The tumor necrosis factor (TNF)-receptor 1–associated death domain protein (TRADD) mediates induction of apoptosis as well as activation of NF-κB by cellular TNF-receptor 1 (TNFR1). TRADD is also recruited by the latent membrane protein 1 (LMP1) oncoprotein of Epstein-Barr virus, but its role in LMP1 signaling has remained enigmatic. In human B lymphocytes, we have generated, to our knowledge, the first genetic knockout of TRADD to investigate TRADD's role in LMP1 signal transduction. Our data from TRADD-deficient cells demonstrate that TRADD is a critical signaling mediator of LMP1 that is required for LMP1 to recruit and activate I-κB kinase β (IKKβ). However, in contrast to TNFR1, LMP1-induced TRADD signaling does not induce apoptosis. Searching for the molecular basis for this observation, we characterized the 16 C-terminal amino acids of LMP1 as an autonomous and unique virus-derived TRADD-binding domain. Replacing the death domain of TNFR1 by LMP1′s TRADD-binding domain converts TNFR1 into a nonapoptotic receptor that activates NF-κB through a TRAF6-dependent pathway, like LMP1 but unlike wild-type TNFR1. Thus, the unique interaction of LMP1 with TRADD encodes the transforming phenotype of viral TRADD signaling and masks TRADD's pro-apoptotic function.

TNIK has an important function in physiological activation and viral transformation of human B-cells by interacting with the TRAF6 adapter complex and mediating NF-κB and JNK signal transduction.

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The Epstein-Barr virus (EBV) oncoprotein LMP1 (latent membrane protein 1) mimics a constitutively active receptor molecule. It contributes to viral cell transformation by the activation of NF-kappaB, JNK/AP1, MAPK, JAK/STAT and PI3-kinase signaling. LMP1 recruits TRAF1-3, 5 and 6, TRADD and RIP1, which are also known as signaling mediators of Toll-like and tumor necrosis factor-receptors. Here, we established a functional proteomics approach to identify novel interaction partners of the LMP1 signaling domain. This approach led to the characterization of the tyrosine phosphatase SHP1 as a direct binding partner of LMP1. Interaction of SHP1 with LMP1 was verified in primary human B-cells, which had been transformed with a recombinant EBV carrying a HAtagged LMP1 allele. The SHP1 binding site of LMP1 is located within the membrane-proximal region of the LMP1 signaling domain and shows no overlap with known protein interaction domains of LMP1. The unique sequence of this site does not resemble known SHP1 interaction motifs of cellular proteins. Mutation of the SHP1 site caused the loss of SHP1 binding to LMP1 in EBV-transformed human B-cells. SHP1 has previously been described as a negative regulator of growth factor or immune receptor signaling by dephosphorylating e.g. tyrosine kinases such as JAKs or SRC kinases. LMP1 induction of the NF-kappaB pathway was greatly enhanced in SHP1-knockout DT40 B-cells as compared to wildtype cells. This effect was reverted by reconstitution of SHP1 expression in the SHP1-KO cells. Also mutation of the SHP1 interaction site or the co-expression of a dominantnegative SHP1 caused hyperactivation of NF-kappaB signaling and JAK3 hyperphosphorylation by LMP1. Because the SHP1 interaction site of LMP1 mediates inhibitory effects on LMP1 signaling, we named this region CTIR1 (C-terminal inhibitory region 1). In summary, the proteomic analysis of the LMP1 complex revealed a novel autoregulatory mechanism of oncogenic LMP1 signaling, which limits its own activity through the recruitment of a tyrosine phosphatase. This mechanism might be of high relevance for the survival of EBV-transformed cells because LMP1 hyperactivity is known to be toxic for the target cells.