The Germinal Center Kinase TNIK Is Required for Canonical NF-κB and JNK Signaling in B-Cells by the EBV Oncoprotein LMP1 and the CD40 Receptor
Figure 3
TNIK is essential for JNK activation by LMP1.
(A) HEK293 cells were transfected in 6-well plates with siRNA against human TNIK (siTNIK) or non-targeting control siRNA (siCTR). Subsequently, the cells were co-transfected with 1 µg of HA-JNK1 and 1 µg of HA-LMP1 wildtype or HA-LMP1Δ194–386 lacking the LMP1 signaling domain, as indicated. HA-JNK1 was immunoprecipitated from cell lysates and immunocomplex kinase assays were performed using recombinant GST-c-Jun as a substrate. Equal HA-JNK1 immunoprecipitation was confirmed by the anti-JNK1 antibody. TNIK downregulation and LMP1 expression was monitored in cell lysates using anti-TNIK and anti-LMP1 (CS1-4) antibodies. The null control construct LMP1Δ194–386 cannot be detected by the CS1-4 antibody because all CS1-4 epitopes are located within the LMP1 signaling domain. Quantification of four independent experiments ± standard deviations, given as x-fold inductions: lane 1, 1.15±0.32; lane 2, 1.48±0.6; lane 3, 1.0±0.0; lane 4, 3.23±1.03. (B) The knockdown of TNIK in lymphoblastoid cells blocks the JNK pathway. EREB2-5 B-cells were treated with Accell siRNA against human TNIK or non-targeting siRNA for 72 h. Cell lysates were analyzed by immunoblotting using anti-TNIK, anti-phospho-JNK, anti-JNK1, and anti-LMP1 (CS1-4) antibodies. n = 3.