Localization of the Major NF-κB-activating Site and the Sole TRAF3 Binding Site of LMP-1 Defines Two Distinct Signaling Motifs

Tufts University, Бостон, Georgia, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 09/1997; 272(32):19777-84. DOI: 10.1074/jbc.272.32.19777
Source: PubMed


The TRAF3 molecule interacts with the cytoplasmic carboxyl terminus (COOH terminus) of the Epstein-Barr virus-encoded oncogene LMP-1. NF-kappaB activation is a downstream signaling event of tumor necrosis factor receptor-associated factor (TRAF) molecules in other signaling systems (CD40 for example) and is an event caused by LMP-1 expression. One region capable of TRAF3 interaction in LMP-1 is the membrane-proximal 45 amino acids (188-242) of the COOH terminus. We show that this region contains the only site for binding of TRAF3 in the 200-amino acid COOH terminus of LMP-1. The site also binds TRAF2 and TRAF5, but not TRAF6. TRAF3 binds to critical residues localized between amino acids 196 and 212 (HHDDSLPHPQQATDDSG), including the PXQX(T/S) motif, that share limited identity to the CD40 receptor TRAF binding site (TAAPVQETL). Mutation of critical residues in the TRAF3 binding site of LMP-1 that prevents binding of TRAF2, TRAF3, and TRAF5 does not affect NF-kappaB-activating potential. Deletion mapping localized the major NF-kappaB activating region of LMP-1 to critical residues in the distal 4 amino acids of the COOH terminus (383-386). Therefore, TRAF3 binding and NF-kappaB activation occur through two separate motifs at opposite ends of the LMP-1 COOH-terminal sequence.

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    • "Structurally, LMP1 is composed of a cytoplasmic N-terminus, six transmembrane domains and a cytoplasmic C-terminus [5]. The C-terminus contains two major signaling domains, C-terminus activating regions 1 and 2 (CTAR 1, CTAR 2), which activate NF-κB [6-8] and p38 [9] signaling pathways through interaction with the effector proteins tumor necrosis factor receptor-associated factors and tumor necrosis factor receptor-associated death domain. LMP1 can also activate ERK signaling through the CTAR 1 domain [10-13], and c-jun N-terminus kinase (JNK) through the CTAR 2 domain [14,15] in an NF-κB independent manner. "
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    ABSTRACT: The Epstein-Barr virus (EBV) encoded Latent Membrane Protein 1 (LMP1) has been shown to increase the expression of promyelocytic leukemia protein (PML) and the immunofluorescent intensity of promyelocytic leukemia nuclear bodies (PML NBs). PML NBs have been implicated in the modulation of transcription and the association of reporter plasmids with PML NBs has been implicated in repression of reporter activity. Additionally, repression of various reporters in the presence of LMP1 has been noted. This study demonstrates that LMP1 suppresses expression of reporter activity in a dose responsive manner and corresponds with the LMP1 induced increase in PML NB intensity. Disruption of PML NBs with arsenic trioxide or a PML siRNA restores reporter activity. These data offer an explanation for previously conflicting data on LMP1 signaling and calls attention to the possibility of false-positives and false-negatives when using reporter assays as a research tool in cells expressing LMP1.
    Full-text · Article · Oct 2011 · Virology Journal
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    • "Structurally, LMP1 is an integral membrane protein of 386 amino acids (aa) that consists of a short cytoplasmic N-terminal domain (aa 1-24), six transmembrane domains (aa 25-186) and a long cytoplasmic C-terminal tail (aa 187-386) [1] [2] [3]. The cytoplasmic C-terminal tail contains two C-terminal activation regions (CTARs), CTAR1 and CTAR2, which are required for LMP1-transduced signaling through tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) [1] [4]. CTAR1 contains a consensus TRAF-binding motif (PXQXT), and can bind to TRAFs 1, 2, 3 and 5 [1] [5] [6] [7] [8]. "
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    ABSTRACT: Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) activates NF-kappaB signaling pathways through the two C-terminal regions, CTAR1 and CTAR2. BS69 has previously been shown to be involved in LMP1-induced c-Jun N-terminal kinase activation through CTAR2 by interacting with tumor necrosis factor (TNFR) receptor-associated factor 6. In the present study, our manipulation of BS69 expression clearly indicates that BS69 negatively regulates LMP1-mediated NF-kappaB activation and up-regulates IL-6 mRNA expression and IkappaB degradation. Our immunoprecipitation experiments suggest that BS69 decreases complex formation between LMP1 and TNFR-associated death domain protein (TRADD).
    Full-text · Article · May 2009 · FEBS letters
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    • "Further study is needed to identify the factors that are responsible for the residual LMP1 signaling observed in TRAF3−/− B cells. Candidate molecules include other adaptor proteins shown previously to be able to interact with LMP1, such as TRAF5, receptor-interacting protein, and TNF-R–associated DD protein (18, 46), and perhaps TRAF1 and -2 may share overlapping functions with these factors. "
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    ABSTRACT: CD40, a member of the tumor necrosis factor receptor family, and the Epstein-Barr virus-encoded oncoprotein latent membrane protein 1 (LMP1) share several tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins for signaling. Among these, TRAF3 was the first identified to directly bind both receptors, yet its role remains a mystery. To address this, we generated B cell lines deficient in TRAF3 by homologous recombination. We found that CD40 signals were normal in the absence of TRAF3, with the exception of moderately enhanced c-Jun NH2-terminal kinase (JNK) activation and antibody secretion. In sharp contrast, LMP1 signaling was markedly defective in TRAF3-/- B cells. LMP1-induced activation of JNK and nuclear factor kappaB, up-regulation of CD23 and CD80, and antibody secretion were substantially affected by TRAF3 deficiency. Reconstitution of TRAF3 expression decreased CD40-induced JNK activation and antibody secretion, and fully restored LMP1 signaling. Although TRAF2 is widely believed to be important for LMP1 function, LMP1 signaling was intact in TRAF2-/- B cells. Our data reveal that CD40 and LMP1 unexpectedly use TRAF3 in different ways, and that TRAF3 is required for LMP1-mediated activation of B cells.
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