George Mosialos

The Epstein-Barr virus transforming protein LMP1 engages signaling proteins for the tumor necrosis factor receptor family

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.

CYLD is a deubiquitinating enzyme that negatively regulates NF-κB activation by TNFR family members

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-κB by specific tumour-necrosis factor receptors (TNFRs). Loss of the deubiquitinating activity of CYLD correlates with tumorigenesis. CYLD inhibits activation of NF-κB 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-κB. The inhibition of NF-κB 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-κB that is required for appropriate cellular homeostasis of skin appendages.

Association of TRAF1, TRAF2, and TRAF3 with an Epstein-Barr virus LMP1 domain important for B-lymphocyte transformation: role in NF-kappaB activation.

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). Further deletional and alanine mutagenesis analyses and comparison with TRAF binding sequences in CD40, in CD30, and in the LMP1 of other lymphycryptoviruses provide the first evidence that PXQXT/S is a core TRAF binding motif. The negative effects of point mutations in the LMP1(1-231) core TRAF binding motif on TRAF binding and NF-kappaB activation genetically link the TRAFs to LMP1(1-231)-mediated NF-kappaB activation. NF-kappaB activation by LMP1(1-231) is likely to be mediated by TRAF1/TRAF2 heteroaggregates since TRAF1 is unique among the TRAFs in coactivating NF-kappaB with LMP1(1-231), a TRAF2 dominant-negative mutant can block LMP1(1-231)-mediated NF-kappaB activation as well as TRAF1 coactivation, and 30% of TRAF2 is associated with TRAF1 in EBV-transformed B cells. TRAF3 is a negative modulator of LMP1(1-231)-mediated NF-kappaB activation. Surprisingly, TRAF1, -2, or -3 does not interact with the terminal LMP1 CT aa 333 to 386 which can independently mediate NF-kappaB activation. The constitutive association of TRAFs with LMP1 through the aa 187 to 231 domain which is important in NF-kappaB activation and primary B-lymphocyte growth transformation implicates TRAF aggregation in LMP1 signaling.

Identification of TRAF6, a Novel Tumor Necrosis Factor Receptor-associated Factor Protein That Mediates Signaling from an Amino-terminal Domain of the CD40 Cytoplasmic Region

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-β 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 NFκB, and its TRAF-C domain suppresses NFκB 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.

The Epstein-Barr Virus Nuclear Protein 2 Acidic Domain Forms a Complex with a Novel Cellular Coactivator That Can Interact with TFIIE

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 domain-mediated 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 transformation: involvement of latent membrane protein 1-mediated activation of NF-κB

Epstein-Barr virus (EBV) transforms resting primary human B lymphocytes into indefinitely proliferating lymphoblastoid cell lines in vitro and is associated with several human malignancies in vivo. Recombinant EBV genetic analyses combined with in vitro B lymphocyte transformation assays demonstrate that latent infection membrane protein 1 (LMP1) is essential for EBV-mediated lymphocyte transformation. LMP1 has no intrinsic enzymatic activity but instead aggregates cellular proteins of the tumor necrosis factor receptor signaling pathway to activate transcription factor NF-κB. Mutants rendering LMP1 defective in these protein interactions are impaired in their abilities to activate NF-κB in reporter gene assays. Concordantly, EBV recombinants with LMP1 mutations that are compromised for NF-κB activation are impaired for growth transformation. Thus, EBV-mediated growth transformation is genetically and biochemically linked to LMP1-mediated activation of NF-κB.

Epstein–Barr virus latent membrane protein 1 activation of NF-κB through IRAK1 and TRAF6

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.

A protein kinase-A recognition sequence is structurally linked to transformation by p59v-rel and cytoplasmic retention of p68c-rel.

The Rel family of proteins includes a number of proteins involved in transcriptional control, such as the retroviral oncoprotein v-Rel, c-Rel, the Drosophila melanogaster developmental protein Dorsal, and subunits of the transcription factor NF-kappa B. These proteins are related through a highly conserved domain of approximately 300 amino acids, called the Rel homology domain, that contains dimerization, DNA binding, and nuclear targeting functions. Also within the Rel homology domain, there is a conserved consensus sequence (Arg-Arg-Pro-Ser) for phosphorylation by cyclic AMP-dependent protein kinase (PKA). We used linker insertion mutagenesis and site-directed mutagenesis to determine the importance of this sequence for the transformation of avian spleen cells by v-Rel and the subcellular localization of c-Rel in chicken embryo fibroblasts (CEF). The insertion of 2 amino acids (Pro-Trp) within this sequence completely abolished transformation and transcriptional repression by v-Rel and resulted in a shift in the localization of c-Rel from cytoplasmic to nuclear in CEF. When the conserved Ser within the PKA recognition sequence was replaced by Ala, there was no significant effect on transformation and transcriptional repression by v-Rel or on cytoplasmic retention of c-Rel. However, when this Ser was changed to Asp or Glu, transformation and transcriptional repression by v-Rel were significantly inhibited and c-Rel showed a diffuse nuclear and cytoplasmic localization in CEF. Although a peptide containing the recognition sequence from v-Rel can be phosphorylated by PKA in vitro, this site is not constitutively phosphorylated to a high degree in vivo in transformed spleen cells incubated with okadaic acid. Our results indicate that the transforming and transcriptional repressing activities of v-Rel and the cytoplasmic retention of c-Rel are dependent on the structure of the conserved PKA recognition motif. In addition, they suggest that phosphorylation at the conserved PKA site could have a negative effect on transformation and transcriptional repression by v-Rel and induce the nuclear localization of c-Rel.

Inactivation of the Deubiquitinase CYLD in Hepatocytes Causes Apoptosis, Inflammation, Fibrosis, and Cancer

The tumor suppressor cylindromatosis (CYLD) inhibits the NFκB and mitogen-activated protein kinase (MAPK) activation pathways by deubiquitinating upstream regulatory factors. Here we show that liver-specific disruption of CYLD triggers hepatocyte cell death in the periportal area via spontaneous and chronic activation of TGF-β activated kinase 1 (TAK1) and c-Jun N-terminal kinase (JNK). This is followed by hepatic stellate cell and Kupffer cell activation, which promotes progressive fibrosis, inflammation, tumor necrosis factor (TNF) production, and expansion of hepatocyte apoptosis toward the central veins. At later stages, compensatory proliferation results in the development of cancer foci featuring re-expression of oncofetal hepatic and stem cell-specific genes. The results demonstrate that, in the liver, CYLD acts as an important regulator of hepatocyte homeostasis, protecting cells from spontaneous apoptosis by preventing uncontrolled TAK1 and JNK activation.

The BRG1- and hBRM-Associated Factor BAF57 Induces Apoptosis by Stimulating Expression of the Cylindromatosis Tumor Suppressor Gene

ABSTRACT Mutation of BRG1, hBRM, and their associated factors, INI1 and BAF57, in primary human tumors has suggested that inactivation of human SWI/SNF (hSWI/SNF) complexes may be involved in neoplastic transformation. BT549 is an invasive human breast carcinoma cell line that lacks expression of BAF57, a key hSWI/SNF subunit that mediates interaction with transcriptional activators and corepressors. In this study we investigated the role of BAF57 in suppressing tumorigenesis by establishing BT549 stable cell lines that expresses full-length BAF57 protein. BT549 clones expressing BAF57 demonstrated marked phenotypic changes, slow growth kinetics, and restoration of contact inhibition. Altered growth was found to be due in part to cell cycle arrest and induction of apoptosis. Furthermore, microarray analysis revealed that BAF57-mediated cell death was associated with up-regulation of proapoptotic genes including the tumor suppressor familial cylindromatosis (CYLD), which was found to be a direct target of BAF57 as determined by chromatin immunoprecipitation analysis. Increased expression of CYLD in BT549 cells induced apoptosis, while its suppression by small interfering RNA inhibited cell death in BAF57 expressing BT549 cells. These findings demonstrate the importance of BAF57 in cell growth regulation and provide a novel link between hSWI/SNF chromatin remodelers and apoptosis.