©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Protein That Interacts with the Death Domain of Fas/APO1 Contains a Sequence Motif Related to the Death Domain (*)

(Received for publication, January 30, 1995)

Mark P. Boldin (§) Eugene E. Varfolomeev (§) Zeev Pancer Igor L. Mett Jacques H. Camonis (1) David Wallach (¶)

From the Department of Membrane Research and Biophysics, The Weizmann Institute of Science, Rehovot, Israel 76100 and Denis Diderot University, Inserm Unit 248, Paris 75010, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

Part of the immune cytolytic processes is receptor-initiated. The tumor necrosis factor (TNF) (^1)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.


EXPERIMENTAL PROCEDURES

Two-hybrid Screen and Two-hybrid beta-Galactosidase Expression Test

Two-hybrid screening (13) of a Gal4 activation domain-tagged HeLa cell cDNA library (Clontech, Palo Alto, CA), using the intracellular domain of Fas/APO1 (residues 175-319, Fas-IC) as ``bait,'' and beta-galactosidase expression tests were performed as described previously(12) , except that the expression of beta-galactosidase was assessed by a filter assay. In the screening, 5 of approximately 3 times 10^6 cDNAs were found to contain the MORT1 insert. Residue numbering in the proteins encoded by the cDNA inserts is as in the Swiss-Prot data bank. Deletion mutants were produced by polymerase chain reaction and point mutants by oligonucleotide-directed mutagenesis(14) .

Induced Expression, Metabolic Labeling, and Immunoprecipitation of Proteins

MORT1, N-linked to the FLAG octapeptide (FLAG-MORT1, Eastman Kodak Co.), Fas-IC, Fas/APO1, p55-R, a chimera comprised of the extracellular domain of p55-R (amino acids 1-168) fused to the transmembrane and intracellular domain of Fas/APO1 (amino acids 153-319), and the luciferase cDNA, which served as a control, were expressed in HeLa cells. Expression was done using a tetracycline-controlled expression vector in a HeLa cell clone (HtTA-1) that expresses a tetracycline-controlled transactivator(15) , as described before(12) . Metabolic labeling with [S]Met and [S]Cys (DuPont, Wilmington, DE, and Amersham, Buckinghamshire, United Kingdom) was performed 18 h after transfection by an additional 4-h incubation at 37 °C in Dulbecco's modified Eagle's medium lacking Met and Cys, supplemented with 2% dialyzed fetal calf serum. The cells were then lysed in radioimmune precipitation buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, and 1 mM EDTA; 1 ml/5 times 10^5 cells), and the lysate was precleared by incubation with irrelevant rabbit antiserum (3 µl/ml) and Protein G-Sepharose beads (Pharmacia, Uppsala, Sweden; 60 µl/ml). Immunoprecipitation was performed by 1-h incubation at 4 °C of 0.3-ml aliquots of lysate with mouse monoclonal antibodies (5 µg/aliquot) against the FLAG octapeptide (M2; Kodak), p55-R (nos. 18 and 20(16) ), Fas/APO1 (ZB4; Kamiya, Thousand Oaks, CA), or the p75 TNF receptor (no. 9(17) ) as a control, followed by an additional 1-h incubation with Protein G-Sepharose beads (30 µl/aliquot).

In Vitro Binding

Glutathione S-transferase (GST) fusions with the wild type or a mutated Fas-IC were produced and adsorbed to glutathione-agarose beads as described(12, 14, 18) . Binding of metabolically labeled FLAG-MORT1 fusion protein and luciferase to GST-Fas-IC was assessed by incubating the beads for 2 h at 4 °C with extracts of HeLa cells, metabolically labeled with [S]Met (60 µCi/ml), that express FLAG-MORT1 or luciferase. The extracts were prepared in a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM dithiothreitol, 1 mM EDTA, 5% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotonin, 20 µg/ml leupeptin, 10 mM sodium fluoride, and 0.1 mM sodium vanadate (1 ml/5 times 10^5 cells).

Assessment of the Cytotoxicity Triggered by Induced Expression of MORT1

MORT1, Fas-IC, p55-IC, and luciferase cDNAs were inserted into a tetracycline-controlled expression vector and transfected to HtTA-1 cells (15) together with the secreted placental alkaline phosphatase cDNA, placed under control of SV40 promoter (the pSBC-2 vector(19) ). Cell death was assessed 40 h after transfection, either by the neutral red uptake assay (20) or, for specifically assessing the death in those cells that expressed the transfected cDNAs, by determining the amounts of placental alkaline phosphatase (21) secreted into the growth medium during the last 5 h of incubation.

Northern and Sequence Analyses

Poly(A) RNA was isolated from total RNA of HeLa cells (Oligotex-dT mRNA kit, Qiagen, Hilden, Germany). Northern analysis, using the MORT1 cDNA as a probe, was performed by conventional methods as described before (12) . The nucleotide sequence of MORT1 was determined in both directions by the dideoxy chain termination method.


RESULTS AND DISCUSSION

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.


FOOTNOTES

*
This work was supported in part by grants from Inter-Lab Ltd., Ness-Ziona, Israel, from Ares Trading S.A., Switzerland, and from the Israeli Ministry of Arts and Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X84709[GenBank].

§
Equivalent contributions were made by these two authors.

To whom correspondence should be addressed. Tel.: 972-8-343941; Fax: 972-8-343165; LVWALACH{at}WEIZMANN.WEIZMANN.AC.IL.

(^1)
The abbreviations used are: TNF, tumor necrosis factor; NGF, nerve growth factor; GST, glutathione S-transferase; Fas-IC, intracellular domain of Fas/APO1; FLAG-MORT1, MORT1 N-linked to the FLAG octapeptide; IC, Intracellular domain; p55-R, p55 tumor necrosis factor receptor; p55-Fas, chimera comprised of the extracellular domain of p55-R and transmembrane and intracellular domains of Fas/APO1.


ACKNOWLEDGEMENTS

We thank Ada Dibeman for careful handling of the cultured cells and Tanya Goncharov for help in establishing the alkaline phosphatase secretion assay for cytotoxicity evaluation.

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.