TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.     

Interleukin-1-induced NF-kappaB activation is NEMO-dependent but does not require IKKbeta

Activation of NF-kappaB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IkappaB kinase (IKK) complex, which contains two kinases named IKKalpha and IKKbeta and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKbeta is required for TNF-induced NF-kappaB activation. To determine whether NEMO and IKKalpha can form a functional IKK complex capable of activating the classical NF-kappaB pathway in the absence of IKKbeta, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IkappaBalpha degradation absolutely requires NEMO and IKKbeta. In contrast, we consistently observed intact IkappaBalpha degradation and NF-kappaB activation in response to IL-1 in two separate cell lines lacking IKKbeta. Furthermore, exogenously expressed, catalytically inactive IKKbeta blocked TNF- but not IL-1-induced IkappaBalpha degradation in wild-type MEFs, and reconstitution of IKKalpha/beta double knockout cells with IKKalpha rescued IL-1- but not TNF-induced NF-kappaB activation. Finally, we have shown that incubation of IKKbeta-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-kappaB activation. Our results therefore demonstrate that NEMO and IKKalpha can form a functional IKK complex that activates the classical NF-kappaB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways.     

TAB4 stimulates TAK1-TAB1 phosphorylation and binds polyubiquitin to direct signaling to NF-kappaB

Responses to transforming growth factor beta and multiple cytokines involve activation of transforming growth factor beta-activated kinase-1 (TAK1) kinase, which activates kinases IkappaB kinase (IKK) and MKK3/6, leading to the parallel activation of NF-kappaB and p38 MAPK. Activation of TAK1 by autophosphorylation is known to involve three different TAK1-binding proteins (TABs). Here we report a protein phosphatase subunit known as type 2A phosphatase-interacting protein (TIP) that also acts as a TAB because it co-precipitates with and directly binds to TAK1, enhances TAK1 autophosphorylation at unique sites, and promotes TAK1 phosphorylation of IKKbeta and signaling to NF-kappaB. Mass spectrometry demonstrated that co-expression of TAB4 protein significantly increased phosphorylation of four sites in TAK1, in a linker region between the kinase and TAB2/3 binding domains, and two sites in TAB1. Recombinant GST-TAB4 bound in an overlay assay directly to inactive TAK1 and activated TAK1 but not TAK1 phosphorylated in the linker sites, suggesting a bind and release mechanism. In kinase assays using TAK1 immune complexes, added GST-TAB4 selectively stimulated IKK phosphorylation. TAB4 co-precipitated polyubiquitinated proteins dependent on a Phe-Pro motif that was required to enhance phosphorylation of TAK1. TAB4 mutated at Phe-Pro dominantly interfered with IL-1beta activation of NF-kappaB involving IKK-dependent but not p38 MAPK-dependent signaling. The results show that TAB4 binds TAK1 and polyubiquitin chains to promote specific sites of phosphorylation in TAK1-TAB1, which activates IKK signaling to NF-kappaB.     

Hydrogen peroxide activates IkappaB kinases through phosphorylation of serine residues in the activation loops

The cellular redox state regulates nuclear factor-kappaB (NF-kappaB) signaling systems. We investigated the effects of H2O2 on inhibitor of NF-kappaB (IkappaB) kinases (IKKalpha and IKKbeta), which phosphorylate IkappaB leading to its degradation and NF-kappaB activation. Tumor necrosis factor (TNF) stimulation increased IKK activity within 10 min, and then IKK activity decreased gradually within 30 min in HeLa cells. Stimulation of the cells with H2O2 induced a slight activation of IKK within 30 min. Furthermore, co-stimulation with TNF suppressed the downregulation of IKK and sustained the activation for more than 30 min. H2O2 also markedly activated IKK in cells that were pretreated with TNF or phorbol myristate acetate. Electrophoretic mobility shift assay revealed that H2O2 enhanced TNF-induced NF-kappaB activation. Studies using IKK mutants and an antibody against phosphorylated IKK proteins revealed that phosphorylation of serine residues, Ser180 of IKKalpha and Ser181 of IKKbeta, in the activation loops was essential for the H2O2-mediated activation of IKK. H2O2-induced activation of IKKalpha and IKKbeta was reduced by IKKbeta and IKKalpha kinase-negative mutants, respectively, indicating that IKKalpha and IKKbeta were stimulated by H2O2 in an interdependent manner. These results suggest that oxidative radical stress has stimulatory effects on NF-kappaB through the activation of IKK, which is mediated by the phosphorylation of serine residues in the activation loops.     

Tumor necrosis factor-alpha-induced IKK phosphorylation of NF-kappaB p65 on serine 536 is mediated through the TRAF2, TRAF5, and TAK1 signaling pathway

The activation of NF-kappaB has been shown to be regulated by multiple phosphorylations of IkappaBs and the NF-kappaB p65 subunit. Here, we characterized the intracellular signaling pathway leading to phosphorylation of p65 on Ser-536 using a novel anti-phospho-p65 (Ser-536) antibody. The Ser-536 of endogenous p65 was rapidly phosphorylated in response to a wide variety of NF-kappaB stimulants including TNF-alpha in the cytoplasm and rapidly dephosphorylated in the nucleus. The TNF-alpha-but not IL-1beta-induced Ser-536 phosphorylation was severely impaired in murine embryonic fibroblasts derived from traf2-/-traf5-/- mice. Bay 11-7082, an inhibitor of IkappaB phosphorylation, inhibited the TNF-alpha-induced phosphorylation in vivo. In addition, overexpression of TGF-beta-activated kinase 1 (TAK1), IKKalpha and IKKbeta stimulated the phosphorylation, and their dominant negative mutants blocked the TNF-alpha-induced phosphorylation. Moreover, small interfering RNAs (siRNAs) against TAK1, IKKalpha and IKKbeta blocked the phosphorylation of endogenous p65. On the other hand, calyculin-A, a protein phosphatase inhibitor, blocked the dephosphorylation in the nucleus in vivo. These results indicate that similar signaling pathways were utilized for the phosphorylations of IkappaBalpha and p65, which further support the idea that both IkappaB and NF-kappaB are substrates for the IKK complex in the activation of NF-kappaB.     

TAK1-binding protein 1, TAB1, mediates osmotic stress-induced TAK1 activation but is dispensable for TAK1-mediated cytokine signaling

TAK1 kinase is an indispensable intermediate in several cytokine signaling pathways including tumor necrosis factor, interleukin-1, and transforming growth factor-beta signaling pathways. TAK1 also participates in stress-activated intracellular signaling pathways such as osmotic stress signaling pathway. TAK1-binding protein 1 (TAB1) is constitutively associated with TAK1 through its C-terminal region. Although TAB1 is known to augment TAK1 catalytic activity when it is overexpressed, the role of TAB1 under physiological conditions has not yet been identified. In this study, we determined the role of TAB1 in TAK1 signaling by analyzing TAB1-deficient mouse embryonic fibroblasts (MEFs). Tumor necrosis factor- and interleukin-1-induced activation of TAK1 was entirely normal in Tab1-deficient MEFs and could activate both mitogen-activated protein kinases and NF-kappaB. In contrast, we found that osmotic stress-induced activation of TAK1 was largely impaired in Tab1-deficient MEFs. Furthermore, we showed that the C-terminal 68 amino acids of TAB1 were sufficient to mediate osmotic stress-induced TAK1 activation. Finally, we attempted to determine the mechanism by which TAB1 activates TAK1. We found that TAK1 is spontaneously activated when the concentration is increased and that it is totally dependent on TAB1. Cell shrinkage under the osmotic stress condition increases the concentration of TAB1-TAK1 and may oligomerize and activate TAK1 in a TAB1-dependent manner. These results demonstrate that TAB1 mediates TAK1 activation only in a subset of TAK1 pathways that are mediated through spontaneous oligomerization of TAB1-TAK1.     

TAK1 is a component of the Epstein-Barr virus LMP1 complex and is essential for activation of JNK but not of NF-kappaB

Epstein-Barr virus latent membrane protein 1 (LMP1) activates NF-kappaB and c-Jun N-terminal kinase (JNK), which is essential for LMP1 oncogenic activity. Genetic analysis has revealed that tumor necrosis factor receptor-associated factor 6 (TRAF6) is an indispensable intermediate of LMP1 signaling leading to activation of both NF-kappaB and JNK. However, the mechanism by which LMP1 engages TRAF6 for activation of NF-kappaB and JNK is not well understood. Here we demonstrate that TAK1 mitogen-activated protein kinase kinase kinase and TAK1-binding protein 2 (TAB2), together with TRAF6, are recruited to LMP1 through its N-terminal transmembrane region. The C-terminal cytoplasmic region of LMP1 facilitates the assembly of this complex and enhances activation of JNK. In contrast, IkappaB kinase gamma is recruited through the C-terminal cytoplasmic region and this is essential for activation of NF-kappaB. Furthermore, we found that ablation of TAK1 resulted in the loss of LMP1-induced activation of JNK but not of NF-kappaB. These results suggest that an LMP1-associated complex containing TRAF6, TAB2, and TAK1 plays an essential role in the activation of JNK. However, TAK1 is not an exclusive intermediate for NF-kappaB activation in LMP1 signaling.     

FAF1 suppresses IkappaB kinase (IKK) activation by disrupting the IKK complex assembly

This study presents a molecular inhibitory mechanism by Fas-associated factor 1 (FAF1) on IkappaB kinase (IKK) activation, where divergent NF-kappaB-activating stimuli converge. FAF1 interacts with IKKbeta in response to proinflammatory stimuli (such as tumor necrosis factor-alpha, interleukin-1beta, and lipopolysaccharide) and suppresses IKK activation. Interaction of the leucine-zipper domain of IKKbeta with FAF1 affected the IKK heterocomplex (IKKalpha/beta) and homocomplex (IKKalpha/alpha, IKKbeta/beta) formations and attenuated IKKgamma recruitment to IKKbeta. Overexpression of FAF1 reduced the level of IKKbeta activity, whereas FAF1 depletion increased the activity. These results indicate that FAF1 inhibits IKK activation and its downstream signaling by interrupting the IKK complex assembly through physical interaction with IKKbeta. Taken together, FAF1 robustly suppresses NF-kappaB activation through the inhibition of IKK activation in combination with previously reported cytoplasmic retention of NF-kappaB p65 (Park, M. Y., Jang, H. D., Lee, S. Y., Lee, K. J., and Kim, E. (2004) J. Biol. Chem. 279, 2544-2549). Such redundant suppression would prevent inadvertent activation of the NF-kappaB pathway.