The protein kinase Tpl2 (tumour progression locus 2) is activated by LPS (lipopolysaccharide), TNFalpha (tumour necrosis factor alpha) and IL (interleukin)-1. Activation of the native Tpl2 complex by these agonists requires the IKKbeta {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase beta}-catalysed phosphorylation of the p105/NF-kappaB1 subunit and is accompanied by the release of the catalytic subunit from both p105/NF-kappaB1 and another subunit ABIN2 (A20-binding inhibitor of NF-kappaB 2). In the present study we report that IL-1 activates the transfected Tpl2 catalytic subunit in an HEK (human embryonic kidney)-293 cell line that stably expresses the IL-1R (IL-1 receptor), but does not express the protein kinase IRAK1 (IL-1R-associated kinase). In these cells IL-1 does not activate IKKbeta or induce the phosphorylation of p105/NF-kappaB1, and nor does the IKKbeta inhibitor PS1145 prevent the IL-1-induced activation of transfected Tpl2. However, the IL-1-stimulated activation of transfected Tpl2 in IRAK1-null cells or activation of the endogenous Tpl2 complex in IRAK1-expressing cells is suppressed by the protein kinase inhibitor PP2 by a mechanism that does not involve inhibition of Src family protein tyrosine kinases. The IL-1-stimulated activation of transfected Tpl2 is accompanied by its phosphorylation at Thr290 and Ser400 and by enhanced phosphorylation of Ser62, which we demonstrate are autophosphorylation events catalysed by Tpl2 itself. We further show that IL-1 triggers the dissociation of Tpl2 from co-transfected ABIN2 in IRAK1-null IL-1R cells, which is not suppressed by PP2 or by the inhibition of Tpl2 or IKKbeta. These studies identify two new signalling events involved in activation of the native Tpl2 complex by IL-1. First, the IRAK1-, IKKbeta- and PP2-independent dissociation of Tpl2 from ABIN2; secondly, the IRAK1- and IKKbeta-independent, but PP2-sensitive, activation of the Tpl2 catalytic subunit.