Roles of TRAF2 and TRAF3 in Epstein-Barr virus latent membrane protein 1-induced alternative NF-κB activation
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- Song, YJ. & Kang, MS. Virus Genes (2010) 41: 174. doi:10.1007/s11262-010-0505-4
Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1)-induced NF-κB activation is essential for EBV-transformed B cell survival. LMP1 has two C-terminal cytoplasmic domains referred to as C-Terminal Activation Regions (CTAR) 1 and 2 that activate the alternative and canonical NF-κB pathways, respectively. While CTAR2 activates TRAF6, IKKβ and IKKγ-dependent canonical NF-κB pathway, CTAR1 interacts with TRAF2 and TRAF3 and activates NIK and IKKα-dependent alternative NF-κB pathway involving p100 processing into functional p52. Using IKKα−/−, IKKβ−/−, IKKγ−/−, TRAF2−/−, TRAF3−/−, TRAF6−/−, and NIKaly/aly mouse embryonic fibroblasts (MEFs), potential roles of these proteins in LMP1-induced alternative NF-κB activation were investigated. Deficiency in IKKα or functional NIK, but not in IKKβ, IKKγ, or TRAF6, severely impaired LMP1-induced p100 processing. Notably, p100 was constitutively processed in TRAF2−/− or TRAF3−/− MEFs independently of LMP1 suggesting that TRAF2 or TRAF3 may play a regulatory role in p100 processing. Subsequently, TRAF2 or TRAF3 over-expression in HEK293 cells significantly blocked LMP1-induced p100 processing. The LMP1 CTAR1 expression in 293HEK cells activated the alternative p65/p52 complex while CTAR2 failed to do so. Taken together, LMP1 activates alternative NF-κB pathway through functional NIK and IKKα that is regulated by TRAF2 or TRAF3.
KeywordsEpstein-Barr virus Latent membrane protein 1 NF-κB p100 processing
Tumor necrosis factor
NF-κB is a family of transcription factors that mediate a wide range of cellular functions including cell proliferation, differentiation, and apoptosis (reviewed in [1, 2]). The NF-κB family includes RelA (p65), RelB, c-Rel, p105/p50 (NF-κB1), and p100/p52 (NF-κB2) that form homo or heterodimers to transactivate gene expression. In a canonical pathway for NF-κB activation, the p65/p50 complexes are retained in the cytoplasm by inhibitor of κB (IκB) proteins. Upon activation, IκB proteins are phosphorylated by IκB kinase β (IKKβ) and degraded via the ubiquitin–proteasome pathway allowing the p65/p50 complexes to translocate into the nucleus. An alternative pathway for NF-κB activation involves NF-κB inducing kinase (NIK)- and IKKα-mediated proteolytic processing of p100 to produce p52 and nuclear translocation of the RelB/p52 complexes.
Members of the Tumor Necrosis Factor (TNF) Receptor (TNFR)-associated factors (TRAFs) are adaptor proteins that interact directly or indirectly with members of the TNFR superfamily (reviewed in ). TRAFs function as signal transducers to activate transcription factors of the NF-κB and AP1 family upon TNFR ligation. To date, seven members (TRAF1 to 7) of the TRAF family have been identified in mammals.
Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1) is essential for EBV-infected B lymphocyte conversion to proliferating lymphoblastoid cell lines (LCLs), and functionally mimics CD40, a member of the TNFR superfamily (reviewed in ). LMP1 consists of a 24 amino acid (aa) cytoplasmic N-terminus, six hydrophobic transmembrane domains (aa 25–186), and a 200 aa cytoplasmic C-terminus (aa 187–386). LMP1 self-aggregates in plasma membrane lipid rafts through transmembrane domains and constitutively activate NF-κB, p38 Mitogen-Activated Protein Kinase (MAPK), and c-Jun N-terminal Kinase through two C-terminal cytoplasm signaling domains referred to as C-Terminal Activation Regions (CTAR) 1 and 2 [5, 6, 7, 8, 9, 10, 11, 12]. CTAR1 engages TRAF1, 2, 3, and 5 through a consensus PXQXT motif found in the CD40 and activates the alternative pathway for NF-κB activation. CTAR2 engages TNFR-associated death domain protein (TRADD) and Receptor Interacting Protein 1 (RIP), and activate the canonical pathway for NF-κB activation [13, 14, 15, 16, 17]. Since NF-κB activation is essential for EBV–LCL survival [18, 19], delineation of LMP1-induced NF-κB activation pathway may contribute to the discovery of potential inhibitor(s) to treat EBV-associated cancers.
This study was designed to delineate the mechanism of LMP1-induced alternative NF-κB activation. Using various knock out (KO) mouse embryonic fibroblasts (MEFs), we investigated the roles of TRAF2, TRAF3, TRAF6, NIK, IKKα, IKKβ, and IKKγ in LMP1-induced p100 processing and found previously unrecognized regulatory roles of TRAF2 and TRAF3 in LMP1-induced alternative NF-κB activation. In addition, we found that LMP1–CTAR1-induced NF-κB activation comprises the alternative p65/p52 complex.
Materials and methods
Cells, retrovirus, plasmids, and transfections
TRAF3−/− MEF was a gift from Dr. Genhong Cheng (UCLA), and IKKα−/−, IKKβ−/−, IKKγ−/−, TRAF2−/− and TRAF6−/− MEFs was previously described . NIKaly/aly MEF was a gift from Dr. Tasuku Honjo (Kyoto University). Retrovirus expressing GFP or LMP1 has been previously described . The pcDNA3FLAG-LMP1 wild type (WT), 1-231 only (CTAR1), and Δ187-351(CTAR2) have been previously described . Effectene for transient transfection was used according to the manufacturer’s directions (Qiagen, Valencia, CA).
Sub-cellular fractionation and western blot analysis
Cells were collected, fractionated, and transferred to nitrocellulose membranes as previously described . Polyclonal rabbit antibody to p100/p52 was a kind gift from Dr. Ulrich Siebenlist (NIH). Antibodies to p65 and alpha–tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Sigma-Aldrich (St. Louis, MO), respectively. Enhanced chemiluminescence detection reagents (Pierce, Rockford, IL) and secondary peroxidase-labeled anti-mouse or anti-rabbit immunoglobulin G antibody (Amersham Biosciences, Piscataway, NJ) were used according to the manufacturer’s directions.
Five million cells were lysed with NP-40 lysis buffer [25 mM Tris–HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, 1% NP-40] containing 50 mM sodium fluoride, 1 mM sodium orthovanadate, and protease inhibitor cocktail (Roche, Indianapolis, IN). Cell lysates were precleared with protein A/G agarose beads (Santa Cruz, CA) and incubated at 4°C for overnight with anti-p65 antibody (Santa Cruz). Immune complexes were collected on protein A/G agarose beads and washed three times with NP-40 lysis buffer. Immunoprecipitates were eluted by addition of an equal volume of 2× sodium dodecyl sulfate–polyacrylamide gel electrophoresis loading buffer [100 mM Tris–HCl (pH 6.8) containing 4% SDS, 0.02% bromophenol blue, and 2% ß-mercaptoethanol] and subjected to western blot analysis with either anti-p100/p52 antibody or anti-p65 antibody.
NIK and IKKα positively regulate LMP1-induced p100 processing to p52
In order to confirm the result obtained using transient transfection, WT, IKKα−/−, or NIKaly/aly MEFs were transduced with retroviruses expressing either GFP (−) or LMP1(+), and equal amounts of cell extracts were subjected to western blot analysis with anti-p100/p52 antibody at 6 days after transduction. Similar results to those obtained using transient transfection were obtained using retrovirus transduction, indicating critical roles of IKKα and NIK in LMP1-induced p100 processing (Supplementary Figure 1). Unlike in transient-transfected WT MEFs expressing LMP1 for 2 days, p100 rapidly underwent processing to p52 in retrovirus transduced WT MEFs expressing LMP1 for 6 days (Supplementary Figure 1, lane 2).
In order to further determine roles of IKKβ and IKKγ in LMP1-induced p100 processing, WT, IKKβ−/−, or IKKγ−/− MEFs were transduced with retroviruses expressing either GFP or LMP1, and equal amounts of cell extracts were subjected to western blot analysis with anti-p100/p52 antibody at 6 days after transduction. In IKKβ−/− or IKKγ−/− MEFs, LMP1-induced p100 processing to p52, despite only 25–50% of LMP1 expression in WT MEFs due to low transduction efficiency (Fig. 1b, compare lane 2 with lanes 4 and 6). Since IKKγ is dispensable for LMP1-induced canonical NF-κB activation , LMP1-induced p100 expression by activating a canonical NF-κB pathway in IKKγ−/− MEFs (Fig. 1b, compare lane 6 with lane 5). In this particular experiment, LMP1 did not strongly induce p100 expression in WT MEFs probably due to cell senescence (Fig. 1b, compare lane 2 with lane 1). Taken together, these data suggest that LMP1-induced p100 processing is dependent on NIK-IKKα, but not IKKβ-IKKγ.
TRAF2 and TRAF3 regulate LMP1-induced p100 processing
TRAF6 is not required for LMP1-induced p100 processing
Since A20 is an ubiquitin-modifying enzyme which targets TRAF6 and blocks NF-κB activation from both CTAR1 and CTAR2 [32, 33, 34, 35], we further investigated the effect of A20 on LMP1-induced p100 processing. Human embryonic kidney (HEK) 293 cells were co-transfected with LMP1 and HA-tagged A20 expression vectors, and p100 processing was determined at 48 h after transfection. LMP1-induced p100 processing was not affected by the over-expression of A20 in HEK293 cells (Fig. 3b, compare lane 2 with lane 4). Thus, A20 had no effect on LMP1-induced p100 processing. Taken together, these data indicate that TRAF6 and A20 are dispensable for LMP1-induced p100 processing.
LMP1–CTAR1 activates alternative p65/p52 complex
LMP1–CTAR1 interacts with TRAF1, 2, 3, and 5 and induces NIK/IKKα-dependent p100 processing to p52 [7, 8, 9, 11, 23, 24, 25, 36]. Since LMP1–CTAR1-induced NF-κB activation is critical for EBV-mediated transformation of B lymphocytes [8, 11, 36], delineation of LMP1-induced p100 processing may contribute to the discovery of novel therapeutic strategies in treating EBV-associated cancers. In this study, we determined roles of IKKα, IKKβ, IKKγ, NIK, TRAF2, TRAF3, and TRAF6 in LMP1-induced p100 processing to p52. Although roles of IKKα, IKKβ, IKKγ, and NIK were previously reported [23, 24, 25], we employed KO MEFs to revisit the roles of these proteins in LMP1-induced p100 processing and to validate the p100 processing assay. The novel findings in this study are that (i) the over-expression of TRAF2 or TRAF3 blocked the LMP1-induced p100 processing in HEK293 cells suggesting negative roles of these proteins in LMP1-induced alternative NF-κB activation, (ii) a deficiency of TRAF6, an essential adaptor protein in a canonical pathway of NF-κB activation, did not abolish LMP1-induced p100 processing in MEFs, (iii) the over-expression A20, a critical regulator of a canonical pathway of NF-κB activation, did not inhibit the LMP1-induced p100 processing; and (iv) LMP1–CTAR1, but not CTAR2, induced the formation of the alternative p65/p52 complex.
TRAF2 and TRAF3 negatively regulate p100 processing by inducing degradation of NIK [27, 28, 29]. TRAF3 recruits a TRAF2–cIAP1–cIAP2 E3 ubiquitin ligase complex to NIK and facilitates its degradation [29, 37]. Thus, lack of TRAF2 or TRAF3 enhances NIK protein levels and might induce robust p100 processing to p52 [28, 38]. The basal protein level of p100 in TRAF3−/− MEFs was significantly lower than in TRAF2−/− MEFs (Fig. 2a, compare lanes 3 and 4 with lanes 5 and 6). Decreased p100 expression may be due to impaired canonical pathway of NF-κB activation in TRAF3−/− cells [39, 40, 41, 42]. Furthermore, TRAF3 may be a major negative regulator of p100 processing by recruiting other E3 ubiquitin ligase complexes in addition to the TRAF2–cIAP1–cIAP2 to NIK. Since p100 processing to p52 was constitutively active in TRAF2−/− or TRAF3−/− MEFs independent of LMP1 expression (Fig. 2a, lanes 3 and 5), we employed the over-expression of TRAF2 or TRAF3 to determine roles of these proteins in LMP1-induced p100 processing. Interestingly, TRAF2 or TRAF3 over-expression in HEK293 cells significantly inhibited LMP1-induced p100 processing. LMP1 induces NIK–IKKα-dependent p100 processing possibly by binding to TRAF2 and TRAF3, down-regulating or sequestering TRAF2 and TRAF3 away from NIK and, subsequently, activating NIK. How LMP1 regulates TRAF2 and TRAF3 to activate NIK and IKKα is under investigation.
Although TRAF6 is essential for LMP1–CTAR2-induced canonical IKKβ activation , it is not required for LMP1–CTAR1-induced alternative p100 processing. Therefore, LMP1–CTAR1 or CTAR2 utilizes a unique set of adaptor proteins to activate the alternative or canonical NF-κB pathway. In consistent with the result obtained using TRAF6−/− MEFs, over-expression of A20, which down-regulates TRAF6, has no effect on LMP1-induced p100 processing. Interestingly, A20 inhibits both LMP1–CTAR1 and CTAR2-induced NF-κB activation . Since LMP1–CTAR1 activates NF-κB activation by inducing p100 processing, how A20 blocks LMP1–CTAR1-induced NF-κB activation is unclear. LMP1–CTAR1 induces the nuclear localization of p65 and activates p65/p52 heterodimers in addition to RelB/p52 heterodimers. Therefore, A20 may affect the alternative p65/p52 complex to down-regulate LMP1–CTAR1-induced NF-κB activation. A20 may have additional functions to regulate the alternative NF-κB activation pathway downstream of p100 processing that are currently unknown.
We are grateful to Dr. Elliott Kieff for discussion. M.-S. K. was supported by the National Research Foundation of Korea, and The Korean Federation of Science and Technology Societies Grant by Korean Government (MEST, Basic Research Promotion Fund). Y.-J. S. was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2010-0003301), and by the Kyungwon University Research Fund in 2010. This work was in part supported by 5R01CA085180-10 granted to Elliott Kieff, Channing Laboratory, Brigham and Women’s Hospital.