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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

LMP1 induces TRAF1 association with M1-pUb chain sensors.A. 293 cells were transfected with LMP1, FLAG-TRAF1 and/or UBAN-GFP (a fusion protein that contains the IKK-gamma M1-pUb binding domain and GFP), as indicated. 24 hours later, cells were stained for TRAF1 and LMP1, and imaged by confocal microscopy. Additional confocal images are shown in S5B Fig) 293 cells were transfected with FLAG-TRAF1 or TRAF2, and after 24 hours, induced for LMP1 1–231 expression for 16 hours. FLAG immuno-purified material and lysates were blotted, as indicated. C) FLAG immuno-purified complexes and lysates from stable GM12878 FLAG-GFP or FLAG-TRAF1 cells were immuno-blotted, as indicated. A-C are representative of three independent experiments.
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ppat.1004890.g004: LMP1 induces TRAF1 association with M1-pUb chain sensors.A. 293 cells were transfected with LMP1, FLAG-TRAF1 and/or UBAN-GFP (a fusion protein that contains the IKK-gamma M1-pUb binding domain and GFP), as indicated. 24 hours later, cells were stained for TRAF1 and LMP1, and imaged by confocal microscopy. Additional confocal images are shown in S5B Fig) 293 cells were transfected with FLAG-TRAF1 or TRAF2, and after 24 hours, induced for LMP1 1–231 expression for 16 hours. FLAG immuno-purified material and lysates were blotted, as indicated. C) FLAG immuno-purified complexes and lysates from stable GM12878 FLAG-GFP or FLAG-TRAF1 cells were immuno-blotted, as indicated. A-C are representative of three independent experiments.

Mentions: To further establish that LMP1 and TRAF1 complexes are decorated by M1-pUb chains in vivo, we next tested whether LMP1 and TRAF1 co-localize with a M1-pUb chain biosensor. The biosensor is comprised of a fusion protein between GFP and the Ubiquitin Binding of ABIN1 and NEMO/IKK-gamma (UBAN) domain. The UBAN-GFP biosensor selectively visualizes the localization of M1-pUb chains in mammalian cells activated by multiple independent stimuli [71,72]. 293 cells were transiently co-transfected with UBAN-GFP, TRAF1 and or/ LMP1. The characteristic 293 cell LMP1 punctate staining pattern was observed by confocal microscopy analysis of LMP1-transfected cells. By contrast, TRAF1 and UBAN-GFP exhibited diffuse cystosolic staining patterns in the absence of LMP1 co-expression (Fig 4A). Interestingly, LMP1 co-expression with TRAF1 and UBAN-GFP altered the TRAF1 and UBAN-GFP patterns, and induced marked co-localized of all three into punctate foci (Figs 4A and S5). We did not observe similar punctate foci of UBAN-GFP or co-localization with LMP1 in the absence of TRAF1 co-expression. Of note, TRAF1 and the UBAN-GFP sensor colocalized to a lesser extent, even in the absence of LMP1. Taken together with the proteomic and biochemical data presented above, these results are further suggest that in cells with LMP1 and TRAF1 co-expression, LMP1 and TRAF1 are present in complexes modified by M1-pUb chains.


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)

LMP1 induces TRAF1 association with M1-pUb chain sensors.A. 293 cells were transfected with LMP1, FLAG-TRAF1 and/or UBAN-GFP (a fusion protein that contains the IKK-gamma M1-pUb binding domain and GFP), as indicated. 24 hours later, cells were stained for TRAF1 and LMP1, and imaged by confocal microscopy. Additional confocal images are shown in S5B Fig) 293 cells were transfected with FLAG-TRAF1 or TRAF2, and after 24 hours, induced for LMP1 1–231 expression for 16 hours. FLAG immuno-purified material and lysates were blotted, as indicated. C) FLAG immuno-purified complexes and lysates from stable GM12878 FLAG-GFP or FLAG-TRAF1 cells were immuno-blotted, as indicated. A-C are representative of three independent experiments.
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ppat.1004890.g004: LMP1 induces TRAF1 association with M1-pUb chain sensors.A. 293 cells were transfected with LMP1, FLAG-TRAF1 and/or UBAN-GFP (a fusion protein that contains the IKK-gamma M1-pUb binding domain and GFP), as indicated. 24 hours later, cells were stained for TRAF1 and LMP1, and imaged by confocal microscopy. Additional confocal images are shown in S5B Fig) 293 cells were transfected with FLAG-TRAF1 or TRAF2, and after 24 hours, induced for LMP1 1–231 expression for 16 hours. FLAG immuno-purified material and lysates were blotted, as indicated. C) FLAG immuno-purified complexes and lysates from stable GM12878 FLAG-GFP or FLAG-TRAF1 cells were immuno-blotted, as indicated. A-C are representative of three independent experiments.
Mentions: To further establish that LMP1 and TRAF1 complexes are decorated by M1-pUb chains in vivo, we next tested whether LMP1 and TRAF1 co-localize with a M1-pUb chain biosensor. The biosensor is comprised of a fusion protein between GFP and the Ubiquitin Binding of ABIN1 and NEMO/IKK-gamma (UBAN) domain. The UBAN-GFP biosensor selectively visualizes the localization of M1-pUb chains in mammalian cells activated by multiple independent stimuli [71,72]. 293 cells were transiently co-transfected with UBAN-GFP, TRAF1 and or/ LMP1. The characteristic 293 cell LMP1 punctate staining pattern was observed by confocal microscopy analysis of LMP1-transfected cells. By contrast, TRAF1 and UBAN-GFP exhibited diffuse cystosolic staining patterns in the absence of LMP1 co-expression (Fig 4A). Interestingly, LMP1 co-expression with TRAF1 and UBAN-GFP altered the TRAF1 and UBAN-GFP patterns, and induced marked co-localized of all three into punctate foci (Figs 4A and S5). We did not observe similar punctate foci of UBAN-GFP or co-localization with LMP1 in the absence of TRAF1 co-expression. Of note, TRAF1 and the UBAN-GFP sensor colocalized to a lesser extent, even in the absence of LMP1. Taken together with the proteomic and biochemical data presented above, these results are further suggest that in cells with LMP1 and TRAF1 co-expression, LMP1 and TRAF1 are present in complexes modified by M1-pUb chains.

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