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-kappaB 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-kappaB 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-kappaB, 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.

FIG. 1

Model depicting the molecular structures and locations of functional domains in LMP1. LMP1 contains a 24-amino-acid cytoplasmic amino terminus, a transmembrane hydrophobic domain, and a 200-amino-acid cytoplasmic carboxy terminus. The carboxy terminus contains the major signaling domains in LMP1. CTAR1 mediates interaction with the TRAFs, induces EGFR expression, and is the minor NF-κB-activating region; CTAR2 is the major NF-κB-activating region. The location of the TRAF-interacting motif, PXQXT, is also indicated.

FIG. 2

Mutational analysis of LMP1 mutants with respect to NF-κB activation and EGFR induction. (A) Diagram of LMP1 mutants used in this experiment. The domains of LMP1 retained by the individual mutants are depicted. (B) EMSA analysis of nuclear NF-κB binding activity in extracts from C33A cells stably transfected with the indicated LMP1 constructs. The arrows indicate the specific complexes induced by LMP1. (C) EMSA analysis of NF-κB binding activity of LMP1 compared to CD40 and TRAF2.

FIG. 3

EGFR induction is decreased in the presence of a constitutively active IκBα. LMP1 or CD40 was transfected into C33A cells in both the absence and the presence of a constitutively active IκBα(SS32/36AA). The resulting cell lines were analyzed by immunoblotting for EGFR expression (top) and the constitutively active IκBα(SS32/36AA) (bottom). Migration of molecular mass standards (in kilodaltons) is shown.

FIG. 4

Effects of mutations in the LMP1 TRAF binding motif PXQXT on NF-κB activation and EGFR induction. (A) Amino acids 200 to 227 of the carboxy-terminal domain of LMP1. The TRAF binding PXQXT motif is indicated by a box, and asterisks denote the residues of the core element. Amino acids 221 to 225 show some similarity with a region in the CD40 TRAF interaction domain and are also indicated by a box. The shaded residues indicate those that were mutated and analyzed in the experiments. (B) EMSA of nuclear NF-κB binding activity in extracts from C33A cells stably transfected with the indicated LMP1 constructs. Induced complexes are indicated by arrows. (C) Immunoblot analysis of EGFR and A20 induction by LMP1 mutants. Migration of molecular mass standards (in kilodaltons) is shown.

FIG. 5

Effects of mutations in the CD40 TRAF binding motif PXQXT on EGFR induction. (A) Alignment of the amino acid sequences of several proteins known to interact with the TRAF molecules reveals a common motif, PXQXT, which is essential for interaction with the TRAFs. (B) The effect of replacement of Thr-234 with Ala in the PXQXT motif in CD40 on the induction of EGFR expression was analyzed by immunoblotting (top). Both wild-type and mutant CD40 proteins were expressed at equal levels as determined by immunoblotting (bottom). Migration of molecular mass standards (in kilodaltons) is shown.

FIG. 6

In vivo analysis of TRAF binding to LMP1 proteins containing mutations in the PXQXT TRAF binding domain. (A) C33A cells were transfected with FLAG-LMP1 expression constructs containing mutations in the PXQXT motif. (B) C33A cells were transfected with FLAG-LMP1 and TRAF expression constructs. LMP1 was immunoprecipitated with anti-FLAG affinity gel, and TRAF binding to each mutant was analyzed by immunoblotting. Immunoblots were also reprobed with the anti-LMP1 monoclonal antibody S12 to ensure that equal amounts of LMP1 were immunoprecipitated in all reactions.

FIG. 7

Effects of overexpression of individual TRAF proteins on EGFR induction. (A) Establishment of a TRAF3-expressing cell line. pMEP4 or pMEP4-TRAF3 was transfected into C33A cells, and following selection in hygromycin, the cell lines were analyzed by immunoblotting for TRAF3 expression. The cell lines were either left untreated or treated with 50 μM CdCl₂ for 6 h to induce expression of the metallothionein-driven TRAF3 expression construct. (B) TRAF1 and TRAF2 were stably overexpressed in the pMEP4 and pMEP4-TRAF3 cell lines, and extracts were analyzed by immunoblotting. + and − indicate the pMEP4-TRAF3 and pMEP4 cell lines. The upper panel is an immunoblot for EGFR expression, and the lower panels are immunoblots for TRAF1 and TRAF2 expression. (C) The LMP1(WT), LMP1(1-231), or CD40 expression construct was transfected into either the pMEP4 or pMEP4-TRAF3 cell line. Resulting cell lines were analyzed by immunoblotting for EGFR expression (top) or TRAF3 expression (bottom). The immunoblot shown in the upper left panel with FLAG-LMP1(WT) is a longer exposure reflecting the somewhat weaker induction of EGFR expression by FLAG-LMP1(WT) than by FLAG-LMP1(1-231) or CD40. Migration of molecular mass standards (in kilodaltons) is shown.