Localization of the major NF-kappa B-activating site and the sole TRAF3 binding site of LMP-1 defines two distinct signaling motifs

The TRAF3 molecule interacts with the cytoplasmic carboxyl terminus (COOH terminus) of the Epstein Barr virus-encoded oncogene LMP-1, NF-kappa B 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-I, The site also binds TRAF2 and TRAF5, brit not TRAF6. TRAF3 binds to critical residues localized between amino acids 196 and 212 (HHDDSLPHPQQAT-DDSG), 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-kappa B-activating potential, Deletion mapping localized the major NF-kappa B activating region of LMP-1 to critical residues in the distal 4 amino acids of the COOH terminus (385-386). Therefore, TRAF3 binding and NF-KB activation occur through two separate motifs at opposite ends of the LMP-1 COOH-terminal sequence.