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

ABSTRACT

The Epstein-Barr virus latent membrane protein 1 (LMP1) oncoprotein causes multiple cellular changes, including induction of epidermal growth factor receptor (EGFR) expression and activation of the NF-κB transcription factor. LMP1 and the cellular protein CD40, which also induces EGFR expression, interact with the tumor necrosis factor receptor-associated factor (TRAF) proteins. The LMP1 carboxy-terminal activation region 1 signaling domain interacts specifically with the TRAFs and is essential for EGFR induction through a mechanism independent of NF-κB alone. LMP1 and CD40 share a common TRAF binding motif, PXQXT. In this study, the PXQXT motifs in both LMP1 and CD40 were altered and mutant proteins were analyzed for induction of EGFR expression. Replacement of the T residue with A in CD40 completely blocked induction of the EGFR, while the same mutation in LMP1 did not affect EGFR induction. Replacement of both P and Q residues with A’s in LMP1 reduced EGFR induction by >75%, while deletion of PXQXT blocked EGFR induction. These results genetically link EGFR induction by LMP1 to the TRAF signaling pathway. Overexpression of TRAF2 potently activates NF-κB, although TRAF2 did not induce expression of the EGFR either alone or in combination with TRAF1 and TRAF3. In vivo analyses of the interaction of the TRAFs with LMP1 variants mutated in the PXQXT domain indicate that high-level induction of EGFR expression requires interaction with TRAF1, -2, and -3. However, exogenous expression of TRAF3 decreased EGFR induction mediated by either LMP1 or CD40. These data suggest that TRAF-mediated activation of EGFR expression requires assembly of a complex containing the appropriate stoichiometry of TRAF proteins clustered at the cell membrane with LMP1.

ACKNOWLEDGMENTS

We thank Katherine Fries and Frank Scholle for critical reading of the manuscript, H. Shelton Earp for the anti-ERCT rabbit antiserum, D. A. Thorley Lawson for the anti-LMP1 monoclonal antibody S12, Vishva Dixit for the anti-A20 monoclonal antibody, Dean Ballard for the IκB(SS32/36AA) construct, George Mosialos for TRAF1 and TRAF3 constructs, Ken Kaye for the TRAF2 construct, and Hitoshi Kikutani for the CD40 cDNA construct. J.L.C. acknowledges the support of A. S. Baldwin, Jr.

This work was supported by Public Health Service grants CA19014, CA32979, and CA52406 from the National Institutes of Health to N.R.-T. and CA72771 to A.S.B. W.E.M. was supported in part by predoctoral NIH National Service Award AI07419-02, and J.L.C. was supported by NIH/NRSA fellowship 1 F32 AG05745-01.