Phosphorylation of the Tumor Necrosis Factor Receptor CD120a (p55) Recruits Bcl-2 and Protects against Apoptosis*

Vincent CottinDagger §, Annemie A. Van LindenDagger , and David W. H. RichesDagger ||**Dagger Dagger §§

From the Dagger  Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado, 80206 and the || Department of Biochemistry and Molecular Genetics, ** Division of Pulmonary and Critical Care Medicine, Department of Medicine, Dagger Dagger  Department of Pharmacology and  Department of Immunology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Received for publication, November 27, 2000, and in revised form, January 19, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ligation of the tumor necrosis factor alpha  receptor CD120a initiates responses as diverse as apoptosis and the expression of NF-kappa B-dependent pro-survival genes. How these opposing responses are controlled remains poorly understood. Here we demonstrate that phosphorylation by p42mapk/erk2 inhibits the apoptotic activity of CD120a while preserving its ability to activate NF-kappa B. Phosphorylated CD120a is re-localized from the Golgi complex to tubular structures of the endoplasmic reticulum wherein it recruits Bcl-2. Antisense-mediated down-regulation of Bcl-2 antagonized the localization of CD120a to tubular structures and reversed the protection from apoptosis conferred by receptor phosphorylation. We propose that phosphorylation of CD120a represents a novel, Bcl-2-dependent mechanism by which the apoptotic activity of the receptor may be regulated. Thus, oncogenic activation of p42mapk/erk2 may serve to inhibit the apoptotic activity of this death receptor while preserving NF-kappa B-dependent responses and may thus indirectly contribute to a failure to eliminate cells bearing oncogenes of the Ras-Raf-MEK-p42mapk/erk2 pathway.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Engagement of the tumor necrosis factor-alpha (TNFalpha )1 receptor CD120a (p55) sets in motion the activation of signaling pathways that lead to apoptosis in certain cells (1). Interaction with TNFalpha leads to trimerization of the receptor and to the binding of the adapter molecule TRADD through death domain·death domain interactions (2). The complex then recruits several signaling molecules, including TRAF2 and RIP, which stimulate MAPK and NF-kappa B activation pathways (3-5), and FADD, which mediates activation of apoptosis by recruitment of caspase-8 (3). The requirement of the FADD-caspase-8 pathway in CD120a-mediated apoptosis has been elegantly demonstrated by the resistance of cells derived from FADD-knock-out mouse embryos to TNFalpha -induced apoptosis (6). Activation of caspase-8 leads to the activation of caspase-3 and the cleavage of Bid to generate a truncated, pro-apoptotic fragment (tBid) capable of inducing the release of cytochrome c from mitochondria (7) and thereby promoting apoptosis.

Fas ligand (Fas-L) and to a lesser extent TNFalpha -mediated apoptosis have been shown to be negatively regulated by several molecules, including Casper/FLICE-inhibitory protein (FLIP) (8, 9), Toso (10), the inhibitor of apoptosis family of proteins (IAP), including c-IAP1, c-IAP2, XIAP, and survivin/TIAP (11), as well as several members of the Bcl-2 family (12). Casper/FLIP, a homolog of caspase-8, interacts with FADD (8) and prevents the recruitment and activation of caspase-8, thereby inhibiting the signaling cascade initiated by death receptors (9), whereas Toso inhibits Fas-dependent apoptosis through the induction of Casper/FLIP expression (10). In contrast, IAP proteins have been shown to interact with, and inhibit, caspases 3, 7, and 9. In addition, c-IAP1 and c-IAP2 are capable of binding to TRAF1 and TRAF2, although the mechanism by which they promote survival is poorly understood (11).

The family of Bcl-2-related proteins comprises both death-inducing (Bax, Bak, Bcl-xS, Bad, Bid, Bik, and Hrk) and death-inhibiting (Bcl-2, Bcl-xL, Bcl-w, Bfl-1, Mcl-1, and Boo) members (13), and, although the mechanism of action of death-inducing Bcl-2 family members is rapidly emerging, the mechanism underlying the death-inhibiting properties of Bcl-2 family members is only partly understood. The ratio of death antagonists to agonists has been proposed to regulate the death-life rheostat within the cell (13). In addition, several Bcl-2 family members are regulated by subcellular localization (7, 14-16). Although Bcl-2 and Bcl-xL are essential components of the general apoptosis regulatory machinery, they are relatively poor inhibitors of the death signals induced by TNFalpha and Fas-L in cells that efficiently activate caspase-8 at the death-inducing signaling complex (DISC) (so-called "type I" cells), but effectively block apoptosis in cells that poorly activate caspase-8 at the DISC ("type II" cells) (13, 17-20). When present, the survival influence of Bcl-2 family proteins in Fas-L or TNFalpha -induced apoptosis generally occurs downstream of the activation of caspase-8 through the inhibition of cytochrome c release from mitochondria (12, 21-23).

The MAPKs comprise three major sub-families in mammalian cells: 1) the p38mapk subfamily; 2) the extracellular signal-regulated kinases (ERK) p44mapk/erk1 and p42mapk/erk2; and 3) the c-Jun NH2-terminal kinases (JNK). In work previously reported from this laboratory, we have shown that specific members of each MAPK sub-family are rapidly and transiently activated in response to stimulation of mouse macrophages with TNFalpha (24-26). Moreover, whereas the targets of these kinases include transcription factors involved in the inflammatory response, recent studies have also shown these targets to include CD120a itself (27). In addition, we have shown that activation of p42mapk/erk2 by other receptors, e.g. growth factor receptors, also promotes phosphorylation of CD120a. (28). As a result of phosphorylation, CD120a expression, especially in the Golgi complex, is down-regulated and the receptor is redistributed to tubular structures associated with the endoplasmic reticulum (27). Thus, the phosphorylation of CD120a by p42mapk/erk2 may represent a broad mechanism by which TNFalpha , growth factors, and other stimuli may regulate the activity of CD120a.

Recent studies have suggested that the activation of p42mapk/erk2 in the context of other pro-apoptotic signals confers a dominant pro-survival advantage (29, 30). For example, activation of MEK1, an upstream activator of p42mapk/erk2, has been shown to antagonize Fas-triggered cell death through the up-regulation of Casper/FLIP (29). Activation of p42mapk/erk2 has also been shown to be protective against TNFalpha -induced apoptosis of L929 cells (31), serum deprivation-induced apoptosis in PC12 cells (32), and UV-induced apoptosis in human primary neutrophils (33). However, the p42mapk/erk2 substrates involved in the survival responses are not known. Given the finding that CD120a itself is phosphorylated upon activation of p42mapk/erk2 (27), we have investigated the influence of CD120a phosphorylation on TNFalpha -induced apoptosis. We report herein that the phosphorylation of CD120a by p42mapk/erk2 inhibits TNFalpha -induced apoptosis in HeLa cells through a Bcl-2-dependent mechanism. In contrast, the ability of the receptor to activate NF-kappa B is unaffected by phosphorylation. As we will show, these findings shed new light on how phosphorylation of CD120a may contribute to the regulation of the multiple functions of this death receptor.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Rabbit polyclonal anti-Bcl-xL (Ab 1690), goat polyclonal anti-CD120a (Ab 1069), anti-TRADD (Ab 1165), and mouse monoclonal anti-Bcl-2 (Ab 7382) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The hamster monoclonal agonistic (Ab 80-4004-01) and antagonistic anti-CD120a (Ab 80-4005-01) antibodies and the monoclonal anti-CD120b antibody (Ab 80-4009-01) were purchased from R&D Systems (Minneapolis, MN). Anti-myc and anti-tubulin mouse monoclonal antibodies were from Sigma Chemical Co. (St. Louis, MO). Mouse monoclonal anti-caspase-8 (Ab 66231A) and anti-FADD (Ab 65751A) were from PharMingen (San Diego, CA). The mouse monoclonal anti-FLAG antibody (M2) was from Kodak-Scientific Imaging Systems (Rochester, NY). The mouse monoclonal phosphospecific anti-ERK antibody (E10) was from New England BioLabs (Beverly, MA). Fluorescent secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The expression vector encoding CD120a has been described previously (27). All deletion and point mutants were constructed using overlapping polymerase chain reaction (34) and verified by restriction enzyme analysis and nucleotide sequencing. The expression vectors for p42mapk/erk2 and MEK1.ca were a generous gift from Dr. Lynn Heasley, University of Colorado Health Sciences Center, Denver, CO. The vector expressing Bcl-xL was kindly provided by Dr. Hong Zhou, Boston, MA. The vector expressing Bcl-2 in pcDNA3 was a gift from Dr. Gary Johnson, National Jewish Medical and Research Center, Denver, CO. The vectors expressing myc-TRADD and FLAG-FADD were provided by Dr. Hong-Bing Shu, National Jewish Medical and Research Center, Denver, CO.

Transfections and Confocal Immunofluorescence Microscopy-- HeLa cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine. Approximately 6 × 104 cells/well were seeded in 12-well plates containing 18-mm glass coverslips and grown in 5% CO2. Cells were transfected with 250 ng of DNA the following day using LipofectAMINE reagent (Life Technologies). 14 h after transfection, the cells were washed with PBS, fixed for 15 min at room temperature in a solution containing 3% (w/v) paraformaldehyde and 3% (w/v) sucrose in PBS (pH 7.5), washed again, and permeabilized with 0.2% (v/v) Triton X-100 for 10 min. The cells were then washed, blocked for 30 min in Hank's balanced salt solution (without Mg2+, Ca2+, or phenol red, pH 7.2) containing 5% normal donkey serum and then incubated with the primary antibodies (1:100) in blocking solution for 2 h. After washing with PBS, the cells were incubated for 1 h with Cy3- and/or fluorescein-conjugated donkey secondary antibodies (1:200). Staining for CD120a was performed using a primary hamster monoclonal antibody (1:200), and a Cy3-conjugated F(ab')2 goat anti-hamster IgG in blocking solution containing 5% normal goat serum. The coverslips were incubated overnight in PBS supplemented with 0.02% sodium azide and mounted in a solution containing 90% glycerol, 10% Tris-HCl 0.1 M, pH 8.5, and 20 mg/ml o-phenylenediamine as an anti-fading agent. To visualize the nuclei, cells were incubated with 10 µg/ml Hoechst 33342 together with the secondary antibodies. Cells were observed with a Leica DMR/XA fluorescence microscope using a 100× plan objective. Digital images were captured using a SensiCam camera, deconvolved using the software Slidebook 2.6 (Intelligent Imaging Innovations, Inc., Denver, CO) to remove fluorescence that was not in focus, and processed using Adobe Photoshop 5.0 (Adobe Systems, Inc.).

TUNEL Assay-- HeLa cells grown on coverslips and transfected 18-24 h earlier with the appropriate expression vectors were washed with PBS, fixed and permeabilized as described above, and incubated with terminal transferase reaction solution containing fluorescein-conjugated dUTP for 1 h at 37 °C as recommended by the manufacturer (Roche Diagnostics Corp., Indianapolis, IN). The cells were washed three times with 0.03 M sodium citrate, pH 7.4, containing 0.3 M sodium chloride, to remove unbound nucleotides, then washed with PBS. Cells were then blocked and incubated with antibodies as above. The percentage of TUNEL-positive cells among transfected cells was determined by counting at least 200 cells with a confocal microscope.

Caspase-8 Cleavage-- 2 × 105 cells grown in 6-well plates were transfected with 500 ng of DNA using the LipofectAMINE reagent. 18 h after transfection, the cells were washed and lysed in Nonidet P-40 lysis buffer (50 mM Tris buffer, pH 8.0, containing 1% Nonidet P-40, 137 mM NaCl, and protease inhibitors). Post-nuclear supernatants were subjected to SDS-PAGE and transfer to nitrocellulose membranes. Procaspase-8 was detected by Western blotting, and quantified by scanning densitometry using the IMAGE 1.6 software (National Institutes of Health).

NF-kappa B Reporter Gene Assays-- Activation of NF-kappa B was determined by a reporter gene assay. Briefly, 2.1 × 105 HEK293GT cells/well were seeded in 6-well cluster plates. The following day, the media were replaced 1 h prior to transfection with 0.5 µg/ml NF-kappa B-luciferase reporter gene plasmid and varying amounts of CD120a constructs using the calcium phosphate method as previously described (35). The cells were then maintained for 18-24 h prior to stimulation in fresh media with 0.5 µg/ml agonist hamster anti-CD120a monoclonal antibody or an irrelevant IgG as control for 6 h prior to lysis and determination of luciferase activity (36).

Co-immunoprecipitation and Western Analysis-- Approximately 1.25 × 106 HEK293GT cells/well were seeded on 100-mm plates and grown in 5% CO2. Cells were transfected the following day by the calcium phosphate precipitation method. Transfected cells were lysed in 1 ml of immunoprecipitation buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM Na3VO4). Post-nuclear supernatants were precleared for 1 h with 25 µl of protein A/G+ beads (Santa Cruz Biotechnology), then incubated for 1 h at 4 °C with 3 µg of monoclonal hamster anti-CD120a, or 3 µg of monoclonal hamster anti-CD120b or non-immune hamster antibody as controls. Immune complexes were precipitated with 50 µl of protein A/G+ beads for 1 h at 4 °C. The Sepharose beads were washed four times with immunoprecipitation buffer. The precipitates were fractionated on SDS-PAGE under non-reducing conditions, and subsequent Western blotting analysis was performed as described previously (27).

Phosphorothioate Oligonucleotides-- Antisense DNA oligonucleotides with a phosphorothioate backbone were synthesized and purified by high pressure liquid chromatography (BIOSOURCE International, Camarillo, CA). The oligonucleotides were 3'-biotinylated to allow staining and to prevent them from interfering with the TUNEL assay. The 20-mer antisense oligonucleotide (2009) targeted against the coding region of the bcl-2 gene, and the scrambled-sequence oligonucleotide with a similar nucleotide content (sc48), were described previously (37). A BLASTN search of the NCBI DNA data base revealed no homology of the oligonucleotides to other human genes. The sequences were as follows: antisense, 5'-AATCCTCCCCCAGTTCACCC-3'; scrambled, 5'-CTCATTCCTACCGACACCCC-3'. Oligonucleotides were transfected for 24 h using LipofectAMINE Plus reagent as recommended by the manufacturer (Life Technologies). Medium was changed 7 h after the transfection.

Results shown for all experiments are representative of at least three separate experiments.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphorylation of CD120a Protects from Apoptosis-- Given the striking changes in the subcellular distribution of CD120a that result from its phosphorylation by p42mapk/erk2 (27), we investigated the effect of phosphorylation on receptor function and signaling, including CD120a-mediated apoptosis and NF-kappa B activation. To address this question, we used HeLa cells, because previous studies have shown these cells to undergo apoptosis through the endogenous receptor (38) and hence they express the necessary signaling pathways to mediate this response. In previous studies we induced receptor phosphorylation by co-transfection with constitutively active MEK1 and p42mapk/erk2. However, these kinases have been shown to affect the balance between apoptosis and survival (29-32) and thus the activation of p42mapk/erk2, per se, may affect the ability of CD120a to induce apoptosis in a CD120a phosphorylation-independent fashion in cells co-transfected with p42mapk/erk2 and MEK1.ca. Therefore, to address the role of phosphorylation of CD120a on TNFalpha -induced apoptosis in the absence of potential artifacts introduced by activated p42mapk/erk2, HeLa cells were transfected with expression vectors encoding wild type CD120a and CD120a.4D/E, a mutant receptor that we have previously shown to mimic the phosphorylated receptor by being redistributed to the endoplasmic reticulum in the absence of p42mapk/erk2 (27). 14 h later, the cells were stimulated with TNFalpha and cycloheximide (50 ng/ml and 10 µg/ml, respectively) for 4 h, fixed, permeabilized, and stained for CD120a under conditions that did not permit the detection of the endogenous receptor. Apoptotic cells were quantified on the basis of characteristic changes in the morphology of the nucleus stained with the Hoechst dye 33342 (Fig. 1a). As expected, transfection of wild type CD120a induced a robust increase in the proportion of apoptotic cells, both in the presence and in the absence of TNFalpha and cycloheximide. In contrast, transfection of the CD120a.4D/E mutant receptor completely abolished apoptosis as compared with that induced by the transfected wild type receptor. To confirm these results, apoptotic cells were also identified by TUNEL staining. Similar results were obtained when the percentage of TUNEL-positive cells among CD120a-transfected cells was quantified by confocal fluorescence microscopy (Fig. 1b). In addition, the inability of the transfected CD120a.4D/E mutant to induce apoptosis was similar to that of CD120a.L351N, a receptor bearing a point mutation in the death domain known to abolish the apoptotic activity of the receptor (39). In contrast, the CD120a.4A (T236A, S240A, S244A, S270A) mutant receptor, in which the four p42mapk/erk2 consensus sites had been mutated to Ala residues, did not protect the cells from the induction of apoptosis (data not shown). To further confirm the effect of phosphorylation of CD120a on the activation of the apoptotic process, transfected HeLa cells were lysed and equal amounts of proteins from post-nuclear lysates were resolved by SDS-PAGE and subjected to Western blotting for procaspase-8 using an antibody that specifically recognizes uncleaved and inactive procaspase-8. As shown in Fig. 1c and as expected, expression of wild type CD120a induced the processing of procaspase-8 (as did TRADD, which was used as an alternative positive control). In contrast, expression of the CD120a.4D/E mutant receptor did not induce the processing of procaspase-8.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1.   Phosphorylation of CD120a protects from the induction of apoptosis. HeLa cells were transfected with wild-type CD120a or mutants of CD120a as indicated. 14 h after transfection, the cells were stimulated with TNFalpha and cycloheximide (CHX) (50 ng/ml and 10 µg/ml, respectively, for 4 h), subjected to the TUNEL assay, and stained for CD120a. Nucleic acids were stained with the Hoechst 33342 dye. The percentage of apoptotic cells among CD120a-transfected cells was quantified using a confocal fluorescence microscope, on the basis of characteristic changes in the morphology of the nucleus (a) or by the TUNEL assay (b). c, transfected cells were lysed, and equal amounts of proteins were subjected to Western blotting for uncleaved procaspase-8, or alpha -tubulin as a control. Signals were quantified by scanning densitometry.

To determine if the phosphorylation of CD120a simply rendered the receptor non-functional we investigated the effect of phosphorylation on the ability of CD120a to activate NF-kappa B using an NF-kappa B-dependent luciferase reporter gene assay. Three approaches were taken. First, HEK293 cells were transfected with expression constructs for wild type CD120a and CD120a.4D/E under conditions that led to constitutive activation of NF-kappa B as a result of receptor overexpression. As can be seen in Fig. 2a, transfection of equal amounts of both wild type CD120a and the phosphorylation mimic CD120a.4D/E led to approximately equivalent levels of NF-kappa B-dependent luciferase reporter gene expression. Second, we expressed low levels of both receptors in HEK293 and specifically activated the transfected receptors by cross-linking with an agonistic hamster anti-mouse CD120a monoclonal antibody. As can be seen in Fig. 2b, cross-linking of the transfected receptors also resulted in approximately equivalent increases in luciferase reporter gene activity, whereas a control hamster monoclonal antibody (anti-mouse CD120b) was without effect. Third, HEK293 cells were co-transfected with the NF-kappa B-luciferase reporter construct and equivalent amounts of either wild type CD120a, CD120a.4D/E, or empty vector (2.5 ng/ml) for 18 h prior to stimulation with mouse TNFalpha (10 ng/ml) for 6 h. As can be seen in Fig. 2c, although transfection with the reporter gene and empty vector led to a low level of luciferase expression, transfection with wild type CD120a or CD120a.4D/E led to a similar increase in basal luciferase expression. However, stimulation with mouse TNFalpha resulted in a ligand-dependent increase in reporter gene expression that was equivalent for both receptors. Thus, although the induction of apoptotic activity was lost as a consequence of CD120a phosphorylation, the ability to activate NF-kappa B was preserved. Furthermore, specific ligation of the transfected receptors using both agonistic antibodies and TNFalpha resulted in the activation of NF-kappa B thus indicating that both the wild type and the phosphorylated receptors are available for signaling this response.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 2.   Activation of NF-kappa B is unaffected by the state of phosphorylation of CD120a. a, HEK293 cells were co-transfected with either wild type CD120a or CD120 4D/E (200 ng of each construct) and the NF-kappa B luciferase reporter gene construct. 18 h after transfection, the cells were lysed and assayed for luciferase activity. b, wild type CD120a or CD120a.4D/E were expressed at low level in HEK293 cells by co-transfection with 2.5 ng of each construct with the NF-kappa B luciferase reporter gene construct. 18 h after transfection, the cells were stimulated with 0.5 µg of either agonistic hamster anti-mouse CD120a, hamster anti-CD120b as a control, or medium alone. Luciferase activity was quantified in cell lysates after 6 h of stimulation with the indicated antibodies. c, HEK293 cells were transfected as in b but were stimulated with mouse TNFalpha (10 ng/ml for 6 h) before determining luciferase activity.

TRADD, FADD, TRAF2, Casper/FLIP, and Caspase-8 Are Not Recruited by CD120a.4D/E-- Following the binding of TNFalpha to CD120a, the adapter molecule TRADD binds through its death domain to the death domain of CD120a (2) thereby forming a platform for the assembly of other signaling molecules of the apoptosis cascade, including FADD (3) and caspase-8 (3). Other molecules with pro-survival activity such as TRAF2 and Casper/FLIP are also recruited to the receptor complex. Thus, protection from apoptosis by phosphorylated CD120a may result from sequestration of the pro-apoptotic molecules TRADD and FADD, thereby removing them from their defined sites of action, or from the recruitment of pro-survival molecules to phosphorylated CD120a-associated tubules. To address this question, HeLa cells were transfected with the CD120a.4D/E mutant receptor and were co-stained for CD120a, and TRADD or FADD. As shown in Fig. 3, both endogenous TRADD and FADD showed a homogenous intracellular staining pattern, which was not affected by expression of the CD120a.4D/E mutant receptor (Fig. 3, rows a and b). TRAF2, was also absent from the tubular structures containing CD120a.4D/E (Fig. 3, row c), rendering the involvement of cIAP proteins in the survival effect of the phosphorylated receptor unlikely. Similarly, co-expression of myc-tagged Casper/FLIP and CD120a.4D/E resulted in a lack of co-localization (Fig. 3, row d), consistent with the failure of FADD to be recruited by CD120a.4D/E and with the conclusion that Casper/FLIP is unlikely to be involved in the pro-survival effect of CD120a.4D/E. Thus, the adapters TRADD, FADD, and TRAF2, and the pro-survival molecule Casper/FLIP, are not recruited to the tubular structures formed by phosphorylated CD120a.


View larger version (87K):
[in this window]
[in a new window]
 
Fig. 3.   TRADD, FADD, TRAF2, Casper, and caspase-8 are not recruited by CD120a.4D/E. HeLa cells were transfected with CD120a.4D/E (a-c, e), cotransfected with CD120a.4D/E and myc-Casper (d), or transfected with myc-TRADD (f) or FLAG-FADD (g). The cells were fixed, permeabilized, stained as indicated, and observed by confocal fluorescence microscopy. Text in italics refers to staining; roman text refers to transfection.

To verify that we could detect the interaction of these proteins by confocal microscopy, we tested whether caspase-8 was recruited to "filaments" formed by the overexpression of FADD and TRADD as previously reported (40) as well as to the CD120a-associated tubular structures. Cells were transfected with the CD120a.4D/E mutant receptor, and stained for endogenous caspase-8. As a control, cells were transfected with FLAG-tagged FADD or myc-tagged TRADD, and then co-stained for caspase-8 and either the FLAG or myc epitopes. As shown in Fig. 3 (row e), caspase-8 exhibited a diffuse staining pattern in untransfected cells and this was not affected by transfection of the CD120a.4D/E mutant receptor. Consistent with previous reports (21, 40), a proportion of cells transfected with FADD showed thick and extensive filaments in the cytoplasm as well as in (or across) the nucleus, the latter imprinting the surface of the nuclear envelope as seen by Nomarski imaging or Hoechst staining (Fig. 3, row g). Cells transfected with TRADD also showed cytoplasmic filaments (Fig. 3, row f). However, in contrast to what was seen with the phosphorylation mimic CD120a.4D/E, caspase-8 was co-localized with the filaments formed by FADD and TRADD (Fig. 3, rows f and g). Thus, caspase-8 is recruited to FADD and TRADD filaments but not to CD120a-associated tubular structures. Collectively, these results suggest that the down-regulation of apoptosis associated with phosphorylation of CD120a does not involve the recruitment of TRADD, FADD, TRAF2, Casper/FLIP, or caspase-8.

Recruitment of Bcl-2 but Not of Bcl-xL by Phosphorylated CD120a-- Because the mechanism underlying the protection from apoptosis by phosphorylated CD120a did not appear to involve sequestration of the pro-apoptotic adapter molecules TRADD, FADD, or caspase-8, we next explored the hypothesis that protection may be mediated by the recruitment of pro-survival factors of the Bcl-2 family, especially because Bcl-2 is associated with intracellular membranes especially mitochondria and the endoplasmic reticulum (41). HeLa cells were transfected with the CD120a.4D/E mutant receptor and stained for endogenous Bcl-2 and CD120a. Endogenous Bcl-2 was detected in the cytoplasm and was associated with small granular structures (Fig. 4, a and b), consistent with previous reports (42). This pattern was not significantly affected by transfection of wild type CD120a (Fig. 4, e-h). However, transfection of the CD120a.4D/E mutant receptor induced a dramatic redistribution of Bcl-2 to cytoplasmic tubular structures (Fig. 4, c and d). Double-staining experiments showed precise co-localization of the phosphorylation mimic CD120a.4D/E and Bcl-2, indicating that endogenous Bcl-2 is recruited to the tubules formed by phosphorylated CD120a (Fig. 4, i-l). Cotransfection of vectors encoding Bcl-2 and CD120a.4D/E also resulted in the recruitment of Bcl-2 to CD120a-containing tubules (data not shown). Bcl-2 was also recruited to the tubular structures formed when wild type CD120a was phosphorylated by cotransfection with MEK1.ca and p42mapk/erk2 (Fig. 4, m-p). Colocalization of Bcl-2 and CD120a.4D/E was also observed in cells fixed and permeabilized with methanol instead of paraformaldehyde and Triton X-100, respectively, excluding the possibility that this association could be the result of the use of detergents (43).


View larger version (77K):
[in this window]
[in a new window]
 
Fig. 4.   CD120a.4D/E and ERK-phosphorylated CD120a but not CD120a.wt recruit Bcl-2. HeLa cells were transfected with CD120a.wt (e-h), CD120a.4D/E (c, d, j-l), or cotransfected with CD120a.wt, p42mapk/erk2, constitutively active MEK1 and Bcl-2 (m-p). The cells were stained for CD120a and Bcl-2, and observed by confocal fluorescence microscopy. Text in italics refers to staining; roman text refers to transfection. q, Bcl-2 interacts with ERK-phosphorylated CD120a in intact cells. Bcl-2 was coexpressed with empty vector (lane 1), CD120a.wt (lanes 2-5, 8, 9), CD120a.4D/E (lane 6), CD120a.L351N (lane 7), or CD120a.wt together with p42mapk/erk2 and constitutively active MEK1 (lane 10) in human HEK293 cells. CD120a was immunoprecipitated as indicated under "Experimental Procedures" using hamster non-immune IgG (lanes 1, 3, 8), no antibody (lane 2), hamster monoclonal anti-CD120b antibody as control (lane 4), or hamster monoclonal anti-CD120a antibody (lanes 5-7, 9, 10). Lysates and immunoprecipitates were separated by SDS-PAGE and subjected to Western blotting with anti-CD120a and anti-Bcl-2 antibodies. The upper arrow indicates the gel shift of P-CD120a on SDS-PAGE.

We next tested whether the interaction between Bcl-2 and phosphorylated CD120a could be detected as a protein complex by co-immunoprecipitation. CD120a or the CD120a phosphorylation mutants were co-expressed with Bcl-2 in HEK293 cells. As can be seen in Fig. 4q, Bcl-2 was co-immunoprecipitated with the CD120a.4D/E mutant receptor but not with wild type CD120a.wt. Bcl-2 was also co-immunoprecipitated with CD120a when phosphorylated by co-expression with MEK1.ca and p42mapk/erk2, a point confirmed by the gel shift of the phosphorylated receptor upon SDS-PAGE (Fig. 4q). No interaction was observed between Bcl-2 and the death domain-inactive CD120a.L351N, suggesting that different mechanisms account for the inability of this receptor mutant and the phosphorylated CD120a to protect against the induction of apoptosis.

To determine if the recruitment of Bcl-2 by phosphorylated CD120a was specific, we also stained for Bcl-xL. Bcl-xL was mainly expressed in cytoplasmic structures (Fig. 5) (mostly mitochondria, as assessed by co-localization with Mitotracker orange, data not shown), but was not recruited to the tubular structures containing CD120a.4D/E (Fig. 5). Bcl-xL was also absent from CD120a-containing tubules in HeLa cells that had been cotransfected with CD120a.wt, MEK1.ca, and p42mapk/erk2 (Fig. 5). These findings thus indicate that phosphorylated CD120a is targeted to the endoplasmic reticulum where it preferentially interacts with Bcl-2.


View larger version (53K):
[in this window]
[in a new window]
 
Fig. 5.   Bcl-xL is not recruited by phosphorylated CD120a. HeLa cells were cotransfected with Bcl-xL and CD120a.4D/E, or CD120a.wt, p42mapk/erk2, constitutively active MEK1 and Bcl-xL. The cells were stained for CD120a and Bcl-xL and observed by confocal fluorescence microscopy. Text in italics refers to staining; roman text refers to transfection.

Bcl-2 Is a Critical Determinant in the Regulation of Apoptosis by Phosphorylated CD120a-- We next examined whether the protection against apoptosis afforded by phosphorylated CD120a was mediated by Bcl-2. HeLa cells were transfected for 24 h with a 3'-biotinylated Bcl-2 antisense phosphorothioate oligonucleotide, or an oligonucleotide of the same nucleotide content but with a scrambled sequence as a control. Staining with Cy5-streptavidin confirmed the efficient nuclear delivery of the oligonucleotides in 100% of the cells (Fig. 6a). Western blot analyses showed decreased Bcl-2 protein levels in cells treated with the Bcl-2 antisense oligonucleotide at concentrations of at least 300 nM, whereas treatment with the control oligonucleotide had little effect (Fig. 6b). Protein levels of Bcl-xL and alpha -tubulin were unchanged, indicating that the effect of the antisense oligonucleotide was specific for Bcl-2 (Fig. 6b). To examine whether Bcl-2 accounts for some of the pro-survival effect of phosphorylated CD120a, HeLa cells were cotransfected with the CD120a.4D/E mutant receptor together with the Bcl-2 antisense or control oligonucleotides. HeLa cells were also transfected with wild type CD120a as a control. The conditions for both receptors were carefully selected to yield a low level of apoptosis in the absence of antisense Bcl-2 oligonucleotide thereby ensuring that we would be able to detect any increase in the degree of apoptosis in the presence of the oligonucleotide. It was also for this reason that we elected to use modest receptor overexpression to induce the necessary low level of apoptosis in the controls as opposed to inducing apoptosis with ligand exposure. 24 h after transfection, the cells were fixed, subjected to TUNEL assay, and stained for CD120a. In cells transfected with the antisense Bcl-2 oligonucleotide, the level of expression of CD120a was greatly reduced in apoptotic cells and the localization of CD120a.4D/E to cytoplasmic tubules was lost. Instead, the receptor exhibited a dim intracytoplasmic punctate staining (Fig. 6c), suggesting that Bcl-2 may be required for the localization of the phosphorylated receptor to the tubular structures associated with the endoplasmic reticulum. Importantly, treatment with the Bcl-2 antisense oligonucleotide (300-450 nM) also induced a significant increase in the level of apoptosis induced by CD120a.4D/E, as shown in Fig. 6d. The Bcl-2 antisense oligonucleotide also potentiated apoptosis in cells transfected with the wild type receptor, although to a lesser extent than that seen with CD120a.4D/E. The apoptosis observed was CD120a-specific, because the antisense oligonucleotide did not promote cell death in cells transfected with the empty vector. In contrast, treatment with the control oligonucleotide induced little increase in apoptosis, indicating that most of the effect of the Bcl-2 antisense oligonucleotide was sequence specific. However, some sequence-independent effects of the oligonucleotides may also have occurred to a much lower extent, as reported (44). Taken together, these data demonstrate that Bcl-2 forms a complex with phosphorylated CD120a and participates in the protection against apoptosis conferred by these events.


View larger version (68K):
[in this window]
[in a new window]
 
Fig. 6.   Bcl-2 participates in the survival activity of phosphorylated CD120a. HeLa cells were cotransfected for 24 h with CD120a mutants as indicated, together with 300-450 nM 3'-biotinylated Bcl-2 antisense phosphorothioate oligonucleotides, or a control oligonucleotide of the same nucleotide content but scrambled sequence. a, staining with Cy5-conjugated streptavidin showed the efficient nuclear delivery of the oligonucleotides. b, equal amounts of proteins from cells lysates were subjected to Western blot analysis for Bcl-2, Bcl-xL, and alpha -tubulin. Concentration of antisense (lanes 1-4) or scrambled (lanes 5-8) oligonucleotides was 0 (lanes 1 and 5), 150 nM (lanes 2 and 6), 300 nM (lanes 3 and 7), 450 nM (lanes 4 and 8). c, 24 h after the transfection, cells were subjected to the TUNEL assay and stained for CD120a. The upper cell was transfected with the scrambled oligonucleotide, whereas the lower cell was transfected with the antisense oligonucleotide. d, cotransfection of the antisense oligonucleotide restores the ability of P-CD120a to induce apoptosis. The percentage of TUNEL-positive cells among CD120a-transfected cells was quantitated using a confocal fluorescent microscope.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CD120a, a death receptor of the TNF receptor superfamily, induces apoptosis in certain cell types upon ligation by TNFalpha . In the present study we show that phosphorylation of CD120a by p42mapk/erk2 differentially regulates its function as a death receptor. When phosphorylated, CD120a loses its ability to induce apoptosis and thus confers a survival advantage to cells expressing the phosphorylated receptor. However, the receptor retains its ability to stimulate the activation of NF-kappa B either by high level overexpression or by agonist antibody- or TNFalpha -mediated ligation of the transfected receptor following low level expression. In addition, we show that Bcl-2 is specifically recruited by phosphorylated CD120a to tubular structures associated with elements of the endoplasmic reticulum and contributes to the survival activity of the phosphorylated receptor. Thus, the phosphorylated CD120a·Bcl-2 complex localized within endoplasmic reticulum tubules may represent an alternative signaling complex that is protective against apoptosis in contrast to the previously described pro-apoptotic CD120a·TRADD complex.

Recently reported studies have established the concept that activation of p42mapk/erk2 confers survival advantages to cells in the face of activation of apoptotic pathways. For example, Xia et al. (32) showed that apoptosis induced by growth factor withdrawal from PC12 cells could be overcome by dominant activation of p42mapk/erk2. Similarly, the activation of T-cells by concanavalin A was shown by Yeh et al. (29) to antagonize Fas-triggered cell death through the MEK1-dependent up-regulation of Casper/FLIP, indicating that cross-talk between p42mapk/erk2 and the pro-apoptotic signals induced by Fas ligation results in a dominant survival response. Therefore, to distinguish between (i) direct inhibition of CD120a-induced apoptosis by the phosphorylated receptor and (ii) potential secondary (or additional) CD120a-independent pro-survival effects of activated p42mapk/erk2 we studied the mechanism of protection against apoptosis by phosphorylated CD120a with a mutant receptor in which the p42mapk/erk2 phosphorylation sites were mutated to Glu and Asp residues (CD120a.4D/E) to mimic the effects of phosphorylation on receptor function (27). The findings from the present study reveal a novel mechanism by which p42mapk/erk2 protects against CD120a-mediated apoptosis, namely, redistribution of the phosphorylated receptor to tubular structures associated with the endoplasmic reticulum wherein it serves as a platform (or signal) for the co-recruitment of Bcl-2.

The mechanism underlying the protective effect conferred by the phosphorylated receptor may be attributable to the loss of the dephosphorylated receptor from its primary site of apoptotic signaling, and/or to a pro-survival effect mediated by Bcl-2. We propose that both mechanisms contribute a role in the protection from apoptosis by the phosphorylated receptor. First, work reported by Schutze et al. (45) has shown that the endocytic inhibitor monodansylcadaverine inhibits the induction of death following cross-linking of CD120a, and studies initially reported by Bradley et al. (46) and confirmed by other groups including our own have conclusively shown that an extensive pool of CD120a exists in the trans-Golgi network (27, 47). Likewise, Bennett and colleagues (48) have reported that Fas is localized within the Golgi complex and that disruption of the Golgi complex with brefeldin A inhibits the ability of Fas to induce apoptosis. Thus, although ligation of the cell surface receptor would intuitively seem likely to be necessary for the initiation of apoptosis, these recent studies support the contention that the initiation of apoptotic signaling by death receptors may involve assembly of the signaling complex within the broad spatial confines of the Golgi complex. In contrast, other functions of CD120a, such as the activation of proline-directed protein kinases, occur independently of receptor internalization (45). Because phosphorylation of CD120a by p42mapk/erk2 promotes its translocation from the Golgi complex to the endoplasmic reticulum while preserving a level of receptor expression at the cell surface, we speculate that the inability of the phosphorylated receptor to signal apoptosis may result in part from selective receptor loss from the Golgi complex while the fraction preserved at the cell surface may be responsible for signaling NF-kappa B activation.

Second, although initial reports suggested that Bcl-2 was incapable of protecting against apoptosis induced by death receptors, recent studies have clarified the mechanisms and conditions under which Bcl-2 is protective against death receptors ligation (17). In particular, in type II cells, of which HeLa cells are a representative (49, 50), Bcl-2 inhibits apoptosis by blocking the release of cytochrome c from mitochondria (51, 52). Our findings also show that Bcl-2 is involved in the protection against apoptosis that results from CD120a phosphorylation, because antisense oligonucleotide-mediated down-regulation of Bcl-2 protein reversed the protection against apoptosis afforded by the CD120a.4D/E expression. In addition, bulk cleavage of pro-caspase-8 in type II cells has been shown to occur as a consequence of cytochrome c release from mitochondria and the ensuing activation of caspase-9 (17). We also observed an absence of bulk pro-caspase-8 processing in response to CD120a.4D/E expression, a finding that is consistent with the conclusion that Bcl-2 serves to inhibit the activation of the mitochondrial amplification pathway in these cells. Thus, Bcl-2 is involved in the protection against apoptosis conferred by CD120a phosphorylation, and this likely is mediated by the prevention of cytochrome c release from mitochondria.

The mechanism by which the phosphorylated CD120a·Bcl-2 complex is formed upon CD120a phosphorylation and how it specifically protects against CD120a-induced apoptosis remains to be investigated. We speculate that the recruitment of Bcl-2 to the CD120a-associated endoplasmic reticulum tubules serves to recruit other pro-apoptotic Bcl-2 family members thereby preventing these molecules from interacting with mitochondria in initiating cytochrome c release. A similar mechanism has recently been proposed to explain the pro-survival effect of the adenoviral protein E1B 19K, a distantly related Bcl-2 homolog that has been shown to inhibit FADD-induced death upstream of caspase-8 (21) and to interact with tBid-activated Bax, thereby preventing Bax from promoting cytochrome c release from mitochondria (50). Other pro-apoptotic molecules such as Bak, Bim, and Bid are also potential candidates that could potentially be sequestered by the phosphorylated CD120a·Bcl-2 complex. Although an attractive hypothesis, we consider it less likely that the phosphorylated CD120a·Bcl-2 complex would recruit non-Bcl-2 family pro-apoptotic proteins such as Apaf-1 or effector caspases, because it has been quite difficult to detect interactions between these proteins in mammalian cells (53, 54). Our findings also raise the question of the possible role of the endoplasmic reticulum in the protection against apoptosis conferred by CD120a phosphorylation. Previous work has shown the endoplasmic reticulum to be a major site of Bcl-2 localization (41) and protection against apoptosis. Deletion of the C-terminal 20-residue hydrophobic membrane insertion domain abrogates or diminishes the death-inhibitory effects of Bcl-2 (42), indicating that the subcellular localization of Bcl-2 may contribute to its anti-apoptotic function. Similarly, the pro-survival effect of Bcl-2 is affected by its localization to either the endoplasmic reticulum or mitochondria in a cell-dependent fashion (55). Furthermore, Hacki and colleagues (56) have recently shown that the endoplasmic reticulum-targeted expression of Bcl-2 also prevented the release of cytochrome c from mitochondria thus emphasizing the potential of cross-talk between these two organelles in the regulation of apoptosis. Unexpectedly, although Bcl-2 was recruited to phosphorylated CD120a, Bcl-xL was not. The significance of this finding is currently unclear. However, while in many situations Bcl-2 and Bcl-xL exhibit similar activities, differences in their activities have previously been noted. For example, Bcl-xL, but not Bcl-2, has been found to redistribute from the cytosol to intracellular membranes upon induction of thymocyte apoptosis in response to dexamethasone (57), whereas FK506 and cyclosporin-induced apoptosis in WEHI-231.7 cells is prevented by enforced expression of Bcl-xL but not by Bcl-2 (58).

In conclusion, we have shown that phosphorylated CD120a recruits Bcl-2 to tubular structures associated with the endoplasmic reticulum and that Bcl-2 contributes to the protection against apoptosis that arises as a consequence of CD120a phosphorylation. Phosphorylation of CD120a may thus contribute to the promotion of resistance to death receptor-induced apoptosis in tumor cells while at the same time preserving the ability of the receptor to activate NF-kappa B, a transcription factor that regulates the expression of both pro-survival molecules, including c-IAPs as well as pro-inflammatory cytokines (59). Whether similar events regulate the apoptotic pathway induced by other death receptors such as Fas, or whether this mechanism is specific for CD120a signaling, remains to be elucidated. However, these findings suggest that post-translational modification of the cytoplasmic domain of CD120a may play an important role in the regulation of receptor function and may thus have significant implications in cell survival and function in cancer and in chronic inflammatory diseases, which are associated with persistent activation of the Ras-Raf-MEK-p42mapk/erk2 pathway by activated oncogenes and cytokines, respectively.

    ACKNOWLEDGEMENTS

We acknowledge Linda Remigio and Cheryl Leu for outstanding technical assistance. We also wish to thank Dr. Gary L. Johnson and Nancy Johnson for their generous help with the confocal microscopy; Dr. Hong-Bing Shu for helpful discussions and for advice for co-immunoprecipitation experiments; and Drs. Lynn Heasley, Gary L. Johnson, Gabriel Nunez, Hong-Bing Shu, and Hong Zhou for providing us with expression vectors.

    FOOTNOTES

* This work was supported in part by United States Public Health Services Grants HL55549 and SCOR HL 56556 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by Traveling Fellowship grants from the Société de Pneumologie de Langue Française, the Association pour la Recherche contre le Cancer, Fondation Alain Philippe, Fondation Lavoisier du Ministère des Affaires Etrangères, and a Michael and Eleanore Stobin 1999 Pediatric Fellowship from National Jewish Medical and Research Center, Denver, CO.

§§ To whom correspondence should be addressed: Dept. of Pediatrics, National Jewish Medical and Research Center, Neustadt Rm. D405, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1188; Fax: 303-398-1381; E-mail: richesd@njc.org.

Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M010681200

    ABBREVIATIONS

The abbreviations used are: TNFalpha , tumor necrosis factor-alpha ; TRADD, TNF receptor-associated death domain protein; FADD, Fas-associated death domain proteins; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; MEK, MAPK/ERK kinase; MEK1.ca, constitutively active MEK1; FLIP, FLICE-inhibitory protein; IAP, inhibitor of apoptosis family of proteins; Fas-L, Fas ligand; DISC, death-inducing signaling complex; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; TRAF2, TNF receptor-associated factor-2.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Ashkenazi, A., and Dixit, V. M. (1998) Science 281, 1305-1308[Abstract/Free Full Text]
2. Hsu, H., Xiong, J., and Goeddel, D. V. (1995) Cell 81, 495-504[Medline] [Order article via Infotrieve]
3. Hsu, H., Shu, H.-B., Pan, M.-G., and Goeddel, D. V. (1996) Cell 84, 299-308[Medline] [Order article via Infotrieve]
4. Hsu, H., Huang, J., Shu, H.-B., Baichwal, V., and Goeddel, D. V. (1996) Immunity 4, 387-396[Medline] [Order article via Infotrieve]
5. Ting, A. T., Pimentel-Muinos, F. X., and Seed, B. (1996) EMBO J. 15, 6189-6196[Abstract]
6. Yeh, W. C., Pompa, J. L., McCurrach, M. E., Shu, H. B., Elia, A. J., Shahinian, A., Ng, M., Wakeham, A., Khoo, W., Mitchell, K., El-Deiry, W. S., Lowe, S. W., Goeddel, D. V., and Mak, T. W. (1998) Science 279, 1954-1958[Abstract/Free Full Text]
7. Li, H., Zhu, H., Xu, C. J., and Yuan, J. (1998) Cell 94, 491-501[Medline] [Order article via Infotrieve]
8. Shu, H. B., Halpin, D. R., and Goeddel, D. V. (1997) Immunity 6, 751-763[Medline] [Order article via Infotrieve]
9. Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J. L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L. E., and Tschopp, J. (1997) Nature 388, 190-195[CrossRef][Medline] [Order article via Infotrieve]
10. Hitoshi, Y., Lorens, J., Kitada, S. I., Fisher, J., LaBarge, M., Ring, H. Z., Francke, U., Reed, J. C., Kinoshita, S., and Nolan, G. P. (1998) Immunity 8, 461-471[Medline] [Order article via Infotrieve]
11. Deveraux, Q. L., and Reed, J. C. (1999) Genes Dev. 13, 239-252[Free Full Text]
12. Kawahara, A., Kobayashi, T., and Nagata, S. (1998) Oncogene 17, 2549-2554[CrossRef][Medline] [Order article via Infotrieve]
13. Kroemer, G. (1997) Nat. Med. 3, 614-620[Medline] [Order article via Infotrieve]
14. Zha, J. P., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S. J. (1996) Cell 87, 619-628[Medline] [Order article via Infotrieve]
15. Datta, S. R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M. E. (1997) Cell 91, 231-241[Medline] [Order article via Infotrieve]
16. del Peso, L., Gonzalez-Garcia, M., Page, C., Herrera, R., and Nunez, G. (1997) Science 278, 687-689[Abstract/Free Full Text]
17. 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[Abstract/Free Full Text]
18. Adams, J. M., and Cory, S. (1998) Science 281, 1322-1326[Abstract/Free Full Text]
19. Huang, D. C., Cory, S., and Strasser, A. (1997) Oncogene 14, 405-414[CrossRef][Medline] [Order article via Infotrieve]
20. Jaattela, M., Benedict, M., Tewari, M., Shayman, J. A., and Dixit, V. M. (1995) Oncogene 10, 2297-2305[Medline] [Order article via Infotrieve]
21. Perez, D., and White, E. (1998) J. Cell Biol. 141, 1255-1266[Abstract/Free Full Text]
22. Yasuhara, N., Sahara, S., Kamada, S., Eguchi, Y., and Tsujimoto, Y. (1997) Oncogene 15, 1921-1928[CrossRef][Medline] [Order article via Infotrieve]
23. Srinivasan, A., Li, F., Wong, A., Kodandapani, L., Smidt, R., Jr., Krebs, J. F., Fritz, L. C., Wu, J. C., and Tomaselli, K. J. (1998) J. Biol. Chem. 273, 4523-4529[Abstract/Free Full Text]
24. Winston, B. W., and Riches, D. W. H. (1995) J. Immunol. 155, 1525-1533[Abstract]
25. Winston, B. W., Chan, E. D., Johnson, G. L., and Riches, D. W. H. (1997) J. Immunol. 159, 4491-4497[Abstract]
26. Chan, E. D., Winston, B. W., Jarpe, M. B., Wynes, M. W., and Riches, D. W. H. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13169-13174[Abstract/Free Full Text]
27. Cottin, V., Van Linden, A., and Riches, D. W. H. (1999) J. Biol. Chem. 274, 32975-32987[Abstract/Free Full Text]
28. Van Linden, A. A., Cottin, V., Leu, C., and Riches, D. W. H. (2000) J. Biol. Chem. 275, 6996-7003[Abstract/Free Full Text]
29. Yeh, J. H., Hsu, S. C., Han, S. H., and Lai, M. Z. (1998) J. Exp. Med. 188, 1795-1802[Abstract/Free Full Text]
30. Yujiri, T., Sather, S., Fanger, G. R., and Johnson, G. L. (1998) Science 282, 1911-4[Abstract/Free Full Text]
31. Gardner, A. M., and Johnson, G. L. (1996) J. Biol. Chem. 271, 14560-14566[Abstract/Free Full Text]
32. Xia, Z., Dickens, M., Raingeaud, J., Davis, R. J., and Greenberg, M. E. (1995) Science 270, 1326-1331[Abstract]
33. Frasch, S. C., Nick, J. A., Fadok, V. A., Bratton, D. L., Worthen, G. S., and Henson, P. M. (1998) J. Biol. Chem. 273, 8389-8397[Abstract/Free Full Text]
34. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene 77, 51-59[CrossRef][Medline] [Order article via Infotrieve]
35. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752[Medline] [Order article via Infotrieve]
36. Brasier, A. R., Tate, J. E., and Habener, J. F. (1989) BioTechniques 7, 1116-1122[Medline] [Order article via Infotrieve]
37. Ziegler, A., Luedke, G. H., Fabbro, D., Altmann, K. H., Stahel, R. A., and Zangemeister-Wittke, U. (1997) J. Natl. Cancer Inst. 89, 1027-1036[Abstract/Free Full Text]
38. Miura, M., Friedlander, R. M., and Yuan, J. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8318-8322[Abstract]
39. Tartaglia, L. A., Merrill Ayres, T., Wong, G. H. W., and Goeddel, D. V. (1993) Cell 74, 845-853[Medline] [Order article via Infotrieve]
40. Siegel, R. M., Martin, D. A., Zheng, L., Ng, S. Y., Bertin, J., Cohen, J., and Lenardo, M. J. (1998) J. Cell Biol. 141, 1243-1253[Abstract/Free Full Text]
41. Krajewski, S., Tanaka, S., Takamaya, S., Schibler, M., Fenton, W., and Reed, J. C. (1993) Cancer Res. 53, 4701-4714[Abstract]
42. Zamzami, N., Brenner, C., Marzo, I., Susin, S. A., and Kroemer, G. (1998) Oncogene 16, 2265-2282[CrossRef][Medline] [Order article via Infotrieve]
43. Hsu, Y. T., and Youle, R. J. (1997) J. Biol. Chem. 272, 13829-13834[Abstract/Free Full Text]
44. Wagner, R. W. (1994) Nature 372, 333-335[CrossRef][Medline] [Order article via Infotrieve]
45. Schutze, S., Machleidt, T., Adam, D., Schwandner, R., Wiegmann, K., Kruse, M. L., Heinrich, M., Wickel, M., and Kronke, M. (1999) J. Biol. Chem. 274, 10203-10212[Abstract/Free Full Text]
46. Bradley, J. R., Thiru, S., and Pober, J. S. (1995) Am. J. Pathol. 146, 27-32[Abstract]
47. Jones, S. J., Ledgerwood, E. C., Prins, J. B., Galbraith, J., Johnson, D. R., Pober, J. S., and Bradley, J. R. (1999) J. Immunol. 162, 1042-1048[Abstract/Free Full Text]
48. Bennett, M., Macdonald, K., Chan, S. W., Luzio, J. P., Simari, R., and Weissberg, P. (1998) Science 282, 290-293[Abstract/Free Full Text]
49. Grullich, C., Sullards, M. C., Fuks, Z., Merrill, A. H., Jr., and Kolesnick, R. (2000) J. Biol. Chem. 275, 8650-8656[Abstract/Free Full Text]
50. Perez, D., and White, E. (2000) Mol. Cell 6, 53-63[Medline] [Order article via Infotrieve]
51. Kluck, R. M., Bossy-Wetzel, E., Green, D. R., and Newmeyer, D. D. (1997) Science 275, 1132-1136[Abstract/Free Full Text]
52. Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T. I., Jones, D. P., and Wang, X. (1997) Science 275, 1129-1132[Abstract/Free Full Text]
53. Hengartner, M. O. (2000) Nature 407, 770-776[CrossRef][Medline] [Order article via Infotrieve]
54. Hausmann, G., O'Reilly, L. A., van Driel, R., Beaumont, J. G., Strasser, A., Adams, J. M., and Huang, D. C. (2000) J. Cell Biol. 149, 623-634[Abstract/Free Full Text]
55. Zhu, W., Cowie, A., Wasfy, G. W., Penn, L. Z., Leber, B., and Andrews, D. W. (1996) EMBO J. 15, 4130-4141[Abstract]
56. Hacki, J., Egger, L., Monney, L., Conus, S., Rosse, T., Fellay, I., and Borner, C. (2000) Oncogene 19, 2286-2295[CrossRef][Medline] [Order article via Infotrieve]
57. Hsu, Y. T., Wolter, K. G., and Youle, R. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 3668-3672[Abstract/Free Full Text]
58. Gottschalk, A. R., Boise, L. H., Thompson, C. B., and Quintans, J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 7350-7354[Abstract]
59. Wang, C. Y., Mayo, M. W., Korneluk, R. G., Goeddel, D. V., and Baldwin, A. S., Jr. (1998) Science 281, 1680-1683[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.