(Received for publication, November 21, 1995; and in revised form, January 16, 1996)
From the
Tumor necrosis factor receptor 1 (TNF-R1) mediates most of the
biological properties of TNF including activation of the transcription
factor NF-B and programmed cell death. An
80-amino acid
region within the intracellular domain of the receptor, termed the
death domain, is required for signaling NF-
B activation and
cytotoxicity. A TNF-R1-associated protein TRADD has been discovered
that interacts with the death domain of the receptor. Elevated
expression of TRADD in cells triggers both NF-
B activation and
programmed cell death pathways. The biological activities of TRADD have
been mapped to a 111-amino acid region within the carboxyl-terminal
half of the protein. This region shows sequence similarity to the death
domain of TNF-R1 and can self-associate and bind to the TNF-R1 death
domain. We have performed an alanine scanning mutagenesis of
TRADD's death domain to explore the relationship among its
various functional properties. Mutations affecting the different
activities of TRADD do not map to discrete regions but rather are
spread over the entire death domain, suggesting that the death domain
is a multifunctional unit. A mutant that separates cell killing from
NF-
B activation by the TRADD death domain has been identified
indicating that these two signaling pathways diverge with TRADD.
Additionally, one of the TRADD mutants that fails to activate NF-
B
was found to act as dominant negative mutant capable of preventing
induction of NF-
B by TNF
. Such observations provide evidence
that TRADD performs an obligate role in TNF-induced NF-
B
activation.
Tumor necrosis factor (TNF
) (
)is a
pro-inflammatory cytokine that performs varied biological functions
including antiviral activity, cytotoxicity, and reprogramming of gene
expression (reviewed in (1, 2, 3) ). The
different effects of TNF
are elicited by binding to two distinct
cell surface receptors: TNF receptor 1 (TNF-R1,
55 kDa) and TNF
receptor 2 (TNF-R2,
75 kDa). Although most cell types express both
TNF receptors, TNF-R1 appears to be primarily responsible for induction
of gene expression and programmed cell death by TNF
(reviewed in (4) and (5) ). Binding of the trimeric TNF
ligand
to the receptor results in receptor aggregation, which is thought to be
a critical event triggering downstream signaling pathways either by
activating receptor-associated effector molecules or by recruiting
effector molecules(6, 7) . Several signal transduction
pathways and signaling molecules have been associated with TNF
action including generation of oxygen radicals and activation of
protein kinases and sphingomyelinases (reviewed in (8) ).
However, it is not clear if these events reflect primary cellular
changes in response to receptor occupancy or secondary events that are
a consequence of other more immediate postreceptor activation steps
triggered by TNF
.
Analysis of the regulation of gene expression
by TNF has revealed binding sites for the transcription factor
NF-B in the promoter region of most genes induced by TNF and has
also established that TNF is a potent activator of latent NF-
B
activity in cells (reviewed in (9) and (10) ). A
significant advance in understanding the mechanism of NF-
B
activation by TNF
has resulted from the isolation of proteins that
interact with the cytoplasmic domains of TNF-R1 and TNF-R2 and mimic
some of the actions of TNF
(11, 12) .
Consistent with the observation that the primary amino acid sequence
of the intracellular domains of TNF-R1 and TNF-R2 displays no
detectable sequence similarity, distinct proteins have been found to
interact with the cytoplasmic region of the two receptors. In the case
of TNF-R2, two related proteins, TRAF-1 and TRAF-2, form a heteromeric
complex with the cytoplasmic domains of the receptor, and
overexpression of TRAF-2 has been shown to activate
NF-B(11, 13) . The TRAF proteins define a new
family of signaling molecules that includes CD40bp or CRAF-1, a protein
that interacts with the cytoplasmic domain of another TNF receptor
family member CD40(14, 15) .
Structure-function
analysis of signaling by TNF-R1 has demonstrated that an 80-amino
acid region within the cytoplasmic domain is required for initiation of
programmed cell death and NF-
B activation (16) . This
region, designated the death domain, is 28% identical over a stretch of
65 amino acids to an intracellular region of the FAS
antigen(17) , another member of the TNF receptor family that
can activate programmed cell death. Insight into signal transduction by
TNF-R1 has resulted from identification of a 34-kDa protein, designated
TRADD, in a yeast two-hybrid screen for proteins interacting with the
intracellular domain of TNF-R1(12) . TRADD interacts with the
death domain of TNF-R1, and TRADD overexpression causes NF-
B
activation and initiates programmed cell death, implicating it as a
signaling molecule downstream of the receptor. Two additional TNF-R1
interacting proteins have been isolated using a two-hybrid screen.
These two proteins interact with a membrane-proximal region of the
receptor that does not include the death domain, and their role in
TNF-R1 signaling is unclear(18) .
Mutational studies of
TRADD have demonstrated that 111 amino acids within the
carboxyl-terminal half of the molecule are necessary and sufficient for
self-association and binding to TNF-R1 as well as for inducing
NF-B activation and cell death(12) . The functionally
important part of TRADD appears to harbor a death domain, since a
68-amino acid stretch within this region shows 32% sequence identity
with the TNF-R1 death domain. The death domain of TRADD is more related
to that of TNF-R1 than FAS, and it does not interact with the death
domain in FAS. Regions homologous to the death domains of TNF-R1 and
FAS have been found in two other proteins including MORT1/FADD and RIP (19, 20, 21) . Both of these proteins
interact via their death homology regions with the death domain of FAS
but only weakly with TNF-R1, and both induce apoptosis when
overexpressed in mammalian cells. With FADD, the region that interacts
with FAS is separable from the one that activates the cell death
pathway. The carboxyl-terminal part of MORT1/FADD is similar in primary
amino acid sequence to the FAS death domain and is capable of direct
interaction with it. However, expression of the amino-terminal 117
amino acids, lacking most of the death homology region of MORT-1/FADD,
triggers cell death(20) . First identified as a protein-protein
interaction motif employed by members of the TNF-R1 family involved in
signaling cell death and NF-
B activation, the death domains may be
present in other proteins with diverse
functions(22, 23) . The rules governing interactions
of the different death domains are unclear. Likewise it is not known
how certain death domains signal cell death when overexpressed, whereas
others do not.
The TRADD death domain performs multiple functions
including self-association, binding to TNF-R1, activation of NF-B,
and induction of cell death(12) . To determine whether these
various activities specified by TRADD map to distinct regions within
the death domain and to gain a better understanding of TRADD's
role in signal transduction by TNF-R1 we have performed an alanine scan
mutagenesis of TRADD's death domain. Alanine substitution mutants
have been surveyed for their capacity to oligomerize and interact with
TNF-R1 in binding assays and to activate NF-
B and induce cell
death in transfection assays. The entire death domain appears to be
involved in self-association of TRADD and binding to TNF-R1. It is
shown that the regions of the death domain mediating NF-
B
activation and induction of cell death overlap. However, one mutant was
identified that kills cells but does not activate NF-
B.
Furthermore, a TRADD mutant with alanines in positions 296-299
behaves as a dominant negative variant capable of preventing induction
of a NF-
B reporter plasmid by TNF
.
Figure 1:
In vitro binding assays with
TRADD death domain mutants. A, sequence of TRADD death domain
from amino acid 196 to 305(12) . The amino acids substituted to
alanine in the individual mutants are underlined. Numbers
above the sequence correspond to the first amino acid changed in
each mutant. B, percent binding of each mutant to GST-TRADD (top panel) and GST-TNF-R1-(aa214-426) (bottom
panel) is shown as a bar graph. Binding was determined by
precipitation of in vitro synthesized S-labeled
TRADD mutant proteins with the GST fusion proteins coupled to
glutathione-Sepharose. The precipitated proteins were resolved on a
SDS-polyacrylamide gel, and the labeled protein brought down was
quantitated with a Phosphoimager. The amount of in vitro translated TRADD-(aa196-312) bound to each wild type GST
fusion protein was considered 100%. The numbers below the bottom panel correspond to amino acid positions in
TRADD.
We first examined TRADD mutants for their ability to self-associate or bind TNF-R1. In vitro synthesized TRADD associates specifically with either TRADD or the intracellular domain of TNF-R1 fused to GST. The GST fusions are attached to glutathione-agarose beads, which facilitates separation of the TRADD bound to the fusion protein from unbound TRADD(12) . This in vitro biochemical assay was used to compare the binding of TRADD-(aa196-312) and various substitution mutants to GST-TRADD and GST-TNF-R1. Mutations resulting in reduced binding to GST-TRADD and GST-TNF-R1 were distributed over the entire death domain, indicating that there were no subregions dedicated exclusively to TRADD multimerization or TNF-R1 binding (Fig. 1B). TRADD self-association may be essential for interaction with TNF-R1, since all mutations that reduced binding to GST-TRADD also compromised GST-TNF-R1 binding. Consistent with this interpretation, alanine substitutions at more positions affected binding to GST-TNF-R1 than to GST-TRADD. The TRADD-TNF-R1 interaction detected here may therefore reflect the binding of an oligomerized TRADD molecule to TNF-R1, and the interface involved in TRADD-TNF-R1 contact is likely to be different from that involved in TRADD-TRADD contact.
Figure 2:
Biological activity of TRADD death domain
mutants. A, bar graph displaying capacity of TRADD
death domain mutants to activate expression of a NF-B-dependent
reporter gene. 293 cells transfected with expression vectors for the
mutants and an E-selectin luciferase plasmid (24) were either
unstimulated or stimulated with TNF
for approximately 12 h before
harvest. Cells were assayed for luciferase activity as detailed under
``Materials and Methods.'' Activation observed with
TRADD-(aa196-312) in unstimulated cells was set at 100%, and
activation observed with the mutants is expressed relative to it. The numbers below the graph correspond to amino acid positions in
TRADD. The upper panel presents the activity observed in
unstimulated cells and the lower panel the activity seen in
cells stimulated with TNF
. The dashed line in the lower panel indicates the base-line activity observed in cells
co-transfected with the E-selectin reporter and an expression vector
lacking TRADD sequences and stimulated with TNF. TNF
enhanced
expression of this reporter gene by approximately 20-fold. B,
cell killing activity of TRADD death domain mutants presented as a bar
graph. HtTA cells were co-transfected with the various TRADD mutants
and a
-galactosidase expression vector. Cells were stained for
-galactosidase, and the percentage of blue cells that were flat
and adherent were counted microscopically and considered live. Cell
killing observed with TRADD-(aa196-312) was set at 100%, and
killing activity of the mutants is expressed relative to it. A
background killing (10%) that was observed when an expression vector
lacking TRADD was transfected has been subtracted. The data presented
are that observed with unstimulated cells. Similar results were
observed when the transfected cells were stimulated with TNF for
approximately 12 h.
Mutant aa296-299 showed
detectable interaction in vitro with both GST-TNF-R1 and
GST-TRADD yet did not activate NF-B in transiently transfected
cells (Fig. 1B and Fig. 2A). Moreover,
expression of the E-selectin luciferase reporter was consistently lower
when cells transfected with this mutant were stimulated with TNF than
in cells transfected with a control vector lacking TRADD sequences.
These observations indicate that the mutant might interfere with
TNF-induced NF-
B activation, perhaps by preventing endogenous
TRADD and/or TNF-R1 from interacting with downstream effector
molecules. The aa296-299 change was reconstructed in a TRADD
protein starting at amino acid 25 to confirm and extend the findings
with the mutant in the death domain background. Both
TRADD-(aa196-312) and TRADD-(aa25-312) with the
aa296-299 substitution potently prevented induction of the
NF-
B reporter gene by TNF in a dose-dependent manner without
significantly affecting induction by IL-1 (Fig. 3A). As
a control, another mutant that does not activate NF-
B (aa210) was
tested for its capacity to prevent induction of the NF-
B reporter
plasmid by TNF. Unlike mutant aa296-299, mutant aa210 failed to
block TNF-induced reporter gene expression.
Taken together,
these findings provide evidence that changing amino acids 296-299
to alanine results in a dominant negative TRADD mutant. To demonstrate
that TRADD with the aa296-299 substitution could associate with
native TRADD and interact with TNF-R1 in cells, a
co-immunoprecipitation experiment was performed (Fig. 3C). 293 cells were co-transfected with
expression vectors for myc epitope-tagged TRADD having the
wild type sequence or alanine substitution at aa296-299 and
expression vectors for either TNF-R1 or flag epitope-tagged TRADD.
TNF-R1 and flag-TRADD were immunoprecipitated from the transfected
cells, and the myc-tagged TRADD brought down with these
proteins was determined with a Western blot (Fig. 3C).
Immunoprecipitation with irrelevant antibodies confirmed that
co-precipitation of myc-tagged TRADDs was specific for the
anti-TNF-R1 and anti-Flag antibodies.
Alanine substitutions
at 296-299 did not affect association with TNF-R1 or TRADD. Taken
together the results of the transfection assays confirm the involvement
of TRADD in the NF-
B activation pathway and furthermore
demonstrate an obligate role for TRADD function in TNF-initiated
NF-
B activation.
Figure 3:
Effect of mutant aa296-299 on
NF-B activation. A, mutant aa296-299 blocks
NF-
B activation induced by TNF but not IL-1. 293 cells were
co-transfected with the NF-
B-dependent reporter plasmid and
varying amounts of TRADD-(aa196-312) with alanines at amino acids
296-299 (296S) or TRADD-(25-312) with alanines at amino
acids 296-299 (296L) as indicated. For each data point the amount
of expression plasmid was made up to 1 µg by addition of expression
vector lacking TRADD sequences. Forty hours after transfection, cells
were stimulated with TNF
or IL-1
for 8 h, and the cells were
then harvested and assayed for luciferase activity. The -fold increase
in reporter gene expression observed when 1 µg of empty expression
vector was co-transfected is set at 100 for both TNF
and IL-1
treatment. The actual increase observed was 20.5-fold for TNF and
8.5-fold for IL-1
. B, activation of NF-
B-dependent
reporter gene expression by amino-terminal truncations of TRADD and
TRADD with the aa296-299 substitution. The E-selectin luciferase
reporter was co-transfected with full-length (aa1-312), truncated
(aa25-312), or just the death domain (aa196-312) of TRADD
with the native sequence or with alanines at aa296-299 (296FL,
296L, and 296S). Luciferase activity was determined in unstimulated
(-TNF) or cells stimulated with TNF for 8 h (+TNF). The numbers given are relative to the luciferase activity observed
for cells transfected with the empty expression vector and left
unstimulated. The asterisks denote the substitution of
alanines at amino acids 296-299. C,
co-immunoprecipitation of TRADD having the aa296-299 substitution
with TNF-R1 and native TRADD. Expression vectors lacking TRADD
sequences(-) or encoding myc epitope-tagged
TRADD-(aa112-312) with the wild type sequence (WT) or with
alanines at aa296-299 (296) were co-transfected into 293 cells
along with expression vectors encoding TNF-R1 (lanes 1-3) and
native TRADD with a flag tag (lanes 4-6). Cell lysates
were immunoprecipitated with a polyclonal rabbit antiserum to TNF-R1 (lanes 1-3) or anti-flag monoclonal antibody (lanes
4-6). Immunoprecipitates were resolved on a
SDS-polyacrylamide gel, and the myc epitope-tagged TRADD
brought down was detected in a protein immunoblot with an anti-myc monoclonal antibody.
Analysis of NF-B activation by
aa296-299 mutants provided an additional insight into TRADD
function. A full-length TRADD protein with the aa296-299
substitution (296FL) reproducibly enhanced expression from a
co-transfected NF-
B reporter plasmid to a small extent,
approximately 6-fold compared with 37-fold with native TRADD, even
though this same mutation inactivated the death domain (Fig. 3B). Similar results were obtained with the
mutation aa300-302. In the context of the death domain alone
substituting amino acids 300-302 with alanine inactivated TRADD;
however, full-length TRADD with the same substitutions showed low
levels of NF-
B-inducing activity.
Removal of 24 amino
acids from the amino terminus of 296FL abolishes the residual NF-
B
inducing activity and unmasks the dominant negative effect of this
mutation on TNF-induced NF-
B activation. This supplementary role
of the amino terminus is most readily observed in mutants that
compromise the activity of the death domain. However, amino-terminal
deletions of wild type TRADD also show a 2-4-fold reduction in
NF-
B activation confirming the importance of this region (Fig. 3B). All of these observations suggest that the
amino-terminal half of TRADD contributes to NF-
B activation by the
death domain. Additional experiments are needed to determine whether
the amino-terminal sequences facilitate NF-
B activation by
stabilizing the interaction of effector proteins with the death domain
or by being a site for interaction of additional factors.
In summary, our mutational analyses indicate that the TRADD death
domain cannot be subdivided into discrete regions that are individually
responsible for the different functions of TRADD. It appears therefore
that this domain is not composed of independent subdomains that are
individually dedicated to different functions of TRADD such as
oligomerization, TNF-R1 binding, NF-B activation, and cell killing
but rather that the TRADD death domain is a multifunctional unit
performing all of these functions. Further insight into a
structure-function relationship will require knowledge of the
three-dimensional structure of the TRADD death domain. The protein
interfaces involved in multimerization of TRADD and binding to TNF-R1
appear distinct but overlapping. TRADD self-association is likely to be
a prerequisite for binding to TNF-R1 suggesting that a multimer of
TRADD interacts with TNF-R1. TNF
is thought to induce receptor
clustering on binding to TNF-R1, and it has been proposed that TRADD
may preferentially bind to aggregated TNF-R1(12) . It is
possible then that interaction between TRADD and TNF-R1 involves
binding of two multimerized death domains, and, in general,
self-association of a death domain is different from the interaction of
death domains from two different proteins.
The amino acid residues
involved in signaling NF-B activation and apoptosis are largely
coincident, but these two activities are separable. The tight link
between NF-
B activation and induction of cell death may reflect
the interaction with TRADD of distinct but related proteins to mediate
the two functions or alternately the existence of a single complex that
can execute the signals required to activate both pathways. Most
importantly, the mutagenesis has identified a dominant negative mutant,
which indicates that the TNF-induced NF-
B activation pathway
proceeds through TRADD. Finally, the TRADD mutants described here
should prove useful in deciphering the role of any new
TRADD-interacting proteins that emerge from future studies.