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Mol Cell Biol, January 1998, p. 608-615, Vol. 18, No. 1
Howard Hughes Medical Institute, Gwen Knapp
Center for Lupus and Immunology Research, and Department of Medicine,
The University of Chicago, Chicago, Illinois
60637,1 and
IDUN Pharmaceuticals
Inc., La Jolla, California 920372
Received 30 July 1997/Accepted 18 September 1997
The gene encoding human IAP-like protein (hILP) is one
of several mammalian genes with sequence homology to the baculovirus inhibitor-of-apoptosis protein (iap) genes. Here we show
that hILP can block apoptosis induced by a variety of extracellular stimuli, including UV light, chemotoxic drugs, and activation of the
tumor necrosis factor and Fas receptors. hILP also protected against
cell death induced by members of the caspase family, cysteine proteases
which are thought to be the principal effectors of apoptosis. hILP and
Bcl-xL were compared for their ability to affect several steps in the apoptotic pathway. Redistribution of cytochrome
c from mitochondria, an early event in apoptosis, was not
blocked by overexpression of hILP but was inhibited by
Bcl-xL. In contrast, hILP, but not Bcl-xL,
inhibited apoptosis induced by microinjection of cytochrome
c. These data suggest that while Bcl-xL may
control mitochondrial integrity, hILP can function downstream of
mitochondrial events to inhibit apoptosis.
Programmed cell death (apoptosis) is
an evolutionarily conserved cellular suicide process by which
extraneous or damaged cells are eliminated from an organism (38,
68). Deregulation of the apoptotic pathway has been implicated in
the pathogenesis of a wide variety of human diseases, including cancer,
autoimmune diseases, virus infections, neurodegenerative diseases, and
AIDS (65). Based largely on genetic studies with the
nematode Caenorhabditis elegans (33), two
mammalian gene families whose products play pivotal roles in the cell
death pathway have been identified. The caspases (cysteine aspartic
proteases), a family of at least 10 mammalian proteases with homology
to the nematode ced-3 gene product (1), are
activated during apoptosis (2, 8, 14, 26, 27, 58) and are
thought to be the principal executors of the apoptotic process
(28). The bcl-2 gene family encodes a group of
CED-9-related proteins which are central regulators of the cellular
apoptotic threshold (5). These proteins have been localized
to the outer nuclear, endoplasmic reticular, and outer mitochondrial
membranes (13, 41, 43, 51). In the nematode, genetic
analysis has placed the ced-9 gene upstream of
ced-3 function. Similarly, in several mammalian models of
apoptosis, overexpression of Bcl-2 has been shown to inhibit the
activation of cellular caspases, suggesting that caspases act
downstream of Bcl-2 protein function (2, 8, 14, 58). More
recent studies have suggested that Bcl-2 family members can function both downstream and upstream of caspase activity (60a). One
potential Bcl-2-regulated event in the apoptotic process is the loss of mitochondrial membrane potential (56), followed by the
release into the cytosol of at least two mitochondrial proteins,
apoptosis-initiating factor (62) and cytochrome c
(44). In cell-free systems, cytochrome c has been
shown to activate caspases. Bcl-2 and related proteins can block
mitochondrial disruption and the release of cytochrome c in
response to a variety of apoptotic stimuli (40, 71).
Cellular apoptosis is a defense mechanism utilized by the host to
eliminate virally infected cells. To counter the host apoptotic response, many DNA viruses encode proteins that interfere with key
regulatory steps in the apoptotic pathway. These proteins include
homologs of known cellular apoptotic modulators such as Bcl-2-related
proteins (32, 50), soluble cytokine receptors (60), and antagonists of cytokine receptor-induced
protein-protein interaction-mediated cell death signals (3, 35,
64). Two baculovirus antiapoptotic genes have been identified,
the caspase inhibitor gene p35 and the iap
(inhibitor-of-apoptosis) gene (17). Although iap
genes were discovered first in insect virus genomes (19),
cellular homologs have recently been identified in the genomes of
insects, birds, and mammals (22, 25, 31, 42, 53, 55, 66).
Certain baculovirus IAPs (Cp-IAP and Op-IAP) can functionally replace
the baculovirus caspase inhibitor p35, as their identification was
achieved by phenotypic rescue of p35-deficient virus (16, 18,
20).
Three bona fide mammalian IAP-related proteins, designated human
IAP-like protein (hILP), c-IAP1, and c-IAP2, have been identified (15). These proteins contain two major elements: (i) an
amino-terminal domain containing three imperfect repeats of an
~65-amino-acid cysteine- and histidine-rich sequence termed the
baculovirus IAP repeat (BIR) and (ii) a carboxy-terminal RING finger, a
zinc-binding domain which has been identified in a number of proteins
that function in cellular differentiation and proliferation (7, 74). In the mammalian IAP-related proteins the BIR and RING domains are separated by an amphipathic region of 120 to 170 residues, whose role has not been defined. This region is not found in the baculovirus IAPs. A fourth mammalian gene has also been identified, encoding a protein termed neuronal apoptosis-inhibitory protein (55) which possesses limited homology to the IAPs. Aside
from the BIR domains, however, neuronal apoptosis-inhibitory protein does not resemble the classical IAPs described above, and so its relationship to the prototype IAPs is unclear.
hilp (25), also called xiap
(42) and MIHA (66), is widely
expressed in human tissues and encodes a 57-kDa cytoplasmic protein
(25). hILP can impede apoptotic cell death induced by virus
infection and by overexpression of caspases (25). c-IAP1 and
c-IAP2 were identified as components of the type 2 tumor necrosis factor (TNF) receptor (TNFR2) signaling complex. These factors have
been shown to exist in the cell associated with the signaling molecule,
TRAF2 (53). Activation of TNFR2 by its ligand, TNF alpha
(TNF- In this study we examined the properties of hILP. hILP was found to
impede apoptotic cell death induced by a wide variety of extracellular
stimuli. The BIR-containing domain was sufficient for this protection.
hILP did not interact with any of the six known TRAF proteins, implying
that its role may be distinct from those of the c-IAPs. Furthermore,
redistribution of cytochrome c associated with the induction
of apoptosis was blocked by the Bcl-2 family member Bcl-xL
but not by hILP. In contrast, overexpression of hILP, but not
Bcl-xL, protected cells from apoptotic cell death induced
by the microinjection of cytochrome c. These findings demonstrate that Bcl-xL and hILP function upstream and
downstream, respectively, of cytochrome c in the apoptotic
cascade.
Cells and transfections.
Human embryonic kidney 293 cells
were grown at 37°C in 5% CO2 in Dulbecco's modified
Eagle's medium with 10% fetal bovine serum, 2 mM glutamine, 100 U of
penicillin per ml, and 100 µg of streptomycin per ml. For
transfections, 10-cm-diameter dishes were seeded with 5 × 105 cells. The medium was replaced the following day, and
cells were transfected by the calcium phosphate procedure as previously
described (24). MCF7 cells expressing the Fas receptor
(MCF7F cells) (63) (a gift of V. Dixit) were grown in RPMI
1640 containing 10% fetal bovine serum, 200 µg of G418 per ml, and
100 µg of hygromycin per ml at 37°C in 5% CO2. For
transfections, each well of a six-well plate was seeded with 2.5 × 105 cells, and 24 h after plating, the cells were
transfected with Lipofectin (Life Technologies). A 1:5 ratio of
pCMV-lacZ or pGreenLantern (pCMV-GLP; Life Technologies) to test
expression vector was used to ensure that every Plasmids.
The expression vectors encoding TRAF1, -2, and -3 have been described previously (24). The Myc epitope-tagged
human TRAF4/CART1 expression vector was kindly provided by C. Rudin and
J. Van Dongen. The murine TRAF5 vector was kindly provided by R. Arch;
the amino terminus was modified to incorporate the Myc epitope tag
(25) prior to subcloning into the pcDNA3 mammalian
expression vector (Invitrogen). Human TRAF6 was obtained by PCR
amplification from a human T-cell cDNA library followed by subcloning
into the pFLAG-CMV-2 expression vector (Kodak).
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Human IAP-Like Protein Regulates Programmed Cell
Death Downstream of Bcl-xL and Cytochrome c

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ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
![]()
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
), is thought to recruit the TRAF2-c-IAP complex to the
cytoplasmic domain of TNFR2. While TRAF2 has been shown to bind
directly to the TNFR2 cytoplasmic domain, the c-IAPs do not bind
directly but are thought to be recruited through their association with
TRAF2. The TRAF2-c-IAP1 heteromer has also been identified in the type
1 TNF receptor (TNFR1) signaling complex (59).
![]()
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-galactosidase- or
GreenLantern protein (GLP)-positive cell had also taken up the test
expression vector. For apoptosis assays, MCF7 cells were treated with
either anti-Fas (150 ng/ml) plus cycloheximide (1 µg/ml), recombinant
human TNF-
(rhTNF-
) (40 ng/ml), cisplatin (20 µg/ml), or UV
light (5 min on a 312-nm transilluminator [Fisherbiotech]). After
12 h, cells were fixed, stained for
-galactosidase expression,
and scored for apoptosis based on morphology.
Ring vector is a deletion construct encoding residues 1 to 449. The
loop vector was constructed by PCR-mediated fusion, using
Pfu polymerase, of residues 1 to 342 to residues 445 to 497. The 3×BIR vector encodes residues 1 to 399 and was a kind gift of R. Clem and J. M. Hardwick.
Precipitation and immunoblot analysis. Cells were cotransfected with 5 µg each of TRAF expression vector and mammalian GST expression vector by the calcium phosphate procedure. Medium was replaced 24 h following transfection. Lysates were prepared 48 h after transfection as follows. Cells were washed once in phosphate-buffered saline and lysed for 10 min at room temperature in 1 ml of lysis buffer (25 mM HEPES [pH 7.9], 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol, 0.1 mM phenylmethanesulfonyl fluoride, 1.0 µg of chymostatin per ml, 0.5 µg of pepstatin per ml, 0.5 µg of bestatin per ml, 0.5 µg of leupeptin per ml, 0.5 µg of aprotinin per ml, and 3 µg of antipain per ml). Lysates were clarified by microcentrifugation at 4°C for 15 min at 16,000 × g, and the protein concentrations in the supernatants were determined by using the Bradford reagent (Bio-Rad). For GST precipitation experiments, samples were incubated with 10 µl of a 50% slurry of glutathione agarose beads, equilibrated in lysis buffer and preblocked with bovine serum albumin, for at least 30 min at 4°C on a rotating platform. The beads were washed four times with 0.5 ml of lysis buffer at 4°C. Aliquots were separated by electrophoresis on sodium dodecyl sulfate-9.5% polyacrylamide gels and immunoblotted with commercial rabbit polyclonal antibodies to human TRAF1, TRAF2, or TRAF3 (Santa Cruz Biotechnologies) or with murine monoclonal antibodies to the Myc epitope (Pharmingen clone 9E10) or FLAG (Kodak M2). Blots were subsequently probed with horseradish peroxidase-conjugated anti-rabbit or anti-mouse monoclonal antibodies (Amersham) as required and developed with an enhanced chemiluminescence detection system (Amersham).
Microinjection. Cell microinjection was performed on the stage of a Nikon Diaphot 300 inverted microscope with an Eppendorf pressure injector (model 5246) and micromanipulator (model 5171). Microinjection needles (about 0.1-µm inner diameter) were pulled from glass capillaries with a horizontal electrode puller (Sutter Instrument model P-97) and loaded with Eppendorf microloaders. Cells were plated on glass cellocate coverslips (Eppendorf) 24 h prior to transfection. Twenty-four to 48 hours after transfection, GLP-positive cells were chosen for injection. To identify injected cells, the injectate contained 0.3% dextran-conjugated Texas red (10,000 molecular weight, lysine fixable; Molecular Probes). Dye alone or dye plus cytochrome c was injected into the cytoplasm of 293 cells (pressure, 80 to 100 hPa; time, 0.3 s). Cells were switched into fresh medium immediately after injection. The concentration of cytochrome c (Sigma no. C7752 from horse heart) in the pipette was 3 mg/ml. The intracellular concentrations of microinjected proteins are estimated to represent a 10- to 100-fold dilution of the amount delivered into the cell. Minaschek et al. (46), using similar equipment, demonstrated that the injection parameters used in their study, (pressure, 100 hPa; time, 0.5 s) deliver about 0.05 pl into the cytosol of 3T3 cells. We estimate the volume of 293 cells to be about 5 pl, similar to that of 3T3 cells. Based on the approximate volume delivered per cell (0.05 to 0.5 pl), the concentration of cytochrome c delivered per cell is estimated to be 150 to 1,500 fg and equivalent to 2.4 to 24 µM. On average, 100 to 150 cells were injected. Two hours after injection, cells were scored for apoptosis, and 35-mm photographs were taken with a Nikon Diaphot 300 inverted microscope.
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RESULTS |
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hILP inhibits apoptosis induced by diverse stimuli.
The
ability of hILP to block different apoptotic stimuli was tested by
transient transfection into the MCF7F human breast carcinoma cell line.
These cells have previously been shown to undergo apoptosis when
exposed to UV light or when treated with TNF-
or agonistic
antibodies to Fas (3). An expression vector encoding FLAG
epitope-tagged hILP was transfected, together with a LacZ expression
vector, and cells were treated with TNF-
, anti-Fas, UV light, or the
cytotoxic drug cisplatin. Twelve hours later, the cells were stained
for
-galactosidase expression, and the viability of
-galactosidase-positive cells was determined by morphological
examination under light microscopy as described previously
(3). As shown in Fig. 1A,
expression of hILP almost completely reversed the apoptotic effects of
each of these stimuli. The degree of inhibition was comparable to that
achieved by expression of the antiapoptotic Bcl-xL gene
(Fig. 1A).
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and Fas
(6, 23, 48). Therefore, MCF7F cells were transfected with
expression vectors encoding caspase 2 or caspase 8, and the effects of
hILP were examined by cotransfection with either the hILP expression
vector or a vector control. As shown in Fig. 1B, the apoptotic cell
death induced by either caspase 2 or caspase 8 was markedly impaired by
expression of hILP.
The BIR domains of hILP are necessary for inhibition of
apoptosis.
To define elements in hILP which are required for its
protective effects, deletion mutants which lacked the RING finger, the linker region between the BIRs and the RING domain, or both the linker
region and the RING finger were constructed, as summarized in Fig.
2A. These plasmids were transfected into
MCF7F cells, which were subsequently stimulated with recombinant
TNF-
. As shown in Fig. 2B, each of the deletion mutants still
protected against TNF-
-induced death, indicating that the BIRs are
sufficient for the protective effects of hILP. A deletion mutant
lacking the BIRs was not expressed to detectable levels under the
conditions used and so could not be assessed in this assay.
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hILP does not possess the TRAF-binding properties of the c-IAPs. The experiments described above revealed that the major elements required for the protective effects of hILP are the three amino-terminal BIR domains. Since the BIR domains of c-IAP1 mediate protein-protein interactions with TRAF proteins (53), the ability of hILP to interact with TRAFs was tested. Human embryonic kidney 293 cells were cotransfected with mammalian expression vectors encoding the six known TRAF proteins (10, 12, 34, 36, 37, 49, 52, 54) together with a mammalian expression vector encoding hILP fused to the GST protein (pEBG-hILP). Control transfections were also performed with either the parental GST vector alone (pEBG) or a GST-c-IAP1 chimera (pEBG-cIAP-1). Lysates were prepared from transfected cells, and complexes were coprecipitated by using glutathione agarose beads and examined by immunoblot analysis with anti-TRAF antibodies. hILP did not coprecipitate any of the six known TRAFs (Fig. 3). Under the same conditions, however, c-IAP1 coprecipitated with TRAF1 and TRAF2, as observed previously (53). High levels of GST-hILP chimeric protein were expressed, as observed by immunoblot analysis with a monoclonal antibody specific to GST (data not shown). These data suggest that unlike the c-IAPs, hILP does not directly associate with TRAFs.
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Effects of Bcl-xL and hILP on cytochrome c
immunolocalization during apoptosis.
The induction of apoptosis by
the engagement of cell surface receptors such as Fas or TNFR1 has been
shown to trigger a number of morphological and biochemical changes in
the cell. One of the earliest detectable markers during cell death is
the loss of mitochondrial function (56). Furthermore,
apoptotic cell death has recently been shown to correlate with the
release of cytochrome c from the mitochondria into the
cytosol, an event which is blocked by overexpression of Bcl-2 family
members (40, 44). To determine whether hILP can block
TNF-
-mediated release of cytochrome c, we monitored
cytochrome c immunolocalization in cells that had been
transfected with either hILP or Bcl-xL. As shown in Fig. 4A, TNF-
treatment induced a change in
the cytochrome c staining pattern from a punctuate
mitochondrial profile in untreated cells to a diffuse cytosolic
distribution. Expression of hILP had no effect on the redistribution of
cytochrome c after TNF-
treatment, while expression of
Bcl-xL nearly completely blocked the redistribution. Quantitative analysis of these experiments is presented in Fig. 4B.
These results suggest that hILP exerts its protective effects without
affecting TNF-
-induced cytochrome c redistribution and that the mechanism by which hILP inhibits apoptosis is distinct from
that utilized by Bcl-xL.
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hILP exerts its antiapoptotic effects at a point downstream of cytochrome c. The inability of hILP to block the redistribution of cytochrome c suggests that the antiapoptotic function of hILP is localized to a point downstream of cytochrome c action. To examine whether hILP could inhibit cytochrome c-mediated death, 293 cells were transfected with an hILP or Bcl-xL expression vector, or with a vector control, and subsequently microinjected with cytochrome c. As shown in Fig. 5, microinjection of cytochrome c into control transfected cells induced a number of morphological changes consistent with apoptosis, including membrane blebbing, cell shrinkage, and nuclear condensation and fragmentation. Control microinjections did not produce these effects. However, microinjection of cytochrome c into cells which had been transfected with the hILP expression vector completely protected the cells from cytochrome c-induced apoptosis. In contrast, expression of Bcl-xL had little or no effect on the cytochrome c-induced changes. These findings indicate that while the induction of apoptosis by cytosolic cytochrome c is not subject to regulation by Bcl-xL, it is efficiently inhibited by hILP, suggesting that the protective effects of hILP are focused downstream of cytochrome c-mediated activation of caspases.
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DISCUSSION |
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Since the discovery of the first iap genes in insect viruses, the mode of action of these proteins in the apoptotic pathway has remained elusive. One potentially important clue to the role of the IAPs was provided by an understanding of the relationship between the baculovirus IAP and the baculovirus apoptosis inhibitor P35 (18). Although IAP and P35 possess no obvious sequence homology, in the context of the virus these proteins are functionally interchangeable, suggesting a redundant role in cell death. Since P35 is known to exert its effects by binding to and competitively inhibiting members of the caspase family of cysteine proteases (4, 9, 53, 70), IAPs might function in an analogous manner. In fact, a recent report indicates that hILP indeed functions to inhibit certain caspases (21). The mechanism of hILP inhibition of caspases may differ from that of P35, however, as we can easily detect cleavage of P35 by recombinant caspases (4) but have been unable to detect cleavage of hILP under similar conditions (3a). Similarly, the baculoviral IAP homolog Op-IAP has been shown to exert its effects at a point downstream in the apoptotic cascade, although it does not appear to affect active capases (44a), suggesting a mechanism distinct from that of p35.
Of the three human IAP-related proteins identified to date, hILP
possesses the most potent antiapoptotic properties (25, 42, 53,
66). To gain insight into the mechanism of protection by hILP, we
examined the ability of hILP to protect against apoptosis induced by a
variety of apoptotic stimuli. As shown in Fig. 1, hILP efficiently
blocks cell death induced by TNF-
, Fas, UV light, and genotoxic
agents, indicating that hILP functions downstream of a point of
convergence of these diverse stimuli. While the apoptotic events
triggered by TNF-
and agonistic antibodies to Fas are cell surface
receptor mediated, DNA damage induced by exposure to UV and cisplatin
is thought to trigger apoptosis in response to these insults. The
ability of hILP to inhibit apoptosis by all of these stimuli
distinguishes it from other antiapoptotic proteins such as the viral
DED-containing proteins E8 and MC159 and the endogenous DED-containing
protein Flame-1, which target cell surface-associated activation of
caspase 8 and are unable to inhibit UV-induced apoptosis (3,
61). Further, while the apoptotic effects of anti-Fas are not
dependent on p53 (29), those of cisplatin are p53 dependent
(67, 69). Thus, hILP functions downstream of
stimulus-dependent pathways that initiate the apoptotic cascade.
Deletion analysis of hILP revealed that the BIR domains of hILP are sufficient to confer protection against these apoptotic stimuli (Fig. 2). This result is in accord with studies with Drosophila in which expression of the BIR domains of DIAP1 was sufficient to rescue cells from normally occurring cell death in the eye (31). The BIR domains of other members of the IAP family have been shown to play important roles in protein-protein interactions. For example, the BIR domains of the baculovirus IAP Op-IAP are required for interaction with the proapoptotic Drosophila protein Doom (30). Similarly, the BIRs of the two other known mammalian IAPs (c-IAP1 and c-IAP2) mediate the interactions with TRAF proteins (53). However, we did not detect any interaction between hILP and any of the six known TRAFs (Fig. 3). Although it is possible that hILP might block apoptosis through an interaction with an as-yet-unknown TRAF, it is more likely that hILP is not a TRAF binding protein and that the BIR domains mediate interactions with other proteins.
Recent reports have described the involvement of mitochondria in the cell death cascade (62, 72, 73). For example, mitochondrial dysfunction, including disruption of the electron transport chain, is a necessary event in the induction of apoptosis (57). Consistent with a critical role for mitochondria in apoptosis, it has been reported that cytochrome c is released from the mitochondria into the cytosol and that cytochrome c itself can effect apoptosis by inducing the activation of caspases (44). These findings imply a sequential order of events leading to the apoptotic death of the cell and also suggest a number of distinct levels of the cell death pathway which might be subject to regulation. Our observations that Bcl-xL and hILP have differential effects on cytochrome c release and cytochrome c-mediated apoptosis support this sequential ordering of the apoptotic pathway.
Recent findings have led to several potential mechanisms by which Bcl-xL may maintain mitochondrial integrity. The recent finding that Bcl-xL can insert into membranes and form ion channels (47) is consistent with a potential role for Bcl-xL in maintaining mitochondrial integrity by contributing to the maintenance of mitochondrial membrane potential. Alternatively, the report of Kharbanda et al. (39) demonstrating that Bcl-xL can bind directly to cytochrome c suggests a more direct role for Bcl-xL in maintaining cytochrome c in its mitochondrial compartment. Our observation that hILP is unable to inhibit the redistribution of cytochrome c suggests that it may function at a more downstream level in the cell death cascade. hILP, in contrast to Bcl-xL, was able to protect against death induced by microinjection of cytochrome c. One effect of cytosolic cytochrome c is the activation of downstream caspases (40, 71). The placement of hILP downstream of cytochrome c release suggests that hILP may function in the cell death pathway by controlling the processing of downstream caspases from zymogens or, by analogy with P35, by direct interaction with and inhibition of the mature protease. Further studies will be required to distinguish between these possibilities.
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ACKNOWLEDGMENTS |
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We thank V. Dixit for MCF7F cells; J. Yuan for the caspase 2 cDNA; R. Arch, C. Rudin, and J. Van Dongen for providing TRAF4 and TRAF5 cDNAs; B. Mayer for providing pEBB and pEBG vectors; and R. Clem and J. M. Hardwick for providing IAP cDNAs. We thank A. Srinivasan, R. Gedrich, C. Rudin, and members of the Thompson and Tomaselli labs for insightful discussions. We also thank C. Mazur and L. Trout for assistance in the preparation of the manuscript.
This work was supported in part by research grant P01 DK49799 (to C.B.T.) from the National Institutes of Health. C.S.D. is a Special Fellow of the Leukemia Society of America.
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FOOTNOTES |
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* Corresponding author. Mailing address: IDUN Pharmaceuticals Inc., 11085 N. Torrey Pines Rd., Suite 300, La Jolla, CA 92037. Phone: (619) 646-8121. Fax: (619) 625-2677. E-mail: barmstro{at}idun.com.
Present address: Metabolism Branch, National Cancer Institute,
National Institutes of Health, Bethesda, MD 20892.
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