scholarly journals TRAF6 and the Three C-Terminal Lysine Sites on IRF7 Are Required for Its Ubiquitination-Mediated Activation by the Tumor Necrosis Factor Receptor Family Member Latent Membrane Protein 1

2008 ◽  
Vol 28 (20) ◽  
pp. 6536-6546 ◽  
Author(s):  
Shunbin Ning ◽  
Alex D. Campos ◽  
Bryant G. Darnay ◽  
Gretchen L. Bentz ◽  
Joseph S. Pagano
Keyword(s):  
Tumor Necrosis Factor ◽  
Membrane Protein ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Receptor ◽  
E3 Ligase ◽  
Latent Membrane Protein ◽  
Latent Membrane Protein 1 ◽  
Iκb Kinase ◽  
Necrosis Factor

ABSTRACT We have recently shown that interferon regulatory factor 7 (IRF7) is activated by Epstein-Barr virus latent membrane protein 1 (LMP1), a member of the tumor necrosis factor receptor (TNFR) superfamily, through receptor-interacting protein-dependent K63-linked ubiquitination (L. E. Huye, S. Ning, M. Kelliher, and J. S. Pagano, Mol. Cell. Biol. 27:2910-2918, 2007). In this study, with the use of small interfering RNA and TNFR-associated factor 6 (TRAF6) knockout cells, we first show that TRAF6 and its E3 ligase activity are required for LMP1-stimulated IRF7 ubiquitination. In Raji cells which are latently infected and express high levels of LMP1 and IRF7 endogenously, expression of a TRAF6 small hairpin RNA construct reduces endogenous ubiquitination and endogenous activity of IRF7. In TRAF6−/− mouse embryonic fibroblasts, reconstitution with TRAF6 expression, but not with TRAF6(C70A), which lacks the E3 ligase activity, recovers LMP1's ability to stimulate K63-linked ubiquitination of IRF7. Further, we identify IRF7 as a substrate for TRAF6 E3 ligase and show that IRF7 is ubiquitinated by TRAF6 at multiple sites both in vitro and in vivo. Most important, we determine that the last three C-terminal lysine sites (positions 444, 446, and 452) of human IRF7 variant A are essential for activation of IRF7; these are the first such sites identified. A ubiquitination-deficient mutant of IRF7 with these sites mutated to arginines completely loses transactivational ability in response not only to LMP1 but also to the IRF7 kinase IκB kinase ε. In addition, we find that K63-linked ubiquitination of IRF7 occurs independently of its C-terminal functional phosphorylation sites. These data support our hypothesis that regulatory ubiquitination of IRF7 is a prerequisite for its phosphorylation. This is the first evidence to imply that ubiquitination is required for phosphorylation and activation of a transcription factor.

Inhibition of tumor necrosis factor-induced phenotypes by short intracellular versions of latent membrane protein-1

2010 ◽  
Vol 22 (2) ◽  
pp. 303-313 ◽  
Author(s):  
Papa Alioune Ndour ◽  
Tan-Sothéa Ouk ◽  
Guillaume Brocqueville ◽  
Alexandra Mougel ◽  
Elsa Vanhecke ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Membrane Protein ◽  
Tumor Necrosis ◽  
Latent Membrane Protein ◽  
Latent Membrane Protein 1 ◽  
Necrosis Factor

Latent Membrane Protein 1, Tumor Necrosis Factor Receptor–Associated Factor (TRAF) 1, TRAF-2, TRAF-3, and Nuclear Factor Kappa B Expression in Posttransplantation Lymphoproliferative Disorders

2003 ◽  
Vol 127 (10) ◽  
pp. 1335-1339
Author(s):  
Preetha Ramalingam ◽  
Wei-Sing Chu ◽  
Raymond Tubbs ◽  
Lisa Rybicki ◽  
James Pettay ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Nuclear Factor Kappa B ◽  
Tumor Necrosis Factor Receptor ◽  
Immunohistochemical Analysis ◽  
Latent Membrane Protein ◽  
Lymphoproliferative Disorders ◽  
Latent Membrane Protein 1 ◽  
Nuclear Factor ◽  
Necrosis Factor

Abstract Context.—Most posttransplantation lymphoproliferative disorders (PTLDs) are associated with Epstein-Barr virus (EBV) infection. The EBV latent membrane protein 1 (LMP-1) is important in the transformation of B lymphocytes through its interaction with intracellular tumor necrosis factor receptor–associated factors (TRAFs) that, in turn, can activate transcription factors such as nuclear factor kappa B (NFκB) and Jun-N-kinase. Of the 6 members of the TRAF family, TRAF-1, TRAF-2, and TRAF-3 are most commonly associated with LMP-1. Recently, it has been suggested that LMP-1–induced TRAF activation is important in the pathogenesis of PTLDs. Objective.—To characterize the expression patterns of these proteins in PTLDs, we studied a series of well-characterized cases for expression of LMP-1, TRAF-1, TRAF-2, TRAF-3, and NFκB by immunohistochemical analysis. Methods.—A total of 27 specimens from 25 patients were analyzed for LMP-1, TRAF-1, TRAF-2, TRAF-3, and NFκB (active form) by immunohistochemical analysis. Expression of EBV-encoded RNA (EBER) was evaluated by in situ hybridization. Correlation between the expression of the different markers was performed using the Mantel-Haenszel χ2 test. Cox proportional hazards analysis and Kaplan-Meier analysis with log-rank testing were used to analyze antigen expression and clinical outcome. Results.—Ninety-six percent of PTLDs expressed NFκB, 74% to 84% expressed TRAFs, 78% expressed EBER, and 77% expressed LMP-1. TRAF-1, TRAF-2, and TRAF-3 expression did not correlate with either EBER or LMP-1 expression. TRAF-2, but not TRAF-1 or TRAF-3, expression correlated with NFκB expression (P = .02). Conclusions.—These results suggest that TRAF molecules and active NFκB are expressed in PTLDs regardless of EBV positivity. Given the association of TRAF-2 and active NFκB expression, TRAF-2 may play an important role in regulating this transcription factor in PTLD.

scholarly journals Molecular Mechanisms of TNFR-associated Factor 6 (TRAF6) Utilization by the Oncogenic Viral Mimic of CD40, Latent Membrane Protein 1 (LMP1)

2011 ◽  
Vol 286 (12) ◽  
pp. 9948-9955 ◽  
Author(s):  
Kelly M. Arcipowski ◽  
Laura L. Stunz ◽  
John P. Graham ◽  
Zachary J. Kraus ◽  
Tony J. Vanden Bush ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Necrosis Factor ◽  
B Cell ◽  
Tumor Necrosis ◽  
Cell Activation ◽  
Tumor Necrosis Factor Receptor ◽  
Latent Membrane Protein ◽  
Latent Membrane Protein 1 ◽  
B Cell Activation ◽  
Necrosis Factor

Latent membrane protein 1 (LMP1), encoded by Epstein-Barr virus, is required for EBV-mediated B cell transformation and plays a significant role in the development of posttransplant B cell lymphomas. LMP1 has also been implicated in exacerbation of autoimmune diseases such as systemic lupus erythematosus. LMP1 is a constitutively active functional mimic of the tumor necrosis factor receptor superfamily member CD40, utilizing tumor necrosis factor receptor-associated factor (TRAF) adaptor proteins to induce signaling. However, LMP1-mediated B cell activation is amplified and sustained compared with CD40. We have previously shown that LMP1 and CD40 use TRAFs 1, 2, 3, and 5 differently. TRAF6 is important for CD40 signaling, but the role of TRAF6 in LMP1 signaling in B cells is not clear. Although TRAF6 binds directly to CD40, TRAF6 interaction with LMP1 in B cells has not been characterized. Here we tested the hypothesis that TRAF6 is a critical regulator of LMP1 signaling in B cells, either as part of a receptor-associated complex and/or as a cytoplasmic adaptor protein. Using TRAF6-deficient B cells, we determined that TRAF6 was critical for LMP1-mediated B cell activation. Although CD40-mediated TRAF6-dependent signaling does not require the TRAF6 receptor-binding domain, we found that LMP1 signaling required the presence of this domain. Furthermore, TRAF6 was recruited to the LMP1 signaling complex via the TRAF1/2/3/5 binding site within the cytoplasmic domain of LMP1.

scholarly journals 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

1998 ◽  
Vol 18 (5) ◽  
pp. 2835-2844 ◽  
Author(s):  
William E. Miller ◽  
Jeanette L. Cheshire ◽  
Nancy Raab-Traub
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Tumor Necrosis ◽  
Tumor Necrosis Factor Receptor ◽  
Growth Factor Receptor ◽  
Latent Membrane Protein ◽  
Latent Membrane Protein 1 ◽  
Egfr Expression ◽  
Epidermal Growth ◽  
Necrosis Factor

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.

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