Epstein-Barr latent membrane protein 1 transformation site 2 activates NF-kappaB in the absence of NF-kappaB essential modifier residues 133-224 or 373-419 - PubMed (original) (raw)

Epstein-Barr latent membrane protein 1 transformation site 2 activates NF-kappaB in the absence of NF-kappaB essential modifier residues 133-224 or 373-419

Daniela Boehm et al. Proc Natl Acad Sci U S A. 2010.

Abstract

Epstein Barr virus latent membrane protein 1 (LMP1) induces NF-κB activation through transformation effector sites (TES) 1 and 2, both of which are critical for B-lymphocyte transformation. TES2 principally activates canonical NF-κB, which we confirm is NF-κB essential modifier (NEMO)-dependent and requires an intact ubiquitin binding in A20 binding inhibitor of NF-κB and NEMO (UBAN) domain. LMP1 TES2 activated NF-κB in Jurkat cell lines harboring NEMO truncated at 372 (A45) or NEMO with an in-frame deletion of 133-224 (2C), whereas TNFα, 12-O-Tetradecanoylphorbol-13-acetate, human T-cell leukemia virus 1 Tax, and CD40 did not. In both A45 and 2C Jurkat cell lines, LMP1 TES2-mediated NF-κB activation was blocked by siRNAs to TNFα receptor-associated factor 6 and NEMO, by IκB kinase inhibitors, and by the IκBα superrepressor, indicating that the NEMO mutants function to support canonical NF-κB activation. Expression of A45 or 2C mutants in NEMO-deficient murine embryonic fibroblasts reproduced the Jurkat phenotypes: LMP1 TES2 activated NF-κB in fibroblasts lacking NEMO amino acids 133-224 or 373-419, but TNFα and Tax did not. Further analysis indicated that TES2 did not activate NF-κB in cells expressing the double deletion mutant Δ133-224/Δ372-419. These data provide further evidence of the essential role for NEMO in LMP1 TES2 NF-κB activation and highlight the importance of unique domains within NEMO for sensing distinct NF-κB stimuli.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

NEMO is required for LMP1 TES2-mediated NF-κB activation in MEFs and 293 cells. (A) Schematic of NEMO: coiled-coil (CC) 1, CC2, ubiquitin binding in ABIN and NEMO (UBAN) domain, leucine zipper (LZ), zinc finger domain (ZF), and residues as indicated. (B) WT or NEMO− murine embryonic fibroblasts were transfected with an NF-κB–luciferase reporter, PGKβ-gal, and cotransfected with PGK2 control vector (ctrl), flag LMP1 (LMP1), flag-LMP1 TES2 (P204A/Q206A TES1 mutation; TES2), or HTLV1 TAX (Tax) as indicated. Vector control cells were either untreated or treated with TNFα (10 ng/mL) or IL-1β (10 ng/mL) for 18 or 6 h posttransfection. Luciferase and β-gal activity was determined at 24 h. (C) 293 cells with regulated expression of LMP1 TES2 and an integrated NF-κB–EGFP reporter plasmid were transfected with nontargeting siRNA (siCtrl) or a pool of four siRNAs to NEMO (siNEMO); 3 d later, LMP1 TES2 expression was induced for 24 h, and GFP was determined by FACS. (D) Western blot analysis of NEMO and tubulin of lysates from C.

Fig. 2.

LMP1 and LMP1 TES2 induce NF-κB in NEMO mutant Jurkat cells. NF-κB reporter assays were performed in (A) 2C (NEMO mutant) and reconstituted 2Cγ or (B) A45 (NEMO mutant) and parental Jurkat cells. As in Fig. 1, cells were cotransfected with reporter constructs and either PGK2 (Ctrl), HTLV1 TAX (TAX), Flag-LMP1 (LMP1), Flag LMP1 TES2 (TES2), or a ligand-independent FLAG-LMP1 CD40 chimera (CD40). Duplicate PGK2 transfected cells were treated with TNFα (10 ng/mL) or TPA (2.5 ng/mL) for 18 or 30 h posttransfection. Luciferase and β-gal were measured 48 h posttransfection. (C) Western blot analysis of Jurkat cell lines with polyclonal antibody to NEMO detects hypomorphic proteins on long exposure (NEMO*). (D) Schematic of encoded NEMO mutants in A45 and 2C Jurkat cells in relation to WT NEMO.

Fig. 3.

NEMO Δ133–224 and 1–372 function to support LMP1- and LMP1 TES2-mediated NF-κB activation. Control or NEMO-specific siRNA was transfected into (A) 2C (Δ133–224) or (B) A45 (1–372) Jurkat cells simultaneously with reporter plasmids and either PGK2 (Ctrl), Flag-LMP1 (LMP1), or Flag LMP1 TES2 (TES2). Luciferase and β-gal were measured 72 h posttransfection. (C) Western blots for NEMO, LMP1, and tubulin from B.

Fig. 4.

TRAF6 is essential for LMP1 TES2-mediated NF-κB activation in NEMO 2C (Δ133–224)- and A45 (1–372)-expressing cells. Control or TRAF6-specific siRNA was transfected into (A) 2C (Δ133–224) or (B) A45 (1–372) Jurkat cells simultaneously with reporter plasmids and PGK2 (Ctrl), Flag-LMP1 (LMP1), or Flag LMP1 TES2 (TES2). Luciferase and β-gal were measured 72 h posttransfection. (C) Western blots for TRAF6, LMP1, and tubulin from B.

Fig. 5.

LMP1 and LMP1 TES2 activate IKKβ in NEMO Δ133–224- and 1–372-expressing cells. (A) 2C (Δ133–224) or (B) A45 (1–372) Jurkat cells were treated with DMSO or an IKKβ inhibitor IV (10 uM) 6 h posttransfection with reporter plasmids and either PGK2 (Ctrl), Flag-LMP1 (LMP1), Flag LMP1 TES2 (TES2), or Flag LMP1 TES1 (TES2 mutant YYD386 to ID386). (C) IKKβ inhibitor IV had no effect on IKKα-mediated p100 processing. Lysates from B were examined for LMP1, p100/p52 NF-κB2, and tubulin expression by Western blot analysis.

Fig. 6.

LMP1 TES2-mediated NF-κB activation requires 303–372 encompassing the NEMO UBAN domain; Δ133–224 and 373–419 are individually dispensable for LMP1 TES2 signaling. Both are required for TNFα and TAX. (A) Schematic of FLAG-HA-NEMO constructs used to reconstitute NEMO− MEFs indicating the positions of coiled-coil domain 1 (CC1), CC2, the ubiquitin binding in ABIN and NEMO domain (UBAN), leucine zipper (LZ), and zinc finger (ZF). (B) Anti-HA Western blot analysis of WT MEFs or NEMO- MEFs stably expressing PGK2 (Ctrl) or NEMO constructs from A as indicated. (C) LMP1 TES2-, TNFα-, and TAX-mediated NF-κB reporter assays in the indicated cell lines from B. Assay is as in Fig. 1. *Significant (P < 0.01). **Not significant (_P_ > 0.05) in Student t test comparison with 1–419 reconstituted NEMO− MEFs.

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Epstein-Barr latent membrane protein 1 transformation site 2 activates NF-kappaB in the absence of NF-kappaB essential modifier residues 133-224 or 373-419 - PubMed (original) (raw)