IKKα, β, ε and TBK1 in CD154-induced NF-κB activation

Objective: To investigate the role of IKKs in CD154-induced NF-κB activation and their relationship with TRAF family proteins. Methods: Using recombinant CD154 to stimulate EBV/LMP1 negative Ramos B cell and to observe the roles of IKKα, β with vectors expressing constitutively active (SS→EE) or kinase dead (K→M) versions of IKKα or IKKβ. Activity of IKK was determined by IKK immunocomplex kinase assay and estimated as the ability to phosphorylate glutathione-S-transferase (GST)-IκB- α. Immunoprecipitation (IP) experiments were performed using specific anti-IKKα, β, ε and TBK1 antibodies conjugated with protein-A beads to examine IKK's correlation with TRAF. Results: CD154 stimulation on Ramos B cells induced activation of IKKα, β, ε and TBK1, increasing of TRAF2's IP with both IKKε and TBK1, TRAF5's IP with TBK1 and decreasing of TANK's IP with TBK1. IKKα/β did not show any direct IP with TRAF proteins. Conclusion: CD154 stimulation on Ramos B cells induces activation of IKKα, β, ε and TBK1, TRAF2 association with both IKKε and TBK1, TRAF5 association with TBK1 but TANK dissociation from TBK1. IKKα/β lie in downstream of this signal pathway.