J Bacteriol Virol.  2012 Mar;42(1):63-68. 10.4167/jbv.2012.42.1.63.

ASK1 is Involved in EBV LMP1-induced NF-kappaB Activation

• Song YJ

• KMID: 1434774
• DOI: http://doi.org/10.4167/jbv.2012.42.1.63

Abstract

Epstein-Barr virus (EBV) latent infection transforms B lymphocytes into proliferating lymphoblastoid cell lines (LCLs). EBV latent infection membrane protein 1 (LMP1) is required for EBV-mediated B lymphocyte transformation, and LMP1-induced NF-kappaB activation is essential for LCL survival. Previously, it was reported that the level of reactive oxygen species (ROS) and the expression of apoptosis signal-regulating kinase 1 (ASK1) are elevated in EBV-positive Burkitt's lymphoma (BL) cells, the potential role of ASK1 in LMP1-induced NF-kappaB activation was thus investigated in this study. In EBV-positive BL cells, ASK1 was highly expressed and activated. In addition, TRAF6-ASK1 interaction was significantly increased in EBV-positive BL cells. Interestingly, the expression of LMP1 alone facilitated ASK1 activation. The expression of a dominant negative ASK1 mutant (ASK1KM) strongly blocked LMP1-induced NF-kappaB activation. Furthermore, LMP1-induced NF-kappaB activation was significantly reduced in ASK1 knock out (ASK1-/-) mouse embryonic fibroblasts (MEFs). Taken together, these results demonstrate that ASK1 is activated by LMP1 and is critical for LMP1-induced NF-kappaB activation.

Keyword

Epstein-Barr virus; Latent membrane protein 1; ASK1; NF-kappaB

MeSH Terms

Animals
B-Lymphocytes
Burkitt Lymphoma
Cell Line
Fibroblasts
Herpesvirus 4, Human
Lymphocyte Activation
MAP Kinase Kinase Kinase 5
Membrane Proteins
Mice
NF-kappa B
Reactive Oxygen Species
MAP Kinase Kinase Kinase 5
Membrane Proteins
NF-kappa B
Reactive Oxygen Species

Figure

• 

  Figure 1 ASK1 is activated in BL41-B958 cells. Equal amounts of BL41 or BL41-B958 cell extracts were subjected to Western blot analysis with anti-ASK1, anti-phopho-ASK1 Thr845 (P-ASK1), anti-LMP1, or anti-tubulin antibody.

• 

  Figure 2 TRAF6-ASK1 or TRAF6-IRAK1 interaction is induced in BL41-B958 cells. BL41 or BL41-B958 cells were lysed, and each cell lysate was immunoprecipitated with anti-TRAF6 antibody. The immunoprecipitates were subjected to Western blot analysis with anti-ASK1, anti-IRAK1 or anti-TRAF6 antibody.

• 

  Figure 3 LMP1 activates ASK1. HeLa cells were transfected with either pSG5 or pSG5-FLAG-LMP1 plus pcDNA3-HA-ASK1. After 24 h, equal amounts of cell extracts were subjected to Western blot analysis with anti-phospho-ASK1 Thr845 antibody (P-ASK1), anti-HA antibody, or anti-LMP1 antibody.

• 

  Figure 4 ASK1 plays an important role in LMP1-induced NF-κB activation. (A) HEK293 cells were transfected with either pSG5 (lanes 1, 3 and 5) or pSG5-FLAG-LMP1 (lanes 2, 4 and 6) with increasing amounts of ASK1KM (lanes 3 to 6). At 24 h after transfection, LMP1-induced NF-κB activation was determined using an NF-κB-dependent luciferase reporter assay. LMP1 protein levels were determined by Western blot analysis with anti-LMP1 antibody. (B) ASK1 wild-type (ASK1+/+) or knock-out (ASK1-/-) mouse embryonic fibroblasts (MEFs) were transfected with either pSG5 or pSG5-FLAG-LMP1, and then NF-κB activation was determined using an NF-κB-dependent luciferase reporter assay at 24 h after transfection. LMP1 protein levels were determined by Western blot analysis with anti-LMP1 antibody.

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