suggest that FLASH may directly interact with TRAF2 through its NAD.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B activation pathways pass through TRADD, a death
domain-containing adaptor protein interacting with TNF-R in a
TNF-dependent process; TRADD-FADD-caspase-8 and
TRADD-TRAF2-RIP-NIK-IKKs cascades lead to the induction of apoptosis
and activation of an anti-apoptotic transcription factor NF-
B,
respectively. Whereas FLASH may be required for the activation of
caspase-8 during Fas- and TNF-R-mediated apoptosis (26, 27), the data
presented here provide the first evidence for involvement of FLASH in
NF-
B activation by TNF-R. The physical interaction of FLASH with
TRAF2, demonstrated here in vitro and in vivo
(Figs. 5 and 6), lends further support to the idea that FLASH
transduces TNF-
signals via a TRAF2-dependent pathway of
NF-
B activation. Though dominant negative mutants of TRAF5 and 6 also partially suppressed FLASH- and TNF-
-mediated activation of
NF-
B (Fig. 4, A and B), complex formation of
FLASH with additional signal mediators leading to NF-
B signaling
such as other TRAFs, TRADD, or RIP remains to be
elucidated.2
B may explain the observation that in many cell types, TNF treatment did not induce
apoptosis in the absence of gene expression. With respect to the role
of FLASH as an activator interacting with caspase-8 and FADD, the lack
of a significant increase of apoptosis following overexpression of both
FLASH and caspase-8 or the inability of TNF-
to induce apoptosis in
a subset of tumor cells may be attributed to FLASH-mediated activation
of NF-
B. NAD-mediated activation of NF-
B may antagonize
DRD-mediated apoptotic signals by encoding inhibitory proteins such
as IAPs and IEX-1L (5, 35, 36). Moreover, recent reports that FADD and
caspase-8 may be required for cell survival and proliferation during
heart and thymus development may be explained by our observations of
FLASH-mediated activation of NF-
B. This speculation is reinforced by
our observation that FLASH transduced NF-
B signaling evoked by
caspase-8 (data not shown).
and most likely has a dual
function in apoptosis and NF-
B signaling. We have additional
evidences that FLASH is also an indispensable component in
receptor-mediated apoptosis. As a component of DISC during
apoptosis and also of the protein complex including TRAFs leading
to NF-
B signaling, FLASH needs to be further characterized for the
stoichiometry of protein-protein interactions and for a fine-tuning
activity balancing survival and apoptosis.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. S. Yonehara (University of Kyoto, Japan) for FLASH cDNA and Dr. N. Spoerel for critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported by the Molecular Medicine Research Group Program (98-MM-01-01-A-03), the KOSEF (Protein Network Research Center), and the National Research Laboratory program (to Y. K. J.).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.
Supported by the Brain Korea 21 project. These authors contributed
equally to this work.
¶ To whom correspondence should be addressed: Department of Life Science, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju 500-712, Korea. Tel.: 82-62-970-2492; Fax: 82-62-970-2484; E-mail: ykjung@eunhasu.kjist.ac.kr.
Published, JBC Papers in Press, May 4, 2001, DOI 10.1074/jbc.M102941200
2 Y. H. Choi, K. B. Kim, B. J. Kim, and Y. K. Jung, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
TNF-, tumor necrosis factor-
;
FLASH, FLICE-associated huge protein;
AS, antisense oligonucleotide;
DED, death effector domain;
DRD, DED-recruiting domain;
NAD, NF-
B-activating domain;
DISC, death-inducing signaling complex;
RT-PCR, reverse
transcription-polymerase chain reaction;
PAGE, polyacrylamide gel
electrophoresis;
HA, hemagglutinin;
-gal,
-galactosidase;
HEK, human embryonic kidney cells;
GST, glutathione
S-transferase;
TRAF, TNF receptor-associated factor;
NIK, NF-
B-inducing kinase;
Ikk, I
B kinase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Beg, A. A.,
and Baltimore, D.
(1996)
Science
274,
782-784 |
2. | Serfas, M. S., Goufman, E., Feuerman, M. H., Gartel, A. L., and Tyner, A. L. (1997) Cell Growth and Differ. 8, 951-961[Abstract] |
3. | Hsu, H., Xiong, J., and Goeddel, D. V. (1995) Cell 81, 495-504[Medline] [Order article via Infotrieve] |
4. | Hsu, H., Shu, H. B., Pan, M. G., and Goeddel, D. V. (1996) Cell 84, 299-308[Medline] [Order article via Infotrieve] |
5. |
Wang, C. Y.,
Mayo, M. W.,
Korneluk, R. G.,
Goeddel, D. V.,
and Baldwin Jr, A. S.
(1998)
Science
281,
1680-1683 |
6. |
Shu, H. B.,
Takeuchi, M.,
and Goeddel, D. V.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
13973-13978 |
7. |
Hu, W. H.,
Johnson, H.,
and Shu, H. B.
(2000)
J. Biol. Chem.
275,
10838-10844 |
8. | Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803-815[Medline] [Order article via Infotrieve] |
9. | Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O'Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) Cell 85, 817-827[Medline] [Order article via Infotrieve] |
10. | Yuan, J., Shaham, S., Ledoux, S., Ellis, H. M., and Horvitz, H. R. (1993) Cell 75, 641-652[Medline] [Order article via Infotrieve] |
11. |
Boldin, M. P.,
Mett, I. L.,
Varfolomeev, E. E.,
Chumakov, I.,
Shemer-Avni, Y.,
Camonis, J. H.,
and Wallach, D.
(1995)
J. Biol. Chem.
270,
387-391 |
12. | Coa, Z., Xiong, J., Takeuchi, M., Kurama, T., and Goeddel, D. V. (1996) Nature 383, 443-446[CrossRef][Medline] [Order article via Infotrieve] |
13. | Rothe, M., Sarma, V., Dixit, V. M., and Goeddel, D. V. (1995) Science 269, 1424-1427[Medline] [Order article via Infotrieve] |
14. |
Nakano, H.,
Oshima, H.,
Chung, W.,
Williams-Abbott, L.,
Ware, C. F.,
Yagita, H.,
and Okumura, K.
(1996)
J. Biol. Chem.
271,
14661-14664 |
15. |
Ishida, T.,
Mizushima, S.,
Azuma, S.,
Kobayashi, N.,
Tojo, T.,
Suzuki, K.,
Aizawa, S.,
Watanabe, T.,
Mosialos, G.,
Kieff, E.,
Yamamoto, T.,
and Inoue, J.
(1996)
J. Biol. Chem.
271,
28745-28748 |
16. |
Ishida, T.,
Tojo, T.,
Aoki, T.,
Kobayashi, N.,
Ohishi, T.,
Watanabe, T.,
Yamamoto, T.,
and Inoue, J.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
9437-9442 |
17. |
Ling, L.,
and Goeddel, D. V.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
9567-9572 |
18. |
Bertin, J.,
Nir, W. J.,
Fischer, C. M.,
Tayber, O. V.,
Errada, P. R.,
Grant, J. R.,
Keilty, J. J.,
Gosselin, M. L.,
Robison, K. E.,
Wong, G. H. W.,
Glucksmann, M. A.,
and DiStefano, P. S.
(1999)
J. Biol. Chem.
274,
12955-12958 |
19. | Liu, Z. G., Hsu, H., Goeddel, D. V., and Karin, M. (1996) Cell 87, 565-576[Medline] [Order article via Infotrieve] |
20. | Malinin, N. L., Boldin, M. P., Kovalenko, A. V., and Wallach, D. (1997) Nature 385, 540-544[CrossRef][Medline] [Order article via Infotrieve] |
21. | Didonato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M. (1997) Nature 388, 548-554[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Mercurio, F.,
Zhu, H.,
Murray, B. W.,
Shevchenko, A.,
Bennett, B. L.,
Li, J. W.,
Young, D. B.,
Barbosa, M.,
Mann, M.,
Manning, A.,
and Rao, A.
(1997)
Science
278,
860-866 |
23. | Régnier, C. H., Song, H. Y., Gao, X., Goeddel, D. V., Cao, Z., and Rothe, M. (1997) Cell 90, 373-383[Medline] [Order article via Infotrieve] |
24. |
Woronicz, J. D.,
Gao, X.,
Cao, Z.,
Rothe, M.,
and Goeddel, D. V.
(1997)
Science
278,
866-870 |
25. | Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91, 243-252[Medline] [Order article via Infotrieve] |
26. | Imai, Y., Kimura, T., Murakami, A., Yajima, N., Sakamaki, K., and Yonehara, S. (1999) Nature 398, 777-785[CrossRef][Medline] [Order article via Infotrieve] |
27. | Medema, J. P. (1999) Nature 398, 756-757[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Inohara, N.,
Koseki, T.,
Lin, J.,
Peso, L.,
Lucas, P. C.,
Chen, F. F.,
Ogura, Y.,
and Núñez, G.
(2000)
J. Biol. Chem.
275,
27823-27831 |
29. |
Newton, K.,
Harris, A. W.,
Bath, M. L.,
Smith, K. G. C.,
and Strasser, A.
(1998)
EMBO J.
17,
706-718 |
30. |
Wong, B. R.,
Josien, R.,
Lee, S. Y.,
Vologodskaia, M.,
Steinman, R. M.,
and Choi, Y.
(1998)
J. Biol. Chem.
273,
28355-28359 |
31. |
Jung, Y.,
Miura, M.,
and Yuan, J.
(1996)
J. Biol. Chem.
271,
5112-5117 |
32. |
Vassar, R.,
Bennett, B. D.,
Babu-Khan, S.,
Kahn, S.,
Mendiaz, E. A.,
Denis, P.,
Teplow, D. B.,
Ross, S.,
Amarante, P.,
Loeloff, R.,
Luo, Y.,
Fisher, S.,
Fuller, J.,
Edenson, S.,
Lile, J.,
Jarosinski, M. A.,
Biere, A. L.,
Curran, E.,
Burgess, T.,
Louis, J. C.,
Collins, F.,
Treanor, J.,
Rogers, G.,
and Citron, M.
(1999)
Science
286,
735-741 |
33. | Baeuerle, P. A., and Henkel, T. (1994) Annu. Rev. Immunol. 12, 141-179[CrossRef][Medline] [Order article via Infotrieve] |
34. |
Reuther, J. Y.,
and Baldwin, A. S.
(1999)
J. Biol. Chem.
274,
20664-20670 |
35. | LaCasse, E. C., Baird, S., Korneluk, R. G., and MacKenzie, A. E. (1998) Oncogene 17, 3247-3259[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Chu, Z. L.,
McKinsey, T. A.,
Liu, L.,
Genty, J. J.,
Malim, M. H.,
and Ballard, D. W.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
10057-10062 |
37. | Kim, I. K., Chung, C. W., Woo, H. N., Hong, G. S., Nagata, S. J., and Jung, Y. K. (2000) Biochem. Biophys. Res. Commun. 277, 311-316[CrossRef][Medline] [Order article via Infotrieve] |
38. | Chung, C. W., Song, Y. H., Kim, I. K., Yoon, W. J., Ryu, B. R., Jo, D. G., Woo, H. N., Kwon, Y. K., Kim, H. H., Gwag, B. J., Mook-Jung, I. H., and Jung, Y. K. (2001) Neurobiol. Disease 8, 162-172[CrossRef][Medline] [Order article via Infotrieve] |