Endogenous Association of TRAF2, TRAF3, cIAP1, and Smac with Lymphotoxin beta  Receptor Reveals a Novel Mechanism of Apoptosis*

Jun KuaiDagger , Elliott Nickbarg§, Joe Wooters§, Yongchang Qiu§, Jack Wang§, and Lih-Ling LinDagger ||

From the Dagger  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

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Lymphotoxin-beta receptor (LTbeta 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 LTalpha 1beta 2 and homotrimer LIGHT, and activates multiple signaling pathways including transcriptional factor NFkappa B, c-Jun N-terminal kinase, and cell death. However, the molecular mechanism of the activation of these signaling pathways by LTbeta R is not clear. Because there is no enzymatic activity associated with the receptor itself, the signal transduction of LTbeta R is mediated by cytoplasmic proteins recruited to receptors. To identify these proteins, we took a proteomic approach. The endogenous LIGHT·LTbeta 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 LTbeta R was further confirmed by coimmunoprecipitation in U937 cells and HEK293 cells. The presence of cIAP1 and Smac in LIGHT·LTbeta R complex revealed a novel mechanism of LIGHT·LTbeta R-induced apoptosis.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Lymphotoxin-beta receptor (LTbeta 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 LTalpha 1beta 2 and homotrimer LIGHT. LIGHT is a newly identified TNF family cytokine that is homologous to lymphotoxins and binds to both LTbeta R and HVEM (herpes virus entry mediator also named as TR2) (5). LTbeta 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 LTalpha , LTbeta , or LTbeta R lack lymph nodes and Peyer's patches demonstrating the role of LT/LTbeta 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 LTbeta R-Ig fusion protein can prevent colitis and collagen-induced arthritis in mice (12, 15). Therefore, LT/LTbeta R is an important therapeutic target.

Similar to other members of the TNFR family, the engagement of ligands to LTbeta R induces receptor aggregation and subsequent activation of multiple signaling pathways including transcription factor NFkappa 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 LTbeta 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 LTbeta R knock-out mice and the diverse activity of LT cytokines, additional signaling proteins are yet to be identified.

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, LTbeta R does not contain a death domain nor can it recruit FADD (36). Nevertheless, LTbeta R induces cell death in some carcinoma cell lines (17, 18). Although TRAF3 has been shown to play a critical role in LTbeta R-mediated apoptosis (24, 37), the precise mechanism of LTbeta R-mediated apoptosis remains to be elucidated.

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 LTbeta R signaling, we applied this approach to identify proteins associated with an affinity-purified endogenous LIGHT·LTbeta 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·LTbeta R complex and reveals a novel mechanism of apoptosis utilized by LIGHT·LTbeta R.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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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 LTbeta R in pFLAG-CMV2 vector (Eastman Kodak, Co.) was a kind gift from Dr. Shie-Liang Hsieh (18). The full-length and Delta 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.

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-LTbeta R, pcDNA3-Smac-HA or pcDNA3-Delta 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 LTbeta R or with HA monoclonal antibody for Smac or Delta 76Smac. The presence of Smac, LTbeta R, TRAF2, and cIAP1 in the immune complex were then analyzed by Western blots.

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-Delta 76Smac-HA expression constructs together with pEMC-beta -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 LTalpha 1beta 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Identification of TRAF3, TRAF2, cIAP1, and Smac in LIGHT·LTbeta R Complex from U937 Cells-- The binding of either homotrimer LIGHT or heterotrimer LTalpha 1beta 2 to LTbeta R induces aggregation of the receptors and subsequent recruitment of cytosolic signaling proteins, resulting in the formation of ligand-LTbeta R complex that initiates the downstream signal transduction. Similar to other members of the TNFR family, LTbeta R activates multiple signaling pathways but so far only endogenous TRAF3 was shown to be recruited to LTbeta 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.

LIGHT binds to both LTbeta R and TR2/HVEM. In U937 cells, LTbeta R is constitutively expressed while the expression of TR2/HVEM is induced by differentiating agents (5, 6, 39). To form a specific ligand-LTbeta 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·LTbeta 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·LTbeta 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).

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 LTbeta R has been reported previously in the overexpression system (23, 25). Our detection of TRAF2 in the endogenous LIGHT·LTbeta R complex confirmed the previous observation and further demonstrated that the endogenous TRAF2 is a signal transducer of LTbeta 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.


                              
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Table I
List of proteins identified in LIGHT · LTbeta R complex
Proteins listed in table were only detected in LIGHT-treated sample but not in control.

As expected, LTbeta R was detected as the receptor in the complex (Table I). The detection of LIGHT and LTbeta R in the complex further confirmed our success in the isolation of LIGHT·LTbeta 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·LTbeta R complex reveals the role of these proteins in the pathway of LIGHT·LTbeta 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.

It should also be noted that we did not detect any peptides of TRAF4, TRAF5, or NFkappa B-inducing kinase by either mass spectrometry or Western blot analysis, even though their roles have been suggested in the LTbeta 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.

Association of TRAF2, TRAF3, cIAP1, and Smac with LTbeta R-- Among the four proteins identified here, TRAF3 is the only protein that was previously shown to interact with endogenous LTbeta R (24). The interaction of TRAF2 with LTbeta 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·LTbeta 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.

As shown in Fig. 3, the recruitment of endogenous TRAF2, TRAF3, and cIAP1 to LIGHT·LTbeta 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 LTbeta 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 LTbeta R upon LIGHT treatment. In contrast, the recruitment of cIAP1 to LTbeta 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-beta -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·LTbeta 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.

Although we detected two peptides from Smac in the LIGHT·LTbeta 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 LTbeta R by overexpression in HEK293 cells that do not have endogenous LTbeta 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 LTbeta 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 LTbeta R. The observation that the full-length Smac was coimmunoprecipitated with LTbeta 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 LTbeta R. HEK293 cells were cotransfected with plasmids expressing FLAG-LTbeta R and Smac-HA or Delta 76Smac-HA as indicated. After 48 h, cells were harvested and immunoprecipitation (IP) was performed with either M2 antibody for LTbeta 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-LTbeta R, Smac-HA, or Delta 76Smac-HA in each sample.

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 LTbeta R, resulting from overexpression. In the reciprocal immunoprecipitation of Smac using HA antibody, LTbeta 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 LTbeta 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 LTbeta R·TRAF2·cIAP1·Smac. Taken together, these data strongly support the physiological association of TRAF2, TRAF3, cIAP1, and Smac with LTbeta R.

In contrast to the full-length Smac, the deletion mutant of Smac (Delta 76Smac) that lacks both the cIAP1 binding site (amino acids 56-75) and mitochondrial localization signal lost the ability to bind to LTbeta 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 LTbeta R remained the same (Fig. 4A, lane 4), suggesting that the recruitment of Smac occurs after the recruitment of TRAF2, TRAF3, and cIAP1.

Smac Potentiates LTbeta 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 LTbeta R. To assess the function of Smac in LTbeta R-mediated apoptosis, we cotransfected MCF7 cells, which express endogenous LTbeta R, with full-length Smac or mutant Delta 76Smac together with pEMC-beta -galactosidase and then treated with LIGHT or LTalpha 1beta 2. Apoptosis was assessed by the morphological analysis of the beta -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 LTalpha 1beta 2-stimulated cells. Interestingly, mutant Delta 76Smac that lost the ability to recruit to the receptor (Fig. 4A) could still potentiate LTbeta R-mediated apoptosis to a degree similar to the full-length Smac, implying that the association of Smac with the LTbeta 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, Smacbeta , 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 LTbeta 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 beta -galactosidase (pEMC-beta -galactosidase) and Smac-HA, Delta 76Smac-HA, or empty vector. After 24 h, cells were treated with phosphate-buffered saline, LIGHT (20 ng/ml), or LTalpha 1beta 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.

In summary, combining the reported evidence for direct interactions among LTbeta R and TRAF3 (37), TRAF2 and cIAP1 (40), and cIAP1 and Smac (34) together with our data demonstrating the association of these molecules with LTbeta R, the kinetics of the recruitment (Fig. 3), the formation of the LTbeta 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 LTbeta R-induced apoptosis as diagrammed in Fig. 6. Upon the binding of LIGHT to LTbeta 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·LTbeta 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 LTbeta R-induced apoptosis required the addition of interferon-gamma (17). Consistent with the role of Smac in our model, interferon-gamma 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 LTbeta 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.


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Fig. 6.   Proposed model of LTbeta R-mediated apoptosis. Upon the binding of LIGHT to LTbeta 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·LTbeta 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 LTbeta R-induced apoptosis.


    ACKNOWLEDGEMENTS

We thank Dr. Shie-Liang Hsieh (National Yang-Ming University School of Medicine, Taipei, Taiwan) for the kind gift of pCMV2-FLAG-LTbeta R 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.

    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

    ABBREVIATIONS

The abbreviations used are: LTbeta R, lymphotoxin-beta receptor; TNF, tumor necrosis factor; TNFR, TNF receptor; X-gal, 5-bromo-4-chloro-3-indolyl-beta -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.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

1. Rennert, P. D., James, D., Mackay, F., Browning, J. L., and Hochman, P. S. (1998) Immunity 9, 71-79[Medline] [Order article via Infotrieve]
2. Futterer, A., Mink, K., Luz, A., Kosco-Vilbois, M. H., and Pfeffer, K. (1998) Immunity 9, 59-70[Medline] [Order article via Infotrieve]
3. Ettinger, R., Browning, J. L., Michie, S. A., van Ewijk, W., and McDevitt, H. O. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13102-13107[Abstract/Free Full Text]
4. Kuprash, D. V., Alimzhanov, M. B., Tumanov, A. V., Anderson, A. O., Pfeffer, K., and Nedospasov, S. A. (1999) J. Immunol. 163, 6575-6580[Abstract/Free Full Text]
5. Zhai, Y., Guo, R., Hsu, T. L., Yu, G. L., Ni, J., Kwon, B. S., Jiang, G. W., Lu, J., Tan, J., Ugustus, M., Carter, K., Rojas, L., Zhu, F., Lincoln, C., Endress, G., Xing, L., Wang, S., Oh, K. O., Gentz, R., Ruben, S., Lippman, M. E., Hsieh, S. L., and Yang, D. (1998) J. Clin. Invest. 102, 1142-1151[Abstract/Free Full Text]
6. Murphy, M., Walter, B. N., Pike-Nobile, L., Fanger, N. A., Guyre, P. M., Browning, J. L., Ware, C. F., and Epstein, L. B. (1998) Cell Death Differ. 5, 497-505[CrossRef][Medline] [Order article via Infotrieve]
7. Banks, T. A., Rouse, B. T., Kerley, M. K., Blair, P. J., Godfrey, V. L., Kuklin, N. A., Bouley, D. M., Thomas, J., Kanangat, S., and Mucenski, M. L. (1995) J. Immunol. 155, 1685-1693[Abstract]
8. De Togni, P., Goellner, J., Ruddle, N. H., Streeter, P. R., Fick, A., Mariathasan, S., Smith, S. C., Carlson, R., Shornick, L. P., and Strauss-Schoenberger, J. (1994) Science 264, 703-707[Medline] [Order article via Infotrieve]
9. Alimzhanov, M. B., Kuprash, D. V., Kosco-Vilbois, M. H., Luz, A., Turetskaya, R. L., Tarakhovsky, A., Rajewsky, K., Nedospasov, S. A., and Pfeffer, K. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9302-9307[Abstract/Free Full Text]
10. Koni, P. A., Sacca, R., Lawton, P., Browning, J. L., Ruddle, N. H., and Flavell, R. A. (1997) Immunity 6, 491-500[Medline] [Order article via Infotrieve]
11. Takemura, S., Braun, A., Crowson, C., Kurtin, P. J., Cofield, R. H., O'Fallon, W. M., Goronzy, J. J., and Weyand, C. M. (2001) J. Immunol. 167, 1072-1080[Abstract/Free Full Text]
12. Dohi, T., Rennert, P. D., Fujihashi, K., Kiyono, H., Shirai, Y., Kawamura, Y. I., Browning, J. L., and McGhee, J. R. (2001) J. Immunol. 167, 2781-2790[Abstract/Free Full Text]
13. Ettinger, R., Munson, S. H., Chao, C. C., Vadeboncoeur, M., Toma, J., and McDevitt, H. O. (2001) J. Exp. Med. 193, 1333-1340[Abstract/Free Full Text]
14. Wu, Q., Salomon, B., Chen, M., Wang, Y., Hoffman, L. M., Bluestone, J. A., and Fu, Y. X. (2001) J. Exp. Med. 193, 1327-1332[Abstract/Free Full Text]
15. Fava, R., Gonzales, M., Szanya, V., Hunt, J., Diegel, R., and Browning, J. (1998) J. Interferon Cytokine Res. 18, 95[Medline] [Order article via Infotrieve] (abstr.)
16. Mackay, F., Majeau, G. R., Hochman, P. S., and Browning, J. L. (1996) J. Biol. Chem. 271, 24934-24938[Abstract/Free Full Text]
17. Browning, J. L., Miatkowski, K., Sizing, I., Griffiths, D., Zafari, M., Benjamin, C. D., Meier, W., and Mackay, F. (1996) J. Exp. Med. 183, 867-878[Abstract]
18. Wu, M. Y., Wang, P. Y., Han, S. H., and Hsieh, S. L. (1999) J. Biol. Chem. 274, 11868-11873[Abstract/Free Full Text]
19. Tartaglia, L. A., Ayres, T. M., Wong, G. H., and Goeddel, D. V. (1993) Cell 74, 845-853[Medline] [Order article via Infotrieve]
20. Rothe, M., Wong, S. C., Henzel, W. J., and Goeddel, D. V. (1994) Cell 78, 681-692[Medline] [Order article via Infotrieve]
21. Hsu, H., Shu, H. B., Pan, M. G., and Goeddel, D. V. (1996) Cell 84, 299-308[Medline] [Order article via Infotrieve]
22. Ye, X., Mehlen, P., Rabizadeh, S., VanArsdale, T., Zhang, H., Shin, H., Wang, J. J., Leo, E., Zapata, J., Hauser, C. A., Reed, J. C., and Bredesen, D. E. (1999) J. Biol. Chem. 274, 30202-30208[Abstract/Free Full Text]
23. Nakano, H., Oshima, H., Chung, W., Williams-Abbott, L., Ware, C. F., Yagita, H., and Okumura, K. (1996) J. Biol. Chem. 271, 14661-14664[Abstract/Free Full Text]
24. VanArsdale, T. L., VanArsdale, S. L., Force, W. R., Walter, B. N., Mosialos, G., Kieff, E., Reed, J. C., and Ware, C. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2460-2465[Abstract/Free Full Text]
25. Marsters, S. A., Ayres, T. M., Skubatch, M., Gray, C. L., Rothe, M., and Ashkenazi, A. (1997) J. Biol. Chem. 272, 14029-14032[Abstract/Free Full Text]
26. Krajewska, M., Krajewski, S., Zapata, J. M., Van Arsdale, T., Gascoyne, R. D., Berern, K., McFadden, D., Shabaik, A., Hugh, J., Reynolds, A., Clevenger, C. V., and Reed, J. C. (1998) Am. J. Pathol. 152, 1549-1561[Abstract]
27. Darnay, B. G., and Aggarwal, B. B. (1999) Ann. Rheum. Dis. 58, I2-I13[Medline] [Order article via Infotrieve]
28. Baud, V., and Karin, M. (2001) Trends Cell Biol. 11, 372-377[CrossRef][Medline] [Order article via Infotrieve]
29. Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998) Cell 94, 491-501[Medline] [Order article via Infotrieve]
30. Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998) Cell 94, 481-490[Medline] [Order article via Infotrieve]
31. Zhang, X. D., Zhang, X. Y., Gray, C. P., Nguyen, T., and Hersey, P. (2001) Cancer Res. 61, 7339-7348[Abstract/Free Full Text]
32. Deng, Y., Lin, Y., and Wu, X. (2002) Genes Dev. 16, 33-45[Abstract/Free Full Text]
33. Madesh, M., Antonsson, B., Srinivasula, S. M., Alnemri, E. S., and Hajnoczky, G. (2002) J. Biol. Chem. 277, 5651-5659[Abstract/Free Full Text]
34. Du, C., Fang, M., Li, Y., Li, L., and Wang, X. (2000) Cell 102, 33-42[Medline] [Order article via Infotrieve]
35. Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G. E., Moritz, R. L., Simpson, R. J., and Vaux, D. L. (2000) Cell 102, 43-53[Medline] [Order article via Infotrieve]
36. Wajant, H., Grell, M., and Scheurich, P. (1999) Cytokine Growth Factor Rev. 10, 15-26[CrossRef][Medline] [Order article via Infotrieve]
37. Force, W. R., Cheung, T. C., and Ware, C. F. (1997) J. Biol. Chem. 272, 30835-30840[Abstract/Free Full Text]
38. Houthaeve, T., Gausepohl, H., Ashman, K., Nillson, T., and Mann, M. (1997) J. Protein Chem. 16, 343-348[Medline] [Order article via Infotrieve]
39. Kwon, B. S., Tan, K. B., Ni, J., Oh, K. O., Lee, Z. H., Kim, K. K., Kim, Y. J., Wang, S., Gentz, R., Yu, G. L., Harrop, J., Lyn, S. D., Silverman, C., Porter, T. G., Truneh, A., and Young, P. R. (1997) J. Biol. Chem. 272, 14272-14276[Abstract/Free Full Text]
40. Rothe, M., Pan, M. G., Henzel, W. J., Ayres, T. M., and Goeddel, D. V. (1995) Cell 83, 1243-1252[Medline] [Order article via Infotrieve]
41. Chai, J., Du, C., Wu, J. W., Kyin, S., Wang, X., and Shi, Y. (2000) Nature 406, 855-862[CrossRef][Medline] [Order article via Infotrieve]
42. Liu, Z., Sun, C., Olejniczak, E. T., Meadows, R. P., Betz, S. F., Oost, T., Herrmann, J., Wu, J. C., and Fesik, S. W. (2000) Nature 408, 1004-1008[CrossRef][Medline] [Order article via Infotrieve]
43. Wu, G., Chai, J., Suber, T. L., Wu, J. W., Du, C., Wang, X., and Shi, Y. (2000) Nature 408, 1008-1012[CrossRef][Medline] [Order article via Infotrieve]
44. Smith, C., Andreakos, E., Crawley, J. B., Brennan, F. M., Feldmann, M., and Foxwell, B. M. (2001) J. Immunol. 167, 5895-5903[Abstract/Free Full Text]
45. Shu, H. B., Takeuchi, M., and Goeddel, D. V. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 13973-13978[Abstract/Free Full Text]
46. Herrera, B., Fernandez, M., Benito, M., Fabregat, I., Salvesen, G. S., Duckett, C. S., Gozani, O., Boyce, M., Yoo, L., Karuman, P., Yuan, J., Cain, K., Bratton, S. B., Cohen, G. M., Guo, F., Nimmanapalli, R., Paranawithana, S., Wittman, S., Griffin, D., Bali, P., O'Bryan, E., Fumero, C., Wang, H. G., Bhalla, K., Taurin, S., Ryazhsky, G. G., Maximova, N. V., Chuchalin, A. G., Hamet, P., Pshezhetsky, A. V., Orlov, S. N., Verhagen, A. M., and Vaux, D. L. (2002) FEBS Lett. 520, 93-96[CrossRef][Medline] [Order article via Infotrieve]
47. Clem, R. J., Sheu, T. T., Richter, B. W., He, W. W., Thornberry, N. A., Duckett, C. S., and Hardwick, J. M. (2001) J. Biol. Chem. 276, 7602-7608[Abstract/Free Full Text]
48. Roberts, D. L., Merrison, W., MacFarlane, M., and Cohen, G. M. (2001) J. Cell Biol. 153, 221-228[Abstract/Free Full Text]
49. Yoshikawa, H., Nakajima, Y., and Tasaka, K. (2001) J. Immunol. 167, 2487-2495[Abstract/Free Full Text]
50. Biemann, K. (1988) Biomed. Environ. Mass Spectrom. 16, 99-111[Medline] [Order article via Infotrieve]


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