Familial cylindromatosis is an autosomal dominant predisposition to tumours of skin appendages called cylindromas. Familial cylindromatosis is caused by mutations in a gene encoding the CYLD protein of previously unknown function. Here we show that CYLD is a deubiquitinating enzyme that negatively regulates activation of the transcription factor NF-kappaB by specific tumour-necrosis factor receptors (TNFRs). Loss of the deubiquitinating activity of CYLD correlates with tumorigenesis. CYLD inhibits activation of NF-kappaB by the TNFR family members CD40, XEDAR and EDAR in a manner that depends on the deubiquitinating activity of CYLD. Downregulation of CYLD by RNA-mediated interference augments both basal and CD40-mediated activation of NF-kappaB. The inhibition of NF-kappaB activation by CYLD is mediated, at least in part, by the deubiquitination and inactivation of TNFR-associated factor 2 (TRAF2) and, to a lesser extent, TRAF6. These results indicate that CYLD is a negative regulator of the cytokine-mediated activation of NF-kappaB that is required for appropriate cellular homeostasis of skin appendages.

The cytoplasmic C-terminus of Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) is essential for B lymphocyte growth transformation and is now shown to interact with a novel human protein (LMP1-associated protein 1 [LAP1]). LAP1 is homologous to a murine protein, tumor necrosis factor receptor-associated factor 2 (TRAF2), implicated in growth signaling from the p80 TNFR. A second novel protein (EBI6), induced by EBV infection, is the human homolog of a second murine TNFR-associated protein (TRAF1). LMP1 expression causes LAP1 and EBI6 to localize to LMP1 clusters in lymphoblast plasma membranes, and LMP1 coimmunoprecipitates with these proteins. LAP1 binds to the p80 TNFR, CD40, and the lymphotoxin-beta receptor, while EBI6 associates with the p80 TNFR. The interaction of LMP1 with these TNFR family-associated proteins is further evidence for their role in signaling and links LMP1-mediated transformation to signal transduction from the TNFR family.

The Epstein-Barr virus (EBV) transforming protein LMP1 appears to be a constitutively activated tumor necrosis factor receptor (TNFR) on the basis of an intrinsic ability to aggregate in the plasma membrane and an association of its cytoplasmic carboxyl terminus (CT) with TNFR-associated factors (TRAFs). We now show that in EBV-transformed B lymphocytes most of TRAF1 or TRAF3 and 5% of TRAF2 are associated with LMP1 and that most of LMP1 is associated with TRAF1 or TRAF3. TRAF1, TRAF2, and TRAF3 bind to a single site in the LMP1 CT corresponding to amino acids (aa) 199 to 214, within a domain which is important for B-lymphocyte growth transformation (aa 187 to 231). Latent-infection membrane protein 1 (LMP1) of EpsteinBarr virus (EBV) is essential for the ability of EBV to induce continuous proliferation of primary human B lymphocytes (20). LMP1 comprises an N-terminal cytoplasmic domain (amino acids [aa] 1 to 24), six markedly hydrophobic transmembrane domains separated by short reverse turns (aa 25 to 186), and a long cytoplasmic carboxyl terminus (CT) (aa 187 to 386) (6) (Fig. 1). Recombinant EBV genetic analyses indicate that the six transmembrane domains and the CT first 45 aa are sufficient for primary B-lymphocyte growth transformation in the context of a specifically mutated EBV recombinant (18,20,21,23).Several lines of evidence indicate that LMP1 is a constitutively activated tumor necrosis factor family receptor (TNFR) similar to activated CD40. First, expression of LMP1 in B lymphoblasts or in primary B lymphocytes results in NF-B activation and expression of Bcl-2, activation markers, adhesion molecules, and autocrine growth factors, all of which are induced in normal B lymphocytes after stimulation by 11,16,24,26,30,33,41,42). Second, a significant fraction of LMP1 constitutively aggregates in a patch in the plasma membrane of lymphoblastoid cell lines (LCLs) (12,26,27,29). The aggregation is dependent on the six hydrophobic transmembrane domains and is essential for transformation. Thus, LMP1 comprising only the last five transmembrane domains and the entire CT, i.e., LMP1 aa 44 to 386, diffusely distributes in the plasma membrane and is nontransforming (20, 26), whereas LMP1 comprising the last two transmembrane domains and the CT, i.e., LMP1 aa 129 to 386, diffusely distributes in all cytoplasmic membranes and has no effect on lymphoblasts (16,26,41). Third, the LMP1 CT aa 187 to 231, which are sufficient for transformation when linked to the six transmembrane domains [LMP1(1-231)] (23), interact with a protein that also interacts with the CD40 cytoplasmic domain (3,15,31,39); a CD40 cytoplasmic domain nonsignaling mutant fails to interact with this protein (15). This protein (previously called LAP1, CD40bp, CRAF1, or CAP1) is now designated TRAF3 because of its extensive C-terminal "TRAF domain" homology to TNFR-associated factor 2 (TRAF2) and TRAF1 (also called EBI6) (36, 37). The LMP1 CT can also bind to TRAF2 in vitro and associates with TRAF1, TRAF2, and TRAF3 in the plasma membrane of tran...

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.

Epstein-Barr virus nuclear antigen 2 (EBNA 2) activates transcription of specific genes and is essential for B-lymphocyte transformation. EBNA 2 has an acidic activation domain which interacts with general transcription factors TFIIB, TFIIH, and TAF40. We now show that EBNA 2 is specifically bound to a novel nuclear protein, p100, and that p100 can coactivate gene expression mediated by the EBNA 2 acidic domain. The EBNA 2 acidic domain was used to affinity purify p100. cDNA clones encoding the p100 open reading frame were identified on the basis of peptide sequences of the purified protein. Antibody against p100 coimmunoprecipitated p100 and EBNA 2 from Epstein-Barr virus-transformed lymphocyte extracts, indicating that EBNA 2 and p100 are complexed in vivo. p100 overexpression in cells specifically augmented EBNA 2 acidic domainmediated activation. The coactivating effect is probably mediated by p100 interaction with TFIIE. Bacterially expressed p100 specifically adsorbs TFIIE from nuclear extracts, and in vitro-translated p56 or p34 TFIIE subunit can independently bind to p100. p100 also appears to be essential for normal cell growth, since cell viability was reduced by antisense p100 RNA and restored by sense p100 RNA expression.Epstein-Barr virus (EBV) is a human herpesvirus which establishes latent infection in B lymphocytes (for reviews, see references 31 and 32). Latently infected lymphocytes are growth transformed by virus infection, and their proliferation can be lethal in immunocompromised patients. The EBV genome encodes six nuclear proteins (EBNAs) and two integral membrane proteins (LMPs) in latently infected B lymphocytes.EBNA 2 is one of the first two genes expressed in EBVinfected B lymphocytes (2, 47). In these cells, EBNA 2 activates transcription of the viral genes encoding LMP1 (1, 60) and LMP2 (64) and of the cellular genes CD21 (9), CD23 (56-58), and c-fgr (34). Molecular genetic analyses indicate that EBNA 2 is essential for EBV-mediated B-lymphocyte transformation (8,24). Further analyses define two essential domains in EBNA 2 that are linked to its role as a transcription activator: an acidic activation domain (6, 7) and a domain that interacts with at least two cellular sequence-specific DNAbinding proteins, J (CBF1) and PU.1 (22,25,28,61). EBNA 2 response elements near the LMP1 (17, 55), LMP2A (63), Cp (50), and cellular CD23 (58) promoters usually have nearby J and PU.1 sites. Thus, genetic and biochemical data indicate that EBNA 2 is brought to response elements by interaction with sequence-specific DNA-binding proteins and EBNA 2 then activates transcription through its C-terminal acidic domain (amino acids 426 to 483).The experiments reported here focus on the identification of cellular proteins that interact with the EBNA 2 acidic domain. The EBNA 2 acidic domain (amino acids 426 to 483) has a core region (amino acids 449 to 462) which has about 25% of the activity of the larger domain (6). Mutation of Trp-454 to Thr (in the protein designated EBNA 2,T 454 ) is a null mutation fo...

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-KcB activation, we have evaluated the role of TRAF2 in LMPl-mediated NF-ucB activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF2A6-86) inhibits NF-KB activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2A6-86 inhibits NF-,cB activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-ucB activation through the last 55 amino acids of the CT by less than 40%o. A TRAF interacting protein, TANK, inhibits NF-.cB activation by more than 70%16 from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-cB activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-KB activation by the last 55 amino acids of the LMP1 CT.

Epstein–Barr virus latent membrane protein 1 (LMP1) activation of NF-κB is critical for Epstein–Barr virus-infected B lymphocyte survival. LMP1 activates the IκB kinase complex and NF-κB through two cytoplasmic signaling domains that engage tumor necrosis factor receptor-associated factor (TRAF)1/2/3/5 or TRADD and RIP. We now use cells lacking expression of TRAF2, TRAF5, TRAF6, IKKα, IKKβ, IKKγ, TAB2, IL-1 receptor-associated kinase (IRAK)1, or IRAK4 to assess their roles in LMP1-mediated NF-κB activation. LMP1-induced RelA nuclear translocation was similar in IKKα knockout (KO) and WT murine embryo fibroblasts (MEFs) but substantially deficient in IKKβ KO MEFs. NF-κB-dependent promoter responses were also substantially deficient in IKKβ KO MEFs but were hyperactive in IKKα KO MEFs. More surprisingly, NF-κB responses were near normal in TRAF2 and TRAF5 double-KO MEFs, IKKγ KO MEFs, TAB2 KO MEFs, and IRAK4 KO MEFs but were highly deficient in TRAF6 KO MEFs and IRAK1 KO HEK293 cells. Consistent with the importance of TRAF6, LMP1-induced NF-κB activation in HEK293 cells was inhibited by expression of dominant-negative TAB2 and Ubc13 alleles. These data extend a role for IKKα in IKKβ regulation, identify an unusual IKKβ-dependent and IKKγ-independent NF-κB activation, and indicate that IRAK1 and TRAF6 are essential for LMP1-induced NF-κB activation.

The binding of heterotrimeric lymphotoxin, LT␣ 1 ␤ 2 , to the LT␤ receptor (LT␤R), a member of the tumor necrosis factor receptor (TNFR) superfamily, induces nuclear factor B (NF-B) activation and cell death in HT29 adenocarcinoma cells. We now show that treatment with LT␣ 1 ␤ 2 or agonistic LT␤R antibodies causes rapid recruitment of TNFRassociated factor 3 (TRAF3) to the LT␤R cytoplasmic domain. Further, stable overexpression of a TRAF3 mutant that lacks the RING and zinc finger domains inhibits LT␤R-mediated cell death. The inhibition is specific for LT␤R cell death signaling, since NF-B activation by LT␣ 1 ␤ 2 and Fasmediated apoptosis are not inhibited in the same cells. The mutant and endogenous TRAF3s are both recruited at equimolar amounts to the LT␤R, suggesting that the mutant disrupts the function of the signaling complex. These results implicate TRAF3 as a critical component of the LT␤R death signaling complex and indicate that at least two independent signaling pathways are initiated by LT␤R ligation.