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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 and TRAF1 complexes are modified by M1-pUb chains.A) FLAG-LMP1 complexes were immuno-purified from LCLs that express FLAG-LMP1 from the EBV genome at physiologic levels [18]. GM12878 LCLs were used as a negative control. M1-linked polyubiquitin chain (M1-pUb) content was analyzed by WB, using a highly chain specific antibody. * indicates antibody heavy chain background. FLAG-LMP1 migrates at a slightly higher than untagged LMP1. B) FLAG complexes were immuno-purified from GM12878 LCLs that stably express either FLAG- GFP or FLAG-TRAF1, and were immuno-blotted, as indicated. C) 293 cells were co-transfected with FLAG- GFP, FLAG-TRAF1, or FLAG-TRAF2, and either empty PSG5-vector control or untagged LMP1 1–386 for 24 hours. Purified FLAG complexes or whole cell lysates were blotted for FLAG or LMP1, as indicated. D) FLAG-LMP1 LCLs were transduced with lentiviruses that express control shGFP or an independent shRNA against HOIP. Following puromycin selection, FLAG LMP1-immuno-purified complexes and lysates were blotted, as indicated. E) Whole cell extracts from 293 TRAF1 cells, uninduced or induced for LMP1 1–231 expression for 16 hours, were blotted for M1-pUb, LMP1, or tubulin. A-E are representative of three independent experiments.
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ppat.1004890.g003: LMP1 and TRAF1 complexes are modified by M1-pUb chains.A) FLAG-LMP1 complexes were immuno-purified from LCLs that express FLAG-LMP1 from the EBV genome at physiologic levels [18]. GM12878 LCLs were used as a negative control. M1-linked polyubiquitin chain (M1-pUb) content was analyzed by WB, using a highly chain specific antibody. * indicates antibody heavy chain background. FLAG-LMP1 migrates at a slightly higher than untagged LMP1. B) FLAG complexes were immuno-purified from GM12878 LCLs that stably express either FLAG- GFP or FLAG-TRAF1, and were immuno-blotted, as indicated. C) 293 cells were co-transfected with FLAG- GFP, FLAG-TRAF1, or FLAG-TRAF2, and either empty PSG5-vector control or untagged LMP1 1–386 for 24 hours. Purified FLAG complexes or whole cell lysates were blotted for FLAG or LMP1, as indicated. D) FLAG-LMP1 LCLs were transduced with lentiviruses that express control shGFP or an independent shRNA against HOIP. Following puromycin selection, FLAG LMP1-immuno-purified complexes and lysates were blotted, as indicated. E) Whole cell extracts from 293 TRAF1 cells, uninduced or induced for LMP1 1–231 expression for 16 hours, were blotted for M1-pUb, LMP1, or tubulin. A-E are representative of three independent experiments.

Mentions: Most TRAF1 in LCLs is associated with LMP1 [29]. We therefore investigated whether LMP1 complexes are modified by M1-linked pUb chains in LCL extracts. FLAG-LMP1 was immuno-purified from LCLs established from recombinant EBV, in which FLAG-tagged LMP1 is expressed at physiological levels from the EBV genome [18]. As a negative control with physiologic LMP1 expression from the EBV genome, we used GM12878 LCLs (where LMP1 is untagged). FLAG immuno-purified material was subjected to western blot analysis, using a M1-linked pUb (M1-pUb) chain specific monoclonal antibody [81]. M1-pUb chains were readily detected in FLAG-LMP1 purified from FLAG-LMP1 LCL extracts, but not from GM12878 extracts, suggesting that either LMP1, or an LMP1 signalosome protein, was modified by LUBAC-catalyzed M1-pUb chains (Fig 3A).


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 and TRAF1 complexes are modified by M1-pUb chains.A) FLAG-LMP1 complexes were immuno-purified from LCLs that express FLAG-LMP1 from the EBV genome at physiologic levels [18]. GM12878 LCLs were used as a negative control. M1-linked polyubiquitin chain (M1-pUb) content was analyzed by WB, using a highly chain specific antibody. * indicates antibody heavy chain background. FLAG-LMP1 migrates at a slightly higher than untagged LMP1. B) FLAG complexes were immuno-purified from GM12878 LCLs that stably express either FLAG- GFP or FLAG-TRAF1, and were immuno-blotted, as indicated. C) 293 cells were co-transfected with FLAG- GFP, FLAG-TRAF1, or FLAG-TRAF2, and either empty PSG5-vector control or untagged LMP1 1–386 for 24 hours. Purified FLAG complexes or whole cell lysates were blotted for FLAG or LMP1, as indicated. D) FLAG-LMP1 LCLs were transduced with lentiviruses that express control shGFP or an independent shRNA against HOIP. Following puromycin selection, FLAG LMP1-immuno-purified complexes and lysates were blotted, as indicated. E) Whole cell extracts from 293 TRAF1 cells, uninduced or induced for LMP1 1–231 expression for 16 hours, were blotted for M1-pUb, LMP1, or tubulin. A-E are representative of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440769&req=5

ppat.1004890.g003: LMP1 and TRAF1 complexes are modified by M1-pUb chains.A) FLAG-LMP1 complexes were immuno-purified from LCLs that express FLAG-LMP1 from the EBV genome at physiologic levels [18]. GM12878 LCLs were used as a negative control. M1-linked polyubiquitin chain (M1-pUb) content was analyzed by WB, using a highly chain specific antibody. * indicates antibody heavy chain background. FLAG-LMP1 migrates at a slightly higher than untagged LMP1. B) FLAG complexes were immuno-purified from GM12878 LCLs that stably express either FLAG- GFP or FLAG-TRAF1, and were immuno-blotted, as indicated. C) 293 cells were co-transfected with FLAG- GFP, FLAG-TRAF1, or FLAG-TRAF2, and either empty PSG5-vector control or untagged LMP1 1–386 for 24 hours. Purified FLAG complexes or whole cell lysates were blotted for FLAG or LMP1, as indicated. D) FLAG-LMP1 LCLs were transduced with lentiviruses that express control shGFP or an independent shRNA against HOIP. Following puromycin selection, FLAG LMP1-immuno-purified complexes and lysates were blotted, as indicated. E) Whole cell extracts from 293 TRAF1 cells, uninduced or induced for LMP1 1–231 expression for 16 hours, were blotted for M1-pUb, LMP1, or tubulin. A-E are representative of three independent experiments.
Mentions: Most TRAF1 in LCLs is associated with LMP1 [29]. We therefore investigated whether LMP1 complexes are modified by M1-linked pUb chains in LCL extracts. FLAG-LMP1 was immuno-purified from LCLs established from recombinant EBV, in which FLAG-tagged LMP1 is expressed at physiological levels from the EBV genome [18]. As a negative control with physiologic LMP1 expression from the EBV genome, we used GM12878 LCLs (where LMP1 is untagged). FLAG immuno-purified material was subjected to western blot analysis, using a M1-linked pUb (M1-pUb) chain specific monoclonal antibody [81]. M1-pUb chains were readily detected in FLAG-LMP1 purified from FLAG-LMP1 LCL extracts, but not from GM12878 extracts, suggesting that either LMP1, or an LMP1 signalosome protein, was modified by LUBAC-catalyzed M1-pUb chains (Fig 3A).

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