MADD, a Novel Death Domain Protein That Interacts with the Type 1 Tumor Necrosis Factor Receptor and Activates Mitogen-activated Protein Kinase*

(Received for publication, November 26, 1996, and in revised form, February 18, 1997)

Andrea R. Schievella , Jennifer H. Chen , James R. Graham and Lih-Ling Lin Dagger

From the Small Molecule Drug Discovery Group, Genetics Institute, Inc., Cambridge, Massachusetts 02140

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The death domain of the type 1 tumor necrosis factor receptor (TNFR1) mediates interactions with several proteins involved in signaling the downstream effects of TNF. We have used the yeast interaction trap to isolate a protein, MADD, that associates with the death domain of TNFR1 through its own C-terminal death domain. MADD interacts with TNFR1 residues that are critical for signal generation and coimmunoprecipitates with TNFR1, implicating MADD as a component of the TNFR1 signaling complex. Importantly, we have found that overexpression of MADD activates the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK), and expression of the MADD death domain stimulates both the ERK and c-JUN N-terminal kinase MAP kinases and induces the phosphorylation of cytosolic phospholipase A2. These data indicate that MADD links TNFR1 with MAP kinase activation and arachidonic acid release and provide further insight into the mechanisms by which TNF exerts its pleiotropic effects.


INTRODUCTION

Tumor necrosis factor (TNF)1 is a central player in the regulation of immune responses. Produced mainly by activated macrophages, TNF promotes a wide variety of cellular activities, including the initiation of inflammation, the induction of antiviral responses, and apoptosis. Two receptors for TNF have been cloned, both of which belong to a superfamily of cell surface receptors that includes the Fas antigen and CD40 (1). The TNF receptors share 30% homology in their extracellular domains but are unrelated in their intracellular domains. The intracellular domains of the Fas antigen and the 55-kDa TNF receptor (TNFR1), both of which can trigger apoptosis, share approximately 28% identity in a region known as the death domain (2, 3). This region of TNFR1 has been shown to be necessary and sufficient for signaling cytotoxicity (3, 4).

TNFR1 mediates most of the biological effects of TNF (3, 5-8). Engagement of this receptor activates a diverse group of intracellular signaling pathways. Among the early downstream effects of TNF are the activation of kinases, including members of the MAP kinase family (9-14), and phospholipases, including cytosolic phospholipase A2 (cPLA2) (reviewed in Ref. 15). The activation of cPLA2 results in the release of arachidonic acid, which is metabolized into the potent proinflammatory mediators prostaglandins and leukotrienes. TNF also activates several transcription factors, including NF-kappa B and c-JUN/AP-1, leading to the up-regulation of a large number of genes involved in the inflammatory response (16). The signaling elements involved in initiating these pathways were not discovered until recently, when the yeast two-hybrid system was used to identify proteins that associate directly with TNFR1. Briefly, TRADD (17) is a death domain-containing protein that interacts directly with the death domain of TNFR1. TRADD is believed to act as an adaptor protein that recruits two other proteins, TRAF2 and RIP, to the receptor (18, 19). TRAF2 has been implicated in the pathway leading to the activation of NF-kappa B (18, 20), while RIP seems to mediate both apoptosis and NK-kappa B activation (19, 21). TRADD also interacts with MORT1/FADD (22, 23), which in turn associates with the ICE-like protease MACH/FLICE (24, 25), providing a mechanism by which TNFR1 activates key downstream mediators of the apoptotic response. The death domain motif plays a central role in these interactions, mediating associations between TNFR1, TRADD, MORT1/FADD, and RIP.

We have performed a yeast interaction trap screen and isolated a 176-kDa protein called MADD, for AP kinase-ctivating eath omain protein, which also interacts with TNFR1. Here we show that MADD associates with TNFR1 through a death domain-death domain interaction and that overexpression of MADD activates the mitogen-activated protein (MAP) kinase ERK. Interestingly, expression of a truncated form of MADD, containing the C-terminal death domain, activates both the ERK and JNK MAP kinases and induces the phosphorylation of cPLA2. These data suggest that MADD provides a physical link between TNFR1 and the induction of MAP kinase activation and arachidonic acid release.


EXPERIMENTAL PROCEDURES

Yeast Interaction Trap

Screening was based on the methods of Gyuris et al. (31). Briefly, the death domain of TNFR1 (amino acids 326-413; TNFR1-DD) was subcloned into pEG202, resulting in a DNA-binding fusion between the bacterial repressor LexA and the TNFR1 death domain. One million transformants from U937 or WI38 cDNA libraries in pJG4-5 (encoding proteins fused to the B42 transcriptional activation domain; see Ref. 26) were screened for TNFR1-DD binding proteins, using an EGY48 yeast reporter strain containing chromosomal lexAop-leu2 and carrying lexAop-lacZ on the plasmid pSH18-34.

Mutagenesis and Plasmid Construction

TNFR1-DD was obtained by PCR using four oligonucleotides encoding overlapping TNFR1-DD sequences. TNFR1-DD was subcloned into pEG202 for screening and M13mp18 (27) for mutagenesis. For mutagenesis, each of five death domain residues was mutated individually to alanine using the Muta-Gene M13 mutagenesis kit (Bio-Rad), and the mutations were confirmed by sequencing. The mutated fragments were subcloned into the pEG202 vector for testing in the yeast interaction trap. The death domains of Fas (amino acids 213-299), MADD (amino acids 1281-1356), TRADD (amino acids 222-289), and the intracellular domains of TNFR1 (amino acids 204-426) and TNFR2 (amino acids 288-461) were obtained by PCR and confirmed by sequencing. 15Delta DD (amino acids 1396-1588) was constructed by digesting 15TU with EcoRV and XhoI, converting the EcoRV site to EcoRI with a linker and subcloning into pJG4-5 as an EcoRI-XhoI fragment. Vectors FLAG-15TU and FLAG-MADD were constructed by cloning NotI/SalI fragments of the respective cDNAs into the mammalian expression vector pED-FLAG, a variant of pED (28) containing the FLAG peptide DYKDDDDK. A NotI fragment of MADD was subcloned into pED to generate pED-MADD.

Northern Analysis and cDNA Cloning

Human tissue and cell line blots (CLONTECH, Palo Alto, CA) containing 2 µg/lane of poly(A)+ mRNA were probed with the partial MADD cDNA 27TU. Hybridization was performed at 65 °C in 5 × Denhardt's solution, 5 × SSC, 0.1% SDS, and 50 µg/ml tRNA from yeast. Washing was at 65 °C in 0.2 × SSC and 0.1% SDS. To obtain the gene encoding full-length MADD, a poly(dT)-primed U937 cDNA library was probed with the partial clone 27TU, yielding a clone of 4.2 kilobase pairs (clone 4a). A 1.5-kilobase fragment corresponding to the 5' region of clone 4a was used to screen a U937 random-primed cDNA library, yielding a cDNA (clone 15) that overlapped clone 4a by 400 base pairs. A PCR fragment encoding the 5' end of MADD was isolated using a method for the rapid amplification of cDNA ends (RACE) (Marathon kit, CLONTECH), with an oligonucleotide corresponding to sequence from the 5' end of clone 4a. This PCR fragment was used to screen a MADD-specific primer-extended U937 library to obtain a cDNA corresponding to the PCR fragment. Ligation of this 5' cDNA to clones 15 and 4a yielded a gene encoding full-length MADD.

Antibodies

MADD polyclonal antibodies 6007 and 6008 were raised in rabbits by immunization using the partial MADD clone 27TU to express a fusion protein with maltose binding protein (vector pMAL, New England Biolabs, Beverly, MA) (MBP-27TU). TNFR1 antibody 4013 was produced by immunizing rabbits with the death domain of human TNFR1 expressed as glutathione S-transferase fusion protein (vector pGEX, Pharmacia Biotech Inc.) (GST-DD). cPLA2 polyclonal antibody 7905 was generated against cPLA2 produced in Escherichia coli, as described previously (29). MBP antibody was purchased from New England Biolabs, HA antibody 12CA5 from Boehringer Mannheim, and FLAG antibody M2 from Kodak.

In Vitro Binding Assays

GST-DD and MBP-27TU were expressed in bacteria and purified as described by the manufacturer. 3 µg of MBP-27TU (or MBP alone) was mixed with 3 µg of GST-DD (or GST alone) immobilized on glutathione-agarose beads and incubated for 2 h in binding buffer (20 mM Tris-Cl, pH 7.5, 0.2% Triton X-100, 140 mM NaCl, 0.1 mM EDTA, 10 mM dithiothreitol, 5% (v/v) glycerol). The beads were washed four times with binding buffer, and bound and unbound proteins were immunoblotted with anti-MBP antibody. Filters were developed by chemiluminescence (Amersham Corp.).

Immunoprecipitations

To immunoprecipitate endogenous MADD, cells were lysed by a 10-min incubation in lysis buffer T (20 mM Tris-Cl, pH 7.5, 1% Triton X-100, 137 mM NaCl, 25 mM beta -glycerophosphate, 2 mM EDTA, 1 mM Na3VO4, 2 mM sodium pyrophosphate, 10% (v/v) glycerol, 10 µg/ml leupeptin, and 2 mM phenylmethylsulfonyl fluoride), and the lysates were centrifuged at 15,000 × g for 10 min. MADD was precipitated with 20 µl of anti-MADD antibody 6008 and protein A-Sepharose (Pharmacia). Immunoprecipitates were washed three times with lysis buffer T and the proteins separated by SDS-polyacrylamide gel eletrophoresis. Immunoblotting was performed as above, using anti-MADD antibody 6007. To monitor coimmunoprecipitation between TNFR1 and MADD, FLAG-MADD was transfected into COS cells using the DEAE-dextran method (30). Two days after transfection, cells were starved for 1 h in Dulbecco's modified Eagles's medium containing 0.1% bovine serum albumin and treated for 15 min with 50 ng/ml human recombinant TNF (Genzyme, Cambridge, MA). Lysis, immunoprecipitation, and immunoblotting were performed as described above, using 20 µl of anti-TNFR1 antibody 4013 for immunoprecipitation and 1 µg/ml anti-FLAG antibody for immunoblotting. Anti-mouse HRP was used as second antibody (Amersham Corp.).

Kinase Assays

COS cells were cotransfected using the DEAE-dextran method with 2-3 µg of HA epitope-tagged kinase and 10-15 µg of either pED-FLAG, FLAG-15TU, or pED-MADD. Two days after transfection, cells were treated with varying concentrations of TNF and lysed as described above. HA-tagged kinase was immunoprecipitated from the supernatant using anti-HA antibody. Kinase assays were performed for 30 min at 30 °C in kinase buffer (25 mM Hepes, pH 7.4, 20 mM MgCl2, 0.1 mM Na3VO4, 2 mM dithiothreitol) containing 50 µM ATP, 5 µCi of [gamma -32P]ATP (3000 Ci/mmol, Amersham Corp.) and 1-2 µg of either myelin basic protein (Sigma) (for ERK assays) or a GST fusion with the N terminus of c-JUN (amino acids 1-79) (for JNK assays) as substrate. Samples were analyzed by autoradiography following electrophoresis.

cPLA2 Phosphorylation Assays

COS cells were cotransfected by DEAE-dextran with 2 µg of pmt-2EMC-cPLA2 (29) and 10 µg of pED-FLAG or FLAG-15TU. Two days after transfection, cells were starved and TNF-treated as above and collected directly into 2 × sample buffer (126 mM Tris-Cl, pH 6.8, 1.4 M beta -mercaptoethanol, 20% (v/v) glycerol, 4% SDS, 0.04% bromphenol blue) for SDS-polyacrylamide gel electrophoresis. Immunoblotting for cPLA2 was performed as described above, using polyclonal antibody 7905.


RESULTS

MADD Interacts with Critical Signaling Residues in the TNFR1 Death Domain

The yeast interaction trap system (31) was used to identify proteins that interact with the death domain of the type 1 tumor necrosis factor receptor. The TNFR1 death domain (amino acids 326-413; TNFR1-DD) was fused to the C-terminal end of the LexA DNA binding domain as a "bait" in the interaction trap screen. This construct was cotransformed into yeast along with a U937 cDNA library in which each cDNA was expressed as a fusion with the B42 transcriptional activation domain under the control of the GAL4 promoter. Approximately one million transformants were screened for their ability to express beta -galactosidase and grow in the absence of leucine. 63 of the 340 LEU+/LacZ+ colonies isolated in the screen demonstrated a galactose-dependent phenotype. Fourteen of these, representing nine independent cDNAs, bound TNFR1-DD selectively, as assessed by comparing the interaction with TNFR1-DD to an unrelated bait, Bicoid.

Two of these clones were partial cDNAs encoding portions of a protein we have termed MADD. These partial clones, called 27TU and 15TU, encoded polypeptides of 607 and 320 amino acids, respectively. A partial MADD cDNA encoding a polypeptide of 410 amino acids was also isolated from a similar screen performed with a WI38 library (not shown). The yeast interaction trap was used to investigate the specificity of the interaction between TNFR1-DD and 27TU/15TU. As shown in Table I, both 27TU and 15TU interacted strongly with the death domain of TNFR1, although neither interacted with the death domain of the Fas antigen. Both clones bound the intracellular domain of TNFR1 but not the type 2 TNF receptor.

Table I. Specificity and mutational analyses of MADD partial clones 27TU and 15TU

The yeast interaction trap was used to compare the ability of 27TU and 15TU (in pJG4-5) to interact with a variety of "baits" (in pEG202), including the TNFR1 death domain (TNFR-DD), the death domain of the Fas antigen (Fas-DD), the intracellular domain of TNFR1 (TNFR1-IC), the intracellular domain of TNFR2 (TNFR2-IC), and the Bicoid protein. 27TU and 15TU were also tested for their ability to bind TNFR1-DD containing mutations at critical signaling residues. Phe-345, Leu-351, Gly-369, Trp-378, and Ile-408 were individually mutated to alanine and subcloned into pEG202 for testing in the interaction trap. Pluses represent relative beta -galactosidase expression, as judged by the color intensity in plates containing 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside. The yeast interaction trap was used to compare the ability of 27TU and 15TU (in pJG4-5) to interact with a variety of "baits" (in pEG202), including the TNFR1 death domain (TNFR-DD), the death domain of the Fas antigen (Fas-DD), the intracellular domain of TNFR1 (TNFR1-IC), the intracellular domain of TNFR2 (TNFR2-IC), and the Bicoid protein. 27TU and 15TU were also tested for their ability to bind TNFR1-DD containing mutations at critical signaling residues. Phe-345, Leu-351, Gly-369, Trp-378, and Ile-408 were individually mutated to alanine and subcloned into pEG202 for testing in the interaction trap. Pluses represent relative beta -galactosidase expression, as judged by the color intensity in plates containing 5-bromo-4-chloro-3-indolyl-beta -D-galactopyranoside.
15TU 27TU

Specificity analysis
  TNFR1-DD +++ +++
  Fas-DD  -  -
  TNFR1-IC +++ +
  TNFR2-IC  -  -
  Bicoid  -  -
Mutational analysis
  TNFR1-DD +++ +++
  F345A + +
  L351A + +
  E369A +++ +++
  W378A ++ +
  I408A ++ ++

In an effort to determine the amino acids within TNFR1-DD involved in this interaction, five of the six death domain residues previously shown to be critical for signaling TNF-induced cytotoxicity (3) were mutated individually to alanine. When assayed in the interaction trap, both N- and C-terminal mutations were found to affect MADD binding (Table I). Several other unrelated clones isolated in the screen did not show differential interaction (data not shown). The interaction of MADD with the death domain of TNFR1, and specifically with several critical signaling residues, supports the relevance of MADD in TNF signaling.

MADD Encodes a Protein of 1588 Amino Acids That Interacts with TNFR1 through a C-terminal Death Domain

Northern analysis revealed MADD mRNA to be expressed in a wide variety of tissues and cell lines as a 7-kilobase transcript (Fig. 1). A full-length MADD cDNA was assembled from partial clones isolated in three library screens. The open reading frame of MADD encodes a novel protein containing 1588 amino acids, with a predicted molecular mass of 176.4 kDa (Fig. 2A). Examination of the MADD sequence revealed several interesting features (Fig. 2B). The most striking is a C-terminal region that bears significant homology to the death domain of TNFR1. In addition, like other death domain-containing proteins (32), MADD contains regions rich in serine and threonine residues. Approximately 25% of the residues in these clusters are serine or threonine. The N terminus of MADD contains a consensus leucine zipper sequence (33), suggesting a mechanism by which MADD might dimerize or interact with other proteins.


Fig. 1. Northern analysis of MADD mRNA. Northern blots containing 2 µg/lane poly(A)+ mRNA from various human tissues (upper panel) and cell lines (lower panel) were probed with partial MADD cDNA 27TU. Kb, kilobase pairs.
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Fig. 2. Structure of full-length MADD. A, the predicted amino acid sequence of MADD. The C-terminal death domain and N-terminal leucine zipper are underlined, with consensus leucine zipper residues shown in bold. The clones isolated from the interaction trap screen, 27TU and 15TU, begin at residues Glu-982 (E982) and Phe-1269 (F1269), respectively, as indicated by arrows. B, diagrammatic representation of MADD. The leucine zipper and death domain homology sequences are shown by the stippled and hatched boxes, respectively. Black boxes represent regions rich in serine and threonine residues.
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Death domains mediate interactions between several proteins involved in TNF signaling. To examine the homology between the death domains of MADD and TNFR1, these sequences were aligned with each other and with Fas and TRADD (Fig. 3). The death domain of MADD is 17% identical to that of TNFR1, 12% identical to Fas, and 14% to TRADD. If conservative amino acid changes are considered, the degree of similarity between MADD and TNFR1, Fas, and TRADD increases to 30, 29, and 22%, respectively. For comparison, TNFR1 and Fas are 21% identical and 35% similar by this alignment. The six residues previously reported to be critical for TNFR1 signaling are indicated with asterisks. MADD is identical to TNFR1 at three of these positions.


Fig. 3. Death domain alignment. The death domain of MADD was aligned with the death domains of human TNFR1, Fas, and TRADD. Residues conserved between MADD and the other death domains are shown boxed, with identical residues in bold. Conserved amino acids were defined as follows: H, K, R; I, L, M, V; D, E; F, Y; S, T; N, Q; A, G; C; P; W. Positions of amino acids previously shown to be critical for signaling (3) are indicated with asterisks.
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To detect endogenous MADD, a fusion protein between partial clone 27TU and maltose binding protein (MBP-27TU) was used as antigen to generate polyclonal antibodies 6007 and 6008. MADD was immunoprecipitated from various cell lines with antibody 6008 and detected by immunoblotting with antibody 6007. As shown in Fig. 4, MADD is widely expressed and migrates as a protein of approximately 200 kDa, similar to overexpressed MADD (see Fig. 5 C). The 100-kDa band is nonspecific and the additional bands (at 170 and 85 kDa) immunoprecipitated from U937 cells represent degradation products of MADD (as judged by immunoprecipitation with preimmune antiserum, data not shown).


Fig. 4. Endogenous MADD expression. MADD was immunoprecipitated from U937, HeLa, CHO, and WI38 cells with anti-MADD antibody 6008. Immunoprecipitated MADD was visualized by blotting with anti-MADD polyclonal 6007. Full-length MADD is indicated with an arrow.
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Fig. 5. Association of TNFR1 and MADD. GST-DD, a fusion protein between GST and TNFR1-DD, was immobilized on glutathione-agarose beads and incubated with purified MBP-27TU fusion protein. MBP fusion protein bound to the beads after extensive washing (A) as well as unbound protein (B) were detected by immunoblotting with anti-MBP antibody. MBP and GST proteins were tested as negative controls. C, association of MADD and TNFR1 in COS. COS cells were transfected with pED-FLAG (V) or FLAG-MADD (M). Two days after transfection, TNFR1 was immunoprecipitated with preimmune or immune polyclonal antibody 4013. Immunoprecipitates (IP) were electrophoresed and immunoblotted with anti-FLAG antibody.
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As discussed above, the death domain is a protein-protein interaction motif found in several proteins implicated in TNFR1 signaling. Both 27TU and 15TU contain this motif, suggesting a mechanism by which they bind the death domain of TNFR1. To investigate whether the death domain of MADD was indeed sufficient for interaction with the death domain of TNFR1, a fragment encoding the 76-amino acid region of death domain homology, residues 1281-1356, was tested in the interaction trap for binding to TNFR1-DD. A death domain deletion mutant of clone 15TU (15Delta DD) was used as a control. As shown in Table II, the MADD death domain is sufficient for interaction with the death domain of TNFR1. Conversely, deletion of the death domain abolishes the ability of 15TU to interact. These data establish that the MADD death domain mediates association with TNFR1. Interestingly, the MADD death domain also mediated interaction with itself, as well as with the death domain of TRADD (Table II). In contrast, no interaction was detected with the death domain of Fas. The observation that MADD can associate with TNFR1 and TRADD, but not with Fas, is consistent with the possibility that MADD is a component of the TNFR1 signaling complex.

Table II. Role of the MADD death domain in mediating protein-protein interactions

MADD, TNFR1, Fas, and TRADD death domains were cloned into pEG202 and cotransformed into yeast with the MADD death domain in pJG4-5. The degree of interaction was assessed by observing relative beta -galactosidase expression. The pEG202 death domain constructs were also tested for interaction with 15Delta DD, corresponding to the partial clone 15TU lacking the death domain and a small amount of flanking sequence. MADD, TNFR1, Fas, and TRADD death domains were cloned into pEG202 and cotransformed into yeast with the MADD death domain in pJG4-5. The degree of interaction was assessed by observing relative beta -galactosidase expression. The pEG202 death domain constructs were also tested for interaction with 15Delta DD, corresponding to the partial clone 15TU lacking the death domain and a small amount of flanking sequence.
TNFR1-DD Fas-DD TRADD-DD MADD-DD

MADD-DD +++  - ++ +++
15Delta DD  -  -  -  -

To confirm the association between MADD and TNFR1, in vitro binding experiments were performed (Fig. 5, A and B). The death domain of TNFR1 was expressed as a fusion protein with glutathione S-transferase (GST-DD) and tested for interaction with MBP-27TU. GST or GST-DD immobilized on glutathione-agarose beads was incubated with purified MBP-27TU or MBP alone. Immunoblotting with alpha -MBP antibody revealed that MBP-27TU bound to GST-DD (Fig. 5A) but not GST, nor did MBP associate with GST-DD.

To test whether MADD associated with TNFR1 in mammalian cells, COS cells were transfected with a plasmid encoding full-length MADD fused at its N terminus to the FLAG epitope (FLAG-MADD). Endogenous TNFR1 was immunoprecipitated using a polyclonal antibody to TNFR1, and the immunoprecipitates were immunoblotted with anti-FLAG antibody. As shown in Fig. 5C, MADD coimmunoprecipitated with TNFR1 immune, but not preimmune, serum. Proteins immunoprecipitated from cells transfected with the pED-FLAG vector (V) showed no immunoreactivity to the FLAG epitope. The coimmunoprecipitation of MADD and TNFR1 was observed in both untreated and TNF-treated cells, suggesting that MADD is constitutively associated with TNFR1.

MADD Clones Activate MAP Kinase and Induce the Phosphorylation of cPLA2

An important signaling pathway in the cellular response to TNF is the activation of the MAP kinases ERK and JNK. These enzymes phosphorylate and activate several transcription factors, including AP-1 (34), ATF2 (35), and ELK-1 (36), leading to the increased transcription of a number of genes involved in inflammation (37-39). ERK has also been shown to activate cPLA2 by phosphorylation, leading to the release of arachidonic acid (40). To explore whether MADD might be involved in these signaling pathways, we tested whether overexpression of this protein stimulated MAP kinase activity. COS cells were cotransfected with HA-epitope tagged ERK2 or JNK1 and cDNAs encoding either intact MADD or partial clone 15TU. After transfection, MAP kinase was immunoprecipitated with anti-HA antibody, and its activity was assessed using myelin basic protein or GST-c-JUN (amino acids 1-79) as substrates for ERK and JNK, respectively. As shown in Fig. 6A, MADD expression stimulated both basal and TNF-induced ERK activity. Immunoblotting of lysates from vector- and MADD-transfected cells with anti-HA antibody confirmed equivalent HA-ERK expression (not shown). Expression of 15TU caused an even greater activation of ERK (Fig. 6B). In addition, 15TU induced a significant activation of JNK even in the absence of TNF, although the stimulation was less robust than that seen with ERK. Expression of intact MADD at levels comparable to 15TU did not stimulate JNK activity (data not shown), possibly because a more modest stimulation would be difficult to detect in this assay. Transfection of the vector (V) or clones unrelated to MADD (not shown) had no effect. The ability of MADD clones to activate ERK and JNK indicates that MADD plays a role in the signaling pathway(s) between TNFR1 and the MAP kinase family of enzymes.


Fig. 6. Effect of MADD and 15TU overexpression on MAP kinase activity. A, COS cells were cotransfected with 3 µg of HA-epitope tagged ERK2 and 15 µg of either pED-FLAG vector (V) or pED-MADD (MADD). After TNF treatment, ERK2 was immunoprecipitated with anti-HA antibody. Immunoprecipitation kinase assays were performed with myelin basic protein as substrate. The reactions were electrophoresed and phosphorylated proteins detected by autoradiography. B, similar to A, but cells were transfected with 2 µg of HA-epitope tagged ERK2 or JNK1 and 10 µg of either pED-FLAG vector (V) or FLAG-15TU (15TU). Myelin basic protein and GST-c-JUN (amino acids 1-79) were used as ERK2 and JNK substrates, respectively. Exposure time was 1 h for both experiments.
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In many cell types, treatment with TNF results in the phosphorylation of cPLA2, a critical step in the activation of this enzyme (41). One of the kinases that can phosphorylate and activate cPLA2 is ERK (40). To test whether MADD might be involved in the signaling pathway leading to cPLA2 phosphorylation, perhaps as a consequence of its ability to activate ERK, we tested whether overexpression of the partial MADD clone 15TU induced the phosphorylation of cPLA2. After cotransfection with cPLA2 and 15TU, COS cells were treated with TNF and the lysates immunoblotted for cPLA2. As phosphorylation significantly reduces the electrophoretic mobility of cPLA2, the phosphorylation state of cPLA2 can be assessed by observing the ratio between the upper and lower bands (42). As shown in Fig. 7, in cells transfected with the vector (V), the majority of cPLA2 was dephosphorylated and became phosphorylated with increasing TNF treatment. Quantitation by phosphoimage analysis revealed the percentage of cPLA2 in the upper band to be 35, 66, 92, and 100% at 0, 0.4, 2, and 10 ng/ml TNF, respectively. In contrast, cPLA2 was heavily phosphorylated in cells expressing 15TU, with 86% of the cPLA2 found in the upper band even in the absence of TNF. The induction of cPLA2 phosphorylation by 15TU suggests that MADD may be involved in the activation of the arachidonic acid cascade by TNF.


Fig. 7. Effect of 15TU overexpression on the phosphorylation of cPLA2. A, COS cells were cotransfected with 2 µg of pmt-2EMC-cPLA2 and 10 µg of either pED-FLAG (Vector) or FLAG-15TU (15TU). Two days after transfection, cells were treated with TNF and the lysates immunoblotted for cPLA2. B, expression of 15TU. Lysates from transfected cells blotted in A were probed with anti-FLAG antibody.
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DISCUSSION

We have identified a protein, MADD, that provides a link between TNFR1 and the activation of MAP kinases and cPLA2. Like TRADD, MADD and TNFR1 associate through a protein-protein binding motif known as the death domain. The interaction between MADD and TNFR1 was first described in the yeast interaction trap and was also demonstrated in in vitro binding experiments using purified proteins. This association was confirmed in mammalian cells by the coprecipitation of MADD with endogenous TNFR1. Two additional lines of evidence implicate MADD as a TNFR1 signaling protein. First, mutation of TNFR1-DD at residues critical for the activation of downstream signaling pathways decreased the ability of MADD to associate, suggesting that the sites of MADD interaction correspond to important signaling residues. Second, and most importantly, overexpression of MADD mimics TNF-induced MAP kinase activation. This effect appears to be mediated through the death domain, as 15TU induced the activation of both ERK and JNK as well as cPLA2 phosphorylation. MADD is the only TNFR1-associated protein reported to activate all of these signaling pathways.

MADD is a 176-kDa protein that interacts with TNFR1 through their respective C-terminal death domains. The death domain is a familiar protein-protein interaction motif in TNF signaling (43), mediating associations between TNFR1, TRADD, MORT1/FADD, RIP, and now MADD. Like other death domains, the death domain of MADD mediates self-interaction as well as interaction with other proteins. This region does not allow indiscriminant binding, however, as evidenced by the ability of MADD to interact with TRADD but not Fas. The interaction of MADD with TNFR1 and TRADD, but not Fas, suggests that MADD is specific to the TNFR1 signaling complex. Interestingly, the N terminus of MADD contains a well-conserved leucine zipper sequence, suggesting a mechanism by which MADD might interact with downstream effector proteins.

MADD coprecipitated with TNFR1 in a TNF-independent manner, suggesting that MADD is constitutively associated with the receptor. However, MADD may exist in an inactive form in the absence of TNF and be activated upon TNF binding, perhaps by aggregation or post-translational modification. Consistent with the latter possibility, MADD is heavily phosphorylated on serine residues.2 We have also found that the high molecular weight band observed upon immunoblotting of 15TU (Fig. 7B) is due to ubiquitination (as assessed by immunoblotting with anti-ubiquitin antibody, not shown). Whether these modifications regulate MADD activity is under investigation. It should be noted that it is also possible that MADD association with the receptor is TNF-dependent under physiological conditions but that the TNFR1 antibody used for immunoprecipitation mimics TNF treatment by inducing aggregation of the receptor.

As discussed above, overexpression of TRADD, through its recruitment of MORT1/FADD, TRAF2 and RIP, induces signaling pathways leading to the initiation of the apoptotic response and the activation of NF-kappa B. No obvious effect on NF-kappa B or cell death was observed upon expression of either full-length or partial MADD clones (data not shown). In addition to these downstream events, TNF has been reported to elicit several, more immediate, cellular responses. The activation of kinases, including the MAP kinases ERK, JNK and p38, and phospholipases, including phospholipase C, neutral and acidic sphingomyelinase and cPLA2, are some of the significant early events in TNF signaling. Overexpression of intact MADD stimulates ERK activity. As mentioned above, this effect is retained in 15TU, a deletion mutant primarily comprised of the death domain. The observation that 15TU activated ERK more potently than did MADD suggests that the N terminus of MADD functions as a negative regulatory domain. 15TU was also observed to induce cPLA2 phosphorylation and JNK activation. Interestingly, although TRAF2 and RIP have recently been shown to stimulate JNK activity, none of the signaling proteins in the TNF receptor complex was able to activate ERK when overexpressed (44). These data, taken together, suggest that MADD is involved in the pathway linking TNFR1 to MAP kinase activation and may play a central role in the stimulation of ERK activity by TNF.

The observation that expression of 15TU is sufficient to stimulate MAP kinase activity suggests that effector proteins for this pathway bind to the MADD death domain. Many proteins involved in the activation of ERK and JNK have been described including the small GTP-binding proteins, such as RAS, CDC42, and RAC (45-49). Examining the relationship between MADD, the small G proteins, and the other proteins in the TNFR1 signaling complex will provide a better understanding of the mechanism(s) by which MADD regulates TNF-induced MAP kinase activation.

As discussed above, phosphorylation of cPLA2 stimulates the intrinsic enzymatic activity of this enzyme (40), leading to the release of arachidonic acid. This phosphorylation can be mediated by ERK (40) and possibly other kinases (50-52). The ability of 15TU to induce cPLA2 phosphorylation may result from its activation of ERK. The induction of cPLA2 phosphorylation by the MADD death domain implicates MADD as a potential regulator of TNF-stimulated arachidonic acid release. Interestingly, in addition to its role in inflammation, cPLA2 has been implicated in TNF-induced apoptosis (53). The activation of JNK has also been proposed to be important in this process (54). The observation that 15TU stimulates both JNK activity and cPLA2 phosphorylation without triggering apoptosis, however, suggests that the activation of other components, such as MACH/FLICE, are required for initiation of this pathway. This is consistent with recent findings demonstrating that JNK activation is insufficient for triggering apoptosis (44).

The identification of MADD adds a new member to the group of death domain-containing proteins in the TNFR1 signaling complex. Clearly, death domains play a central role in regulating the diverse signaling cascades that are initiated when TNF binds its receptor. The death domain of TRADD mediates the recruitment of both RIP and MORT1/FADD to TNFR1, which (with TRAF2) signals the activation of NF-kappa B and the initiation of apoptosis. The effect of MADD expression, in contrast, implicates this protein in the pathway(s) leading to the activation of MAP kinases, particularly ERK, and the release of arachidonic acid. These data, taken together, support the concept that individual death domains mediate signaling through distinct intracellular pathways. The recruitment of this diverse group of proteins to the TNFR1 signaling complex provides a mechanism by which TNF exerts such pleiotropic effects.


FOOTNOTES

*   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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U77352[GenBank].


Dagger    To whom correspondence should be addressed: 87 CambridgePark Dr., Genetics Institute, Inc., Cambridge, MA 02140. Tel.: 617-498-8934; Fax: 617-498-8993.
1   The abbreviations used are: TNF, tumor necrosis factor; TNFR1, tumor necrosis factor receptor type 1; MAP kinase, mitogen-activated protein kinase; cPLA2, cytosolic phospholipase A2; PCR, polymerase chain reaction; GST, glutathione S-transferase; MBP, maltose binding protein; HA, hemagglutinin; ERK, extracellular signal-regulated kinase; JNK, c-JUN N-terminal kinase.
2   A. R. Schievella and L.-L. Lin, unpublished data.

ACKNOWLEDGEMENTS

We are very grateful to Ron Kriz for the construction of cDNA libraries, Kevin Bean and Kerry Kelleher for extensive DNA sequencing, and Wei Cao for technical assistance. We also thank John Knopf, Roger Davis, and James Clark for helpful discussions and Hsiang-Ai Yu for help with the death domain alignment. We also appreciate receiving GST-c-JUN-(1-79), HA-ERK, and HA-JNK constructs from Roger Davis and anti-ubiquitin antibody from Arthur Haas.


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