CaMKII directly binds, phosphorylates, and activates TAK1. (A,B) RAW264.7 cells were stimulated with 0.1 μg/mL LPS for the indicated time. Equal amount cell lysates were immunoprecipitated with CaMKII-α (A) or TAK1 (B) antibody and then immunoblotted (IB) with CaMKII-α and TAK1 antibody. (C) HEK293 cells were transfected with TAK1-HA and wt or mutant CaMKII-α–flag construct as indicated. After 24 hours, cell extracts were immunoprecipitated with anti-flag antibody and then immunoblotted with anti-HA and anti-flag antibody. C indicates catalytic domain (amino acids 1-260); R, regulatory domain (amino acids 261-309); A, association domain (amino acids 310-478). (D) GST pull-down assays were performed with recombinant GST-tagged CaMKII-α and cell extracts of RAW264.7 cells. (E) One microgram of recombinant TAK1 protein was incubated with recombinant active CaMKII-α at 30°C for 30 minutes. Samples were separated by SDS-PAGE followed by autoradiography. (F) MBP was added into the reaction mixture as in panel E followed by incubation for another 20 minutes. Samples were analyzed by immunoblotting with anti–phospho-MBP antibody. Data are representative of 3 independent experiments.