Activation of the p38 Mitogen-activated Protein Kinase Pathway by Epstein-Barr Virus-encoded Latent Membrane Protein 1 Coregulates Interleukin-6 and Interleukin-8 Production

Aristides G Eliopoulos,Neil J Gallagher,Sarah M.S Blake,Christopher W Dawson,Lawrence S Young

doi:10.1074/jbc.274.23.16085

Aristides G Eliopoulos, Neil J Gallagher + Show 3 more

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https://doi.org/10.1074/jbc.274.23.16085

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Abstract

The Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is a pleiotropic protein the activities of which include effects on gene expression and cell transformation, growth, and death. LMP1 has been shown to induce nuclear factor (NF)-kappaB and c-Jun NH2-terminal kinase/AP-1 activities in target cells, and in this study we demonstrate that LMP1 also engages the p38 mitogen-activated protein kinase cascade, leading to activation of the transcription factor ATF2. Mutational analysis of the LMP1 cytoplasmic COOH terminus revealed that p38 activation occurs from both the tumor necrosis factor receptor-associated factor (TRAF)-interacting, membrane-proximal COOH-terminal activating region (CTAR)1 domain (amino acids 186-231) and the extreme tumor necrosis factor receptor-associated death domain (TRADD) binding CTAR2 region (amino acids 351-386). Because LMP1 also engages signaling on the NF-kappaB axis through CTAR1 and CTAR2, we have examined whether these two pathways are overlapping or independent. We have found that inhibition of p38 by the highly specific inhibitor SB203580 did not affect NF-kappaB binding activity. Conversely, although the metabolic inhibitor D609 blocked NF-kappaB activation, it did not impair the ability of LMP1 to signal on the p38 axis, suggesting that these two LMP1-mediated pathways are primarily independent. Divergence of signals must, however, occur downstream of TRAF2 as a dominant negative TRAF2 mutant that blocks LMP1-induced NF-kappaB activation also inhibited p38 signaling. In addition, we have found that p38 inhibition significantly impaired LMP1-mediated interleukin-6 and -8 expression. Thus, p38 may play a significant cooperative role in regulating at least some of the pleiotropic activities of LMP1.

Highlights

Epstein-Barr virus (EBV)1 is a human herpesvirus that is associated with several types of malignancy
Among the nuclear and membrane proteins expressed as a consequence of EBV infection, the latent membrane protein 1 (LMP1) is of particular interest because it induces the oncogenic transformation of rodent fibroblast cell lines [2, 3]
LMP1 Expression Induces Phosphorylation of p38 and ATF2—To determine the effects of LMP1 on the p38 MAPK pathway, an ecdysone-inducible system was used to provide regulatable expression of LMP1. This system is based on the binding of the steroid hormone ecdysone analog ponasterone A to a heterodimeric receptor comprising a modified ecdysone receptor and the retinoid X receptor (RXR) which allows the subsequent activation of an ecdysone-responsive promoter to express the target gene [37]

Summary

Introduction

Epstein-Barr virus (EBV) is a human herpesvirus that is associated with several types of malignancy. The 45 most membrane-proximal residues of the LMP1 COOH terminus (amino acids 186 –231) are critical for EBV-mediated transformation of primary B cells, but recent studies suggest that the long term growth of these cells requires the distal COOH-terminal sequences (amino acids 352–386) [4, 14, 15] These two functional domains of the LMP1 cytoplasmic tail can activate the transcription factor NF␬B, with the extreme COOH-terminal activating region 2 (CTAR2, amino acids 351–386) being the principal contributor to this effect in the majority of cell lines [6, 16]. In addition to NF-␬B, LMP1 expression signals for activation of a Ras/MAPK/ERK pathway [21] and of the JNK (c-Jun NH2-terminal kinase, known as the stressactivated protein kinase, SAPK) cascade [22,23,24], a phenomenon that is mediated through CTAR2 and results in the induction of the transcription factor c-Jun/AP-1 [22, 24]

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