An aliquot of each TCL was analyzed by immunoblotting with an anti-LMP1 or an anti-GFP antibody

An aliquot of each TCL was analyzed by immunoblotting with an anti-LMP1 or an anti-GFP antibody. DISCUSSION Extensive studies using mice with genetic mutations in NF-B components have revealed essential roles for NF-B activation in lymphocyte development, activation, proliferation, and survival (22). human B cells. Furthermore, transient expression of STAP-2 in EBV-positive human B cells decreased cell growth. Finally, STAP-2 knockout mouse embryonic fibroblasts showed enhanced LMP1-induced cell growth. These results suggest that STAP-2 acts as an endogenous negative regulator of EBV LMP1-mediated signaling through TRAF3 and TRADD. Epstein-Barr virus (EBV) belongs to the herpesvirus group Rabbit polyclonal to ZNF697 and can infect most human individuals. Although the majority of infected carriers remain asymptomatic, the virus may sometimes play a role in the pathogenesis of lymphoid and epithelial malignancies, such as Burkitt’s lymphoma, Hodgkin’s lymphoma, and nasopharyngeal carcinoma (42). EBV-infected cells express several latent antigens, including EBV nuclear antigens and latent membrane proteins (LMPs). These EBV-derived antigens activate resting B cells to produce proliferating Entecavir lymphoblasts and to provide survival signals for maintaining infected cells. Among EBV-derived antigens, LMP1 by itself can transform rodent fibroblasts and render them tumorigenic in nude mice (30, 46). Transgenic mice expressing LMP1 under the control of a mouse immunoglobulin H (IgH) enhancer and Entecavir a VH promoter develop B cell lymphomas at an accelerated rate Entecavir as they age (19). In LMP1 transgenic mice, it has been demonstrated that LMP1 is expressed in lymphoma tissues at high levels and that several NF-B-activating oncogenes, including A20 and Bcl-2, are upregulated in the LMP1-expressing lymphomas compared with those in LMP1-negative counterparts (19). Structurally, LMP1 is an integral membrane protein of 386 amino acids (aa) that consists of a short cytoplasmic N-terminal domain (aa 1 to 24), six transmembrane domains (aa 25 to 186), and a long cytoplasmic C-terminal tail (aa 187 to 386) (1, 21, 23). The cytoplasmic C-terminal tail contains two C-terminal activation regions (CTARs), CTAR1 and CTAR2, which are required for LMP1 signaling through tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) (1, 13). CTAR1 contains a consensus TRAF-binding motif (PXQXT ) and can bind to TRAF1, -2, -3, and -5 (1, 2, 6, 18, 31). CTAR2 interacts with the TNFR-associated death domain (TRADD) protein, the receptor-interacting protein (RIP) (1, 14, 15), and the BS69 protein (45). LMP1-induced signals through these two domains induce p100 NF-B (p100/NF-B2) and p105 NF-B (p105/NF-B1) precursors and upregulate their processing into p52 and p50 (13, 20, 25, 29, 32), indicating that LMP1 is involved in both canonical NF-B upregulation and noncanonical processing of p100 into p52. The binding of LMP1 to TRAFs and TRADD also initiates the formation of a signaling complex that leads to the activation of mitogen-activated protein kinase Entecavir p38 (7). In addition, the activation of c-Jun N-terminal kinase (JNK) by LMP1 is mediated through CTAR1 and CTAR2 (3, 8). BS69 has been shown to be involved in LMP1-induced JNK activation by interacting with TRAF6 (45). Recently, we cloned two novel adaptor molecules, signal-transducing adaptor protein 1 (STAP-1) and STAP-2 (26, 27). STAP-1 was identified as a c-was 0.01. Total RNA samples isolated from these cells were subjected to RT-PCR analysis using STAP-2 Entecavir or G3PDH primers. An aliquot of each total cell lysate (TCL) was analyzed by immunoblotting (IB) with an anti-LMP1 antibody. NS, nonspecific band. The STAP-2 expression levels were also quantified by RT and quantitative real-time PCR analysis. Data represent the levels of STAP-2 mRNA normalized by that of G3PDH mRNA as an internal control and are expressed relative to the value at time zero. Data represent the means of duplicate PCR determinations, which generally varied by less than 10%. (b) MEFs in 24-well plates were.