INTERACTION OF TUMOR-NECROSIS-FACTOR RECEPTOR-ASSOCIATED FACTOR SIGNALING PROTEINS WITH THE LATENT MEMBRANE-PROTEIN-1 PXQXT MOTIF IS ESSENTIAL FOR INDUCTION OF EPIDERMAL GROWTH-FACTOR RECEPTOR EXPRESSION

The Epstein-Barr virus latent membrane protein 1 (LMP1) oncoprotein ca uses multiple cellular changes, including induction of epidermal growt h factor receptor (EGFR) expression and activation of the NF-kappa B t ranscription factor. LMP1 and the cellular protein CD40, which also in duces EGFR expression, interact with the tumor necrosis factor recepto r-associated factor (TRAF) proteins. The LMP1 carboxy-terminal activat ion region 1 signaling domain interacts specifically with the TRAFs an d is essential for EGFR induction through a mechanism independent of N F-kappa B alone. LMP1 and CD40 share a common TRAF binding motif, PXQX T. In this study, the PXQXT motifs in both LMP1 and CD40 were altered and mutant proteins were analyzed for induction of EGFR expression. Re placement of the T residue with A in CD40 completely blocked induction of the EGFR, while the same mutation in LMP1 did not affect EGFR indu ction. Replacement of both P and Q residues with A's in LMP1 reduced E GFR induction by >75%, while deletion of PXQXT blocked EGFR induction. These results genetically link EGFR induction by LMP1 to the TRAF sig naling pathway. Overexpression of TRAF2 potently activates NF-kappa B, although TRAF2 did not induce expression of the EGFR either alone or in combination with TRAF1 and TRAF3. In vivo analyses of the interacti on of the TRAFs with LMP1 variants mutated in the PXQXT domain indicat e that high-level induction of EGFR expression requires interaction wi th TRAF1, -2, and -3. However, exogenous expression of TRAF3 decreased EGFR induction mediated by either LMP1 or CD40. These data suggest th at TRAF-mediated activation of EGFR expression requires assembly of a complex containing the appropriate stoichiometry of TRAF proteins clus tered at the cell membrane with LMP1.