Interleukin-1 stimulated activation of the COT catalytic subunit through the phosphorylation of Thr290 and Ser62

The protein kinase COT/Tpl2 is activated by interleukin-1 (IL-1), TNFα and lipopolysaccharide, and its activation by these agonists involves the IκB kinase β (IKKβ) catalysed phosphorylation of the p105 regulatory subunit. Here, we show that COT activation also requires catalytic subunit phosphorylation, since IL-1β induced a 5–10-fold activation of a COT mutant unable to bind p105. Activation was paralleled by the phosphorylation of Thr290 and Ser62 and unaffected by the IKKβ inhibitor PS1145 at concentrations which prevented the degradation of IκBα. Mutagenesis experiments indicated that COT activation is initiated by Thr290 phosphorylation catalysed by an IL-1-stimulated protein kinase distinct from IKKβ, while Ser62 phosphorylation is an autophosphorylation event required for maximal activation.