TI - TRADD Mediates IKKbeta Activation by LMP1 . AB - TRADD overexpression and RNAi studies have suggested that TRADD mediates TNFR1 activation of the NF-kappaB pathway [22,24] . To demonstrate that DG75 TRADD-/- cells are suitable tools to study the role of TRADD in signal transduction , we stimulated TRADD+/+ and TRADD-/- cells with soluble human TNFalpha and monitored the activity of the NF-kappaB pathway (Figure 2A) . DG75 wild-type cells responded to TNFalpha stimulation with a rapid PHOSphorylation and subsequent degradation of I-kappaB . In contrast , the knockout of TRADD completely abolished TNFalpha -induced I-kappaB PHOSphorylation . This result delivered genetic evidence for an essential role of TRADD in TNFR1 signaling to NF-kappaB and showed that canonical NF-kappaB signaling is intact in DG75 cells . TRADD interacts with the CTAR2 domain of LMP1 , which signals through IKKbeta to activate the I-kappaB -dependent NF-kappaB pathway [8,10,11] . To investigate a potential role of TRADD in LMP1 -induced NF-kappaB signaling , we performed IKKbeta kinase assays in DG75 TRADD+/+ and TRADD-/- cells , because IKKbeta activity is the most proximal and specific readout of CTAR2 signaling on the NF-kappaB axis ( Figure 2B ) . Due to the high endogenous NF-kappaB activity levels in DG75 cells , NF-kappaB reporter gene assays did not result in reliable induction levels after LMP1 expression ( unpublished data ) . HA-LMP1Delta371-386 , which lacks the 16 C-terminal amino acids of LMP1 , is defective in CTAR2 signaling and served as null control . Expression of wild-type HA-LMP1 resulted in a 2.8-fold activation of IKKbeta in TRADD+/+ cells , monitored as I-kappaBalpha subSTRate phosphorylaTION and IKKbeta autoPHOSphorylation (Figure 2B) . Thus , CTAR2 triggered canonical NF-kappaB signaling in wild-type cells . Strikingly , CTAR2 lost its potential to induce IKKbeta in TRADD-/- cells , which clearly showed that CTAR2 requires TRADD to activate the IKKbeta pathway (Figure 2B) . To demonstrate specificity of this effect for LMP1 , we analyzed CD40 signaling in wild-type and TRADD-deficient cells . CD40 does not recruit TRADD and was therefore anticipated to signal also in the absence of TRADD . Confirming this presumption , a constitutively active chimera composed of the LMP1 transmembrane domain and the CD40 signaling domain , LMP1-CD40 , induced IKKbeta activity in TRADD+/+ and TRADD-/- cells to similar levels . To exclude that defects unrelated to TRADD had caused the defective LMP1 signaling in DG75 TRADD-/- cells , we tested whether ectopic expression of TRADD restored LMP1 activation of IKKbeta in TRADD-deficient cells (Figure 2C) . To express TRADD at physiological levels , TRADD-/- cells were co-transfected with the pACYC184-1012.4 vector , which carries the complete human TRADD gene under the control of its endogenous promoter ( see Materials and Methods ) . Exogenous TRADD expression alone did not induce IKKbeta , further confirming that TRADD protein levels lay within a normal range which did not result in TRADD autoactivation . Notably , reintroduction of TRADD enabled LMP1 to activate IKKbeta in TRADD-/- cells , verifying TRADD's essential role in CTAR2 signaling on the NF-kappaB axis ( Figure 2C ) . In summary , our results provided strong genetic evidence for a critical role of TRADD in LMP1 signal transduction . In fact , LMP1 requires TRADD to induce the IKKbeta/NF-kappaB pathway .