TRAF1 Coordinates Polyubiquitin Signaling to Enhance Epstein-Barr Virus LMP1-Mediated Growth and Survival Pathway Activation

<div><p>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.</p></div

LMP1 1–231 expression induces K63-pUb chain attachment to TRAF2.

<p>293 cells were co-transfected with FLAG-tagged GFP, TRAF1 (T1), TRAF2 (T2), TRAF3 (T3), or LMP1 constructs, HA-LMP1, and untagged TRAF1 for 24 hours, as indicated. 1% SDS was added to whole cell lysates, and samples were boiled for 5 minutes to denature complexes. SDS was diluted to 0.1%, and anti-FLAG IP was performed. Western blots were performed, as indicated. A-D are representative of three independent experiments.</p

Analysis of LMP1 and TRAF1 domains important for M1-pUb attachment.

<p>A) 293 cells were co-transfected with FLAG-TRAF1, and with either wildtype LMP1 (WT), an LMP1 mutant that signal only from the TES2 domain (TES2), an LMP1 mutant that signals only from the TES1 domain (TES1), or an LMP1 double mutant (DM) that does not signal from either TES1 or TES2. Immuno-purified FLAG-TRAF1 complexes and whole cell lysates were blotted, as indicated. B) 293 cells were transfected with FLAG-tagged WT LMP1, LMP1 1–231, or LMP1 DM and untagged TRAF1. Purified FLAG complexes or lysates were blotted as indicated. C) 293 cells were co-transfected with untagged LMP1 and FLAG-tagged GFP, TRAF1 1–416, TRAF1 183–416, or TRAF1 264–416. FLAG complexes or lysates were blotted, as indicated. A-C are representative of triplicate experiments.</p

TRAF2, HOIP, HOIL-1L, and SHARPIN, but not cIAP1/2, are important for LMP1 1-231-induced M1-pUb chain attachment to TRAF1 complexes.

<p>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.</p

RelB controls Rb phosphorylation, EZH2 expression and senescence through PSMA5 induced regulation of p21^WAF1 and p53 protein stability.

<p>(A) PSMA5 and ANAPC1 regulate p21^WAF1 and p53 protein stability. Western blot analysis of NHD fibroblasts treated with the indicated siRNAs. (B & C) RelB regulates PSMA5 expression. Whole cell protein lysates (B) or RNA (C) was prepared from NHD fibroblasts treated with the indicated siRNAs and western blot or Q-PCR analysis of PSMA5 expression was performed. (D) RelB regulates ANAPC1 expression. RNA was prepared from NHD fibroblasts treated with the indicated siRNAs and Q-PCR analysis of ANAPC1 was performed. (E) siRNA mediated knock down of PSMA5 results in loss of Rb phosphorylation. Western blot analysis of NHD fibroblasts treated with the indicated siRNAs. (F) siRNA mediated knock down of PSMA5 results in loss of EZH2 expression. RNA was prepared from NHD fibroblasts treated with the indicated siRNAs and Q-PCR analysis of EZH2 was performed. Psma5: (*** p≤0.001) Anapc1: (* p≤0.05). (G & H) siRNA mediated knock down of PSMA5 (G) or ANAPC1 (H) induces p53 dependent cellular senescence. NHD fibroblasts were transfected with the listed siRNAs and analyzed for senescence by β-galactosidase staining after 7 days.</p

CD40 stimulation leads to NF-κB activation and CLL induction in Chronic Lymphocytic Leukemia cells.

<p>(A) Analysis of EZH2 mRNA expression in CLL cells. RNA was prepared from CLL cells stimulated with CD40L/IL4 expressing mouse fibroblasts or with untransfected fibroblasts (NTL) and IL4 for the indicated times and Q-PCR analysis of EZH2 expression was performed. (B) Analysis of EZH2 protein level in CLL cells. Western blot analysis of nuclear extracts from CLL cells stimulated with CD40L/IL4 or untransfected fibroblasts (NTL) and IL4 for the indicated times. (C & D) EZH2 protein and RNA levels in CLL cells is NF-κB dependent. Whole cell protein lysates (C) and RNA (D) were prepared from CLL cells stimulated for 24 hours with CD40L/IL4 and treated with the IKKβ inhibitor TPCA-1 where indicated.</p

NF-κB2 controls Rb phosphorylation, EZH2 expression and senescence through CDK4 and CDK6 regulation.

<p>(A & B) NF-κB2 regulates CDK4 & 6 expression. RNA was prepared from NHD fibroblasts treated with the indicated siRNAs and Q-PCR analysis of CDK4 (A) and CDK6 (B) expression was performed. (C) NF-κB2 regulates CDK4 expression. Western blot analysis of NHD fibroblasts treated with the indicated siRNAs. Note that this is a reprobing of blots used in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004642#pgen-1004642-g002" target="_blank">Fig. 2A</a> and the β-actin blot shown here is the same as in that figure. (D) siRNA mediated knock down of CDK4 and CDK6 results in loss of Rb phosphorylation and EZH2 expression. Western blot analysis of NHD fibroblasts treated with the indicated siRNAs. (E) siRNA mediated knock down of CDK4 induces cellular senescence. NHD fibroblasts were transfected with the listed siRNAs and analyzed for senescence by β-galactosidase staining after 7 days. (F) Re-expression of CDK partially recovers the effects of NF-κB2 siRNA depletion. 96 hours after the transfection of NHD fibroblasts treated with the indicated siRNAs, cells were further transfected with CDK4 and CDK6 expression plasmids. After an additional 24 hours, protein extracts were prepared and western blot analysis performed.</p

Summary of the results identified in this manuscript through which NF-κB2 and RelB regulate p53 dependent cellular senescence in primary human NHD fibroblasts.

<p>Depletion of NF-κB2 and RelB leads to a decrease in Rb phosphorylation. Unphosphorylated Rb represses E2F transcriptional activity and consequently inhibits EZH2 expression (which is negatively regulated by p53). However, this occurs through distinct pathways. Depletion of NF-κB2 leads to down regulation of CDK4 and CDK6, which are known to phosphorylate Rb directly. RelB depletion leads to an increase in p53 and p21 protein stability as a consequence of loss of expression of genes such as PSMA5 and ANAPC1. Other gene targets may be involved in these processes. NF-κB subunits also bind directly to the EZH2 promoter and this may also contribute towards its regulation. As a consequence of this pathway, down regulation of EZH2 results in numerous changes in gene expression, including p53 dependent repression of a number of gene targets. These include inhibition of RACGAP1 expression, resulting in Rac1/Cdc42 dependent induction of reactive oxygen species (ROS). Together with other changes, such as suppression of DEK and ultimately induction of p14^ARF and p16^INK4a, the ultimate consequence of NF-κB2 and RelB siRNA depletion and subsequent loss of EZH2 expression is p53 dependent cell senescence. Note, RACGAP1 activity as well as various inducers/suppressors or senescence may also be regulated by p53. Many of these genes may also be direct targets of NF-κB. Not shown is that oxidative stress is required to drive the basal level p53 activity seen in these cells.</p

EZH2 is an NF-κB regulated target gene.

<p>(A) The basal level of p53 protein in NHD fibroblasts is ROS dependent. NHD fibroblasts were grown under normoxia at 3% O2 for 7 days before western blot analysis. (B & C) siRNA mediated knock-down of NF-κB2 and RelB leads to a reduction in EZH2 mRNA and protein levels. RNA (B) or protein (C) was prepared from NHD fibroblasts treated with the indicated siRNAs 48 hours after transfection and Q-PCR or western blot analysis was performed to determine EZH2 expression. *** P≤0.001. (D & E) Lymphotoxin β receptor stimulation leads to induction of EZH2 expression. NHD fibroblasts were treated with LTβR agonist antibody for the times indicated and either Q-PCR (D) or western blot analysis (E) was performed to determine the expression of EZH2 (D) or EZH2, p52/p100, RelB and p53 (E). * P≤0.05.</p

The alternative NF-κB pathway suppresses ROS production through the regulation of RacGAP.

<p>(A) Table summarizing the fold effect on RACGAP1 expression induced by transfection of the listed siRNAs in the microarray analysis. (B) NF-κB2, RelB and EZH2 regulate RACGAP1 expression in NHD fibroblasts. RNA was prepared from NHD fibroblasts treated with the indicated siRNAs and Q-PCR analysis of RACGAP1 expression was performed. (C) siRNA mediated knock-down of RACGAP1 induces ROS production. NHD fibroblasts were transfected with the siRNAs shown and analyzed for ROS production after 4 days. (D) ROS production induced by siRNA mediated knock down of NF-κB2 and RelB is dependent upon Rac1 and Cdc42. NHD fibroblasts were transfected with the siRNAs shown and analyzed for ROS production after 4 days.</p