FIGURE 4.

Ser-412 phosphorylation and the C-terminal coiled-coil domain are essential for TAK1 kinase activities. A, FLAG-tagged TAK1 WT, K63W, S412A, and aa 1–303 and 1–436 truncation mutants were expressed and purified from 293T cells and used in the in vitro kinase assay by using kinase-dead MKK6 purified from E. coli as the substrate. After the reaction, proteins were detected with the indicated antibodies. B, RAW264.7 cells stably expressing FLAG-TAK1 WT or S412A mutant were stimulated for the indicated time. FLAG-TAK1 was then immunoprecipitated by using M2 FLAG antibody-agarose beads and used in the kinase assay as in A. IPKA, immunoprecipitation protein kinase assay. IP, immunoprecipitation; p-, phosphorylated.