Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M408678200 on October 1, 2004

J. Biol. Chem., Vol. 279, Issue 50, 52106-52116, December 10, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
279/50/52106    most recent
M408678200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, Q.
Right arrow Articles by Chui, Y.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, Q.
Right arrow Articles by Chui, Y.-L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

A Death Receptor-associated Anti-apoptotic Protein, BRE, Inhibits Mitochondrial Apoptotic Pathway*{boxs}

Qing Li{ddagger}§, Arthur Kar-Keung Ching{ddagger}§, Ben Chung-Lap Chan{ddagger}||, Stephanie Ka-Yee Chow{ddagger}, Pak-Leong Lim{ddagger}, Tony Cheong-Yip Ho**{ddagger}{ddagger}, Wai-Ki Ip**, Chun-Kwok Wong**, Christopher Wai-Kei Lam**, Kenneth Ka-Ho Lee§§, John Yeuk-Hon Chan¶¶, and Yiu-Loon Chui{ddagger}||||

From the {ddagger}Clinical Immunology Unit and Sir Y. K. Pao Centre for Cancer, the **Department of Chemical Pathology, and the §§Department of Anatomy, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, New Territory, Hong Kong Special Administrative Region, China, and the ¶¶Department of Molecular Pathology, University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030

Received for publication, July 30, 2004 , and in revised form, September 8, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
BRE, brain and reproductive organ-expressed protein, was found previously to bind the intracellular juxtamembrane domain of a ubiquitous death receptor, tumor necrosis factor receptor 1 (TNF-R1), and to down-regulate TNF-{alpha}-induced activation of NF-{kappa}B. Here we show that BRE also binds to another death receptor, Fas, and upon overexpression conferred resistance to apoptosis induced by TNF-{alpha}, anti-Fas agonist antibody, cycloheximide, and a variety of stress-related stimuli. However, down-regulation of the endogenous BRE by small interfering RNA increased apoptosis to TNF-{alpha}, but nottoetoposide, indicating that the physiological antiapoptotic role of this protein is specific to death receptor-mediated apoptosis. We further demonstrate that BRE mediates antiapoptosis by inhibiting the mitochondrial apoptotic machinery but without translocation to the mitochondria or nucleus or down-regulation of the cellular level of truncated Bid. Dissociation of BRE rapidly from TNF-R1, but not from Fas, upon receptor ligation suggests that this protein interacts with the death inducing signaling complex during apoptotic induction. Increased association of BREwith phosphorylated, sumoylated, and ubiquitinated proteins after death receptor stimulation was also detected. We conclude that in contrast to the truncated Bid that integrates mitochondrial apoptosis to death receptor-triggered apoptotic cascade, BRE inhibits the integration. We propose that BRE inhibits, by ubiquitination-like activity, components in or proximal to the death-inducing signaling complexes that are necessary for activation of the mitochondria.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosis plays an essential role in growth development, cellular homeostasis, and defense against abnormal cellular changes (1, 2). Cells receive external signals through surface death receptors, such as the widely expressed TNF-R11 and Fas (3, 4), or internal stress signals, such as DNA damage (5), to undergo extrinsic or intrinsic pathway of apoptosis, respectively (6). The extrinsic apoptosis involves formation of death-inducing signaling complex (DISC) and activation of the apical caspase-8, which in turn starts an activation cascade of executioner caspases to dismantle the cell (7–12). Intrinsic apoptosis involves release of pro-apoptotic factors from the intermembrane compartment of mitochondria mediated by Bcl-2 family members following cellular stress, to trigger both caspase-dependent and -independent apoptosis (13–15). Although cellular apoptosis can occur by either pathway independently, these pathways are linked by tBid, which recruits the mitochondrial pro-apoptotic activities to death receptor-mediated apoptosis (16, 17). In reverse, Smac/DIABLO released from the mitochondria is needed to promote activation of caspase-8 in the DISC generated by TNF-R1 activation (18). Cross-talk between the two pathways has been shown to be essential to death receptor-mediated apoptosis in some cell types (18–21).

Death receptor-mediated apoptosis is regulated by intracellular proteins that include TRAFs, inhibitors of apoptosis, and c-FLIP, induction of some of which are NF-{kappa}B-dependent (22–24). By yeast two-hybrid screens, the anti-apoptotic proteins SODD and SUMO-1 that bind to the death domain (DD) of death receptors were identified (25, 26). The same approach also resulted in identification of BRE as a binding protein of TNF-R1 (27). BRE is a highly conserved and widely expressed putative stress-modulating gene (28–30). However, distinctly unlike SODD and SUMO-1, BRE binds to the juxtamembrane cytoplasmic region of the receptor. Whereas DDs are known to recruit adaptor molecules for signal transduction (31, 32), function of the membrane-proximal cytoplasmic region has not been clearly elucidated, although it may be involved in NO synthase induction (32). Overexpression of this protein by transfection was shown to down-modulate TNF-{alpha}-induced activation of the anti-apoptotic NF-{kappa}B (27). This indicated that BRE could promote apoptosis. Alternatively, BRE might function like SODD to block TNF-R1 signaling at the very proximal end, thus inhibiting both NF-{kappa}B and apoptotic pathways (26). Here we sought to determine whether BRE has a pro- or anti-apoptotic role in the death receptor pathway, and by what mechanism apoptosis is regulated by this protein.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Antibodies (clone or code number) and reagents were purchased from the following sources: rabbit polyclonal anti-TNF-R1 and anti-ubiquitin antibodies (Calbiochem); rabbit polyclonal anti-Fas (C-20), anti-TRADD (H-278), and mouse monoclonal anti-SUMO-1 (D-11) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); mouse monoclonal anti-GAPDH (9.B.88) and anti-{beta}-actin (2A2.1) antibodies (U. S. Biological, Swampscott, MA); mouse monoclonal anti-Fas (13), anti-G28, and anti-BiP antibodies (Transduction Laboratories); mouse monoclonal anti-Fas (DX2), anti-cytochrome c (7H8.2C12), rat monoclonal anti-TGF (A75-2.1), rabbit polyclonal anti-BID, and anti-caspase-8 (poly-1326) antibodies (Pharmingen); rabbit polyclonal anti-cleaved caspase-3, anti-cleaved caspase-9, anti-PARP, and mouse monoclonal anti-caspase-8 (1C12) antibodies (Cell Signaling Technology, Beverly, MA); mouse monoclonal anti-Fas IgM antibody (CH11), anti-Smac/DIABLO (78-1-118), and anti-histone H1 (AE-4) antibodies (Upstate Biotechnology, Inc.); rabbit polyclonal anti-pan-phosphoprotein and mouse monoclonal anti-{beta}-tubulin (2-28-33) antibodies (Zymed Laboratories Inc.); rabbit polyclonal anti-catalase antibody (Abcam Ltd., Cambridge, UK); mouse monoclonal anti-prohibitin (Ab-1) antibody (Lab Vision Corp., Fremont, CA); mouse monoclonal anti-TNF-R1 antibodies (16803) with and without FITC conjugation, and goat anti-TNF-R1 affinity-purified polyclonal antibody (R&D Systems, Minneapolis, MN); mouse monoclonal anti-V5 antibody and the horseradish peroxidase (HRP) conjugate (Invitrogen); HRP-conjugated anti-rabbit Ig secondary antibody (Promega, Madison, WI); FITC-conjugated anti-rabbit, anti-mouse, HRP-conjugated anti-mouse Ig secondary antibodies, and protease inhibitors mixture (Sigma); human recombinant TNF-{alpha} (Invitrogen); {lambda}-protein phosphatase (New England Biolabs, Beverly, MA); GS-BRE (ResGen GeneStorm® Clone for accession number L38616 [GenBank] ), GS alone expression vector, G418, Zeocin, and Lipofectamine 2000 (Invitrogen).

Establishment of Stable Transfectants—HeLa D122 cells were transfected with 3 µg of full-length BRE cloned into pcDNA3.1 expression vector or GS-BRE by Lipofectamine 2000. HeLa-stable transfectants of BRE or GS-BRE were selected for 2 weeks by 2 mg/ml G418 (Invitrogen) or 400 µg/ml Zeocin (Invitrogen), respectively, before being subjected to cloning by limiting dilution. D122 stable transfectants of BRE was likewise obtained, but the concentration of G418 used was 600 µg/ml. Jurkat A3 cells were transfected with 10 µg of BglII-linearized GS-BRE by electroporation using Bio-Rad gene pulsar (1.6 kV at 25 microfarads) and selected for 2 weeks by 50 µg/ml Zeocin before cloning.

Annexin-V Apoptosis Assay—Cells were treated with various death stimuli for the indicated time. Apoptosis as indicated by phosphatidylserine exposure was detected using annexin V-FITC apoptosis detection kit (Pharmingen) or TACSTM annexin V-FITC apoptosis detection kit (Trevigen, Gaithersburg, MD), according to the manufacturer's manual. Flow analysis was conducted using BD Biosciences FACSCalibur flow cytometer and CellQuest software. All time points were measured in triplicate, and results were expressed as mean ± S.E. of multiple (≥3) independent experiments.

Preparation and Transfection of siRNA—Two BRE-specific siRNA, Si1 and Si3, corresponding to TCTGGCTGCACATCATTGA (nucleotides 124–142, nucleotide position number 1 being the start of the initiation codon), and CTGGACTGGTGAATTTTCA (nucleotides 491–509), respectively, were custom designed by and purchased from Qiagen (Valencia, CA). A ready-made {beta}-actin siRNA corresponding to TGAAGATCAAGATCATTGC was directly purchased from Qiagen. siRNA was transfected at 150 nM using Lipofectamine 2000 according to the manufacturer's protocol. At 48 h after the first transfection, a second and third round of transfection with the same siRNA were performed with an interval of 48 h.

Semi-quantitative RT-PCR—Total RNA from cells was prepared by RNeasy mini kit (Qiagen), treated with DNase I (amplification grade) (Invitrogen), and reverse-transcribed using oligo(dT)12–18 (Invitrogen) as primer. All PCRs were carried out in a 25-µl reaction volume containing 1 unit of AmpliTaq Gold (PerkinElmer Life Sciences), 0.2 mM dNTP, 2 mM MgCl2 and 1x AmpliTaq Gold buffer. For amplification of the ectopically expressed BRE, 28–30 cycles at the annealing temperature of 52.4 °C using the forward primer, T7 (5'-TAA TAC GAC TCA CTA TAG GG), and reverse primer 1179L18 (5'-CAT CTG GGG CTG TAC GGA), were performed. For the endogenous BRE, 26 cycles at an annealing temperature of 57.7 °C were performed with 1093U18 (5'-CCT CGA GAC CAG CCA ACT) and 1650L18 (5'-AGT GAA GGG CTC TAG GAT) as the forward and reverse primers, respectively. Clontech primers, forward (5'-ACC ACA GTC CAT GCC ATC AC) and reverse G3PDH3 (5'-TCC ACC ACC CTG TTG CTG TA) were used for 16 cycles at annealing temperature of 61.9 °C for amplifying GAPDH.

Immunoprecipitation and Western Blotting—HeLa cells (1 x 106) at 48 h after transient transfection with 3 µ g of GS-BRE, GS-BRE ({Delta}His6), or V5-BRE using Lipofectamine 2000 (Invitrogen), or the same number of the HeLa stable GS-BRE transfectant, NHGS2, at various time points after treatment with TNF-{alpha} or anti-Fas agonist antibody (CH11) were lysed in 200 µl of lysis buffer (50 mM NaCl, 20 mM Tris, pH 7.6, 1% Nonidet P-40, 1x protease inhibitor mixture) for 1 h. The lysates were cleared by centrifugation at 16,000 x g at 4 °C for 10 min. Crude protein concentration was measured by BCA protein assay kit (Pierce). Two hundred µg of total protein of each sample was transferred to a new microcentrifuge tube, and immunoprecipitation was performed with 2 µg of the indicated antibodies and 50 µl of protein-G slurry for overnight incubation. Ten µl of the precipitated materials were subjected to Western blot analysis with the indicated antibodies. Primary antibody was incubated with the membrane in the dilution recommended by the manufacturer. The bound primary antibody was visualized with HRP-conjugated secondary antibody using LumingenTM PS-3 detection reagent (Amersham Biosciences) with exposure to x-ray film (Kodak).

{lambda}-Phosphatase Treatment—2 x 106 HeLa cells at 48 h after transient transfection with GS-BRE were lysed in 500 µl of lysis buffer containing 1% Nonidet P-40, 20 µl of 10x reaction buffer, 20 µl of 10x MnCl2 (20 mM). For each 155 µl of lysate, 5 µl of {lambda}-protein phosphatase (2,000 units) was added with or without 1 mM sodium vanadate for incubation at 30 °C for 60 min. Reaction was stopped by cooling on ice, and the samples were then subjected to immunoprecipitation by anti-pan-phosphoprotein antibody and Western blotting analysis as above.

Subcellular Fractionation—For preparation of the nuclear fraction, 1 x 107 cells were required using the nuclear/cytosol fractionation kit in accordance with the manufacturer's protocol (BioVision, Inc., Mountain View, CA). For the preparation of cytosolic, microsomal, and mitochondrial fractions, 1 x 108 cells were required using digitonin lysis buffer (20 mM Hepes, pH 7.4, 250 mM sucrose, 0.05% digitonin, 1 mM EDTA, 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol, and protease inhibitors mixture). Nuclei and unbroken cells were removed by centrifugation at 700 x g for 3 min. The supernatant was further centrifuged at 13,000 x g for 2 min to pellet the mitochondria. The remaining supernatant was further centrifuged at 100,000 x g for 1 h to pellet the microsomes, leaving the supernatant as the cytosolic fraction. The mitochondrial and microsomal pellets were subsequently lysed in 1% Nonidet P-40 lysis buffer.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Reduction of TNF-{alpha} and Cycloheximide Apoptosis by BRE Overexpression—To determine the effect of BRE on TNF-{alpha}-induced apoptosis, three cell lines, D122 mouse Lewis lung carcinoma (33), HeLa human cervical carcinoma, and Jurkat A3 human acute T cell leukemia subclone selected for high sensitivity to anti-Fas agonist antibody (34), were stably transfected with human BRE expression constructs and subjected to treatment with TNF-{alpha} in the presence of cycloheximide. Overexpression of BRE (Fig. 1, A and B) or GS-BRE, a C-terminally His6- and V5-tagged form of BRE (Fig. 1C), partially suppressed TNF-{alpha}-induced apoptosis in D122 and HeLa cell lines. The V5 epitope of GS-BRE allows protein expression to be detected in the transfectants using an anti-V5 monoclonal antibody. Although a rabbit polyclonal antibody with specificity to BRE was reported previously (35), we were not able to confirm such specificity by using siRNA to guide identification of the BRE target band in immunoblotting. For the cycloheximide-sensitive Jurkat cells (36, 37), stable transfection with GS-BRE, as shown by GSB1 and GSB2, reduced the apoptosis induced by a low dose of cycloheximide at 0.1 µg/ml (Fig. 1D) and also at a higher dose of 10 µg/ml (Fig. 1E), used in sensitizing HeLa to TNF-{alpha}-induced apoptosis. Combining TNF-{alpha} with the low dose of cycloheximide increased apoptosis of the Jurkat cells, but the transfectant GSB2 remained more resistant to apoptosis than the parent and empty vector controls. GSB1, which expressed a lower level of GS-BRE, was no longer resistant (Fig. 1D).



View larger version (33K):
[in this window]
[in a new window]
  		FIG. 1.
Overexpression of BRE down-modulates apoptosis induced by TNF-{alpha} in the presence of cycloheximide or by cycloheximide alone. A, D122-p3BRE-a4 and -a6 are two D122 stable transfectants expressing full-length human BRE under the control of cytomegalovirus promoter of pcDNA3. Untransfected D122 and a stable transfectant clone of pcDNA3 vector only, D122-pcDNA3-A, are negative controls. Apoptosis was induced by treatment with 20 ng/ml TNF-{alpha} and 5 µg/ml cycloheximide at indicated time points. Expression levels of the transfected BRE gene was estimated by semi-quantitative RT-PCR, with GAPDH as control for mRNA input. B, HeLa-p3BRE-H5 and -H6 are two HeLa stable transfectants of the same BRE expression construct as in A. Apoptosis was induced by treatment with 100 ng/ml TNF-{alpha} and 10 µg/ml cycloheximide at indicated time points. C, GS3, -4, and -6 and NHGS2 and NHGS3 are the HeLa stable transfectants expressing GS-BRE. Apoptosis was induced as in B. GS-BRE levels in the transfectants were analyzed by Western blotting using an anti-V5 monoclonal antibody with anti-{beta}-actin antibody to visualize protein loading. D and E, GSB1 and -2 are two Jurkat stable transfectants expressing GS-BRE. Cells were either untreated or treated with 0.1 or 10 µg/ml cycloheximide (CHX) alone, with or without 100 ng/ml of TNF-{alpha}, or 100 ng/ml TNF-{alpha} alone for 18 h. Anti-GAPDH antibody in Western blotting analysis was for visualizing protein loading. Percentage of cell viability was determined by flow cytometry in conjunction with annexin V-FITC apoptosis detection kit (Pharmingen).

 
Promotion of TNF-{alpha}-induced Apoptosis by Down-regulation of BRE—To determine whether BRE at its endogenous expression level has any anti-apoptotic effect to TNF-{alpha}, endogenous BRE expression in HeLa was down-regulated by siRNA. As shown in Fig. 2, A and B, two BRE siRNAs, Si1 and Si3, were found to suppress significantly both the GS-BRE protein expression in NHGS2 (the HeLa stable transfectant shown above in Fig. 1C with the highest expression level of GS-BRE), and the endogenous BRE transcript levels in NHGS2 and the parent HeLa. Si3 was more effective than Si1. As expected, the minor BRE transcripts, {alpha}b and {alpha}c (29), were also suppressed. As shown in Fig. 2C, down-regulation of BRE in HeLa and BRE and GS-BRE in NHGS2 by Si3 increased susceptibility of the cells to TNF-{alpha}-induced apoptosis at 8 and 16 h of the cytokine treatment. Si1 and the {beta}-actin siRNA (as negative control) had no effect compared with the mock transfection. Interestingly, the basal cell survival (without TNF-{alpha} treatment) was not affected by Si3 treatment. Thus, BRE has a physiological role in inhibiting TNF-{alpha}-induced apoptosis.



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 2.
siRNA-knockdown of BRE increases sensitivity to TNF-{alpha}-induced apoptosis. A, Western blot analysis for the expression levels of GS-BRE in NHGS2 treated with BRE-specific siRNA, Si1, and Si3. Mock (transfection without siRNA) and transfection with {beta}-actin siRNA are negative controls. B, semi-quantitative RT-PCR of the endogenous BRE in HeLa and NHGS2 with or without BRE siRNA treatment, with GAPDH as control for mRNA input. C, percentage of cell viability of the same panel of cells as in B treated with TNF-{alpha} (20 ng/ml) in the presence of cycloheximide (10 µg/ml) at the indicated time points.

 
Reduction of Apoptosis Induced by Fas and Stress-related Stimuli by Overexpression of BRE—Despite the failure of BRE to bind the intracellular region of Fas as reported (27), the effect of BRE overexpression in the Fas-sensitive Jurkat A3 cells in response to the anti-Fas agonist antibody, CH11, was tested. To our surprise, apoptotic response was attenuated in 4 of the 5 Jurkat A3 GS-BRE stable transfectants tested in approximate correlation with the GS-BRE expression levels. As with the apoptotic responses to cycloheximide and TNF-{alpha}, GSB2 was more resistant than GSB1, which in turn was more resistant than the untransfected and empty vector controls (Fig. 3A).



View larger version (35K):
[in this window]
[in a new window]
  		FIG. 3.
BRE reduces apoptosis induced by Fas and stress-related stimuli. A, percentage viability of Jurkat GS-BRE stable transfectants, GSB1, GSB2, 1/6, 1/19, and 1/25 in comparison with the untransfected and empty vector controls, after treatment with anti-Fas agonist antibody for 16 h. B, percentage viability of Jurkat and GS-BRE or empty vector stable transfectants after treatment with stress-related stimuli: 4-nitroquinoline-1-oxide (4NQO) (10 µM for 6 h), staurosporine (0.12 µM for 16 h), and etoposide 1 (68 µM for 6 h) and 2 (10 µM for 16 h). C, percentage viability of HeLa with or without siRNA transfection at 24 and 30 h after treatment with 400 µM of etoposide. BRE transcript levels were estimated by semi-quantitative RT-PCR.

 
The anti-apoptotic effect of BRE overexpression to the stress-related stimuli was also shown by GSB1 and -2 (Fig. 3B). To all three stimuli, 4-nitroquinoline-1-oxide (DNA-damaging agent), etoposide (topoisomerase II inhibitor), and staurosporine (broad spectrum protein kinase inhibitor), GSB2 was again more resistant than GSB1, the untransfected and empty vector control cells. Down-regulation of the endogenous BRE in HeLa by the siRNA, Si3, however, resulted in, at best, only a marginal increase of apoptosis to etoposide at 24 h of treatment (Fig. 3C). This indicates that BRE at its endogenous expression level has little, if any, modulatory effect on apoptosis induced by stress stimulation. This is in contrast with the clear down-modulatory effect of the endogenous BRE on TNF-{alpha}-induced apoptosis in HeLa shown above in Fig. 2.

In summary, the findings above show that BRE, when overexpressed, can impart cross-resistance to apoptosis induced by TNF-{alpha}, cycloheximide, anti-Fas agonist antibody, and stress stimuli in a linked manner. However, the physiological anti-apoptotic role of BRE is likely restricted to the death receptors.

Association of BRE with Fas—The ability of BRE in reducing apoptotic response to both TNF-{alpha} and Fas prompted us to reappraise the interaction between BRE and Fas. HeLa cells transiently transfected with GS-BRE were used in co-immunoprecipitation experiments to determine whether the endogenous TNF-R1 and Fas could be pulled down together by the anti-V5 monoclonal antibody that binds the V5 tag at the C terminus of GS-BRE. As extended from the previously reported work showing co-precipitation of the ectopically expressed recombinant TNF-R1 and BRE (27), endogenous TNF-R1 was found co-immunoprecipitated with GS-BRE (Fig. 4A, lane 1). The same result was obtained by using HeLa transiently transfected with a BRE construct encoding a BRE protein tagged at the N terminus with the V5 epitope, V5-BRE (Fig. 4A, lane 2). Likewise, when anti-TNF-R1 antibody was used to immunoprecipitate the endogenous TNF-R1 of the GS-BRE-transfected cells, GS-BRE was co-immunoprecipitated (Fig. 4B, lane 2). As with the TNF-R1, endogenous Fas was also co-immunoprecipitated with the GS-BRE using anti-V5 antibody (Fig. 4C, lane 1). In reverse, GS-BRE was co-immunoprecipitated with the endogenous Fas by two different anti-Fas antibodies (Fig. 4, D, lanes 2 and 3, and B, lane 3). Thus, our data not only confirm binding of BRE with TNF-R1 but also demonstrate binding of BRE with Fas.



View larger version (53K):
[in this window]
[in a new window]
  		FIG. 4.
BRE associates with TNF-R1 and Fas. A, co-immunoprecipitation of TNF-R1 with GS-BRE and V5-BRE using anti-V5 monoclonal antibody from HeLa transiently transfected with the respective expression constructs. MAB2, a third party mouse monoclonal antibody against phosphorylcholine, is the negative control. Immunoblotting was performed using a rabbit anti-TNF-R1 polyclonal antibody (Calbiochem). B, co-immunoprecipitation of GS-BRE (~52 kDa) with TNF-R1 and Fas using mouse anti-TNF-R1 monoclonal antibody (clone 16803, R & D Systems) and rabbit anti-Fas polyclonal antibodies (C20, Santa Cruz Biotechnology), respectively. MAB2, rat anti-TGF monoclonal antibody (clone A75-2.1, Pharmingen), and normal rabbit serum are negative controls. GS-BRE was immunoblotted by HRP-conjugated anti-V5 monoclonal antibody. C, co-immunoprecipitation of Fas with GS-BRE detected by rabbit anti-Fas polyclonal antibody (C20). Two monoclonal anti-Fas antibodies (clone DX2, Pharmingen, and clone 13, Transduction Laboratories) are positive controls for Fas immunoprecipitation. D, co-precipitation of GS-BRE with Fas immunoprecipitated by 2 anti-Fas antibodies, DX2 and C20. GS-BRE was immunoblotted by HRP-conjugated anti-V5 monoclonal antibody. IP, immunoprecipitation; WB, Western blotting analysis.

 
Inhibition of the Mitochondrial Apoptotic Pathway by Overexpression of BRE—To identify the apoptotic event inhibited by BRE overexpression, HeLa and Jurkat cells with or without stable transfection of GS-BRE were treated with the death receptor stimuli, anti-Fas agonist antibody, or TNF-{alpha}, or a stress stimulus, etoposide. Effects on the defining events encompassing the extrinsic and intrinsic apoptotic pathways in the 8-h time course studies were determined. In the Fas-mediated apoptosis of Jurkat transfectant GSB2 (GS), cleavage of caspase-8 and Bid was similar to that of the parent Jurkat cells. However, the mitochondria-dependent events were attenuated to different degrees, with the reduction in the cleavage of caspase-9 being the most remarkable, and the cytosolic release of Smac/DIABLO and cytochrome c being the least, and intermediate, respectively. Cleavage of the main executioner caspase-3 and substrate, PARP, was also mildly attenuated (Fig. 5A). These findings localize the inhibitory action of BRE to a downstream position of tBid.



View larger version (51K):
[in this window]
[in a new window]
  		FIG. 5.
Overexpression of BRE inhibits mitochondria-dependent apoptotic pathway. Jurkat (J) and its stable GS-BRE transfectant GSB2 (GS) were treated with 20 ng/ml CH11 (A) or 68 µM etoposide (B), and HeLa (H) and its stable transfectant, NHGS2 (N2) were treated with TNF-{alpha} (20 ng/ml) in the presence of cycloheximide (10 µg/ml) (C). Processing of caspase-8, -9, -3, Bid, and PARP was detected in total cell lysates by Western blotting analysis. Detection of cytochrome c and Smac/DIABLO was done in the cytosol. Mitochondrial fraction was also detected in A. Antibodies to {beta}-tubulin and prohibitin are cytosolic and mitochondrial markers, respectively.

 
For the treatment of GSB2 with etoposide, all of the apoptotic events including the cleavage of caspase-8, caspase-9, Bid, caspase-3, and PARP, as well as the mitochondrial release of cytochrome c, Smac/DIABLO were either blocked or markedly delayed compared with the parent Jurkat in the 8-h time course study (Fig. 5B). This broad inhibition of all the apoptotic events is consistent with the block of the mitochondria-dependent apoptotic pathway, with cleavage of caspase-8 and Bid being secondary to activation of the etoposide-induced mitochondrial events (38, 39). The same broad inhibition of all the apoptotic events in the HeLa stable transfectant NHGS2 (N2) treated with TNF-{alpha} was also observed (Fig. 5C). This pattern is expected from a block at the mitochondria, which are involved in both the early receptor-proximal caspase-8 activation and later amplification of the receptor-initiated apoptotic signal in the TNF-{alpha}-treated HeLa cells (18, 40).

Dissociation of BRE from TNF-R1, but Not Fas, upon Receptor Ligation—To understand how BRE, a death-receptor associated protein, could exert its anti-apoptotic effect at the distal mitochondrial events, association of BRE with TNF-R1 at different time points after treatment with TNF-{alpha} was investigated by co-immunoprecipitation experiments using the HeLa stable transfectant NHGS2. Within 2 min after TNF-{alpha} treatment, co-immunoprecipitation of GS-BRE with TNF-R1 became virtually undetectable. Simultaneously, association of TRADD with TNF-R1 was observed. Unlike SODD which reassociates with the TNF-R1 at 10 min after the receptor activation (26), no re-association of GS-BRE with TNF-R1 was found throughout the 1-h time course. As no reduction of GS-BRE in the total cell lysate was found in the same period, degradation of the cellular GS-BRE is unlikely the cause (Fig. 6A). This finding thus indicates that BRE dissociates from TNF-R1 early in the receptor signaling. In contrast, treatment of the same transfectant with anti-Fas agonist antibody, CH11, in the same time course did not lead to dissociation of GS-BRE from the DISC (Fig. 6B). GSB2, a Jurkat GS-BRE transfectant used above for all the experiments involving Fas, was not selected for study in this experiment, as this CD95 type II cell type produces too little DISC for detection with the conventional immunoprecipitation approach (19). The difference between TNF-R1 and Fas in association with GS-BRE during receptor activation could reflect their distinctly different initial ways in forming DISC. Unlike Fas, activated TNF-R1 does not form DISC directly; instead DISC is formed after the initial signaling complex has detached from the receptor (41). Thus, it is possible that BRE upon receptor activation interacts with DISC.



View larger version (65K):
[in this window]
[in a new window]
  		FIG. 6.
BRE dissociates from TNF-R1, but not from Fas, upon receptor ligation. NHGS2 was treated with 100 ng/ml TNF-{alpha} (A) or 200 ng/ml CH11 (B). At the indicated time points, cell lysates of A were subjected to immunoprecipitation (IP) by mouse monoclonal anti-TNF-R1 antibody (clone 16803, R&D Systems), and the precipitates were immunoblotted with anti-V5-HRP, rabbit anti-TRADD, and TNF-R1 polyclonal antibodies. The same result was obtained by using a goat anti-TNF-R1 affinity-purified polyclonal antibody (R&D Systems) for immunoprecipitation. Cell lysates of B were subjected to immunoprecipitation by monoclonal anti-Fas (DX2) antibody, and the precipitates were immunoblotted with anti-V5-HRP, anti-caspase-8, and Fas (C20) antibodies. Immunoprecipitation by anti-GAPDH antibody is the negative control. All the cell lysates were also immunoblotted directly with anti-V5-HRP for GS-BRE to ensure similar amount of starting proteins for immunoprecipitation and to show no degradation of GS-BRE during the time course study.

 
Association of BRE with Phosphorylated, Sumoylated, and Ubiquitinated Proteins after Death Receptor Stimulation— Phosphorylation, ubiquitination, and sumoylation are important post-translational modifications involved in the regulation of apoptosis (42–44). To investigate whether BRE might interact with proteins modified by any of the above mechanisms after death receptor stimulation, NHGS2 cells were treated with TNF-{alpha} or CH11 for 2 h, and the association of GS-BRE with phosphorylated, sumoylated, and ubiquitinated proteins was analyzed by co-immunoprecipitation using antibodies specific to these modifications. As shown in Fig. 7A, lanes 1–3, co-immunoprecipitation of GS-BRE with phosphorylated proteins was enhanced after death receptor stimulation. Enhancement of GS-BRE co-immunoprecipitation was more dramatic with the sumoylated and ubiquitinated proteins, as only little if any GS-BRE could be detected in the immunoprecipitates if the cells were not stimulated (Fig. 7A, lanes 4–9). No change was observed in SDS-PAGE mobility of GS-BRE in all three co-immunoprecipitation experiments, indicating that the protein was not itself modified by any of the three mechanisms.



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 7.
BRE associates with phosphorylated, sumoylated, and ubiquitinated proteins after death receptor stimulation. A, co-immunoprecipitation of GS-BRE with phosphorylated proteins from cell lysates of NHGS2 with or without treatment of 100 ng/ml TNF-{alpha} or 200 ng/ml CH11 for 2 h, using anti-pan-phosphoprotein antibody. The corresponding cell lysates were directly immunoblotted for visualization of the content of GS-BRE. Co-immunoprecipitation of GS-BRE with sumoylated and ubiqitinated proteins was also performed with anti-SUMO-1 and anti-ubiquitin antibodies, respectively, using cell lysates of NHGS2 treated as above. B, cell lysate of the unstimulated GS-BRE transfectant was treated with {lambda}-protein phosphatase in the presence or absence of sodium vanadate, an inhibitor of phosphatase, or not treated, before being subjected to co-immunoprecipitation analysis using the anti-pan-phosphoprotein antibody. The corresponding cell lysates were directly immunoblotted to ascertain no degradation of GS-BRE by the phosphatase treatment. IP, immunoprecipitation; WB, Western blotting analysis.

 
To ascertain the specificity of the association between BRE and phosphorylated proteins in the co-immunoprecipitation experiment, cell lysate with GS-BRE was pretreated with {lambda}-phosphatase with or without the phosphatase inhibitor, sodium vanadate, before being subjected to immunoprecipitation by the anti-pan-phosphoprotein antibody. Removal of phosphate from proteins resulted in no co-precipitation of GS-BRE (Fig. 7B, lane 2). With no pretreatment or with phosphatase treatment in the presence of sodium vanadate, GS-BRE was co-precipitated (Fig. 7B, lanes 1 and 3). These data validate the specific association of BRE with phosphorylated proteins in co-immunoprecipitation.

Subcellular Localization of BRE—Subcellular fractionation of GSB2 revealed co-enrichment of GS-BRE with the cytosol and, to a lesser extent, nuclei but not at all with mitochondria (Fig. 8A). During apoptotic induction by etoposide, the GS-BRE in GSB2 did not translocate to mitochondria (Fig. 8B). To show that the C-terminal V5 and His6 tags in GS-BRE did not alter subcellular localization of the BRE protein, the endogenous BRE in subcellular fractions was immunoblotted with a mouse anti-BRE serum raised against the recombinant BRE protein. As shown in Fig. 9A, the mouse antiserum recognizes a band that can be knocked down by the BRE-specific siRNA, Si3, and has a size consistent with the predicted 44 kDa of BRE. As expected, the mouse serum also recognizes the ectopically expressed GS-BRE in GSB2. However, five nonspecific bands, *1–*5, not affected by Si3 were also stained by the serum. Subcellular fractionation of Jurkat cells before and after apoptotic induction by the anti-Fas agonist antibody, CH11, confirmed the findings based on GS-BRE (Fig. 9B). Furthermore, no co-enrichment with the peroxisomal marker, catalase, was detected for BRE, indicating that this protein is not a peroxisomal protein, despite the presence of a putative signaling sequence at the C terminus (29). No translocation of the cytosolic BRE to nucleus was detected either during apoptotic induction (Fig. 9C).



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 8.
GS-BRE resides in the cytosol and nucleus. A, total GSB2 cell lysate and subcellular fractions were subjected to Western blotting analysis using the indicated antibodies. Each lane was loaded with 30 µg of protein. B, cell lysate of GSB2 at each indicated time point of treatment with 68 µM etoposide was fractionated and immunoblotted with the indicated antibodies. T, unfractionated; N, nuclei; M, mitochondria; Mc, microsomes, and C, cytosol.

 



View larger version (38K):
[in this window]
[in a new window]
  		FIG. 9.
Endogenous BRE resides in the cytosol and nucleus. A, immunoblotting of endogenous BRE and ectopically expressed GS-BRE by a mouse anti-BRE serum in HeLa with or without transfection with BRE-specific siRNA, Si3, and Jurkat transfectant, GSB2, respectively. The asterisks 1–5 indicate nonspecific bands. Similar loading was confirmed by anti-GAPDH antibody. B and C, immunoblotting of endogenous BRE in subcellular fractions of Jurkat cell lysate before and after treatment with anti-Fas agonist antibody, CH11, at indicated time points, with cell viability determined by flow analysis of annexin-V-FITC and propidium iodide staining. The nonspecific bands are numbered asterisks as in A. Subcellular fractions were confirmed by organelle-specific antibodies. T, unfractionated; N, nuclei; M, mitochondria; Mc, microsomes, and C, cytosol.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we report identification of a death receptor-associated protein, BRE, that attenuates TNF-{alpha}- and anti-Fas agonist antibody-induced apoptosis by blocking activation of the mitochondrial apoptotic machinery. BRE is a highly conserved and widely expressed protein (28–30). We have shown that overexpression of BRE in cell lines reduced apoptotic response to death receptor stimuli, TNF-{alpha} and anti-Fas agonist antibody, as well as to cycloheximide alone, and to the stress stimuli 4-NQO, staurosporine, and etoposide. However, the physiological anti-apoptotic role of BRE is specific to the death receptor stimulation, as reduction in the endogenous BRE expression level by siRNA sensitized apoptosis to TNF-{alpha} but not to etoposide. Thus, the attenuation of apoptotic response to stress stimuli by overexpressed BRE is likely to reflect the inhibitory action of the excessive BRE not in association with the death receptors. Basal cell survival was not affected by down-regulation of BRE.

Evidence for the mitochondrial apoptotic machinery being the target for the anti-apoptotic action of BRE is provided by the observations that BRE overexpression inhibited the following: 1) the mitochondria-dependent events downstream of tBid in Fas-initiated apoptosis; 2) the etoposide-induced mitochondria-dependent apoptotic pathway; and 3) both the death receptor- and mitochondria-dependent TNF-{alpha}-induced apoptotic pathways in HeLa, the latter pathway has been shown to promote activation of the apical caspase-8 in DISC (18). Furthermore, the findings of BRE overexpression being able to inhibit apoptosis induced by cycloheximide alone, which has been indicated to activate the mitochondrial apoptotic events (37, 45), and by stress-related stimuli, 4-NQO, etoposide, and stauroporine, are consistent with the above observations. Down-regulation of cell surface expression of TNF-R1 and Fas by BRE overexpression can be ruled out as flow cytometric analysis of the stable GS-BRE transfectants revealed no shift in the fluorescence profile of the surface staining for these death receptors (supplemental Fig. S1). Targeting only the mitochondria-dependent branch of apoptosis by BRE is also consistent with our observation that this protein can only partially inhibit death receptor-mediated apoptosis.

It was shown previously by the yeast two-hybrid assay that BRE binds to the intracellular domain of TNF-R1 but not to Fas (27). In the same report, interaction between BRE and TNF-R1 within mammalian cells was demonstrated by co-immunoprecipitation of the two proteins ectopically expressed in transfected cells. Here, by using the same co-immunoprecipitation approach, we demonstrated not only association between the ectopically expressed BRE with the endogenous TNF-R1 but also with the endogenous Fas. Although it is possible that the binding between BRE and Fas is indirect, a loose homology (score, 36.0) between the BRE-binding region of TNF-R1 and the corresponding region of Fas, second only to that between the conserved DDs of the two proteins (score, 63.0) as revealed by an online local similarity program SIM (expasy.org/tools/sim.html), favors direct association of the two proteins. Site-directed mutagenesis experiments will be required to confirm this point.

We have shown in time course experiments the rapid dissociation of BRE from TNF-R1 within 2 min after treatment with TNF-{alpha} but no dissociation from Fas within the whole 1-h period after treatment with anti-Fas agonist antibody. This discrepancy could be because of the different initial ways in forming DISC by the two death receptors. Activated Fas recruits FADD to the receptor to form DISC in situ within minutes (46, 47). Activated TNF-R1, by contrast, recruits TRADD first to form a platform to recruit other adaptor molecules in forming an NF-{kappa}B-activating complex I. This complex subsequently develops into FADD- and caspase-8-containing DISC (complex II) after detachment from the receptor (41). BRE may detach together with complex II, which has been shown to form rapidly after receptor stimulation by size exclusion chromatography that showed incorporation of caspase-8 and FADD in a high molecular weight complex within 5 min of the treatment (41).

Our further demonstration that BRE showed a marked increase in binding with ubiquitinated, sumoylated, and phosphorylated proteins after death receptor stimulation suggests that BRE is involved in post-translational modification of proteins within or interacting with DISC. Recently, BRE was electronically annotated as having a match at its N-terminal region with ubiquitin-conjugating enzymes (www.ebi.ac.uk/interpro/IEntry?ac=IPR010358 (accessed 2 September 2004)). A recent report (48) also shows BRE forms a nuclear multiprotein complex containing BRCA1 and BARD1 and promotes the ubiquitination activity (ubiquitin-ligating enzyme) of the complex. However, unlike BRCA1, which is a tumor suppressor nuclear protein (49, 50), our subcellular fractionation experiments confirm cytosolic as well as nuclear but not mitochondrial localization of GS-BRE. Our findings also clearly indicate that the majority of BRE is located in the cytosolic compartment. Apoptotic induction induced no translocation of BRE from cytosol to mitochondria nor to the nucleus. Therefore, we hypothesize that the anti-apoptotic role of the cytosolic BRE is independent of its nuclear counterpart and, unlike the Bcl-2 family members, is not dependent on direct targeting of the mitochondria. Rather, the role of BRE in cytosol is to associate with the death receptors. This strategic association allows BRE to react rapidly to receptor ligation and to interact with the proteins within or in close proximity of DISC. Probably by the ubiquitination-like activity of BRE, components that are necessary for activation of the mitochondrial apoptotic machinery are negatively regulated. Although it is well established that Bcl-2 family members are the main players that regulate activation of the mitochondrial apoptotic pathway (51), concerted actions with a multitude of other proteins are necessary for the activation to occur. It has been shown in some cell lines that caspase-2 and cofilin are also required for the occurrence of mitochondrial apoptotic events (52, 53). In the case of BRE, the targeted protein required for mitochondrial activation remains to be identified.

In death receptor-induced apoptosis, the mitochondrial apoptotic pathway can be relevant. In TNF-{alpha}-induced apoptosis in HeLa and mouse embryonic fibroblast at least, early mitochondrial efflux of Smac/DIABLO is necessary for the release of DISC-recruited caspase-8 from the inhibition of cellular inhibitors of apoptosis (18). Full processing of the main executioner caspase-3 may also require cooperation between caspase-8 and the mitochondria-dependent caspase-9 (54). In TRAIL-induced and Fas-induced apoptosis in the Fas type II cells, such as human colon cancer cell line, HCT116, human T leukemia Jurkat cell line, and primary hepatocytes, amplification of the death receptor-initiated apoptotic signal by the mitochondria is necessary for the occurrence of apoptosis (19–21). There are also abundant natural examples of viral proteins that can block apoptosis initiated from the death receptors by inhibition at the mitochondria level. These include the viral homologs of the Bcl-2 family, such as BHRF1 and BALF1 of EB virus (55–57) and E1B 19K of adenovirus (58). Non-Bcl-2 homologous proteins include vMIA of cytomegalovirus (59), M11L of myxoma virus (60), and F1L of vaccinia virus (61). These viral proteins confer immune evasion by preventing apoptosis of the infected cells induced by TNF-{alpha}, FasL, or TRAIL of the host immune system (58, 62–65), indicating the importance of cross-talk between the extrinsic and intrinsic pathways in vivo.

In conclusion, we have identified BRE as a death receptor-associated protein, which specifically down-modulates death receptor-mediated apoptosis by inhibiting activation of the mitochondrial apoptotic pathway. This novel cellular mechanism mediates a functional consequence opposite to that of tBid.


   FOOTNOTES
 
* This work was supported in part by Grant CUHK 4090/02M from Research Grant Committee of Hong Kong. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains Fig. S1. Back

§ Both authors contributed equally to this work. Back

Supported by a postgraduate studentship awarded by The Chinese University of Hong Kong. Back

|| Supported by a postdoctoral fellowship awarded by The Chinese University of Hong Kong. Back

{ddagger}{ddagger} Present address: Dept. of Pharmacy, The Chinese University of Hong Kong, Shatin, New Territory, Hong Kong Special Administrative Region, China. Back

|||| To whom correspondence should be addressed: Clinical Immunology Unit, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, New Territory, Hong Kong. Tel.: 852-2632-2588; Fax: 852-2645-0856; E-mail: yiuloonchui{at}cuhk.edu.hk.

1 The abbreviations used are: TNF-R1, tumor necrosis factor-receptor 1; BRE, brain and reproductive organ-expressed protein; GS-BRE, Research Genetics GeneStormR clone of BRE; DISC, death-inducing signaling complex; DD; death domain; Bid, BH3 interacting domain death agonist; NF-{kappa}B, nuclear factor {kappa}B; SODD, silencer of death domains; SUMO-1, small ubiquitin-like modifier-1; FADD, Fas-associated death domain protein; TRADD, TNF-R1-associated death domain protein; Bcl-2; B-cell lymphoma/leukemia-2; siRNA, small interfering RNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; tBid, truncated Bid; FITC, fluorescein isothiocyanate; RT, reverse transcriptase; pan, pantothenate; PARP, poly(ADP-ribose) polymerase. Back


   ACKNOWLEDGMENTS
 
We are grateful to Peggy H. Y. Fung for clerical help in preparing this manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Vaux, D. L., and Korsmeyer, S. J. (1999) Cell 96, 245–254[CrossRef][Medline] [Order article via Infotrieve]
  2. Strasser, A., O'Connor, L., and Dixit, V. M. (2000) Annu. Rev. Biochem. 69, 217–245[CrossRef][Medline] [Order article via Infotrieve]
  3. Aggarwal, B. B. (2003) Nat. Rev. Immunol. 3, 745–756[CrossRef][Medline] [Order article via Infotrieve]
  4. Nagata, S. (1997) Cell 88, 355–365[CrossRef][Medline] [Order article via Infotrieve]
  5. Dive, C., Evans, C. A., and Whetton, A. D. (1992) Semin. Cancer Biol. 3, 417–427[Medline] [Order article via Infotrieve]
  6. Strasser, A., Harris, A. W., Huang, D. C., Krammer, P. H., and Cory, S. (1995) EMBO J. 14, 6136–6147[Medline] [Order article via Infotrieve]
  7. Martin, S. J., and Green, D. R. (1995) Cell 82, 349–352[CrossRef][Medline] [Order article via Infotrieve]
  8. Enari, M., Talanian, R. V., Wong, W. W., and Nagata, S. (1996) Nature 380, 723–726[CrossRef][Medline] [Order article via Infotrieve]
  9. Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. (1996) Cell 85, 803–815[CrossRef][Medline] [Order article via Infotrieve]
  10. Chinnaiyan, A. M., Tepper, C. G., Seldin, M. F., O'Rourke, K., Kischkel, F. C., Hellbardt, S., Krammer, P. H., Peter, M. E., and Dixit, V. M. (1996) J. Biol. Chem. 271, 4961–4965[Abstract/Free Full Text]
  11. Ashkenazi, A., and Dixit, V. M. (1998) Science 281, 1305–1308[Abstract/Free Full Text]
  12. Fischer, U., Janicke, R. U., and Schulze-Osthoff, K. (2003) Cell Death Differ. 10, 76–100[CrossRef][Medline] [Order article via Infotrieve]
  13. Green, D. R., and Reed, J. C. (1998) Science 281, 1309–1312[Abstract/Free Full Text]
  14. Gross, A., McDonnell, J. M., and Korsmeyer, S. J. (1999) Genes Dev. 13, 1899–1911[Free Full Text]
  15. Wang, X. (2001) Genes Dev. 15, 2922–2933[Free Full Text]
  16. Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998) Cell 94, 491–501[CrossRef][Medline] [Order article via Infotrieve]
  17. Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. (1998) Cell 94, 481–490[CrossRef][Medline] [Order article via Infotrieve]
  18. Deng, Y., Ren, X., Yang, L., Lin, Y., and Wu, X. (2003) Cell 115, 61–70[CrossRef][Medline] [Order article via Infotrieve]
  19. Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, C., Li, F., Tomaselli, K. J., Debatin, K. M., Krammer, P. H., and Peter, M. E. (1998) EMBO J. 17, 1675–1687[CrossRef][Medline] [Order article via Infotrieve]
  20. Deng, Y., Lin, Y., and Wu, X. (2002) Genes Dev. 16, 33–45[Abstract/Free Full Text]
  21. Yin, X. M., Wang, K., Gross, A., Zhao, Y., Zinkel, S., Klocke, B., Roth, K. A., and Korsmeyer, S. J. (1999) Nature 400, 886–891[CrossRef][Medline] [Order article via Infotrieve]
  22. Wang, C. Y., Mayo, M. W., Korneluk, R. G., Goeddel, D. V., and Baldwin, Jr., A. S. (1998) Science 281, 1680–1683[Abstract/Free Full Text]
  23. Aggarwal, B. B. (2000) Annu. Rheum. Dis. 59, Suppl. 1, 6–11
  24. Micheau, O., Lens, S., Gaide, O., Alevizopoulos, K., and Tschopp, J. (2001) Mol. Cell. Biol. 21, 5299–5305[Abstract/Free Full Text]
  25. Okura, T., Gong, L., Kamitani, T., Wada, T., Okura, I., Wei, C. F., Chang, H. M., and Yeh, E. T. (1996) J. Immunol. 157, 4277–4281[Abstract]
  26. Jiang, Y., Woronicz, J. D., Liu, W., and Goeddel, D. V. (1999) Science 283, 543–546[Abstract/Free Full Text]
  27. Gu, C., Castellino, A., Chan, J. Y., and Chao, M. V. (1998) FASEB J. 12, 1101–1108[Abstract/Free Full Text]
  28. Li, L., Yoo, H., Becker, F. F., Ali-Osman, F., and Chan, J. Y. (1995) Biochem. Biophys. Res. Commun. 206, 764–774[CrossRef][Medline] [Order article via Infotrieve]
  29. Ching, A. K., Li, P. S., Li, Q., Chan, B. C., Chan, J. Y., Lim, P. L., Pang, J. C., and Chui, Y. L. (2001) Biochem. Biophys. Res. Commun. 288, 535–545[CrossRef][Medline] [Order article via Infotrieve]
  30. Ching, A. K., Li, Q., Lim, P. L., Chan, J. Y., and Chui, Y. L. (2003) DNA Cell Biol. 22, 497–504[CrossRef][Medline] [Order article via Infotrieve]
  31. Itoh, N., and Nagata, S. (1993) J. Biol. Chem. 268, 10932–10937[Abstract/Free Full Text]
  32. Tartaglia, L. A., Ayres, T. M., Wong, G. H., and Goeddel, D. V. (1993) Cell 74, 845–853[CrossRef][Medline] [Order article via Infotrieve]
  33. Eisenbach, L., Segal, S., and Feldman, M. (1983) Int. J. Cancer 32, 113–120[Medline] [Order article via Infotrieve]
  34. Juo, P., Kuo, C. J., Reynolds, S. E., Konz, R. F., Raingeaud, J., Davis, R. J., Biemann, H. P., and Blenis, J. (1997) Mol. Cell. Biol. 17, 24–35[Abstract]
  35. Miao, J., Panesar, N. S., Chan, K. T., Lai, F. M., Xia, N., Wang, Y., Johnson, P. J., and Chan, J. Y. (2001) J. Histochem. Cytochem. 49, 491–500[Abstract/Free Full Text]
  36. Gottlieb, R. A., Nordberg, J., Skowronski, E., and Babior, B. M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 654–658[Abstract/Free Full Text]
  37. Tang, D., Lahti, J. M., Grenet, J., and Kid, V. J. (1999) J. Biol. Chem. 274, 7245–7252[Abstract/Free Full Text]
  38. Sun, X. M., MacFarlane, M., Zhuang, J., Wolf, B. B., Green, D. R., and Cohen, G. M. (1999) J. Biol. Chem. 274, 5053–5060[Abstract/Free Full Text]
  39. Engels, I. H., Stepczynska, A., Stroh, C., Lauber, K., Berg, C., Schwenzer, R., Wajant, H., Janicke, R. U., Porter, A. G., Belka, C., Gregor, M., Schulze-Osthoff, K., and Wesselborg, S. (2000) Oncogene 19, 4563–4573[CrossRef][Medline] [Order article via Infotrieve]
  40. Shchepina, L. A., Pletjushkina, O. Y., Avetisyan, A. V., Bakeeva, L. E., Fetisova, E. K., Izyumov, D. S., Saprunova, V. B., Vyssokikh, M. Y., Chernyak, B. V., and Skulachev, V. P. (2002) Oncogene 21, 8149–8157[CrossRef][Medline] [Order article via Infotrieve]
  41. Micheau, O., and Tschopp, J. (2003) Cell 114, 181–190[CrossRef][Medline] [Order article via Infotrieve]
  42. Datta, S. R., Brunet, A., and Greenberg, M. E. (1999) Genes Dev. 13, 2905–2927[Free Full Text]
  43. Baier, A., Meineckel, I., Gay, S., and Pap, T. (2003) Curr. Opin. Rheumatol. 15, 274–279[CrossRef][Medline] [Order article via Infotrieve]
  44. Lee, J. C., and Peter, M. E. (2003) Immunol. Rev. 193, 39–47[CrossRef][Medline] [Order article via Infotrieve]
  45. Scaffidi, C., Schmitz, I., Zha, J., Korsmeyer, S. J., Krammer, P. H., and Peter, M. E. (1999) J. Biol. Chem. 274, 22532–22538[Abstract/Free Full Text]
  46. Chinnaiyan, A. M., O'Rourke, K., Tewari, M., and Dixit, V. M. (1995) Cell 81, 505–512[CrossRef][Medline] [Order article via Infotrieve]
  47. 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[CrossRef][Medline] [Order article via Infotrieve]
  48. Dong, Y., Hakimi, M. A., Chen, X., Kumaraswamy, E., Cooch, N. S., Godwin, A. K., and Shiekhattar, R. (2003) Mol. Cell 12, 1087–1099[CrossRef][Medline] [Order article via Infotrieve]
  49. Blackshear, P. E., Goldsworthy, S. M., Foley, J., McAllister, K. A., Bennett, L. M., Collins, N. K., Bunch, D. O., Brown, P., Wiseman, R. W., and Davis, B. J. (1998) Oncogene 16, 61–68[CrossRef][Medline] [Order article via Infotrieve]
  50. Starita, L. M., and Parvin, J. D. (2003) Curr. Opin. Cell Biol. 15, 345–350[CrossRef][Medline] [Order article via Infotrieve]
  51. Cory, S., and Adams, J. M. (2002) Nat. Rev. Cancer 2, 647–656[CrossRef][Medline] [Order article via Infotrieve]
  52. Lassus, P., Opitz-Araya, X., and Lazebnik, Y. (2002) Science 297, 1352–1354[Abstract/Free Full Text]
  53. Chua, B. T., Volbracht, C., Tan, K. O., Li, R., Yu, V. C., and Li, P. (2003) Nat. Cell Biol. 5, 1083–1089[CrossRef][Medline] [Order article via Infotrieve]
  54. Perez, D., and White, E. (2000) Mol. Cell 6, 53–63[CrossRef][Medline] [Order article via Infotrieve]
  55. Henderson, S., Huen, D., Rowe, M., Dawson, C., Johnson, G., and Rickinson, A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8479–8483[Abstract/Free Full Text]
  56. Foghsgaard, L., and Jäättelä, M. (1997) J. Virol. 71, 7509–7517[Abstract]
  57. Kawanishi, M. (1997) J. Virol. 71, 3319–3322[Abstract]
  58. White, E., Sabbatini, P., Debbas, M., Wold, W. S., Kusher, D. I., and Gooding, L. R. (1992) Mol. Cell. Biol. 12, 2570–2580[Abstract/Free Full Text]
  59. Goldmacher, V. S., Bartle, L. M., Skaletskaya, A., Dionne, C. A., Kedersha, N. L., Vater, C. A., Han, J. W., Lutz, R. J., Watanabe, S., McFarland, E. D. C., Kieff, E. D., Mocarski, E. S., and Chittenden, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12536–12541[Abstract/Free Full Text]
  60. Everett, H., Barry, M., Lee, S. F., Sun, X., Graham, K., Stone, J., Bleackley, R. C., and McFadden, G. (2000) J. Exp. Med. 191, 1487–1498[Abstract/Free Full Text]
  61. Wasilenko, S. T., Stewart, T. L., Meyers, A. F., and Barry, M. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 14345–14350[Abstract/Free Full Text]
  62. Gooding, L. R., Aquino, L., Duerksen-Hughes, P. J., Day, D., Horton, T. M., Yei, S. P., and Wold, W. S. (1991) J. Virol. 65, 3083–3094[Abstract/Free Full Text]
  63. Hashimoto, S., Ishii, A., and Yonehara, S. (1991) Int. Immunol. 3, 343–351[Abstract/Free Full Text]
  64. Marshall, W. L., Yim, C., Gustafson, E., Graf, T., Sage, D. R., Hanify, K., Williams, L., Fingeroth, J., and Finberg, R. W. (1999) J. Virol. 73, 5181–5185[Abstract/Free Full Text]
  65. Tollefson, A. E., Toth, K., Doronin, K., Kuppuswamy, M., Doronina, O. A., Lichtenstein, D. L., Hermiston, T. W., Smith, C. A., and Wold, W. S. (2001) J. Virol. 75, 8875–8887[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
B. Schiedlmeier, A. C. Santos, A. Ribeiro, N. Moncaut, D. Lesinski, H. Auer, K. Kornacker, W. Ostertag, C. Baum, M. Mallo, et al.
HOXB4's road map to stem cell expansion
PNAS, October 23, 2007; 104(43): 16952 - 16957.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
279/50/52106    most recent
M408678200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, Q.
Right arrow Articles by Chui, Y.-L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, Q.
Right arrow Articles by Chui, Y.-L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement