CD40 signalings play crucial roles in B-cell function.To identify molecules which transduce CD40 signalings, we have utilized the yeast two-hybrid system to clone cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer, designated TRAF6, has been molecularly cloned. TRAF6 has a tumor necrosis factor receptor (TNFR)-associated factor (TRAF) domain in its carboxyl terminus and has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other TRAF family proteins. TRAF6 does not associate with the cytoplasmic tails of TNFR2, CD30, lymphotoxin-␤ receptor, and LMP1 of Epstein-Barr virus. Deletion analysis showed that residues 246 -269 of CD40 which are required for its association with TRAF2, TRAF3, and TRAF5 are dispensable for its interaction with TRAF6, whereas residues 230 -245 were required. Overexpression of TRAF6 activates transcription factor NFB, and its TRAF-C domain suppresses NFB activation triggered by CD40 lacking residues 246 -277. These results suggest that TRAF6 could mediate the CD40 signal that is transduced by the amino-terminal domain (230 -245) of the CD40 cytoplasmic region and appears to be independent of other known TRAF family proteins.

Definitive risk factors for the development of adult T-cell leukemia (ATL) among asymptomatic human T-cell leukemia vi-

No abstract

The functions of the ecdysone-induced DHR3 and E75B orphan nuclear receptors in the early stages of Drosophila metamorphosis were investigated. DHR3 represses the ecdysone induction of early genes turned on by the pulse of ecdysone that triggers metamorphosis. It also induces betaFTZF1, an orphan nuclear receptor that is essential for the appropriate response to the subsequent prepupal pulse of ecdysone. The E75B receptor, which lacks a complete DNA binding domain, inhibits this inductive function by forming a complex with DHR3 on the betaFTZF1 promoter, thereby providing a timing mechanism for betaFTZF1 induction that is dependent on the disappearance of E75B.

TRAF2 is a RING finger protein that regulates the cellular response to stress and cytokines by controlling JNK, p38 and NF-jB signaling cascades. Here, we demonstrate that TRAF2 ubiquitination is required for TNFa-induced activation of JNK but not of p38 or NF-jB. Intact RING and zinc finger domains are required for TNFa-induced TRAF2 ubiquitination, which is also dependent on Ubc13. TRAF2 ubiquitination coincides with its translocation to the insoluble cellular fraction, resulting in selective activation of JNK. Inhibition of Ubc13 expression by RNAi resulted in inhibition of TNFa-induced TRAF2 translocation and impaired activation of JNK but not of IKK or p38. TRAF2 aggregates in the cytoplasm, as seen in HodgkinReed-Sternberg lymphoma cells, resulting in constitutive NF-jB activity but failure to activate JNK. These findings demonstrate that the TRAF2 RING is required for Ubc13-dependent ubiquitination, resulting in translocation of TRAF2 to an insoluble fraction and activation of JNK, but not of p38 or NF-jB. Altogether, our findings highlight a novel mechanism of TRAF2-dependent activation of diverse signaling cascades that is impaired in HodgkinReed-Sternberg cells.

Signals emanating from CD40 play crucial roles in B-cell function. To identify molecules that transduce CD40 signalings, we have used the yeast two-hybrid system to clone cDNAs encoding proteins that bind the cytoplasmic tail of CD40. A cDNA encoding a putative signal transducer protein, designated TRAF5, has been molecularly cloned.TRAF5 has a tumor necrosis factor receptor-associated factor (TRAF) domain in its carboxyl terminus and is most homologous to TRAF3, also known as CRAF1, CD40bp, or LAP-1, a previously identified CD40-associated factor. The amino terminus has a RING finger domain, a cluster of zinc fingers and a coiled-coil domain, which are also present in other members of the TRAF family protein except for TRAF1. In vitro binding assays revealed that TRAF5 associates with the cytoplasmic tail of CD40, but not with the cytoplasmic tail of tumor receptor factor receptor type 2, which associates with TRAF2. Based on analysis of the association between TRAF5 and various CD40 mutants, residues 230-269 of CD40 are required for the association with TRAF5. In contrast to TRAF3, overexpression of TRAF5 activates transcription factor nuclear factor #cB. Furthermore, amino-terminally truncated forms ofTRAF5 suppress the CD40-mediated induction of CD23 expression, as is the case with TRAF. These results suggest that TRAF5 and TRAF3 could be involved in both common and distinct signaling pathways emanating from CD40.CD40 is expressed in late B cells in bone marrow, mature B cells, and certain accessory cells, including bone-marrow derived dendritic cells and follicular dendritic cells (1-3), and is a receptor for CD40 ligand (CD40L) present on activated CD4+ T cells (4). Signaling through CD40 rescues B cells from apoptosis induced by crosslinking of the surface immunoglobulin M (IgM) complex (5) and also induces B cells to differentiate and to undergo Ig isotype switching (6, 7). CD40L has been shown to be defective in patients with X-linked hyper IgM syndrome, whose B cells do not form germinal centers and produce only IgM and/or IgD (8). In addition, their B cells have the ability to switch from IgM to IgG, IgE, or IgA production in vitro by the stimulation of CD40 (9). This genetic evidence strongly supports the idea that the intercellular communication through CD40-CD40L is essential for germinal center formation and Ig class switching.CD40 is a member of the tumor necrosis factor receptor (TNFR) superfamily, which includes TNFR1 and TNFR2 (10, 11), lymphotoxin 13 receptor (12), Fas antigen (13), OX40 (14), CD30 (15), and the low-affinity nerve growth factor receptor (16), all of which share a ligand-binding domain composed of tandemly repeated cysteine-rich modules. Among these, Fas antigen and TNFR1 have significant similarity in their cytoplasmic domain over 46 amino acids, a part of the domain called a death domain, suggesting that these receptors could have either common or similar signaling mechanisms (13).Biochemical purification of receptor-associated proteins or the recently developed cDNA...

Overexpression of CD30 and constitutive NF-kappaB activation characterizes tumor cells of Hodgkin's disease (HD), Hodgkin and Reed-Sternberg (H-RS) cells. We report that in H-RS cells overexpression of CD30 leads to self-aggregation, recruitment of TRAF2 and TRAF5, and NF-kappaB activation, independent of CD30 ligand. CD30 and TRAF proteins co-localized in H-RS cell lines and in lymph nodes of HD. An adenovirus-vector carrying a decoy CD30 lacking the cytoplasmic region or a dominant negative IkappaBalpha mutant blocks NF-kappaB activation, down regulates IL-13 expression and induces apoptosis. Thus, in H-RS cells, ligand-independent activation of CD30 signaling drives NF-kappaB activation and this leads to constitutive cytokine expression, which provides a molecular basis for HD. Inhibition of NF-kappaB activation by adenovirus vector-mediated gene transfer may provide a novel strategy of cell- and target molecule-specific therapy for patients with HD.

Signals emanated from CD30 can activate the nuclear factor B (NFB). The two conserved subdomains, D1 and D2, in the C-terminal cytoplasmic region of CD30 were tested for interaction with two tumor necrosis factor receptor-associated factor (TRAF) proteins with NFB activating capacity, TRAF2 and TRAF5. TRAF5 is the newest member of the TRAF family that binds to lymphotoxin ␤ receptor and CD40. TRAF5, as well as TRAF2, interacted with the D2 subdomain of CD30 in vitro and in vivo. Deletion analysis by the yeast twohybrid system revealed that the C-terminal 22 and 30 amino acid residues are dispensable for interaction of TRAF5 and TRAF2 with CD30, respectively. Substitution of alanine for threonine at 463 abolished the interaction with TRAF2. Overexpression of the TRAF domain of TRAF2 or TRAF5 showed a dominant negative effect on CD30-mediated NFB activation. Simultaneous expression of these TRAF domains further suppressed the NFB activation, suggesting an interplay of these TRAF proteins. Expression of TRAF2 and TRAF5 mRNA was demonstrated in T-and B-cell lines that express CD30. Taken together, our results indicate that TRAF2 and TRAF5 directly interact with CD30 and are involved in NFB activation by CD30 signaling.CD30 is a member of TNF 1 receptor superfamily that comprises a group of cysteine-rich receptor proteins such as CD27, CD40, and Fas antigen (1-4). Biochemical studies of CD30 as well as functional studies of the ligand for CD30 (CD30L) provided strong evidence to support regulatory roles for CD30 in lymphocytes (5-7). CD30L induces various biological effects on human CD30-positive cell lines such as activation, proliferation, differentiation, and cell death, depending on cell type, stage of differentiation, transformation status, and the presence of other stimuli (7). Recently, it was reported that CD30-deficient mice showed impaired negative selection in the thymus (8) and that CD30 is involved in signaling TCR-mediated cell death of T-cell hybridoma (9). As for signal transduction of CD30, Ellis et al. reported the induction of Ca 2ϩ influx by cross-linking CD30 on Jurkat cells (10), and signals mediated by CD30 were seen to regulate gene expression through activation of NFB (11,12).Because the cytoplasmic tail of receptors of the TNF receptor family does not have intrinsic catalytic activity such as kinase activity, it was considered that molecules that associate with these receptors mediate signal transduction. Putative signal transducing proteins that associate with TNF receptor type II were cloned and named TNF receptor-associated factor (TRAF) 1 and 2 (13). Subsequently, TRAF3 or CRAF1 (CD40 bp, CAP1, LAP1) was identified as the CD40 signal transducing molecule reviewed in Ref. 18). We have recently cloned TRAF5 that associates with lymphotoxin ␤ receptor and CD40 and mediates NFB activating signals (19,20).We have found that an approximately 100-amino acid sequence of the C-terminal region of the CD30 cytoplasmic region was highly conserved among human, rat, and mouse CD30 protein, and in thi...