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. 1998 Aug 18;95(17):10106-11.
doi: 10.1073/pnas.95.17.10106.

Epstein-Barr virus-transforming protein latent infection membrane protein 1 activates transcription factor NF-kappaB through a pathway that includes the NF-kappaB-inducing kinase and the IkappaB kinases IKKalpha and IKKbeta

B S Sylla  1 , S C Hung, D M Davidson, E Hatzivassiliou, N L Malinin, D Wallach, T D Gilmore, E Kieff, G Mosialos
Affiliations

Epstein-Barr virus-transforming protein latent infection membrane protein 1 activates transcription factor NF-kappaB through a pathway that includes the NF-kappaB-inducing kinase and the IkappaB kinases IKKalpha and IKKbeta

B S Sylla et al. Proc Natl Acad Sci U S A. .

Abstract

The Epstein-Barr virus oncoprotein latent infection membrane protein 1 (LMP1) is a constitutively aggregated pseudo-tumor necrosis factor receptor (TNFR) that activates transcription factor NF-kappaB through two sites in its C-terminal cytoplasmic domain. One site is similar to activated TNFRII in associating with TNFR-associated factors TRAF1 and TRAF2, and the second site is similar to TNFRI in associating with the TNFRI death domain interacting protein TRADD. TNFRI has been recently shown to activate NF-kappaB through association with TRADD, RIP, and TRAF2; activation of the NF-kappaB-inducing kinase (NIK); activation of the IkappaB alpha kinases (IKKalpha and IKKbeta); and phosphorylation of IkappaB alpha. IkappaB alpha phosphorylation on Ser-32 and Ser-36 is followed by its degradation and NF-kappaB activation. In this report, we show that NF-kappaB activation by LMP1 or by each of its effector sites is mediated by a pathway that includes NIK, IKKalpha, and IKKbeta. Dominant negative mutants of NIK, IKKalpha, or IKKbeta substantially inhibited NF-kappaB activation by LMP1 or by each of its effector sites.

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Figures

Figure 1
Figure 1
Schematic diagram of aggregated LMP1 in the cell plasma membrane. TRAF1, TRAF2, and TRAF3 constitutively associate with the LMP1-proximal NF-κB-activating region (CTAR1), which is also a key transformation effector site (TES1). TRADD constitutively associates with the LMP1 distal and major NF-κB-activating domain (CTAR2), which is a second transformation effector site (TES2). Aggregation of TRAF2 most likely activates NIK, which in turn phosphorylates and activates IKKα and IKKβ. IKKα and IKKβ then phosphorylate IκBα, leading to its ubiquitination and degradation and NF-κB activation.
Figure 2
Figure 2
Dominant negative mutant of protein kinase NIK (NIKKK429–430AA) inhibits NF-κB activation by LMP1. (A) A luciferase reporter plasmid with three upstream NF-κB binding sites was electroporated into 293 cells with a control β-galactosidase expression construct (GKβgal), and pCDNA3 (Invitrogen)-based expression plasmids for LMP1 (5 μg), F-LMPCTAR1 (10 μg), or F-LMPCTAR2 (10 μg) in the presence or absence of pCDNA3NIK KK429–430AA (referred as INIK) (10 μg). Luciferase activities were normalized for cotransfected β-galactosidase activity. Values of NF-κB activation in the presence of NIKKK429–430AA were normalized to the corresponding activities (set at 100%) obtained in the absence NIKKK429–430AA. Mean values (±SD) of relative luciferase activities from four experiments are shown. In these four experiments, NF-κB activation by full-length LMP1 was an average of 29-fold, whereas LMPCTAR1 induced 9-fold and LMPCTAR2 induced 19-fold NF-κB activation. (B) Western blot of LMP1 expression in the presence and absence of NIKKK429–430AA (INIK). LMP1 was detected with the S12 monoclonal antibody. (C) Western blot of F-LMPCTAR1 and F-LMPCTAR2 expression in the presence and absence of NIKKK429–430AA (INIK). FLAG-tagged proteins were detected with the M5 monoclonal anti-FLAG antibody.
Figure 3
Figure 3
NIK induces IκBα phosphorylation on Ser-32 and Ser-36. In vitro-translated 35S-labeled NIK and 35S-labeled wild-type IκBα, S32/36A, S32A, or S36A mutants were incubated in a standard kinase buffer and were analyzed by SDS/PAGE and autoradiography. The positions of IκBα, and phosphorylated IκBα (PIκBα) are indicated by arrows.
Figure 4
Figure 4
NIK phosphorylates IκBα in the presence but not in the absence of rabbit reticulocyte lysate. In vitro-translated 35S-labeled F-NIK and IκBα in reticulocyte lysate or, after immunoprecipitation from reticulocyte lysate (IPIκBα + IPF-NIK), were incubated in a standard kinase buffer containing 2.5 μM okadaic acid, and samples were then analyzed by SDS/PAGE and autoradiography.
Figure 5
Figure 5
Effects of dominant negative mutants of IKKα (M-IKKαK44A) (A and B) or IKKβ (F-IKKβΔ34) (C and D) on LMP1-, LMPCTAR1-, or LMPCTAR2-induced NF-κB activation in 293 cells. F-LMP1 (4 μg) in pSG5, F-LMPCTAR1(0.5 μg), or F-LMPCTAR2 (1 μg) in pCDNA3 were cotransfected with a 3XκB luciferase reporter gene plasmid, a β-galactosidase expression construct, and increasing amounts (0.1–2 μg) of pRK5-myc-IKKαK44A, pCDN3-F-IKKβΔ34, or pCDNA3, a vector control. Because of the high expression of F-LMPCTAR1 relative to F-LMP1 and LMPCTAR2, 0.5–2 μg of myc-IKKαK44A and IKKβΔ34 were cotransfected with LMPCTAR1. Luciferase activities were normalized for β-galactosidase activities. NF-κB activation by LMP1 constructs cotransfected with IKKαK44A or IKKβΔ34 were normalized to the activities (set at 100%) obtained in the absence of IKKαK44A or IKKβΔ34. In these experiments LMP1-induced NF-κB activation averaged 76-fold, whereas LMPCTAR1-induced activation averaged 17-fold and LMPCTAR2-induced activation averaged 53-fold. Representative relative luciferase activities from at least two experiments are shown. Western blot analysis of F-LMP1, myc-IKKαK44A (B), or F-IKKβΔ34 (D) expression levels. After protein transfer, the membranes were sequentially probed with monoclonal antibodies to detect FLAG (M5) or to detect M-IKKαK44A (9E10).
Figure 6
Figure 6
IKKα and IKKβΔ9 inhibit LMP1-induced NF-κB activation. Luciferase reporter assays are as described in Fig. 5. Cells were transfected with 0.5 μg of expression pCDNA3-LMP1, pRK5-myc-IKKα, and pCDNA3-F-IKKβΔ9.
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