Fig. 3: The role of TAK1 in JNK and NF-κB signaling by LMP1.

a Pharmacological TAK1 inhibition blocks JNK, but not canonical NF-κB activation by LMP1. NGFR-LMP1 was induced by antibody crosslinking in the presence of DMSO or 500 nM of the TAK1 inhibitor (5Z)−7-oxozeaenol (TAK1-IH). b LMP1-induced IKK2 activity depends on the presence of TAK1 protein, but TAK1 kinase activity is dispensable. TAK1-deficient HEK293 cells (clone 3) or corresponding wildtype cells were transfected with Flag-IKK2 together with HA-LMP1, the inactive mutant HA-LMP1(AAA/Y384G), or empty vector. Where indicated, the cells were treated with 500 nM TAK1-IH for 5 h prior to cell lysis. Flag-IKK2 kinase assays were performed. IKK2 activity was measured as radioactive GST-IκBα phosphorylation (topmost panel). Phosphorylation of immunoprecipitated Flag-IKK2 at serines 177/181 was detected on immunoblots. c JNK activation by LMP1 requires both TAK1 protein and its kinase activity. Experiments were essentially performed as described under c, except that HA-JNK1 was transfected and assayed using GST-c-Jun as substrate. For kinase assay quantification and statistics see Supplementary Table 4. d IKK2 activation involves IKK2 autophosphorylation. HEK293 cells were transfected as indicated and treated with DMSO, IKK inhibitor ACHP or TAK1-IH. Phosphorylation of imunoprecipitated Flag-IKK2 at serines 177/181 was detected. e LMP1 depends on TAK1 protein, but not its kinase activity, to induce K63-ubiquitination of NEMO. HEK293 and TAK1^crKOHEK293 cells were transfected with HA-Ubiquitin K63 and Flag-NEMO together with LMP1 wildtype or empty vector (w/o). After 24 h, cells were treated with 500 nM TAK1-IH where indicated. Flag-NEMO was immunoprecipitated (IP) using the Flag (6F7) antibody. K63-linked ubiquitination of NEMO was detected by the HA (3F10) antibody. f TAK1 mediates IKK2 interaction with the LMP1 complex. HEK293 wildtype and TAK1^crKO cells were transfected with Flag-IKK2 together with HA-LMP1 wildtype or AAA/Δ371–386 lacking CTAR2. Flag-IKK2 was precipitated via the Flag (6F7) antibody and interacting LMP1 was detected by the HA (3F10) antibody. CRISPR/Cas9 knockouts were verified by immunoblotting (This Figure) and by sequencing (see Supplementary Table 2). a–f The data are representative of at least two independent experiments. a–b, d–f For immunoblot quantification and statistics see Supplementary Table 3.