Tumor Necrosis Factor Receptor-Associated Factor (TRAF) Family: Adapter Proteins That Mediate Cytokine Signaling

Experimental Cell Research 254, 14 –24 (2000) doi:10.1006/excr.1999.4733, available online at http://www.idealibrary.com on

REVIEW Tumor Necrosis Factor Receptor-Associated Factor (TRAF) Family: Adapter Proteins That Mediate Cytokine Signaling Jun-ichiro Inoue, 1 Takaomi Ishida,* Nobuo Tsukamoto, Norihiko Kobayashi, Asuka Naito, Sakura Azuma, and Tadashi Yamamoto Department of Oncology and *Department of Pathology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan

key molecules in the signals regulating the immune and inflammatory systems.

INTRODUCTION

The first two members of the tumor necrosis factor receptor-associated factor (TRAF) family of proteins, TRAF1 and TRAF2, were identified as signal transducers of tumor necrosis factor (TNF) receptor type II (TNFRII) in 1994 [1]. Since then, four additional members of the TRAF family, TRAF3, TRAF4, TRAF5, and TRAF6, have been identified in human and mouse [2–10]. In addition, one TRAF of the nematode Caenorhabditis elegans [11] and two TRAFs of the Drosophila melanogaster [12] have been reported. As described below, identification of the TRAF family of proteins led to dramatic progress in the elucidation of the molecular mechanisms of signal transduction emanating from the TNFR superfamily and the Toll/interleukin-1 receptor (Toll/IL-1R) family. TRAF2, TRAF5, and TRAF6 serve as adapter proteins that link the cell surface receptors and downstream kinase cascades, which results in activation of transcription factors, nuclear factor kB (NFkB) and activator protein-1 (AP-1). Although neither TRAF1 nor TRAF3 activates any kinase tested so far in in vitro experiments, TRAF1 transgenic mice [13] and TRAF3-deficient mice [14] exhibit marked impairment in their immune systems. Thus, the TRAF-mediated signals apparently play important roles in regulating cell survival, proliferation, and stress responses. While TRAF family was growing, another group of adapter proteins, the death domain (DD) proteins, was shown to mediate the signals emanating from some of the TNFR superfamily and Toll/IL-1R family by binding to their cytoplasmic tails [15–17]. In such DD-protein-mediated signals, TRAFs either directly or indirectly bind to the DD protein to transduce the signals. Thus, the members of the TRAF family are

DISCOVERY OF THE TRAF FAMILY

Membrane-integrated receptor proteins with no intrinsic enzyme activity often use associated proteins to transduce signals. For example, the antigen receptor and T cell receptor activate nonreceptor tyrosine kinases upon ligand binding [18]. Thus, the TNFR family of receptors, which do not have any kinase motif in their cytoplasmic tail, were expected to use associated proteins to transduce signals linked to immune and inflammatory responses. Biochemical purification of receptor-associated proteins and a recently developed cDNA cloning system [19] that uses yeast genetic selection led to the discovery of the TRAF family of proteins as adapter proteins that directly associate with the cytoplasmic tail of the TNFR superfamily. TRAF1 was identified by biochemical purification of an intracellular factor that associated with TNFRII, and TRAF2 cDNA was cloned with a yeast two-hybrid screening using the TNFRII cytoplasmic region as bait [1]. TRAF3, also known as CD40bp, LAP-1, or CRAF1, was identified independently by several groups as a cytoplasmic factor that binds to CD40 and LMP-1, a membrane protein encoded by the Epstein–Barr virus genome [2–5]. TRAF4, originally named CART1 (cysteinerich motif associated with RING and TRAF domains) was identified by differential screening of a cDNA library of lymph nodes that contained metastatic tumor cells [6]. TRAF5 cDNA was cloned by yeast two-hybrid using CD40 cytoplasmic tail as bait [8] and independently by PCR using degenerated primers [7]. TRAF6 was also isolated by yeast two-hybrid screening using CD40 cytoplasmic tail as bait [10] and independently by screening of an EST expression library [9].

1 To whom correspondence and reprint requests should be addressed at Department of Oncology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 1088639, Japan. Fax: 813-5449-5413. E-mail: [email protected].

0014-4827/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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TRAF PROTEINS

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FIG. 1. Structure of the TRAF family of proteins. TRAF2, TRAF3, TRAF5, and TRAF6 have five zinc fingers (5X), whereas TRAF1 and TRAF4 have one (1X) and seven (7X) zinc fingers, respectively. TRAF4 has two potential nuclear localization signals (NLS).

STRUCTURE OF THE TRAF PROTEINS

All members of the TRAF family share a common stretch of amino acids at their carboxyl terminus, which has been designated as the TRAF domain [1] (Fig. 1). The TRAF domain is further divided into two subregions [3]. The carboxyl-terminal half of the TRAF domain, TRAF-C, is highly conserved among members of the TRAF family (Fig. 2, Table 1), and the aminoterminal portion of the TRAF domain, TRAF-N, which was predicted to form a coiled-coil structure, is less conserved. Previous overexpression experiments revealed that the TRAF-C domain mediates homo- and heterodimerization of the TRAF proteins and association of the TRAF proteins with various cell surface receptors that can recruit TRAFs upon stimulation [3, 20]. However, recent crystallographic studies of the TRAF2–TNFRII and TRAF2–CD40 complexes provided evidence that the TRAF domain of TRAF2 forms a mushroom-shaped trimer consisting of both the coiled coil and the TRAF-C, which forms a unique, eight-stranded b-sandwich [21, 22]. Nine amino acids in the TRAF-C domain of TRAF2 (393R, 395Y, 399D, 453S, 454S, 455S, 466A, 467S, and 468G) have been proposed to be involved in hydrogen bonds that mediate interaction of TRAF2 with receptor peptides derived from the TRAF2-binding site of CD40 and TNFRII (Fig. 2, see shaded amino acids in TRAF2). Consistent with this finding, the coiled-coil domain was reported to contribute to receptor binding [23, 24]. All TRAF proteins, except TRAF1, share two additional predicted structural features in their aminoterminal half. The most amino-terminal motif is the

RING finger (Fig. 1). TRAF2 and TRAF3 contain a normal C3HC4-type RING finger, which is found in a number of RING finger proteins. In contrast, TRAF4, TRAF5, and TRAF6 contain an unusual C3HC3D-type RING finger that varies from the C3HC4 RING finger

FIG. 2. Sequence comparison of the TRAF-C domain of the murine TRAF proteins. Consensus amino acids are indicated as bold letters when more than five of six TRAFs have identical amino acids or all TRAFs have identical charged amino acids at the corresponding positions. Nine shaded amino acids in TRAF2 are predicted to contact amino acids within the cytoplasmic tail of CD40 or TNFRI by hydrogen bond.

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TABLE 1 Comparison of the TRAF Family Proteins in the TRAF-C Domain of Homology TRAF1 TRAF1 TRAF2 TRAF3 TRAF4 TRAF5 TRAF6

TRAF2

TRAF3

TRAF4

TRAF5

TRAF6

62

57 59

39 42 38

54 56 65 41

30 28 30 25 29

Note. Data are presented as percentages of amino acid sequence identity in the TRAF-C domain. The following amino acid regions of each TRAF protein were used for the calculation of identity: 260 – 409 of mouse TRAF1, 352–501 of mouse TRAF2, 415–567 of mouse TRAF3, 308 – 470 of mouse TRAF4, 404 –558 of mouse TRAF5, and 359 –530 of mouse TRAF6.

motif by replacement of the last cysteine with an aspartic acid. The other predicted structural feature is the zinc finger motif located between the RING finger and the coiled-coil domain (Fig. 1). TRAF2, TRAF3, TRAF5, and TRAF6 contain five zinc finger repeats, whereas TRAF4 has seven repeats. The relation of the structural differences of the RING finger and the zinc finger motif among TRAFs with the biological functions of TRAFs remains to be elucidated. Interestingly, TRAF4 has two potential nuclear localization signals (Fig. 1) and appears to be located in the nucleus [6], which is definitely different from the observation that other TRAFs are localized in the soluble fraction of the cytoplasm of cells. Consistent with its nuclear localization, TRAF4 is only one mammalian TRAF that has not been demonstrated to associate with any of the known receptors. TRAF4 probably belongs to a subfamily of TRAFs that functions in the nucleus. INTERACTION OF TRAF PROTEINS WITH THE TNFR SUPERFAMILY AND THE IL-1R-ASSOCIATED KINASE (IRAK)

In vitro binding experiments and transient transfection experiments revealed that TRAFs interact with various members of the TNFR superfamily and Toll/ IL-1R family (Table 2). All members of the TNFR superfamily share a ligand-binding domain composed of tandemly repeated cysteine-rich modules. The TRAF-C domain has been shown to mediate, at least in part, the association of TRAF proteins with the receptor tails. Comparison of the TRAF family proteins in the TRAF-C domain of homology indicates that TRAF-C domains of TRAF1, TRAF2, TRAF3, and TRAF5 are quite similar, whereas those of TRAF4 and TRAF6 are divergent from other TRAF proteins (Table 1). Based on these similarities, TRAF4 and TRAF6 would presumably interact with distinct peptides, whereas the

other TRAFs would all recognize similar amino acid sequences. Subsequent studies, as described below, revealed that this could be the case. In most cases, TRAF1, TRAF2, TRAF3, and TRAF5 bind to the same receptors (Table 2). A number of mutational studies have demonstrated the PXQXT motif in the CD40, CD30, RANK, and the membrane-proximal part of the cytoplasmic region of LMP1 as a consensus sequence required for binding to TRAF2, TRAF3, and TRAF5 [25, 26]. Furthermore, it was reported that other motifs such as the EXGKE [27] and the VXXSXXEE [23] could also mediate binding to the TRAF proteins. However, TRAF2 and TRAF3 have been shown to bind overlapping but distinct motifs in the CD40 cytoplasmic tail [28]. TRAF6 binds to CD40 [10, 29], RANK [30 –32], p75 NGF receptor [33], and IRAK that associates with the IL-1R via MyD88 [9]. A characteristic structural feature of Toll/IL-1R family is the Toll/IL-1R homology (TIR) domain within the cytoplasm of these receptors [34], and this domain may recruit MyD88 to other members of the Toll/IL-1R family. Recent experiments using MyD88-deficient mice revealed that MyD88 mediates signals emanating from IL-18 receptor, TLR-2 (Toll-like receptor 2) and TLR-4 as well as IL-1R [35, 36]. TLR-2 and TLR-4 have been suggested to be LPS receptors. Furthermore, NFkB activation but not Jun N-terminal kinase (JNK) activation by TLR-4 signals was shown to be inhibited by a dominant-negative mutant of TRAF6 [37]. Therefore, upon stimulation of these receptors, IRAK and/or IRAK2 [38] could be recruited to the cytoplasmic tail of these receptors via MyD88. IRAK and IRAK2 in turn associate with TRAF6 leading to the activation of downstream kinases. In the cases of CD40 and RANK, TRAF6 as well as other TRAFs including TRAF2, TRAF3, and TRAF5 bind to the cytoplasmic tail [10]. However, the TRAF6binding site is distinct from that of other TRAFs [29, 31]. Although extensive mutational analysis remains to be performed, a putative consensus TRAF6-binding site would be basic–QXPXEX–acidic, which was proposed based on the amino acid sequences of CD40, RANK, IRAK1, and IRAK2 [32]. When the glutamic acid (E), sixth amino acid in the consensus TRAF6binding site, was replaced with an alanine in the cytoplasmic tail of CD40, both TRAF6 binding and NFkB activation through this site were completely abolished [29]. Interestingly, crystallographic studies have shown that amino acids of TRAF2 that make contact with CD40 and TNFRII are identical to the corresponding amino acids of TRAF3 and TRAF5, whereas those of TRAF4 and TRAF6 are almost completely different (Fig. 2) [21, 22]. This finding also agrees with those obtained from the binding experiments described above. However, results obtained from in vitro binding assays or overexpression experiments do not necessar-

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TRAF PROTEINS

TABLE 2 Interaction of the TRAF Family of Proteins with TNFR Superfamily and Toll/IL-1R Family Receptors

Ligands

Signals

Associated TRAFs

TNFa, LTa TNFa, LTa CD27 ligand CD30 ligand CD40 ligand LTa 1b 2, LIGHT OPGL/RANKL/TRANCE/ODF Neutrophins LIGHT, LTa 4-1BB ligand OX40 ligand GITR ligand AITR ligand Fas ligand Apo3 ligand, TWEAK

PA PA P PDB PDB P PD A P P PD B ? A A

2 (via TRADD) 1, 2, 5 2, 5 1, 2, 3, 5 2, 3, 5, 6 3, 5 1, 2, 3, 5, 6 6 1, 2, 3, 5 1, 2, 3 1, 2, 3, 5 2 1, 2, 3 ? ?

TRAIL/Apo2 ligand TRAIL/Apo2 ligand

A A

? ?

TRAIL/Apo2 ligand

B

?

TRAIL/Apo2 ligand

B

?

IL-1

P

TLR2

LPS

P

TLR4

LPS

P

6 (via IRAK and MyD88) 6 (via IRAK and MyD88) 6 (via IRAK and MyD88)

TNFR superfamily TNFRI TNFRII CD27 CD30 CD40 LTb-R RANK p75NGFR ATAR/HVEM 4-1BB/CD137 OX40/CD134 GITR AITR Fas/CD95 DR3/TRAMP/WSL-1/ Apo3/LARD DR4/TRAIL-R1 DR5/TRICK2/KILLER/ TRAIL-R2 TRAIL-R3 (decoy receptor) TRAIL-R4 Toll/IL-1R family IL-1RI

Viral proteins LMP1 CrmD

? ?

P ?

Other associated proteins

TRADD, RIP, FAN

Jak3

FADD TRADD FADD, TRADD, RIP FADD, TRADD, RIP

MyD88 MyD88 MyD88

1, 2, 3, 5 ?

Note. P, proliferation; D, differentiation; A, apoptosis; B, blocking apoptosis.

ily tell us the physiological roles of these proteins. Evidence for the interaction of the endogenous TRAF proteins with the endogenous receptors upon ligand stimulation or the impairment of signaling in cells established from mice deficient in one of the TRAF genes is necessary to determine their physiological roles. Several reports demonstrated the ligand-dependent recruitment of TRAF proteins into receptor signaling complexes: CD40 with TRAF2 and TRAF3 [39], LTbR with TRAF3 [40], and 4-1BB with TRAF2 [41]. CASCADES OF TRAF-MEDIATED SIGNALS

Crystallographic findings of the TRAF2–CD40 complex suggest that a single TRAF2 molecule in the TRAF2 trimer could bind to a single CD40 molecule of the receptor trimer [21, 22]. Since CD40 is thought to be a monomer prior to ligand stimulation and to form a trimer upon binding of CD40 ligand that itself constitutively forms trimer, the trimer–trimer interaction of

TRAF2 and CD40 could partly explain why CD40 recruits TRAF2 upon ligand binding. Most ligands for TNFR superfamily are predicted to form trimers, and some of them have been proven biochemically [42, 43]. Thus, the TRAF trimer–receptor trimer association could generally explain their stimulation-dependent interactions. However, how TRAF transduces signals to downstream molecules remains to be elucidated since the structural changes of TRAF proteins upon stimulation are not known. A recent study reported that oligomerization of the amino-terminal portion of TRAF2 and TRAF6 containing the RING and zinc fingers resulted in the activation of downstream kinases [44]. Thus, it is possible that trimer of endogenous TRAF proteins could further form an oligomer or that the structural changes of the trimer could be induced by stimulation. Signal transduction pathways triggered by TRAF2, TRAF5, and TRAF6 are linked to two distinct families of transcription factor: the AP-1 family and the Rel/

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FIG. 3. Schematic representation of the signal transduction pathways emanating from TNFRI and IL-1R. See text for details.

NFkB family whose activation is achieved by the activation of the mitogen-activated protein kinase (MAPK) family and IkB kinase (IKK), respectively. As described below, accumulating evidence indicates that members of the MAPK kinase kinases (MAP3K) or MAP3K kinase (MAP4K) family, which are upstream of MAPK, could be activated by the TRAF proteins. Activation mechanisms of JNK by TRAF have been studied more than that of other MAPKs such as ERK and p38. JNK is activated by members of the MAPK kinase (MAP2K) family, SEK1 and MKK7 [45– 47]. Three members of the MAP3K, MEKK1 [44], apoptosis-inducing kinase (ASK1) [48], and TGFb-activating kinase (TAK1) [49], and two members of MAP4K, germinal center kinase (GCK) [50] and GCK-related kinase (GCKR) [51], have been shown to be involved in the TRAF-mediated activation of SEK1–JNK pathway. Overexpression of one of these kinases activates JNK. Furthermore, kinaseinactive mutants of MEKK1, ASK1, and GCKR inhibit TRAF2-mediated JNK activation, and inactive TAK1 blocks TRAF6-mediated JNK activation. More importantly, the interaction between ASK1 and TRAF2 and that between TAK1 and TRAF6 have been demonstrated to be dependent on TNFa and IL-1 stimulation, respectively. Since GCK/GCKR are upstream of MEKK1, at least three distinct JNK activation pathways could be triggered by TRAF proteins (Fig. 3). NFkB-inducing kinase (NIK) has been identified as a TRAF2-interacting protein by the yeast two-hybrid screening using TRAF2 as bait [52]. Although NIK is a member of MAP3K, overexpression of NIK activates NFkB but not JNK. MEKK1 and TAK1, also members of MAP3K, activate both NFkB and JNK when they are overexpressed. Cot/TPL-2, another member of MAP3K, mediates CD3/CD28 costimulatory signals to activate NFkB [53, 54]. However, a functional linkage of the

Cot/TPL-2 and the TRAF proteins are unclear. Activity of NFkB is controlled by a specific inhibitory subunit called IkB. Seven species of IkB (IkBa, IkBb, IkBg, IkBe, Bcl-3, p100, and p105) have been identified [55– 57]. With the exception of Bcl-3 [58], the IkB proteins mask the nuclear localization signal of the NFkB proteins, thereby sequestering them in the cytoplasm. In the cases of IkBa, IkBb, and IkBe, activation of NFkB is carried out by a posttranslational mechanism including signal-dependent phosphorylation of the two specific serine residues of IkB proteins and the subsequent rapid degradation of the IkB proteins by the ubiquitin– proteosome pathway [59 – 67]. Biochemical characterization of the IkB kinase (IKK) revealed that it apparently is a large, multisubunit kinase of about 700 kDa [68]. The IkB kinase-1 (IKK1, IKKa), which phosphorylates the two specific serine residues in the aminoterminal portion of IkB, was identified using biochemical purification [69] and yeast two-hybrid screening with NIK as bait [70]. IKK2 (IKKb) was subsequently cloned by several laboratories [71–73]. Based on transient transfection experiments, NIK and MEKK1 appear to be upstream kinases that activate IKK. However, these two kinases seem to mediate distinct signals since MEKK1 preferentially activates IKKb, whereas NIK efficiently activates both IKKa and IKKb [74]. The regulation of MEKK1 and NIK could be different. MEKK1 associates with TRAF2 in a TNFa stimulation-dependent manner. Thus, the direct activator of MEKK1 could be an adapter protein TRAF2. Although NIK has been isolated as a TRAF2-binding protein, this association has not been shown to be signal-dependent. A recent study showed that TAK1 associates with TRAF6 in an IL-1 stimulation-dependent manner and that TAK1 phosphorylates and activates NIK [49]. Thus, two sequential MAP3Ks, TAK1 and NIK, are present in the downstream signal of TRAF6 (Fig. 3). Another description of MEKK1 activator, ECSIT, was reported recently [75]. MEKK1 is a 195-kDa protein that is thought to be activated in part via proteolytic cleavage by a caspase. ECSIT has been identified as a TRAF6-binding protein and its overexpression enhances MEKK1 processing. Thus, TRAF6mediated signals may induce MEKK1 processing to activate IKKb, although this has not yet been proven. NFkB activation prevents apoptosis induced by TNFa or chemical compounds used for cancer therapy [76 –78]. Several reports suggest that a set of antiapoptotic genes is induced by TNFRI-mediated NFkB activation. Based on their NFkB-dependent expression and antiapoptotic function, the c-IAP (inhibitor of apoptosis protein 1), TRAF1, and A20 [79 – 82] have been proposed to play some role in NFkB-mediated prevention of apoptosis. C-IAP1 was shown to inhibit members of the caspase family whose activation leads

TRAF PROTEINS

to cell death. The mechanisms of TRAF1- and A20mediated inhibition of apoptosis are unclear. TRAF-ASSOCIATED PROTEINS

To develop a comprehensive understanding of the molecular mechanism of TRAF-mediated signals, a number of researchers have been looking for and have identified several proteins that associate with TRAF proteins. TANK (TRAF family member-associated NFkB activator) [83] and I-TRAF (TRAF-interacting protein) [84] were identified by the yeast two-hybrid system using TRAF3 and TRAF2, respectively, as bait. I-TRAF and TANK are identical and bind all members of TRAF except TRAF4 through the TRAF-C domain. However, the function of I-TRAF/TANK is not clear due to conflicting results. One report shows that TANK and TRAF2 activate NFkB synergistically, whereas another shows that I-TRAF apparently inhibits TRAF2-mediated NFkB activation. This discrepancy may result from the experimental conditions, but it remains to be resolved. The zinc finger A20 was reported to be induced by NFkB which is activated by the TNFa stimulation, the crosslinking of CD40, and the expression of LMP1 protein [81]. Expression of A20 renders B cell lines resistant to apoptosis [82]. A20 interacts with TRAF1 and TRAF2 to inhibit TRAF2-mediated NFkB activation [85]. A20 also interacts with TRAF6 and interferes with IL-1-induced NFkB activation at the level of TRAF6 [86]. Thus, A20 has a feedback system in which it could inhibit its own expression. TRIP (TRAF-interacting protein) was identified by the yeast two-hybrid using TRAF1 as bait, and TRIP interacts with both TRAF1 and TRAF2 in mammalian cells [87]. TRIP contains an amino-terminal RING finger followed by a putative coiled-coil structure. TRIP inhibits TNFa- and CD30-induced NFkB activation but does not inhibit IL-1-mediated activation. Peg3, a product of a maternally imprinted gene, interacts with TRAF2 but not with other TRAFs [88]. Overexpression of Peg3 activates NFkB. Furthermore, Peg3 synergistically enhances NFkB activation induced by either TRAF2 or TNFa, whereas a dominantnegative mutant of Peg3 significantly inhibits TRAF2and TNFa-induced NFkB activation. Therefore, Peg3 is thought to be the first imprinted participant in the NFkB activation pathway. However, Peg3 is also presumed to be involved in p53/Myc-mediated apoptosis. Thus, the role of Peg3-mediated NFkB activation in the regulation of cell survival remains to be elucidated. C-IAP1 and c-IAP2 have been identified by purification of proteins that bind to the cytoplasmic tail of TNF receptor type II and subsequent peptide sequencing [89]. Casper has sequence similarity with caspase-8 and interacts with TRAF1 and TRAF2 through their

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TRAF-N domain [90]. Casper induces apoptosis and competes with c-IAP1, which also interacts with the TRAF-N domain, for binding to TRAF2. Since c-IAP1 is thought to block apoptosis [79], competition between Casper and c-IAP1 for binding to TRAF2 could be a mechanism by which TRAF-mediated signals regulate cell death or survival. RIP, first identified as a Fas-binding protein, has a DD [91]. RIP was shown to be recruited to TNFRI via another DD containing protein, TRADD, in a stimulation-dependent manner [92]. Overexpression experiments revealed that RIP interacts with TRAF1, TRAF2, and TRAF3. Therefore, it is thought that the cytoplasmic tail of the TNFRI trimer could signal dependently recruit TRADD, which in turn recruits RIP and TRAF2 to transduce the signal leading to JNK and IKK activation (Fig. 3) PHYSIOLOGICAL ROLES OF TRAF PROTEINS

Interaction of TRAF with various receptors has mostly been studied in nonphysiological conditions such as overexpression of TRAFs by transient transfection or use of GST–receptor fusion protein expressed in bacteria. Thus, the results obtained from in vitro studies do not necessarily explain the role of TRAF proteins in vivo. To determine whether the in vitro findings correlate with in vivo findings and to determine whether TRAFs have additional functions, mice with genetic impairment in various TRAF genes have been generated. In TRAF2-deficient (TRAF2 2/2) mice generated by targeting two exons encoding RING finger [93], the frequency of viable TRAF2 2/2 offspring is about 10%, which is less than expected based on the Mendelian ratio and which suggests that TRAF2 could play some role in embryogenesis. Viable TRAF2 2/2 mice are normal at birth but become runted, and most of them die at the age of 10 –14 days. TRAF2 has been thought to mediate TNFa signaling to activate both JNK and NFkB. However, examination of TRAF2 2/2 embryonic fibroblasts revealed a severe reduction in JNK activation and only a mild effect on NFkB activation. TRAF5 may compensate for the loss of TRAF2 in NFkB activation. Mice carrying the transgene in which a dominant-negative type of TRAF2 is expressed under the control of a lymphocyte-specific promoter were generated [94]. In such mice, JNK activation by TNFa or CD40 is significantly reduced, whereas NFkB activation is not altered. Thus, two independent lines of evidence indicate that TNFR signals require TRAF2 for activation of JNK but not of NFkB. TRAF2 2/2 hematopoietic cells and embryonic fibroblast cells were shown to be more sensitive than those from wild-type mice to TNFa-induced cell death, indicating that TRAF2 is essential for antiapoptotic signaling. Devel-

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opment of erythroid and myeloid lineages and of thymocytes is normal in the absence of TRAF2. Although complete blockade of B cell development was not observed, TRAF2 2/2 mice had fewer pre-B cells (B220 1, CD43 2) than wild-type mice. TRAF3 2/2 mice also appear normal at birth but become progressively runted [14] and die by 10 days of age. Although a progressive depletion occurs in all lineages of white cells in the periphery of TRAF3 2/2 mice, fetal liver cells from day 14 TRAF3 2/2 embryos can reconstitute T cell, B cell, granulocytic, and erythroid lineages in lethally irradiated mice. However, in these reconstituted mice, immune responses to T-dependent antigen and T cell response to ovalbumin sensitization are significantly reduced. Although TRAF3 has been identified as a CD40-binding protein [3], the proliferative responses of B cells from wild-type and TRAF3 2/2 mice to CD40 ligand stimulation are not different. Furthermore, CD40-mediated upregulation of CD23 is normal in TRAF3 2/2 mice. Thus, TRAF3 is not required for CD40 signaling. Since TRAF3, TRAF2, and TRAF5 bind to the same domain in CD40 cytoplasmic tail, TRAF2 and/or TRAF5 could compensate for the loss of TRAF3. Disruption of the TRAF5 gene resulted in the much milder impairment in mutant mice than that in mice deficient in other TRAFs including TRAF2, TRAF3, and TRAF6 [95]. TRAF5 2/2 mice are born at the expected Mendelian ratios and are healthy with no obvious abnormalities. Surface marker analysis of thymocytes, splenocytes, and lymph node cells revealed that the composition of lymphocytes is not altered in TRAF5 2/2 mice. Based on the in vitro experiments, TRAF5 is thought to be implicated in the signal emanating from CD27, CD30, CD40, and lymphotoxin-b receptor (LTb-R) [7, 8, 96, 97]. The development of lymph nodes and Peyer’s patches were reported to be normal in TRAF5 2/2 mice, indicating that TRAF5 is not required for formation of secondary lymphoid organs whose generation requires LTb-R signaling [98]. Although activation of JNK and NFkB by TNFa, CD27, and CD40 are not altered, B cells from TRAF5 2/2 mice have defects in proliferation and induction of CD23, CD54, CD80, CD86, and Fas in response to CD40 stimulation. Thus, TRAF5 is required for CD40 signaling. As described above, TRAF6 is divergent from the other TRAFs (Table 1) and is thought to mediate signals emanating from IL-1 receptor, TLRs, CD40, RANK, and p75 NGF receptor [9, 10, 30 –33]. Similar to TRAF2 2/2 mice, the frequency of viable TRAF6 2/2 offspring is about 12%, which is less than expected based on the Mendelian ratio. This suggests that TRAF6 could also play some important roles in embryogenesis. Viable TRAF6 2/2 mice appear normal at birth but become smaller than their normal littermates by day 6. The mutant animals become more runted and die at 17

to 19 days old. Our group [99] and Lomaga et al. [100] have generated TRAF6 2/2 mice. In both studies, TRAF6 2/2 mice exhibited severe osteopetrosis, a disorder of bone remodeling caused by impaired formation or function of osteoclasts, which are multinuclear and come into contact with bone when mature. However, the mechanisms of osteopetrosis appear to be different in each report. In our experiments, staining of bone sections for tartrate-resistant acid phosphatase (TRAP) that is highly expressed in osteoclasts showed only a few weakly TRAP 1 mononuclear cells. Thus, we concluded that TRAF6 is required for differentiation of the osteoclasts from the progenitor cells. However, Lomaga et al. reported a normal number of multinuclear TRAP 1 osteoclasts, which lacked contact with bone surfaces in TRAF6 2/2 mice. Thus, they concluded that TRAF6 is required for the maturation of osteoclasts and is dispensable for differentiation. Although their targeted exon is different from ours, this discrepancy remains to be clarified. Analysis of ODF 2/2 (osteoclast differentiation factor also known as OPGL, osteoprotegrin ligand) mice showed that the interaction of RANK with ODF is essential for osteoclast formation [101]. RANK has been shown to associate with TRAFs 1, 2, 3, 5, and 6 in in vitro experiments [30 –32]. TRAF6 interacted with a distinctive membrane-proximal region of the RANK cytoplasmic tail that differed from the regions binding other TRAFs. Furthermore, deletion of the TRAF6-binding site of RANK completely prevented the RANK-dependent activation of NFkB [31], suggesting that NFkB activation by the TRAF6mediated RANK signaling could be essential for the formation of functional osteoclasts. The lack of functional osteoclast formation reported in the p50/p52 double-knockout mice [102, 103] is consistent with this idea since p50 or p52 is required to form transcriptionally active heterodimers [57]. One possibility is that NFkB activation leads to the expression of one or more antiapoptotic genes, which would be consistent with the report that ODF enhances Bcl-xL expression [104, 105]. The presence of a normal number of osteoclast precursor cells in the spleen of TRAF6 2/2 mice suggests that ODF–RANK interaction leads to apoptosis of precursor cells from TRAF6 2/2 mice during the course of differentiation. It has been shown that signal-dependent interaction of TRAF6 with TAK1 results in the activation of both IKK and JNK [49]. Thus, the role of JNK activation in osteoclast formation cannot be ruled out. IL-1-induced proliferation of thymocytes and the activation of NFkB and JNK of embryonic fibroblasts in response to IL-1 were absent in TRAF6 2/2 mice [99, 100], indicating that TRAF6 is essential in IL-1 signaling. Proliferation and NFkB activation of splenic B cells in response to anti-CD40 antibody and LPS were also significantly reduced in this mutant, indicating that TRAF6 is involved in CD40 and LPS signaling.

TRAF PROTEINS

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Considering these findings in TRAF6 2/2mice, TRAF6 apparently mediates two members of the TNF receptor superfamily, CD40 and RANK, and three members of the Toll/IL-1 receptor family, IL-1 receptor, TLR2, and TLR4. Impairment of p75 NGF receptor in TRAF6 2/2 mice has not been documented. Transgenic mice have been generated that carry TRAF1 expression plasmid under the control of a lymphocyte-specific promoter [13]. TRAF1 overexpression inhibits T-cell-receptor-induced apoptosis of CD8 1 T cells, suggesting that TRAF1 could regulate apoptotic signals. However, TRAF1 2/2 mice must be necessary to elucidate the physiological role of TRAF1. Neither TRAF1 2/2 nor TRAF4 2/2 mice have been reported.

6.

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