Limits...
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 TES1 signaling is important for K63-linked pUb chain attachment to TRAF1 and LMP1 complexes.A) 293 TRAF1 cells were co-transfected with empty pSG5 vector (-) or the indicated LMP1 construct, and with FLAG-TRAF1. FLAG-TRAF1 IPs were immunoblotted for K63-pUb, using a K63-pUb chain-specific monoclonal antibody, or for FLAG, as indicated. Lysates were blotted, as indicated. B) FLAG-IPs from GM12878 or FLAG-LMP1 LCLs were blotted for K63-pUb chains or LMP1, as indicated. Lysates were blotted for LMP1. C) FLAG-tagged LMP1 WT, 1–231 or DM constructs were co-transfected with untagged-TRAF1 vector into 293 cells. FLAG-LMP1 immuno-purified complexes or whole cell lysates were blotted, as indicated.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4440769&req=5

ppat.1004890.g009: LMP1 TES1 signaling is important for K63-linked pUb chain attachment to TRAF1 and LMP1 complexes.A) 293 TRAF1 cells were co-transfected with empty pSG5 vector (-) or the indicated LMP1 construct, and with FLAG-TRAF1. FLAG-TRAF1 IPs were immunoblotted for K63-pUb, using a K63-pUb chain-specific monoclonal antibody, or for FLAG, as indicated. Lysates were blotted, as indicated. B) FLAG-IPs from GM12878 or FLAG-LMP1 LCLs were blotted for K63-pUb chains or LMP1, as indicated. Lysates were blotted for LMP1. C) FLAG-tagged LMP1 WT, 1–231 or DM constructs were co-transfected with untagged-TRAF1 vector into 293 cells. FLAG-LMP1 immuno-purified complexes or whole cell lysates were blotted, as indicated.

Mentions: The LMP1 TES2 domain highly activates TRAF6 K63-pUb ligase activity, but does not associate with TRAF1. To test whether LMP1 TES2 signaling nonetheless stimulates K63-pUb attachment to TRAF1 complexes, we co-transfected 293 cells with FLAG-TRAF1 and either WT LMP1, or LMP1 mutants deficient for TES1 and/or TES2 signaling. FLAG-TRAF1 complexes purified from cells that co-expressed wildtype LMP1, or the TES2 LMP1 mutant, were modified by K63-pUb chains. By contrast, the LMP1 TES1 domain 204AQAAA208 triple point mutant did not stimulate attachment of k63-pUb chains to TRAF1 complexes (Fig 9A). This result is consistent with a model in which TRAF1 recruitment to LMP1 TES1 is important for subsequent K63-pUb attachment to TRAF1 complexes.


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 TES1 signaling is important for K63-linked pUb chain attachment to TRAF1 and LMP1 complexes.A) 293 TRAF1 cells were co-transfected with empty pSG5 vector (-) or the indicated LMP1 construct, and with FLAG-TRAF1. FLAG-TRAF1 IPs were immunoblotted for K63-pUb, using a K63-pUb chain-specific monoclonal antibody, or for FLAG, as indicated. Lysates were blotted, as indicated. B) FLAG-IPs from GM12878 or FLAG-LMP1 LCLs were blotted for K63-pUb chains or LMP1, as indicated. Lysates were blotted for LMP1. C) FLAG-tagged LMP1 WT, 1–231 or DM constructs were co-transfected with untagged-TRAF1 vector into 293 cells. FLAG-LMP1 immuno-purified complexes or whole cell lysates were blotted, as indicated.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004890.g009: LMP1 TES1 signaling is important for K63-linked pUb chain attachment to TRAF1 and LMP1 complexes.A) 293 TRAF1 cells were co-transfected with empty pSG5 vector (-) or the indicated LMP1 construct, and with FLAG-TRAF1. FLAG-TRAF1 IPs were immunoblotted for K63-pUb, using a K63-pUb chain-specific monoclonal antibody, or for FLAG, as indicated. Lysates were blotted, as indicated. B) FLAG-IPs from GM12878 or FLAG-LMP1 LCLs were blotted for K63-pUb chains or LMP1, as indicated. Lysates were blotted for LMP1. C) FLAG-tagged LMP1 WT, 1–231 or DM constructs were co-transfected with untagged-TRAF1 vector into 293 cells. FLAG-LMP1 immuno-purified complexes or whole cell lysates were blotted, as indicated.
Mentions: The LMP1 TES2 domain highly activates TRAF6 K63-pUb ligase activity, but does not associate with TRAF1. To test whether LMP1 TES2 signaling nonetheless stimulates K63-pUb attachment to TRAF1 complexes, we co-transfected 293 cells with FLAG-TRAF1 and either WT LMP1, or LMP1 mutants deficient for TES1 and/or TES2 signaling. FLAG-TRAF1 complexes purified from cells that co-expressed wildtype LMP1, or the TES2 LMP1 mutant, were modified by K63-pUb chains. By contrast, the LMP1 TES1 domain 204AQAAA208 triple point mutant did not stimulate attachment of k63-pUb chains to TRAF1 complexes (Fig 9A). This result is consistent with a model in which TRAF1 recruitment to LMP1 TES1 is important for subsequent K63-pUb attachment to TRAF1 complexes.

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