TNIK is required for JNK and canonical NF-κB signaling by CD40.

(A) The knockdown of TNIK impairs CD40-induced JNK signaling in HEK293 cells. HEK293 cells were transfected in 6-well plates with TNIK or non-targeting siRNA. Subsequently, the cells were transfected with 2 µg of the CD40 expression vector pESBOS-CD40 where indicated. 1 µg of pRK5-HA-JNK1 was co-transfected. HA-JNK activity was monitored in immunocomplex kinase assays. Immunoprecipitated HA-JNK1, expression of CD40, and downregulation of TNIK was detected using the anti-JNK1, anti-CD40, and anti-TNIK antibodies. Quantification of four independent experiments: lane 1, 1.0±0.0; lane 2, 7.18±3.15; lane 3, 0.8±0.59; lane 4, 3.28±1.68. (B) IKKβ activation by CD40 is blocked by TNIK downregulation. HEK293 cells were transfected as in (A) except that Flag-IKKβ was transfected instead of HA-JNK1. Flag-IKKβ immunocomplex kinase assays were performed. Flag-IKKβ, CD40, and TNIK were detected using the indicated antibodies. Quantification of three independent experiments: lane 1, 1.0±0.0; lane 2, 8.17±1.81; lane 3, 0.6±0.16; lane 4, 3.2±1.36. (C) The knockdown of TNIK in B-cells inhibits JNK and canonical NF-κB signaling after stimulation of the endogenous CD40 receptor. BL41 cells were treated with 5 µM Accell TNIK or non-targeting siRNA for 72 h. Cells were stimulated with 500 ng/ml recombinant human CD40 ligand for the indicated times. Cell lysates were subjected to immunoblot analysis using anti-phospho-JNK, anti-JNK1, anti-IκBα, and anti-TNIK antibodies. Equal loading was verified by staining with anti-tubulin antibodies. n = 3. (D) TNIK mediates CD54 upregulation by CD40. BL41 B-cells were incubated with TNIK or non-targeting Accell siRNA and stimulated with 1 µg/ml CD40 ligand as indicated and described in Materials and Methods. CD54 surface levels were analyzed by flow cytometry. n = 2. (E) CD40 stimulation induces the interaction of TNIK and TRAF6 in B-cells. BL41 cells were stimulated with 500 ng/ml CD40 ligand. At the indicated times, cells were lysed and endogenous TNIK was immunoprecipitated by the anti-TNIK antibody. TRAF6 co-precipitation was detected by the anti-TRAF6 (H-274) antibody. Lysate controls were stained with the indicated antibodies. n = 3.