From the Musculoskeletal Science and
§ Protein Chemistry and Proteomics, Wyeth Research,
Cambridge, Massachusetts 02140
Received for publication, August 23, 2002, and in revised form, January 17, 2003
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ABSTRACT |
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Lymphotoxin- Lymphotoxin- Similar to other members of the TNFR family, the engagement of ligands
to LT DD-containing receptors initiate their death signals by recruitment of
FADD to oligomerized receptors, which in turn recruits and activates
caspase 8 (21, 27, 28). Activated caspase 8 then cleaves Bid, a BH3
domain containing pro-apoptotic protein of the Bcl-2 family. The
trans-location of cleaved Bid to mitochondria evokes cytochrome
c release, demonstrating the cross-talk of receptor-induced apoptosis with mitochondria-mediated apoptosis (29, 30). TRAIL-induced apoptosis also induces and requires the mitochondrial release of Smac,
a second mitochondrial protein that is released to cytosol concurrent
with the release of cytochrome c (31-33). The function of
Smac is to antagonize the inhibition of caspases by IAPs and thus
promote apoptosis (34, 35). In contrast to death domain receptors,
LT Since TNF family receptors are at a very low abundance in cells, most
of our knowledge of receptor-signaling complexes is acquired by
enhanced amplification of signals in overexpressed systems. Typically,
little is known regarding the endogenous signaling complex. The
recently developed proteomic approach provides us a powerful means to
detect very low abundant proteins in an endogenous ligand-receptor
complex. To better understand the molecular mechanism of LT Cell Culture, Antibodies, and Reagents--
U937, HEK293, and
MCF7 cells were obtained from American Type Culture Collection (ATCC)
and cultured in RPMI 1640 medium (Invitrogen), Dulbecco's modified
Eagle's medium (Invitrogen), and Eagle's minimum essential
medium (ATCC) with 0.01% insulin, respectively. All media were
supplemented with 10% fetal bovine serum. TRAF2 and TRAF3 antibodies
were purchased from Santa Cruz Biotechnology. cIAP1 antibody was
obtained from R & D Systems. Anti-FLAG (M2) antibody and affinity beads
were obtained from Sigma. HA antibody (3F10) was purchased from Roche
Molecular Biochemicals. Smac antibodies were purchased from Alexis
Biochemicals and Cell Signaling Technology. All chemical reagents
otherwise specified were purchased from Sigma.
Plasmids Construction--
N-terminal FLAG-tagged full-length
LT Purification of Endogenous LIGHT-Receptor Complex--
1 × 1010 U937 cells were washed twice with warm
phosphate-buffered saline (37 °C) and resuspended at a concentration
of 1 × 10 7 cells/ml. Cells were either treated or
left untreated with 20 ng/ml of FLAG-LIGHT (Alexis) for 10 min at
37 °C. Cells were then lysed in 50 ml of lysis buffer (20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% Triton
X-100, 1 mM EDTA, 30 mM NaF, 1 mM
NaVO4, and protease inhibitor cocktails (Roche Molecular
Biochemicals)) and gently rocked at 4 °C for 30 min. Cell debris was
removed by centrifugation twice at 10,000 × g for 30 min. Lysate was preclarified by incubation with Gamma binding beads
(Amersham Biosciences) for 1 h. The resulting lysate was applied
twice to a mini-column (Bio-Rad) of 0.2 ml of M2-affinity beads
(Sigma). The beads were washed twice with high salt (1 M
NaCl) lysis buffer, three times more with lysis buffer, and then
transferred into an Eppendorf tube. The immunocomplex was first eluted
with FLAG peptide (Sigma) at a concentration of 2 mg/ml. The residual
binding proteins were then further eluted with 8 M urea.
50% of the peptide-eluted proteins or one-tenth of the 8 M
urea-eluted proteins were separated on the 4-12% SDS-PAGE gel and
transferred to nitrocellulose membrane for Western blotting using TRAF3
antibody. These samples were also separated on the 4-12% SDS-PAGE gel
and visualized by silver staining.
Mass Spectrometry and Protein Identification--
Protein bands
of interest were manually excised from the gel, reduced, and alkylated
with iodoacetamide and then digested in situ with trypsin
using an automated digestion robot (ABIMED, Langenfeld, Germany)
as described previously (38). The peptide digests were then sequenced
using a high throughput tandem mass spectrometer (LCQ-DECA ion
trap, ThermoFinnigan, San Jose, CA) equipped with a
microelectrospray reversed phase liquid chromatography interface. Data
were acquired in automated MS/MS mode using the data acquisition
software provided with the mass spectrometer to detect and sequence
each peptide as it eluted from the column. The dynamic exclusion and
isotope exclusion functions were employed to increase the number of
peptide ions that were analyzed. During the LS-MS/MS run, typically
>1000 fragmentation spectra are collected from each sample and matched
against the nonredundant databases (NCBI) using the Sequest software
package (ThermoQuest).
Immunoprecipitation and Western Analysis--
For
immunoprecipitation, 1 × 10 8 U937 cells were treated
with FLAG-LIGHT at 20 ng/ml for different times or left untreated. Cells were then harvested and lysed in 4 ml of lysis buffer (see above). Cell debris was removed by centrifugation at 14,000 × g for 10 min, and the resulting lysate was precleared with
Gamma binding beads for 1 h at 4 °C. 20 µl of M2 beads then
were added to cell lysate and incubated at 4 °C for 3 h. After
binding, beads were washed five times with lysis buffer. Immune
complexes bound to the beads were eluted with sample buffer, resolved
on 4-12% SDS-PAGE gels, transferred to polyvinylidene difluoride
membrane, and probed with TRAF2, TRAF3, or cIAP1 antibody. Signals were detected with horseradish peroxidase-conjugated secondary antibody and
ECL detection kits (Amersham Biosciences). For immunoprecipitation in
HEK293 cells, 4 µg of pFLAG-CMV2-LT Apoptosis Assay--
MCF7 cells (5 × 105
cells/well) were seeded on cover slides in 6-well plates 1 day before
transfection. Cells in each well were transfected with 1 µg of
pcDNA3 vector, pcDNA3-Smac-HA, or pcDNA3- Identification of TRAF3, TRAF2, cIAP1, and Smac in LIGHT·LT
LIGHT binds to both LT
As expected, LIGHT was detected in band 6 (Fig. 1) (Table
I). Several peptides of TRAF2 were
detected in band 3. The association of TRAF2 with LT
As expected, LT
It should also be noted that we did not detect any peptides of TRAF4,
TRAF5, or NF Association of TRAF2, TRAF3, cIAP1, and Smac with
LT
As shown in Fig. 3, the recruitment of
endogenous TRAF2, TRAF3, and cIAP1 to LIGHT·LT
Although we detected two peptides from Smac in the LIGHT·LT
There was no further increase of Smac recruitment when stimulated with
LIGHT (data not shown). This is probably because of the aggregation and
activation of LT
In contrast to the full-length Smac, the deletion mutant of Smac
( Smac Potentiates LT
In summary, combining the reported evidence for direct interactions
among LT receptor (LT
R) is a member of
tumor necrosis factor receptor family and plays essential roles
in the embryonic development and organization of secondary lymphoid
tissues. It binds two types of tumor necrosis factor family cytokines,
heterotrimer LT
1
2 and homotrimer LIGHT, and activates multiple
signaling pathways including transcriptional factor NF
B, c-Jun
N-terminal kinase, and cell death. However, the molecular mechanism of
the activation of these signaling pathways by LT
R is not clear.
Because there is no enzymatic activity associated with the receptor
itself, the signal transduction of LT
R is mediated by cytoplasmic
proteins recruited to receptors. To identify these proteins, we took a proteomic approach. The endogenous LIGHT·LT
R complex was
affinity-purified from U937 cells, and proteins associated with the
complex were identified by mass spectrometry. Four of five proteins
identified, TRAF2, TRAF3, cIAP1, and Smac, are reported here. Their
association with LT
R was further confirmed by coimmunoprecipitation
in U937 cells and HEK293 cells. The presence of cIAP1 and Smac in
LIGHT·LT
R complex revealed a novel mechanism of
LIGHT·LT
R-induced apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
receptor
(LT
R),1 a member of the
tumor necrosis factor (TNF) receptor family, plays important roles in
embryonic development of secondary lymphoid tissues and maintenance of
their architecture in adults (1-4). It binds two types of TNF-related cytokines, heterotrimer LT
1
2 and homotrimer LIGHT. LIGHT is a
newly identified TNF family cytokine that is homologous to lymphotoxins and binds to both LT
R and HVEM (herpes virus entry mediator also named as TR2) (5). LT
R is expressed on most cell types including cells of fibroblast, epithelial, and myeloid lineage but not on T or B
lymphocytes, whereas the expression of its ligands is restricted to
activated lymphocytes (5, 6). Knock-out mice of LT
, LT
, or LT
R
lack lymph nodes and Peyer's patches demonstrating the role of
LT/LT
R signaling in lymphoid organogenesis (2, 7-10).
Inflammation-associated lymphoid organogenesis has been found in some
autoimmune diseases such as rheumatoid arthritis, inflammatory bowel
disease, and spontaneous autoimmune diabetes (11-14). Indeed, the
treatment of LT
R-Ig fusion protein can prevent colitis and
collagen-induced arthritis in mice (12, 15). Therefore, LT/LT
R is an
important therapeutic target.
R induces receptor aggregation and subsequent activation of
multiple signaling pathways including transcription factor NF
B,
c-Jun N-terminal kinase, and cell death (16-18). Since there is no
enzymatic activity associated with TNF receptor family, the activation
of downstream signals is mediated by the recruitment of cytosolic
proteins to the intracellular portion of the receptors. To date, two
families of proteins have emerged as candidates of these proteins, the
death domain (DD)-containing proteins and the TNF receptor-associated
factors (TRAFs). Each of these families is defined by a characteristic
sequence homology domain that is involved in protein-protein
interactions. Death domain was also found in the intracellular domains
of a subgroup of TNF family receptors such as TNFR1 and Fas (19). The
DD-containing receptors initiate their signals by DD-mediated
recruitment of DD-containing proteins such as TRADD, FADD, and
receptor-interacting protein kinase to the receptors. TRAFs are
a family of six RING finger (except TRAF1) containing proteins with a
homologous TRAF domain at their C terminus. Different TRAFs are widely
used by the TNF family receptors. They can directly interact with
non-DD receptors; whereas their interaction with DD-containing
receptors are mostly through other DD-containing proteins (20, 21) with
the exception of p75 neurotrophin receptor (22). Four members of TRAFs
have been reported to interact with intracellular domain of LT
R
(23-26). However, only endogenous TRAF3 was shown to be recruited to
the receptor in a ligand-dependent manner (24). Given the
distinct phenotype of LT
R knock-out mice and the diverse activity of
LT cytokines, additional signaling proteins are yet to be identified.
R does not contain a death domain nor can it recruit FADD (36).
Nevertheless, LT
R induces cell death in some carcinoma cell lines
(17, 18). Although TRAF3 has been shown to play a critical role in
LT
R-mediated apoptosis (24, 37), the precise mechanism of
LT
R-mediated apoptosis remains to be elucidated.
R
signaling, we applied this approach to identify proteins associated
with an affinity-purified endogenous LIGHT·LT
R complex from U937
cells. Here we report the identification of TRAF2, TRAF3, cIAP1, and
Smac in this complex. Additional protein identified in this study is
currently under investigation and will be reported elsewhere. Our work
for the first time demonstrates the physiological association of TRAF2,
cIAP1, and Smac in the LIGHT·LT
R complex and reveals a novel
mechanism of apoptosis utilized by LIGHT·LT
R.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
R in pFLAG-CMV2 vector (Eastman Kodak, Co.) was a kind gift from
Dr. Shie-Liang Hsieh (18). The full-length and
76 deletion mutant of
Smac with a C-terminal HA tag were amplified by PCR reaction from a
human ovary cDNA library. The PCR fragments were then cloned into
pcDNA3.1(+) at NdeI and XhoI sites.
R, pcDNA3-Smac-HA or
pcDNA3-
76Smac-HA were transfected into cells on a 100-mm dish
using FuGENE 6 (Roche Molecular Biochemicals) according to the
manufacturer's instruction. Forty-eight hours after transfection,
cells were collected with cell lifters and lysed in 0.5 ml of lysis
buffer. Immunoprecipitation was performed in the same fashion as in
U937 cells with either M2 beads for LT
R or with HA monoclonal
antibody for Smac or
76Smac. The presence of Smac, LT
R, TRAF2,
and cIAP1 in the immune complex were then analyzed by Western blots.
76Smac-HA
expression constructs together with pEMC-
-galactosidase using FuGENE
6 (Roche Molecular Chemicals). Twenty-four hours after transfection,
cells were treated with phosphate-buffered saline (control), LIGHT (20 ng/ml), or LT
1
2 (20 ng/ml) for 6 h and then fixed and
stained with X-gal (Sigma). Apoptosis was assessed by morphological
analysis and expressed as a percentage of apoptotic (round and
detached) cells in the total of transfected blue cells.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
R
Complex from U937 Cells--
The binding of either homotrimer LIGHT or
heterotrimer LT
1
2 to LT
R induces aggregation of the receptors
and subsequent recruitment of cytosolic signaling proteins, resulting
in the formation of ligand-LT
R complex that initiates the downstream signal transduction. Similar to other members of the TNFR family, LT
R activates multiple signaling pathways but so far only endogenous TRAF3 was shown to be recruited to LT
R in a
ligand-dependent manner (16-18, 23-26). Therefore,
additional yet unknown proteins are required for signaling. To identify
these signaling proteins, we have applied a proteomic approach.
R and TR2/HVEM. In U937 cells, LT
R is
constitutively expressed while the expression of TR2/HVEM is induced by
differentiating agents (5, 6, 39). To form a specific ligand-LT
R
complex, we took the advantage of this phenomenon. Undifferentiated
U937 cells were treated with FLAG-tagged LIGHT for 10 min to form the
LIGHT·LT
R complex. Endogenous receptor complex was
affinity-purified with anti-FLAG antibody (M2)-conjugated beads and
then eluted with FLAG peptide. The eluted proteins were resolved on
4-12% SDS-PAGE gels and visualized by silver staining. Approximately,
eight protein bands were found to be present only in the LIGHT-treated
sample but not in the control (Fig.
1A). Consistent with a
previous report (24), we detected TRAF3 in the LIGHT-treated sample by
Western blot using a polyclonal antibody against TRAF3 (24) and
demonstrated the formation of the physiological LIGHT·LT
R complex
(Fig. 1B). To identify proteins in this complex, these eight
bands were excised from the gel and analyzed by liquid chromatography
electrospray ionization mass spectrometry.
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Fig. 1.
Multiple proteins associated with
LIGHT-receptor complex. U937 cells were treated (+) or
untreated ( ) with FLAG-LIGHT. Immunoprecipitation was performed with
anti-FLAG antibody M2-conjugated Sepharose beads. After extensive wash,
immunocomplex was eluted with FLAG peptides. The eluted proteins were
resolved on 4-12% SDS-PAGE gels and visualized by silver staining
(A). The presence of TRAF3 in the complex was detected by
Western blot with anti-TRAF3 antibody (B).
R has been
reported previously in the overexpression system (23, 25). Our
detection of TRAF2 in the endogenous LIGHT·LT
R complex confirmed
the previous observation and further demonstrated that the endogenous
TRAF2 is a signal transducer of LT
R. Proteins in other bands either
could not be determined due to the poor quality of the spectrum or
later turned out to be nonspecific binding proteins such as Hsp90 at
band 1 and actin at band 4. Surprisingly, we were unable to detect
peptides corresponding to TRAF3 at the expected position of band 3. Neither could we detect peptides corresponding to receptors. One
possibility was that the amount of TRAF3 and receptors, if any, was
below the detection limit of mass spectrometry. As indicated by the
Western blot of TRAF3, FLAG peptides only eluted one-tenth of the total TRAF3 protein on the beads (data not shown). This low efficiency was
probably the result of the multimeric and high affinity interactions between antibody and ligand-receptor complex. To increase the recovery
of proteins from the beads, we further eluted the beads with 8 M urea. Samples were then resolved on SDS-PAGE gels, and bands at the expected position of TRAF3 were excised and analyzed by
mass spectrometry. Three peptides of TRAF3 (Table I) were detected in
the LIGHT-treated sample, and these peptides were totally absent in the
sample without LIGHT treatment. Given the success with the
identification of TRAF3 in the urea-eluted sample, we decided to
process all of the bands in both LIGHT-treated and control samples even
though 8 M urea also eluted proteins nonspecifically bound
to the beads, resulting in an indistinguishable pattern between
LIGHT-treated and control samples on SDS-PAGE gels (data not shown).
After protein identification, nonspecific binding proteins in the
control sample were subtracted from those in the LIGHT-treated sample,
resulting in proteins that specifically bind to the LIGHT-receptor
complex. A total of five signaling proteins were identified. Among them
four, are reported here. Table I summarizes the peptides detected and
their assigned proteins.
List of proteins identified in LIGHT · LTR complex
R was detected as the receptor in the complex
(Table I). The detection of LIGHT and LT
R in the complex further confirmed our success in the isolation of LIGHT·LT
R complex. TRAF2
and TRAF3 were detected (Table I). In addition, four peptides of
cIAP1 at the position of band 2 and two peptides of Smac at the
position of band 6 were detected (Table I). Fig.
2 shows two representative mass spectra
of these peptides. cIAP1 is a member of IAP family that inhibits
apoptosis by direct interaction with caspases to block their activity
(40). Smac is normally a mitochondrial protein and is released to the
cytosol concurrent with the cytochrome c release during
apoptosis (34, 35). It was found to interact with the BIR2-3 domain of
IAPs, the same domain to which caspases bind. The binding of Smac to
IAPs relieves the binding of IAPs to caspases, thus promoting
caspase-mediated apoptosis (41-43). The identification of Smac
and cIAP1 in LIGHT·LT
R complex reveals the role of these proteins
in the pathway of LIGHT·LT
R-induced apoptosis.
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Fig. 2.
Representative fragment ion spectra of
peptides identified in cIAP1 (A) and Smac
(B). Peptide mixture from in-gel digestion of
each protein band was subjected to an automated
data-dependent nano-LC-MS/MS analysis during which the mass
spectrometer was constantly alternating between one MS survey scan
followed by three MS/MS scans. Peptide fragments were labeled according
to the nomenclature suggested by Biemann (50). Two peptides, SALEMGFNR
(A) from cIAP1 and LAEAQIEELR (B) from Smac, were
unambiguously identified by the presence of their almost corresponding
complete b- and y-ion series.
B-inducing kinase by either mass spectrometry or
Western blot analysis, even though their roles have been suggested in
the LT
R signaling (25, 26, 44). This could be due to the low
abundance of the proteins and the high complexity of the samples.
Nevertheless, our success of detecting additional signaling proteins
demonstrates that proteomic approach is indeed a powerful mean to
identify signaling proteins in the endogenous receptor complex.
R--
Among the four proteins identified here, TRAF3 is the only
protein that was previously shown to interact with endogenous LT
R (24). The interaction of TRAF2 with LT
R was shown in the
overexpression system (23, 25), and cIAP1 was found to be a component
of TNFR1 and TNFR2 complexes (40, 45). To confirm the association of
these proteins with the LIGHT·LT
R complex, we treated U937 cells
with FLAG-LIGHT for different times and immunoprecipitated with FLAG
antibody followed by Western blot analysis using antibody against
TRAF2, TRAF3, or cIAP1.
R complex was
time-dependent. Recruitment of cIAP1 was gradually
increased within 15 min. A similar pattern was observed for TRAF3.
Recruitment of TRAF2 appeared to be more rapid, peaked between 5 and 10 min (Fig. 3C). The kinetics of recruitment suggests that
TRAF2 is recruited to the receptor prior to TRAF3 and cIAP1. The direct
interaction of TRAF3 with the intracellular domain of LT
R has been
demonstrated using purified proteins (37), and there is no evidence of
interaction between TRAF2 and TRAF3 (36). Thus, TRAF3 is probably
directly recruited to LT
R upon LIGHT treatment. In contrast, the
recruitment of cIAP1 to LT
R is probably through its interaction with
TRAF2 because cIAP1-TRAF2 interaction has been shown in
vitro and there is no evidence of interaction between cIAP1 and
receptor or TRAF3 (40). Interestingly, cIAP1 in the complex recognized
by a polyclonal antibody raised against the C terminus of cIAP1 (AF818,
R&D systems) is ~60 kDa, smaller than the full-length cIAP1 (~70
kDa) in cell lysate (Fig. 3A). This 60-kDa band was not
detected by another antibody raised against a peptide at the BIR1
domain of the cIAP1 (sc-1867, Santa Cruz Biotechnology), which
recognizes 70-kDa cIAP1 in cell lysate, suggesting that the N terminus
of cIAP1 in the complex might be cleaved. In line with our observation,
cIAP1 was found to be cleaved in response to virus- or transforming
growth factor-
-induced apoptosis (46, 47). The role of this cleavage
is presently unclear.
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Fig. 3.
Time-dependent recruitment of
TRAF2, TRAF3, and cIAP1 to the LIGHT·LT R
complex. U937 cells were treated with FLAG-LIGHT for different
times (as indicated) or left untreated. Immunoprecipitation was
performed with M2 antibody as shown in Fig. 1 followed by Western blots
with anti-cIAP1 (A), anti-TRAF3 (B), or
anti-TRAF2 (C) antibody, respectively. D, as a
control, the amount of TRAF2 in the cell lysate was determined by
Western blot analysis using TRAF2 antibody.
R
complex by mass spectrometry, we were unable to detect the endogenous association using antibodies against Smac (Alexis Biochemicals or Cell
Signaling Technology). This is possibly due to the low sensitivity of
Smac antibodies. Therefore, we investigated the association of Smac
with LT
R by overexpression in HEK293 cells that do not have
endogenous LT
R and HVEM (5). Smac was expressed as a C-terminal
HA-tagged fusion protein and appeared as a doublet of 28 and 23 kDa on
the Western blot, which corresponded to full-length Smac with the
N-terminal mitochondrial localization signal peptide (amino acids
1-55) and the mature Smac without its signal peptide (Fig.
4A) (34). Importantly, both
forms were coimmunoprecipitated with FLAG-tagged LT
R (Fig.
4A, lane 3). When we fractionated cytosol and
mitochondria, we found that while all of the full-length Smac resided
in the mitochondria fraction, a significant amount (approximately
one-third) of the mature Smac was in the cytosolic fraction (data not
shown), consistent with previous reports (34, 35). Therefore, these
data suggest that the cytosolic mature form of Smac is the
physiological form of Smac that interacts with LT
R. The observation
that the full-length Smac was coimmunoprecipitated with LT
R may be
artificial because of the disruption of the mitochondrial membrane by
Triton X-100.
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Fig. 4.
Association of Smac with
LT R. HEK293 cells were cotransfected with
plasmids expressing FLAG-LT
R and Smac-HA or
76Smac-HA as
indicated. After 48 h, cells were harvested and
immunoprecipitation (IP) was performed with either M2
antibody for LT
R (A) or with HA antibody for Smac
(B) followed by Western blots (WB) with anti-HA
(for Smac-HA), anti-cIAP1, anti-TRAF2, or anti-TRAF3 antibody,
respectively. Bottom panels indicate the expression of
FLAG-LT
R, Smac-HA, or
76Smac-HA in each sample.
R, resulting from overexpression. In the reciprocal
immunoprecipitation of Smac using HA antibody, LT
R was detected
(Fig. 4B, lane 2) and further confirmed the association. In accordance with our previous observation in U937 cells,
endogenous TRAF2, cIAP1 (60 kDa), and TRAF3 were also recruited to
LT
R overexpressed in HEK293 cells (Fig. 4A, lane
3). Furthermore, endogenous TRAF2 and cIAP1 were detected in the
reciprocal immunoprecipitation of Smac (Fig. 4B, lane
2), indicating the formation of a complex of
LT
R·TRAF2·cIAP1·Smac. Taken together, these data strongly support the physiological association of TRAF2, TRAF3, cIAP1, and Smac
with LT
R.
76Smac) that lacks both the cIAP1 binding site (amino acids 56-75)
and mitochondrial localization signal lost the ability to bind to
LT
R (Fig. 4A, lane 4). This finding suggests
that the cIAP1 binding site of Smac is important for its recruitment to
the receptor. The interaction between the N terminus of Smac and the
BIR3 domain of XIAP has been demonstrated by the x-ray crystal
structure and mutational analysis (41, 43). Because of the high
homology among IAPs, it is likely that the recruitment of Smac is
mediated by its interaction with the BIR3 domain of cIAP1. Despite the
difference between the full-length and the deletion mutant of Smac,
the level of cIAP1, TRAF2, and TRAF3 on LT
R remained the same (Fig.
4A, lane 4), suggesting that the recruitment of
Smac occurs after the recruitment of TRAF2, TRAF3, and cIAP1.
R-induced Apoptosis--
Smac has been shown
to promote apoptosis in response to several stimuli that trigger the
mitochondria-mediated apoptosis pathway such as UV irradiation (34).
Here, we showed for the first time that Smac is recruited to LT
R. To
assess the function of Smac in LT
R-mediated apoptosis, we
cotransfected MCF7 cells, which express endogenous LT
R, with
full-length Smac or mutant
76Smac together with
pEMC-
-galactosidase and then treated with LIGHT or LT
1
2.
Apoptosis was assessed by the morphological analysis of the
-galactosidase-expressing cells (see "Materials and Methods" for
details). The overexpression of full-length Smac-potentiated apoptosis
(Fig. 5) in MCF7 cells and stimulation of
LIGHT further increased apoptosis. A similar effect was observed in the
LT
1
2-stimulated cells. Interestingly, mutant
76Smac that lost
the ability to recruit to the receptor (Fig. 4A) could still
potentiate LT
R-mediated apoptosis to a degree similar to the
full-length Smac, implying that the association of Smac with the LT
R
may not be necessary for the apoptosis. This result also suggests that
C terminus of Smac possesses proapoptotic activity and is independent
of its interaction with cIAP1. Consistent with our data, Cohen and
colleagues (48) reported that a Smac variant, Smac
, lacking the
N-terminal IAP binding domain still potentiated apoptosis (48).
Therefore, under physiological condition where the cIAP1 binding site
is not known to be cleaved from Smac, Smac may have a dual role in LT
R-mediated apoptosis. The N terminus of Smac antagonizes the function of cIAP1 while C terminus of Smac acts in concert with N
terminus to further promote the apoptosis by an unknown mechanism.
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Fig. 5.
Smac potentiates LIGHT-induced
apoptosis. MCF7 cells were cotransfected with plasmids expressing
-galactosidase (pEMC-
-galactosidase) and Smac-HA,
76Smac-HA,
or empty vector. After 24 h, cells were treated with
phosphate-buffered saline, LIGHT (20 ng/ml), or LT
1
2 (20 ng/ml),
respectively, for 6 h and then fixed and stained with X-gal.
Apoptosis was assessed by morphological analysis and expressed as a
percentage of apoptotic cells (round and detached) in total transfected
blue cells. Bars represent the average of duplicate samples in three
separate experiments. In each experiment, >1000 cells were
counted.
R and TRAF3 (37), TRAF2 and cIAP1 (40), and cIAP1 and Smac
(34) together with our data demonstrating the association of these
molecules with LT
R, the kinetics of the recruitment (Fig. 3), the
formation of the LT
R·TRAF2·cIAP·Smac complex (Fig. 4), and the
dependence of cIAP1 binding site for Smac recruitment (Fig.
4A) have led us to propose a model for the LT
R-induced
apoptosis as diagrammed in Fig. 6. Upon
the binding of LIGHT to LT
R, TRAF2 is first recruited to the
receptor followed by TRAF3 and cIAP1, during which the BIR1 domain of
cIAP1 is cleaved. The cIAP1 on the receptor inhibits apoptosis by
direct interaction with caspases. The initial LIGHT·LT
R complex
also triggers the mitochondria-mediated apoptosis pathway by an unknown
mechanism, which induces the release of Smac from mitochondria. The
cytosolic Smac is then recruited to the receptor via its interaction
with cIAP1. The interaction of N terminus of Smac with cIAP1 releases the inhibition of cIAP1 on caspases while the C terminus of Smac works
in concert with N terminus to promote apoptosis by a mechanism yet to
be identified. It has been reported that LT
R-induced apoptosis required the addition of interferon-
(17). Consistent with the role
of Smac in our model, interferon-
was shown to induce de
novo synthesis of Smac in WEHI 279 cells (49). It should be noted
that TRAF3 has been suggested to play an important role in
LT
R-induced apoptosis based on the inhibitory effect of a dominant
negative mutant of TRAF3 (17, 18, 37). However, the mechanism of its
function is unclear. Based on our model, it is tempting to speculate
that TRAF3 promotes the cIAP1-Smac pathway by triggering the release of
Smac from mitochondria. Future studies on the functional interaction
among these signaling proteins including detailed mutagenesis studies
to define the interaction sites and to generate dominant negative
mutants would be necessary to validate the model and elucidate the
precise mechanism underlying this signaling event.
View larger version (22K):
[in a new window]
Fig. 6.
Proposed model of
LT R-mediated apoptosis. Upon the binding
of LIGHT to LT
R, TRAF2 is first recruited to the receptor followed
by TRAF3 and cIAP1, during which the BIR1 domain of cIAP1 is cleaved.
cIAP1 on the receptor may inhibit activity of caspases by direct
interaction with caspases. The initial LIGHT·LT
R complex also
triggers the mitochondria-mediated apoptosis pathway by an unknown
mechanism, which induces the release of Smac from mitochondria. The
cytosolic Smac is then recruited to the receptor via its interaction
with the BIR3 domain of cIAP1. The N terminus of Smac antagonizes the
function of cIAP1 while the C terminus works in concert with N terminus
to promote LT
R-induced apoptosis.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Shie-Liang Hsieh (National
Yang-Ming University School of Medicine, Taipei, Taiwan) for the
kind gift of pCMV2-FLAG-LTR plasmid. We are grateful to Drs. John
Cuozzo and Jean-Baptiste Telliez for their critical reading of the
paper. We thank Yi Zhu for helping with U937 cell culture. We also
thank Dr. Glenn Larsen (Musculoskeletal Science, Wyeth Research) and
Dr. Rod Hewick (Protein Chemistry, Wyeth Research) for their support of
this project.
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: Neogenesis Pharmaceuticals, Inc., 840 Memorial Dr., Cambridge, MA 02139.
To whom correspondence should be addressed: Musculoskeletal
Science, Wyeth Research, 200 Cambridge Park Dr., Cambridge, MA 02140. Tel.: 617-665-5476; Fax: 617-665-5499; E-mail: llin@wyeth.com.
Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M208672200
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ABBREVIATIONS |
---|
The abbreviations used are:
LTR, lymphotoxin-
receptor;
TNF, tumor necrosis factor;
TNFR, TNF
receptor;
X-gal, 5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside;
IAP, inhibitor of apoptosis protein;
cIAP1, cellular inhibitor of apoptosis
protein 1;
BIR domains, baculoviral inhibitory repeat domains;
TRAF2
and TRAF3, TNF receptor-associated factor 2 and 3, respectively;
DD, death domain;
Smac, second mitochondria-derived activator of caspase;
HA, hemagglutinin;
LIGHT, homologous to lymphotoxins, exhibits
inducible expression, and competes with HSV glycoprotein D for herpes
virus entry mediator;
LC, liquid chromatography;
MS, mass spectrometry;
HVEM, herpes virus entry mediator.
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