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The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.

The TPL-2 MEK kinase is essential for activation of the Erk MAP kinase pathway during innate immune responses. TPL-2 is found in complex with ABIN-2 (A20-binding inhibitor of NF-kappaB 2). Here, using antigen-presenting cells from ABIN-2-deficient mice, we show that ABIN-2 was required for optimal activation of Erk induced by receptors that signal via TPL-2, including Toll-like receptor 4 and tumor necrosis factor receptor 1 in macrophages, and CD40 in B cells. ABIN-2 was necessary for the maintenance of TPL-2 protein stability. In contrast, ABIN-2 deficiency did not affect agonist-induced regulation of transcription factor NF-kappaB. Stimulation of ABIN-2-deficient macrophages via Toll-like receptor 4 showed that different thresholds of Erk signaling were required for optimal induction of tumor necrosis factor and interleukin 1beta. Thus, ABIN-2 acts to positively regulate the Erk signaling potential by stabilizing TPL-2.

Activation of the oncogenic potential of the MEK kinase TPL-2 (Cot) requires deletion of its C terminus. This mutation also weakens the interaction of TPL-2 with NF-kappaB1 p105 in vitro, although it is unclear whether this is important for the activation of TPL-2 oncogenicity. It is demonstrated here that TPL-2 stability in vivo relies on its high-affinity, stoichiometric association with NF-kappaB1 p105. Formation of this complex occurs as a result of two distinct interactions. The TPL-2 C terminus binds to a region encompassing residues 497 to 534 of p105, whereas the TPL-2 kinase domain interacts with the p105 death domain. Binding to the p105 death domain inhibits TPL-2 MEK kinase activity in vitro, and this inhibition is significantly augmented by concomitant interaction of the TPL-2 C terminus with p105. In cotransfected cells, both interactions are required for inhibition of TPL-2 MEK kinase activity and, consequently, the catalytic activity of a C-terminally truncated oncogenic mutant of TPL-2 is not affected by p105. Thus, in addition to its role as a precursor for p50 and cytoplasmic inhibitor of NF-kappaB, p105 is a negative regulator of TPL-2. Insensitivity of C-terminally truncated TPL-2 to this regulatory mechanism is likely to contribute to its ability to transform cells.

The transcription factor NF-κB is composed of homodimeric andheterodimeric complexes of Rel/NF-κB-family polypeptides, which include Rel-A, c-Rel, Rel-B, NF-κB1/p50 and NF-κB2/p52 (ref. 1). The NF-κB1 gene encodes a larger precursor protein, p105, from which p50 is produced constitutively by proteasome-mediated removal of the p105 carboxy terminus2,3,4,5. The p105 precursor also acts as an NFκB-inhibitory protein, retaining associated p50, c-Rel and Rel-A proteins in the cytoplasm through its carboxy terminus6,7. Following cell stimulation by agonists, p105 is proteolysed more rapidly and released Rel subunits translocate into the nucleus8,9,10. Here we show that TPL-2 (ref. 11), which ishomologous to MAP-kinase-kinase kinases in its catalytic domain12, forms a complex with the carboxy terminus of p105. TPL-2 was originally identified, in a carboxy-terminal-deleted form, as an oncoprotein in rats11 and is more than 90% identical to the human oncoprotein COT13. Expression of TPL-2 results in phosphorylation and increased degradation of p105 while maintaining p50production. This releases associated Rel subunits or p50–Rel heterodimers to generate active nuclear NF-κB. Furthermore, kinase-inactive TPL-2 blocks the degradation of p105 induced by tumour-necrosis factor-α. TPL-2 is therefore a component of a new signalling pathway that controls proteolysis of NF-κB1 p105.

To investigate the importance of proteolysis of NF-kappaB1 p105 induced by the kinase IKK in activation of the transcription factor NF-kappaB, we generated 'Nfkb1(SSAA/SSAA)' mice, in which the IKK-target serine residues of p105 were substituted with alanine. Nfkb1(SSAA/SSAA) mice had far fewer CD4+ regulatory and memory T cells because of cell-autonomous defects. These T cell subtypes require activation of NF-kappaB by the T cell antigen receptor for their generation, and the Nfkb1(SSAA) mutation resulted in less activation of NF-kappaB in CD4+ T cells and proliferation of CD4+ T cells after stimulation of the T cell antigen receptor. The Nfkb1(SSAA) mutation also blocked the ability of CD4+ T cells to provide help to wild-type B cells during a primary antibody response. IKK-induced p105 proteolysis is therefore essential for optimal T cell antigen receptor-induced activation of NF-kappaB and mature CD4+ T cell function.

The nf-kb2 gene encodes the cytoplasmic NF-kappaB inhibitory protein p100 from which the active p52 NF-kappaB subunit is derived by proteasome-mediated proteolysis. Ligands which stimulate p100 processing to p52 have not been defined. Here, ligation of CD40 on transfected 293 cells is shown to trigger p52 production by stimulating p100 ubiquitylation and subsequent proteasome-mediated proteolysis. CD40-mediated p52 accumulation is dependent on de novo protein synthesis and triggers p52 translocation into the nucleus to generate active NF-kappaB dimers. Endogenous CD40 ligation on primary murine splenic B cells also stimulates p100 processing, which results in the delayed nuclear translocation of p52-RelB dimers. In both 293 cells and primary splenic B cells, the ability of CD40 to trigger p100 processing requires functional NF-kappaB-inducing kinase (NIK). In contrast, NIK activity is not required for CD40 to stimulate the degradation of IkappaBalpha in either cell type. The regulation of p100 processing by CD40 is likely to be important for the transcriptional regulation of CD40 target genes in adaptive immune responses.

Stimulation of cells with tumor necrosis factor alpha (TNFalpha) triggers NF-kappaB1 p105 proteolysis, releasing associated Rel subunits to translocate into the nucleus and modulate target gene expression. Phosphorylation of serine 927 within the p105 PEST region by the IkappaB kinase (IKK) complex is required to promote p105 proteolysis in response to TNFalpha stimulation. In this study, the role of the p105 death domain (DD) in signal-induced p105 proteolysis is investigated. Endogenous p105 is shown to interact with the IKK complex in HeLa cells, and transient transfection experiments in 293 cells indicate that each of the catalytic components of the IKK complex, IKK1 and IKK2, can bind to p105. Interaction of p105 with both IKK1 and IKK2 is substantially reduced by deletion of the p105 DD or introduction of a specific point mutation (L841A) into the p105 DD homologous to the lpr mutation in Fas. Phosphorylation of immunoprecipitated p105 on serine 927 by purified recombinant IKK1 or IKK2 protein in vitro is dramatically reduced in both DD mutants relative to wild type. Furthermore, both of the DD mutations significantly impair the ability of low concentrations of IKK2 to induce p105 serine 927 phosphorylation and proteolysis in transiently transfected 3T3 cells. However, high levels of transiently expressed IKK2 bypass the requirement for the p105 DD to induce p105 serine 927 phosphorylation. Finally, p105 serine 927 phosphorylation by the endogenous IKK complex after TNFalpha stimulation and subsequent p105 proteolysis is blocked in both p105 DD mutants when stably expressed in HeLa cells. Thus, the p105 DD acts as a docking site for IKK, increasing its local concentration in the vicinity of the p105 PEST region and facilitating efficient serine 927 phosphorylation.