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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

TRAF2, HOIP, HOIL-1L, and SHARPIN, but not cIAP1/2, are important for LMP1 1-231-induced M1-pUb chain attachment to TRAF1 complexes.A) 72 hours after siRNA transfection of 293 TRAF1 cells, LMP1 1–231 expression was induced for 16 hours. FLAG-TRAF1 complexes and lysates were WB, as indicated. B) 293 TRAF1 cells were treated with a SMAC mimetic peptide to deplete cells of cIAP ligases, and were then induced for LMP1 1–231 expression in the presence of the SMAC mimetic, as indicated. FLAG-TRAF1 IPs and lysates were blotted as indicated. A-B are representative of three independent experiments.
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ppat.1004890.g006: TRAF2, HOIP, HOIL-1L, and SHARPIN, but not cIAP1/2, are important for LMP1 1-231-induced M1-pUb chain attachment to TRAF1 complexes.A) 72 hours after siRNA transfection of 293 TRAF1 cells, LMP1 1–231 expression was induced for 16 hours. FLAG-TRAF1 complexes and lysates were WB, as indicated. B) 293 TRAF1 cells were treated with a SMAC mimetic peptide to deplete cells of cIAP ligases, and were then induced for LMP1 1–231 expression in the presence of the SMAC mimetic, as indicated. FLAG-TRAF1 IPs and lysates were blotted as indicated. A-B are representative of three independent experiments.

Mentions: Loss-of-function approaches were used to test the importance of LUBAC subunits, TRAF2, TRAF3 and cIAP1/2 in M1-pUb chain attachment to TRAF1 complexes, since each were identified as high-confidence TRAF1 interactors. First, we used an siRNA approach to investigate the role of the three LUBAC components. 72 hours after 293 TRAF1 cell siRNA transfection, LMP1 1–231 expression was induced for 16 hours. The M1-pUb chain content of FLAG- TRAF1 immuno-purified complexes was analyzed by western blot. Knockdown efficiency was measured by western blot, using whole cell lysates (Figs 6A and S7). We were unable to identify commercially available antibodies that recognized endogenous HOIL-1L in our 293 cells, and instead used quantitative PCR analysis to validate HOIL-1L mRNA depletion in a parallel experiment (S8 Fig). Interestingly, we found that depletion of HOIP, HOIL-1L, or SHARPIN each impaired M1-pUb chain attachment to TRAF1 complexes, suggesting that all three LUBAC components play important and non-redundant roles, at least in 293 TRAF1 cells (Fig 6A). TRAF2 depletion likewise reduced M1-pUb chain abundance in purified FLAG-TRAF1 complexes, and also diminished association between TRAF1 and the LUBAC components HOIP and SHARPIN (Figs 6A and S7). By contrast, TRAF3 depletion did not impair M1-pUb chain attachment to purified TRAF1 complexes (S7 Fig). Taken together with our prior observation that TRAF2 complexes are not modified by M1-pUb in cells that lack TRAF1 expression, our results suggest that a TRAF1:TRAF2 heterotrimer, rather than a TRAF1 homotrimer, may be the functional unit that associates with LUBAC. Of note, HOIL-1L knockdown increased TRAF2 steady state levels (Fig 6A), while HOIP and SHARPIN knockdown also increased TRAF2 levels to a lesser extent. To our knowledge, LUBAC has not previously been implicated in control of TRAF2 steady-state levels, though it remains possible that this effect is specific to cells that express LMP1. Depletion of HOIP and HOIL-1L from 293 TRAF1 cells impaired LMP1 1-231-mediated p38, JNK and canonical NF-kB activation, consistent with a role for M1-pUB chains at or above the level of TAK1 kinase activation (S9 Fig).


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)

TRAF2, HOIP, HOIL-1L, and SHARPIN, but not cIAP1/2, are important for LMP1 1-231-induced M1-pUb chain attachment to TRAF1 complexes.A) 72 hours after siRNA transfection of 293 TRAF1 cells, LMP1 1–231 expression was induced for 16 hours. FLAG-TRAF1 complexes and lysates were WB, as indicated. B) 293 TRAF1 cells were treated with a SMAC mimetic peptide to deplete cells of cIAP ligases, and were then induced for LMP1 1–231 expression in the presence of the SMAC mimetic, as indicated. FLAG-TRAF1 IPs and lysates were blotted as indicated. A-B are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440769&req=5

ppat.1004890.g006: TRAF2, HOIP, HOIL-1L, and SHARPIN, but not cIAP1/2, are important for LMP1 1-231-induced M1-pUb chain attachment to TRAF1 complexes.A) 72 hours after siRNA transfection of 293 TRAF1 cells, LMP1 1–231 expression was induced for 16 hours. FLAG-TRAF1 complexes and lysates were WB, as indicated. B) 293 TRAF1 cells were treated with a SMAC mimetic peptide to deplete cells of cIAP ligases, and were then induced for LMP1 1–231 expression in the presence of the SMAC mimetic, as indicated. FLAG-TRAF1 IPs and lysates were blotted as indicated. A-B are representative of three independent experiments.
Mentions: Loss-of-function approaches were used to test the importance of LUBAC subunits, TRAF2, TRAF3 and cIAP1/2 in M1-pUb chain attachment to TRAF1 complexes, since each were identified as high-confidence TRAF1 interactors. First, we used an siRNA approach to investigate the role of the three LUBAC components. 72 hours after 293 TRAF1 cell siRNA transfection, LMP1 1–231 expression was induced for 16 hours. The M1-pUb chain content of FLAG- TRAF1 immuno-purified complexes was analyzed by western blot. Knockdown efficiency was measured by western blot, using whole cell lysates (Figs 6A and S7). We were unable to identify commercially available antibodies that recognized endogenous HOIL-1L in our 293 cells, and instead used quantitative PCR analysis to validate HOIL-1L mRNA depletion in a parallel experiment (S8 Fig). Interestingly, we found that depletion of HOIP, HOIL-1L, or SHARPIN each impaired M1-pUb chain attachment to TRAF1 complexes, suggesting that all three LUBAC components play important and non-redundant roles, at least in 293 TRAF1 cells (Fig 6A). TRAF2 depletion likewise reduced M1-pUb chain abundance in purified FLAG-TRAF1 complexes, and also diminished association between TRAF1 and the LUBAC components HOIP and SHARPIN (Figs 6A and S7). By contrast, TRAF3 depletion did not impair M1-pUb chain attachment to purified TRAF1 complexes (S7 Fig). Taken together with our prior observation that TRAF2 complexes are not modified by M1-pUb in cells that lack TRAF1 expression, our results suggest that a TRAF1:TRAF2 heterotrimer, rather than a TRAF1 homotrimer, may be the functional unit that associates with LUBAC. Of note, HOIL-1L knockdown increased TRAF2 steady state levels (Fig 6A), while HOIP and SHARPIN knockdown also increased TRAF2 levels to a lesser extent. To our knowledge, LUBAC has not previously been implicated in control of TRAF2 steady-state levels, though it remains possible that this effect is specific to cells that express LMP1. Depletion of HOIP and HOIL-1L from 293 TRAF1 cells impaired LMP1 1-231-mediated p38, JNK and canonical NF-kB activation, consistent with a role for M1-pUB chains at or above the level of TAK1 kinase activation (S9 Fig).

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