Localization of the major NF-kappaB-activating site and the sole TRAF3 binding site of LMP-1 defines two distinct signaling motifs.
ABSTRACT 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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ABSTRACT: We have previously shown that CD40 causes strong activation of the c-Jun N-terminal kinase (JNK), the p38 mitogen-activated protein kinases (MAPK) and MAPKAP kinase-2, a downstream target of p38 MAPK. To identify signaling motifs in the CD40 cytoplasmic domain that are responsible for activation of these kinases, we have created a set of 11 chimeric receptors consisting of the extracellular and transmembrane domains of CD8 fused to portions of the murine CD40 cytoplasmic domain. These chimeric receptors were expressed in WEHI-231 B lymphoma cells. We found that amino acids 35–45 of the CD40 cytoplasmic domain constitute an independent signaling motif that is sufficient for activation of the JNK and p38 MAPK pathways, as well as for induction of IκBα phosphorylation and degradation. Amino acids 35–45 were also sufficient to protect WEHI-231 cells from anti-IgM-induced growth arrest. This is the same region of CD40 required for binding the TNF receptor-associated factor-2 (TRAF2), TRAF3, and TRAF5 adapter proteins. These data support the idea that one or more of these TRAF proteins couple CD40 to the kinase cascades that activate NF-κB, JNK, and p38 MAPK.The Journal of Immunology 04/1999; 162(8):4720-4730. · 5.36 Impact Factor
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ABSTRACT: LMP-1 is a constitutively active Tumor Necrosis Factor Receptor analog encoded by Epstein-Barr virus. LMP-1 activation correlates with oligomerization and raft localization, but direct evidence of LMP-1 oligomers is limited. We report that LMP-1 forms multiple high molecular weight native LMP-1 complexes when analyzed by BN-PAGE, the largest of which are enriched in detergent resistant membranes. The largest of these high molecular weight complexes are not formed by purified LMP-1 or by loss of function LMP-1 mutants. Consistent with these results we find a dimeric form of LMP-1 that can be stabilized by disulfide crosslinking. We identify cysteine 238 in the C-terminus of LMP-1 as the crosslinked cysteine. Disulfide crosslinking occurs post-lysis but the dimer can be crosslinked in intact cells with membrane permeable crosslinkers. LMP-1/C238A retains wild type LMP-1 NF-κB activity. LMP-1's TRAF binding, raft association and oligomerization are associated with the dimeric form of LMP-1. Our results suggest the possibility that the observed dimeric species results from inter-oligomeric crosslinking of LMP-1 molecules in adjacent core LMP-1 oligomers.Virus Research 09/2013; · 2.83 Impact Factor