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TRAF1 Coordinates Polyubiquitin Signaling to Enhance Epstein-Barr Virus LMP1-Mediated Growth and Survival Pathway Activation.

Greenfeld H, Takasaki K, Walsh MJ, Ersing I, Bernhardt K, Ma Y, Fu B, Ashbaugh CW, Cabo J, Mollo SB, Zhou H, Li S, Gewurz BE - PLoS Pathog. (2015)

Bottom Line: TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders.We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity.Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.

ABSTRACT
The Epstein-Barr virus (EBV) encoded oncoprotein Latent Membrane Protein 1 (LMP1) signals through two C-terminal tail domains to drive cell growth, survival and transformation. The LMP1 membrane-proximal TES1/CTAR1 domain recruits TRAFs to activate MAP kinase, non-canonical and canonical NF-kB pathways, and is critical for EBV-mediated B-cell transformation. TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders. We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity. To gain insights into how TRAF1 amplifies LMP1 TES1 MAP kinase and canonical NF-kB pathways, we performed proteomic analysis of TRAF1 complexes immuno-purified from cells uninduced or induced for LMP1 TES1 signaling. Unexpectedly, we found that LMP1 TES1 domain signaling induced an association between TRAF1 and the linear ubiquitin chain assembly complex (LUBAC), and stimulated linear (M1)-linked polyubiquitin chain attachment to TRAF1 complexes. LMP1 or TRAF1 complexes isolated from EBV-transformed lymphoblastoid B cell lines (LCLs) were highly modified by M1-linked polyubiqutin chains. The M1-ubiquitin binding proteins IKK-gamma/NEMO, A20 and ABIN1 each associate with TRAF1 in cells that express LMP1. TRAF2, but not the cIAP1 or cIAP2 ubiquitin ligases, plays a key role in LUBAC recruitment and M1-chain attachment to TRAF1 complexes, implicating the TRAF1:TRAF2 heterotrimer in LMP1 TES1-dependent LUBAC activation. Depletion of either TRAF1, or the LUBAC ubiquitin E3 ligase subunit HOIP, markedly impaired LCL growth. Likewise, LMP1 or TRAF1 complexes purified from LCLs were decorated by lysine 63 (K63)-linked polyubiqutin chains. LMP1 TES1 signaling induced K63-polyubiquitin chain attachment to TRAF1 complexes, and TRAF2 was identified as K63-Ub chain target. Co-localization of M1- and K63-linked polyubiquitin chains on LMP1 complexes may facilitate downstream canonical NF-kB pathway activation. Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.

No MeSH data available.


Related in: MedlinePlus

Analysis of LMP1 and TRAF1 domains important for M1-pUb attachment.A) 293 cells were co-transfected with FLAG-TRAF1, and with either wildtype LMP1 (WT), an LMP1 mutant that signal only from the TES2 domain (TES2), an LMP1 mutant that signals only from the TES1 domain (TES1), or an LMP1 double mutant (DM) that does not signal from either TES1 or TES2. Immuno-purified FLAG-TRAF1 complexes and whole cell lysates were blotted, as indicated. B) 293 cells were transfected with FLAG-tagged WT LMP1, LMP1 1–231, or LMP1 DM and untagged TRAF1. Purified FLAG complexes or lysates were blotted as indicated. C) 293 cells were co-transfected with untagged LMP1 and FLAG-tagged GFP, TRAF1 1–416, TRAF1 183–416, or TRAF1 264–416. FLAG complexes or lysates were blotted, as indicated. A-C are representative of triplicate experiments.
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ppat.1004890.g005: Analysis of LMP1 and TRAF1 domains important for M1-pUb attachment.A) 293 cells were co-transfected with FLAG-TRAF1, and with either wildtype LMP1 (WT), an LMP1 mutant that signal only from the TES2 domain (TES2), an LMP1 mutant that signals only from the TES1 domain (TES1), or an LMP1 double mutant (DM) that does not signal from either TES1 or TES2. Immuno-purified FLAG-TRAF1 complexes and whole cell lysates were blotted, as indicated. B) 293 cells were transfected with FLAG-tagged WT LMP1, LMP1 1–231, or LMP1 DM and untagged TRAF1. Purified FLAG complexes or lysates were blotted as indicated. C) 293 cells were co-transfected with untagged LMP1 and FLAG-tagged GFP, TRAF1 1–416, TRAF1 183–416, or TRAF1 264–416. FLAG complexes or lysates were blotted, as indicated. A-C are representative of triplicate experiments.

Mentions: We previously found that HOIP and HOIL-1L are important for LMP1 TES2-mediated canonical NF-kB pathway activation in a genome-wide siRNA screen [38], suggesting that LMP1 TES2 may also activate LUBAC activity. To determine whether signaling by LMP1 TES2 also stimulates addition of M1-pUb chains to TRAF1 complexes, 293 cells were co-transfected with either wildtype (WT) LMP1, or with LMP1 mutants deficient for TES1, TES2 or TES1/TES2 signaling and with FLAG-tagged TRAF1. M1-pUb chains were detectable on FLAG-TRAF1 complexes immuno-purified from cells that co-expressed wildtype LMP1, or the LMP1 384ID385 mutant, which is for TES2 signaling. By contrast, FLAG-TRAF1 complexes purified from cells that co-expressed either a LMP1 TES1 alanine point mutant (LMP1 204PQQAT208-> 204AQAAA208) deficient for TRAF recruitment or a LMP1 double mutant (DM) deficient for TES1 and TES2 signaling, were not modified by M1-pUb chains (Fig 5A). These results suggest that association between TRAF1 and the LMP1 TES1 domain are required for M1-pUb chain attachment to TRAF1 complexes, and that LMP1 TES2-mediated NF-kB activation does not stimulate LUBAC to modify TRAF1.


TRAF1 Coordinates Polyubiquitin Signaling to Enhance Epstein-Barr Virus LMP1-Mediated Growth and Survival Pathway Activation.

Greenfeld H, Takasaki K, Walsh MJ, Ersing I, Bernhardt K, Ma Y, Fu B, Ashbaugh CW, Cabo J, Mollo SB, Zhou H, Li S, Gewurz BE - PLoS Pathog. (2015)

Analysis of LMP1 and TRAF1 domains important for M1-pUb attachment.A) 293 cells were co-transfected with FLAG-TRAF1, and with either wildtype LMP1 (WT), an LMP1 mutant that signal only from the TES2 domain (TES2), an LMP1 mutant that signals only from the TES1 domain (TES1), or an LMP1 double mutant (DM) that does not signal from either TES1 or TES2. Immuno-purified FLAG-TRAF1 complexes and whole cell lysates were blotted, as indicated. B) 293 cells were transfected with FLAG-tagged WT LMP1, LMP1 1–231, or LMP1 DM and untagged TRAF1. Purified FLAG complexes or lysates were blotted as indicated. C) 293 cells were co-transfected with untagged LMP1 and FLAG-tagged GFP, TRAF1 1–416, TRAF1 183–416, or TRAF1 264–416. FLAG complexes or lysates were blotted, as indicated. A-C are representative of triplicate experiments.
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ppat.1004890.g005: Analysis of LMP1 and TRAF1 domains important for M1-pUb attachment.A) 293 cells were co-transfected with FLAG-TRAF1, and with either wildtype LMP1 (WT), an LMP1 mutant that signal only from the TES2 domain (TES2), an LMP1 mutant that signals only from the TES1 domain (TES1), or an LMP1 double mutant (DM) that does not signal from either TES1 or TES2. Immuno-purified FLAG-TRAF1 complexes and whole cell lysates were blotted, as indicated. B) 293 cells were transfected with FLAG-tagged WT LMP1, LMP1 1–231, or LMP1 DM and untagged TRAF1. Purified FLAG complexes or lysates were blotted as indicated. C) 293 cells were co-transfected with untagged LMP1 and FLAG-tagged GFP, TRAF1 1–416, TRAF1 183–416, or TRAF1 264–416. FLAG complexes or lysates were blotted, as indicated. A-C are representative of triplicate experiments.
Mentions: We previously found that HOIP and HOIL-1L are important for LMP1 TES2-mediated canonical NF-kB pathway activation in a genome-wide siRNA screen [38], suggesting that LMP1 TES2 may also activate LUBAC activity. To determine whether signaling by LMP1 TES2 also stimulates addition of M1-pUb chains to TRAF1 complexes, 293 cells were co-transfected with either wildtype (WT) LMP1, or with LMP1 mutants deficient for TES1, TES2 or TES1/TES2 signaling and with FLAG-tagged TRAF1. M1-pUb chains were detectable on FLAG-TRAF1 complexes immuno-purified from cells that co-expressed wildtype LMP1, or the LMP1 384ID385 mutant, which is for TES2 signaling. By contrast, FLAG-TRAF1 complexes purified from cells that co-expressed either a LMP1 TES1 alanine point mutant (LMP1 204PQQAT208-> 204AQAAA208) deficient for TRAF recruitment or a LMP1 double mutant (DM) deficient for TES1 and TES2 signaling, were not modified by M1-pUb chains (Fig 5A). These results suggest that association between TRAF1 and the LMP1 TES1 domain are required for M1-pUb chain attachment to TRAF1 complexes, and that LMP1 TES2-mediated NF-kB activation does not stimulate LUBAC to modify TRAF1.

Bottom Line: TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders.We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity.Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, United States of America.

ABSTRACT
The Epstein-Barr virus (EBV) encoded oncoprotein Latent Membrane Protein 1 (LMP1) signals through two C-terminal tail domains to drive cell growth, survival and transformation. The LMP1 membrane-proximal TES1/CTAR1 domain recruits TRAFs to activate MAP kinase, non-canonical and canonical NF-kB pathways, and is critical for EBV-mediated B-cell transformation. TRAF1 is amongst the most highly TES1-induced target genes and is abundantly expressed in EBV-associated lymphoproliferative disorders. We found that TRAF1 expression enhanced LMP1 TES1 domain-mediated activation of the p38, JNK, ERK and canonical NF-kB pathways, but not non-canonical NF-kB pathway activity. To gain insights into how TRAF1 amplifies LMP1 TES1 MAP kinase and canonical NF-kB pathways, we performed proteomic analysis of TRAF1 complexes immuno-purified from cells uninduced or induced for LMP1 TES1 signaling. Unexpectedly, we found that LMP1 TES1 domain signaling induced an association between TRAF1 and the linear ubiquitin chain assembly complex (LUBAC), and stimulated linear (M1)-linked polyubiquitin chain attachment to TRAF1 complexes. LMP1 or TRAF1 complexes isolated from EBV-transformed lymphoblastoid B cell lines (LCLs) were highly modified by M1-linked polyubiqutin chains. The M1-ubiquitin binding proteins IKK-gamma/NEMO, A20 and ABIN1 each associate with TRAF1 in cells that express LMP1. TRAF2, but not the cIAP1 or cIAP2 ubiquitin ligases, plays a key role in LUBAC recruitment and M1-chain attachment to TRAF1 complexes, implicating the TRAF1:TRAF2 heterotrimer in LMP1 TES1-dependent LUBAC activation. Depletion of either TRAF1, or the LUBAC ubiquitin E3 ligase subunit HOIP, markedly impaired LCL growth. Likewise, LMP1 or TRAF1 complexes purified from LCLs were decorated by lysine 63 (K63)-linked polyubiqutin chains. LMP1 TES1 signaling induced K63-polyubiquitin chain attachment to TRAF1 complexes, and TRAF2 was identified as K63-Ub chain target. Co-localization of M1- and K63-linked polyubiquitin chains on LMP1 complexes may facilitate downstream canonical NF-kB pathway activation. Our results highlight LUBAC as a novel potential therapeutic target in EBV-associated lymphoproliferative disorders.

No MeSH data available.


Related in: MedlinePlus