(Received for publication, January 30, 1995)
From the
Signaling for cell death by Fas/APO1 occurs via a distinct
region in its intracellular domain. This region contains a conserved
sequence motif, the death domain motif, that is also found in the
intracellular domains of the p55 tumor necrosis factor receptor and the
low affinity nerve growth factor receptor, as well as in the regulatory
domain of the ankyrins. A novel protein that specifically binds to the
death domain of Fas/APO1 but not to Fas/APO1 molecules with a loss of
function point mutation occurring in lpr mice was
cloned by a two-hybrid screen of a HeLa cells' cDNA library. The
cloned protein itself contains a death domain motif, and this region
binds to the death domain of Fas/APO1, while the region upstream to the
death domain prompts self-association of the protein. Induced
expression of the protein results in ligand-independent triggering of
cytotoxicity, suggesting that it is involved in cell death induction by
Fas/APO1.
Part of the immune cytolytic processes is receptor-initiated.
The tumor necrosis factor (TNF) ()receptors and the
structurally related receptor Fas/APO1 trigger destructive activities
in cells upon stimulation by leukocyte-produced ligands that lead to
their own
demise(1, 2, 3, 4, 5, 6) .
The mechanisms of this triggering process are poorly understood.
Mutational studies indicate that, in Fas/APO1 and the p55 TNF receptor
(p55-R), signaling for cytotoxicity involves distinct regions within
their intracellular domains(7, 8, 9) . These
regions, the death domains, have sequence similarity and share common
sequence features with some other membrane-associated proteins, the
ankyrins, and the low affinity nerve growth factor (NGF) receptor (see
the present study). The death domains of both Fas/APO1 and p55-R have a
tendency to self-associate. Their self-association apparently promotes
receptor aggregation necessary in the initiation of signaling (10, 11, 12) and, at high levels of receptor
expression, can result in the triggering of ligand-independent
signaling(12) . Since the death domains do not appear to
possess enzymatic activities, it seems likely that in addition to their
self-association, they also bind, either directly or through docking
proteins, to other cellular proteins that possess these activities. We
describe the cloning of a protein that binds to the death domain of
Fas/APO1 and is apparently involved in the cytotoxic triggering
process. This protein itself contains an amino acid sequence similar to
the death domain.
The cDNA described in this study was cloned by a two-hybrid
system screen(13) , using the intracellular domain of human
Fas/APO1 (Fas-IC) as bait. Sequence analysis indicated that it encodes
a novel protein (see below). Applying the two-hybrid test to further
evaluate the specificity of the binding of this protein (which will be
referred to as MORT1, for mediator of receptor-induced toxicity) to
Fas-IC and to define the particular region in Fas-IC to which it binds
led to the following findings (Table 1). (a) The protein
binds both to human and to mouse Fas-IC but not to several other tested
proteins, including three receptors of the TNF/NGF receptor family (p55
and p75 TNF receptors and CD40). (b) Replacement mutations at
position 225 (Ile) in the death domain of mouse Fas/APO1 (the lpr mutation), shown to abolish signaling both in vitro and in vivo(9, 22) , also
prevent binding to MORT1. (c) The MORT1 binding site in
Fas/APO1 occurs within the death domain of this receptor. (d)
MORT1 binds to itself. This self-binding and the binding of MORT1 to
Fas/APO1 involve different regions of the protein. A fragment of MORT1,
corresponding to residues 1-117, binds to the full-length MORT1
but does not bind to Fas/APO1. Conversely, a fragment corresponding to
residues 130-245 binds to Fas/APO1 but does not bind to MORT1 (Table 1).
Expression of MORT1 molecules fused at their N
terminus with the FLAG octapeptide (FLAG-MORT1) in HeLa cells yielded
proteins of four distinct sizes of approximately 27, 28, 32, and 34
kDa. All four proteins bound to Fas-IC upon incubation with a
GST-Fas-IC fusion protein. As in the yeast two-hybrid test, MORT1 did
not bind to a GST-Fas-IC fusion protein with a replacement at the lpr mutation site (I225A, Fig. 1).
Figure 1:
Interaction of MORT1 with Fas-IC in
vitro. S metabolically labeled MORT1, produced in
transfected HeLa cells and fused with the FLAG octapeptide (FLAG-MORT1)
was used in binding studies to GST, GST fused with the human or mouse
Fas-IC (GST-huFas-IC, GST-mFas-IC), or to GST fused with Fas-IC
containing an Ile to Ala replacement mutation at position 225. Also
shown are the proteins in extracts of cells transfected with the
FLAG-MORT1 chimera or, as control, with the luciferase cDNA,
immunoprecipitated using anti-FLAG antibody (Abs). The
proteins were analyzed by SDS-polyacrylamide gel electrophoresis (10%
acrylamide) followed by autoradiography.
FLAG-MORT1 also showed an ability to bind to the intracellular domain of Fas/APO1 as well as to the intracellular domain of a Fas/APO1 chimera whose extracellular domain was replaced with that of p55-R (p55-Fas) when coexpressed with these receptors in HeLa cells. Thus, as shown in Fig. 2, immunoprecipitation of FLAG-MORT1 from extracts of the transfected cells also resulted in precipitation of the coexpressed Fas/APO1 or p55-Fas. Conversely, immunoprecipitation of these receptors resulted in the coprecipitation of FLAG-MORT1.
Figure 2:
Interaction of MORT1 with Fas-IC within
transfected HeLa cells. FLAG-MORT1, human Fas/APO1, Fas/APO1 chimera in
which the extracellular domain of Fas/APO1 was replaced with the
corresponding region in human p55-R (p55-Fas), or human p55-R which
served as a negative control were expressed in HeLa cells and
metabolically labeled with S-Cys (20 mCi/ml) and
S-Met (40 mCi/ml). Cross-immunoprecipitation of MORT1 with
the co-expressed receptor was performed using the indicated antibodies.
The proteins were analyzed by SDS-polyacrylamide gel electrophoresis
(10% acrylamide), followed by
autoradiography.
Northern analysis using MORT1 cDNA as a probe (Fig. 4A) revealed a single hybridizing transcript in HeLa cells. The size of this transcript (approximately 1.8 kilobases) is close to that of the MORT1 cDNA (1670 nucleotides).
Figure 4:
The MORT1 transcript and its encoded
protein. A, Northern analysis of HeLa cells poly(A) RNA (0.3 µg), using the MORT1 cDNA as probe. B,
deduced amino acid sequence of MORT1. The death domain motif is underlined. C, sequence homology of the death domain
motif in MORT1 and in p55-R, Fas/APO1, low affinity NGF receptor, and
the C-terminal part of the regulatory domain in ankyrin 1 (all human)
as defined by the LINEUP and PRETTY programs of the GCG package.
Identical and similar residues in three or more of the proteins are boxed. Gaps introduced to maximize alignment are denoted by dots. The significance of this homology was confirmed as
follows. (a) Multiple alignment of the death domain motif
sequences, using the HSSP program of the PredictProtein
Service(27) , showed sequence identities of 21-38% and
sequence similarities of 30-48%. (b) Searching the
Swiss-Prot data bank with a profile created (using the PILEUP, LINEUP,
and PROFILEMAKE programs of the GCG package) from consensuses of the
death domain motif sequences in the known p55-R and Fas/APO1 (human,
mouse, rat), NGF receptor (human, rat, and chicken), and ankyrin (human
and mouse ankyrin 1 and human ankyrins c and g) sequences and in MORT1
yielded high scores only for the sequences used in creating the profile
(Zscores >8.5 for all of them in search with the
``Bioccelerator'' Compugen,
Israel).
In sequence analysis of MORT1, the cDNA was found to contain an open reading frame of 245 amino acids (Fig. 4B). A homology search using the PredictProtein Service (PHDsec) and the PRODOM program of the GCG package revealed a significant similarity between a region of approximately 65 residues in MORT1, within that part of the molecule that binds to Fas/APO1, and a region of the same length within the death domains of Fas/APO1 and p55-R (Fig. 4C). This part of the death domain also shows similarity to a region in the intracellular domain of the low affinity NGF receptor (23) , a receptor whose extracellular domain is known to contain another conserved sequence motif common to Fas/APO1, the TNF receptors, and other members of the TNF/NGF receptor family. It is also similar to a conserved region in the ankyrins, structural proteins that link spectrin-based membrane skeletal proteins to the cytoplasmic domains of integral plasma membrane proteins(24, 25) . This region is located in the N-terminal part of the ankyrin regulatory domain, just upstream to the section of the domain whose expression is subject to modulation by alternative splicing and below the spectrin binding and membrane binding domains. The death domain motif is distinct from the ankyrin repeat motif that is found in the membrane binding domain of the ankyrins.
High expression of p55-IC results in the triggering of a cytocidal effect(12) . Expression of Fas-IC in HeLa cells also has a small cytotoxic effect, which could be only detected with a sensitive assay (compare Fig. 3A to 3B). These cytotoxic effects are most likely prompted by the propensity of the death domains in the receptors to self-associate. Since MORT1 also self-associates, we examined the effect of its induced expression on cell viability. As shown in Fig. 3, expression of MORT1 in HeLa cells resulted in significant cell death, greater than that caused by Fas-IC expression.
Figure 3: Ligand-independent triggering of cytocidal effects in cells transfected with MORT1. The effect of transient expression of MORT1, human Fas-IC, human p55-IC, or luciferase, which served as a control, on the viability of HeLa cells was assessed using a tetracycline-controlled expression vector. Cell viability was evaluated 40 h after transfecting these cDNAs either in the presence (white bar) or absence (shaded bar) of tetracycline (1 µg/ml, to block expression), together with a cDNA encoding the secreted placental alkaline phosphatase. Cell viability was determined either by the neutral red uptake assay (A) or, for specifically determining the viability of cells that expressed the transfected DNA, by measuring the amounts of placental alkaline phosphatase secreted into the growth medium (B).
The specific association of MORT1
with the death domain in Fas/APO1 and the fact that even a slight
change of structure in that region, which prevents signaling (the lpr mutation), also abolishes the binding of
MORT1 indicate that MORT1 plays a role in triggering cell death. This
idea is further supported by the observation that MORT1 alone can
trigger a cytocidal effect. What kind of role this protein serves is
not clear. It may (i) function as modulator in the self-association of
Fas/APO1 by its binding ability to Fas/APO1 as well as to itself, (ii)
serve as a docking site for additional proteins that are involved in
Fas/APO1 signaling, or (iii) constitute a part of a distinct signaling
system that interacts with Fas/APO1 signaling. Our finding that MORT1
contains a death domain motif cannot yet provide a clue to its
function. The occurrence of this motif in the NGF receptor, which, when
inducing death does so only in the absence of ligand(26) , as
well as in the ankyrins suggests that this motif plays a more general
role than that implied in the name death domain. One general kind of
activity of this motif, found so far in three of the proteins
containing it, Fas/APO1, p55-R, and MORT1, is the ability to
self-interact or interact with other proteins that contain this motif.
Further studies of the identity of the cellular proteins with which
MORT1 interacts and of the contribution of the specific sequence and
structural features of the death domain motif to these interactions
should elucidate the role of MORT1 in signaling as well as the general
functions of the sequence features that define the death domain motif.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X84709[GenBank].
Note Added in Proof-A further 5`-extending sequence of MORT1, cloned from a human monocyte library by polymerase chain reaction (31 nucleotides, included in the submission to the EMBL Data Bank), did not contain ATG. In addition, we found that MORT1 cDNA can be effectively expressed in HeLa cells without an N-linked ATG-containing sequence. In that form it yields two distinct protein sizes of approximately 27 and 28 kDa. These findings suggest that the translation start site of MORT1 is Met-38 (Fig. 4B). The products of 32 and 34 kDa in Fig. 1and Fig. 2may be due to initiation of translation at the translation start site of the FLAG octapeptide.