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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

TRAF1 and LUBAC associate in LMP1-stimulated cells.A) 293 cells were transfected with FLAG-tagged TRAF1 or TRAF3, and LMP1 1–231 expression was induced 24 hours, as indicated. FLAG immuno-purified complexes and lysates were analyzed by western blot (WB), as indicated. B) FLAG immuno-purified complexes and lysates were immuno-purified GM12878 LCLs with stable FLAG-GFP or FLAG-TRAF1 expression, and analyzed by western blot, as indicated. A-B are representative of three independent experiments.
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ppat.1004890.g002: TRAF1 and LUBAC associate in LMP1-stimulated cells.A) 293 cells were transfected with FLAG-tagged TRAF1 or TRAF3, and LMP1 1–231 expression was induced 24 hours, as indicated. FLAG immuno-purified complexes and lysates were analyzed by western blot (WB), as indicated. B) FLAG immuno-purified complexes and lysates were immuno-purified GM12878 LCLs with stable FLAG-GFP or FLAG-TRAF1 expression, and analyzed by western blot, as indicated. A-B are representative of three independent experiments.

Mentions: The LMP1 1-231-induced association between TRAF1 and LUBAC was further validated by IP/western blot analysis, using both conditional 293 cells and GM12878 LCLs with stably FLAG-TRAF1 expression at physiological levels (S1 Fig). First, FLAG-TRAF1 complexes were immuno-purified from conditional 293 cells, either undincued or induced for LMP1 1–231 expression for 16 hours (Fig 2A). LMP1 induction increased the level of HOIP and SHARIPN in FLAG-TRAF1 complexes. By contrast, we did not observe significant co-purification of LUBAC components with FLAG-TRAF3 complexes immuno-purified from conditional LMP1 1–231 293 cells with stable FLAG-TRAF3 expression. Of note, twice as many FLAG-TRAF3 cells were used in this experiment, to achieve similar levels of immuno-purified FLAG-TRAF1 and FLAG-TRAF3. We were unable to find a suitable antibody for analysis of endogenous HOIL-1L. Also of note, LMP1 1–231 induction caused TRAF1 and TRAF3 steady state levels to decrease, perhaps as a result of increased turnover. To validate that TRAF1 associates with LUBAC in LCL extracts, we tested whether FLAG-TRAF1 purified from GM12878 cells also retrieved LUBAC components. Both HOIP and SHARPIN co-immunoprecipitated with FLAG-TRAF1, but not with a FLAG-GFP control (Fig 2B). Likewise, HA-SHARPIN complexes immuno-purified from GM12878 cells with stable HA-SHARPIN expression co-immunoprecipitated TRAF1 and LMP1 (S4 Fig). Taken together, our data suggest that TRAF1 and LUBAC are present together in protein-protein complexes in LMP1+ cells.


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)

TRAF1 and LUBAC associate in LMP1-stimulated cells.A) 293 cells were transfected with FLAG-tagged TRAF1 or TRAF3, and LMP1 1–231 expression was induced 24 hours, as indicated. FLAG immuno-purified complexes and lysates were analyzed by western blot (WB), as indicated. B) FLAG immuno-purified complexes and lysates were immuno-purified GM12878 LCLs with stable FLAG-GFP or FLAG-TRAF1 expression, and analyzed by western blot, 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.g002: TRAF1 and LUBAC associate in LMP1-stimulated cells.A) 293 cells were transfected with FLAG-tagged TRAF1 or TRAF3, and LMP1 1–231 expression was induced 24 hours, as indicated. FLAG immuno-purified complexes and lysates were analyzed by western blot (WB), as indicated. B) FLAG immuno-purified complexes and lysates were immuno-purified GM12878 LCLs with stable FLAG-GFP or FLAG-TRAF1 expression, and analyzed by western blot, as indicated. A-B are representative of three independent experiments.
Mentions: The LMP1 1-231-induced association between TRAF1 and LUBAC was further validated by IP/western blot analysis, using both conditional 293 cells and GM12878 LCLs with stably FLAG-TRAF1 expression at physiological levels (S1 Fig). First, FLAG-TRAF1 complexes were immuno-purified from conditional 293 cells, either undincued or induced for LMP1 1–231 expression for 16 hours (Fig 2A). LMP1 induction increased the level of HOIP and SHARIPN in FLAG-TRAF1 complexes. By contrast, we did not observe significant co-purification of LUBAC components with FLAG-TRAF3 complexes immuno-purified from conditional LMP1 1–231 293 cells with stable FLAG-TRAF3 expression. Of note, twice as many FLAG-TRAF3 cells were used in this experiment, to achieve similar levels of immuno-purified FLAG-TRAF1 and FLAG-TRAF3. We were unable to find a suitable antibody for analysis of endogenous HOIL-1L. Also of note, LMP1 1–231 induction caused TRAF1 and TRAF3 steady state levels to decrease, perhaps as a result of increased turnover. To validate that TRAF1 associates with LUBAC in LCL extracts, we tested whether FLAG-TRAF1 purified from GM12878 cells also retrieved LUBAC components. Both HOIP and SHARPIN co-immunoprecipitated with FLAG-TRAF1, but not with a FLAG-GFP control (Fig 2B). Likewise, HA-SHARPIN complexes immuno-purified from GM12878 cells with stable HA-SHARPIN expression co-immunoprecipitated TRAF1 and LMP1 (S4 Fig). Taken together, our data suggest that TRAF1 and LUBAC are present together in protein-protein complexes in LMP1+ cells.

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