Feedback control of the protein kinase TAK1 by SAPK2a/p38alpha

TAB1, a subunit of the kinase TAK1, was phosphorylated by SAPK2a/p38alpha at Ser423, Thr431 and Ser438 in vitro. TAB1 became phosphorylated at all three sites when cells were exposed to cellular stresses, or stimulated with tumour necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) or lipopolysaccharide (LPS). The phosphorylation of Ser423 and Thr431 was prevented if cells were pre-incubated with SB 203580, while the phosphorylation of Ser438 was partially inhibited by PD 184352. Ser423 is the first residue phosphorylated by SAPK2a/p38alpha that is not followed by proline. The activation of TAK1 was enhanced by SB 203580 in LPS-stimulated macrophages, and by proinflammatory cytokines or osmotic shock in epithelial KB cells or embryonic fibroblasts. The activation of TAK1 by TNF-alpha, IL-1 or osmotic shock was also enhanced in embryonic fibroblasts from SAPK2a/p38alpha-deficient mice, while incubation of these cells with SB 203580 had no effect. Our results suggest that TAB1 participates in a SAPK2a/p38alpha-mediated feedback control of TAK1, which not only limits the activation of SAPK2a/p38alpha but synchronizes its activity with other signalling pathways that lie downstream of TAK1 (JNK and IKK).     

TAB1 modulates IL-1alpha mediated cytokine secretion but is dispensable for TAK1 activation

Biochemical evidence indicates that TGF-beta-activated kinase 1 (TAK1), a key modulator of the inflammatory response, exists in a complex with various adaptor proteins including the TAK1 binding protein 1 (TAB1). However, the physiological importance of TAB1 in TAK1 activation, and in the subsequent induction of proinflammatory mediators, remains unclear. In this study, a critical role for TAK1 in IL-1alpha or TNFalpha stimulated MAPK and NFkappaB activation was confirmed by inhibition of the nuclear accumulation of NFkappaB p65 and phosphorylated forms of c-Jun and p38 following siRNA mediated TAK1 silencing. These effects were associated with significant reductions in IL-1alpha stimulated levels of secreted IL-6, IL-8, MCP-1 and GM-CSF. In contrast, IL-1alpha or TNFalpha dependent cellular redistribution of NFkappaB p65 and phosphorylated c-Jun and p38 was not affected by 80% siRNA mediated knockdown of TAB1 protein levels. Interestingly, IL-6, IL-8 and GM-CSF release from TAB1 siRNA transfected cells was significantly reduced following IL-1alpha treatment, but was unchanged after TNFalpha stimulation, suggesting differential roles for TAB1 in IL-1alpha and TNFalpha signalling pathways. These findings may imply an as yet unidentified role for TAB1 in the inflammatory response, which is independent of the activation of classical TAK1 associated signalling cascades.     

Phosphorylation-dependent activation of TAK1 mitogen-activated protein kinase kinase kinase by TAB1

TAK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that is involved in the c-Jun N-terminal kinase/p38 MAPKs and NF-kappaB signaling pathways. Here, we characterized the molecular mechanisms of TAK1 activation by its specific activator TAB1. Autophosphorylation of two threonine residues in the activation loop of TAK1 was necessary for TAK1 activation. Association with TAK1 and induction of TAK1 autophosphorylation required the C-terminal 24 amino acids of TAB1, but full TAK1 activation required additional C-terminal Ser/Thr rich sequences. These results demonstrated that the association between the kinase domain of TAK1 and the C-terminal TAB1 triggered the phosphorylation-dependent TAK1 activation mechanism.     

TAK1-dependent signaling requires functional interaction with TAB2/TAB3

Transforming growth factor beta-activated kinase 1 (TAK1), a member of the MAPKKK family, was initially described to play an essential role in the transforming growth factor beta-signaling pathway, but recent evidence has emerged implicating TAK1 in the interleukin (IL)-1 and tumor necrosis factor (TNF) pathways. Notably, two homologous proteins, TAB2 and TAB3, have been identified as adaptors linking TAK1 to the upstream adaptors TRAFs. However, it remains unclear whether the interaction between TAB2/TAB3 and TAK1 is necessary for its kinase activation and subsequent activation of the IKK and MAPK pathways. Here, we characterized the TAB2/TAB3-binding domain in TAK1 and further examined the requirement of this interaction for IL-1, TNF, and RANKL signaling. Through deletion mapping experiments, we demonstrated that the binding motif for TAB2/TAB3 is a non-contiguous region located within the last C-terminal 100 residues of TAK1. However, residues 479-553 of TAK1 appear to be necessary and sufficient for TAB2/TAB3 interaction. Conversely, residues 574-693 of TAB2 were shown to interact with TAK1. A green fluorescent protein fusion protein containing the last 100 residues of TAK1 (TAK1-C100) abolished the interaction of endogenous TAB2/TAB3 with TAK1, the phosphorylation of TAK1, and prevented the activation of IKK and MAPK induced by IL-1, TNF, and RANKL. Furthermore, TAK1-C100 blocked RANKL-induced nuclear accumulation of NFATc1 and consequently osteoclast differentiation consistent with the ability of a catalytically inactive TAK1 to block RANKL-mediated signaling. Significantly, our study provides evidence that the TAB2/TAB3 interaction with TAK1 is crucial for the activation of signaling cascades mediated by IL-1, TNF, and RANKL.     

Critical roles of threonine 187 phosphorylation in cellular stress-induced rapid and transient activation of transforming growth factor-beta-activated kinase 1 (TAK1) in a signaling complex containing TAK1-binding protein TAB1 and TAB2

Transforming growth factor-beta-activated kinase 1 (TAK1) mitogen-activated protein kinase kinase kinase has been shown to be activated by cellular stresses including tumor necrosis factor-alpha (TNF-alpha). Here, we characterized the molecular mechanisms of cellular stress-induced TAK1 activation, focusing mainly on the phosphorylation of TAK1 at Thr-187 and Ser-192 in the activation loop. Thr-187 and Ser-192 are conserved among species from Caenorhabditis elegans to human, and their replacement with Ala resulted in inactivation of TAK1. Immunoblotting with a novel phospho-TAK1 antibody revealed that TNF-alpha significantly induced the phosphorylation of endogenous TAK1 at Thr-187, and subsequently the phosphorylated forms of TAK1 rapidly disappeared. Intermolecular autophosphorylation of Thr-187 was essential for TAK1 activation. RNA interference and overexpression experiments demonstrated that TAK1-binding protein TAB1 and TAB2 were involved in the phosphorylation of TAK1, but they regulated TAK1 phosphorylation differentially. Furthermore, SB203580 and p38alpha small interfering RNA enhanced TNF-alpha-induced Thr-187 phosphorylation as well as TAK1 kinase activity, indicating that the phosphorylation is affected by p38alpha/TAB1/TAB2-mediated feedback control of TAK1. These results indicate critical roles of Thr-187 phosphorylation in the stress-induced rapid and transient activation of TAK1 in a signaling complex containing TAB1 and TAB2.     

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.     

The Yersinia enterocolitica effector YopP inhibits host cell signalling by inactivating the protein kinase TAK1 in the IL-1 signalling pathway

The mechanism by which YopP simultaneously inhibits mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB pathways has been elusive. Ectopic expression of YopP inhibits the activity and ubiquitination of a complex consisting of overexpressed TGF-beta-activated kinase 1 (TAK1) and its subunit TAK1-binding protein (TAB)1, but not of MEK kinase 1. YopP, but not the catalytically inactive mutant YopP(C172A), also suppresses basal and interleukin-1-inducible activation of endogenous TAK1, TAB1 and TAB2. YopP does not affect the interaction of TAK1, TAB1 and TAB2 but inhibits autophosphorylation of TAK1 at Thr 187 and phosphorylation of TAB1 at Ser 438. Glutathione S-transferase-tagged YopP (GST-YopP) binds to MAPK kinase (MAPKK)4 and TAB1 but not to TAK1 or TAB2 in vitro. Furthermore, YopP in synergy with a previously described negative regulatory feedback loop inhibits TAK1 by MAPKK6-p38-mediated TAB1 phosphorylation. Taken together, these data strongly suggest that YopP binds to TAB1 and directly inhibits TAK1 activity by affecting constitutive TAK1 and TAB1 ubiquitination that is required for autoactivation of TAK1.     

O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release

Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is a key serine/threonine protein kinase that mediates signals transduced by pro-inflammatory cytokines such as transforming growth factor-β, tumour necrosis factor (TNF), interleukin-1 (IL-1) and wnt family ligands. TAK1 is found in complex with binding partners TAB1-3, phosphorylation and ubiquitination of which has been found to regulate TAK1 activity. In this study, we show that TAB1 is modified with N-acetylglucosamine (O-GlcNAc) on a single site, Ser395. With the help of a novel O-GlcNAc site-specific antibody, we demonstrate that O-GlcNAcylation of TAB1 is induced by IL-1 and osmotic stress, known inducers of the TAK1 signalling cascade. By reintroducing wild-type or an O-GlcNAc-deficient mutant TAB1 (S395A) into Tab1(-/-) mouse embryonic fibroblasts, we determined that O-GlcNAcylation of TAB1 is required for full TAK1 activation upon stimulation with IL-1/osmotic stress, for downstream activation of nuclear factor κB and finally production of IL-6 and TNFα. This is one of the first examples of a single O-GlcNAc site on a signalling protein modulating a key innate immunity signalling pathway.     

TAB1beta (transforming growth factor-beta-activated protein kinase 1-binding protein 1beta ), a novel splicing variant of TAB1 that interacts with p38alpha but not TAK1

The mitogen-activated protein kinases (MAPKs) play an important role in a variety of biological processes. Activation of MAPKs is mediated by phosphorylation on specific regulatory tyrosine and threonine sites. We have recently found that activation of p38alpha MAPK can be carried out not only by its upstream MAPK kinases (MKKs) but also by p38alpha autophosphorylation. p38alpha autoactivation requires an interaction of p38alpha with TAB1 (transforming growth factor-beta-activated protein kinase 1-binding protein 1). The autoactivation mechanism of p38alpha has been found to be important in cellular responses to a number of physiologically relevant stimuli. Here, we report the characterization of a splicing variant of TAB1, TAB1beta. TAB1 and TAB1beta share the first 10 exons. The 11th and 12th exons of TAB1 were spliced out in TAB1beta, and an extra exon, termed exon beta, downstream of exons 11 and 12 in the genome was used as the last exon in TAB1beta. The mRNA of TAB1beta was expressed in all cell lines examined. The TAB1beta mRNA encodes a protein with an identical sequence to TAB1 except the C-terminal 69 amino acids were replaced with an unrelated 27-amino acid sequence. Similar to TAB1, TAB1beta interacts with p38alpha but not other MAPKs and stimulates p38alpha autoactivation. Different from TAB1, TAB1beta does not bind or activate TAK1. Inhibition of TAB1beta expression with RNA interference in MDA231 breast cancer cells resulted in the reduction of basal activity of p38alpha and invasiveness of MDA231 cells, suggesting that TauAlphaBeta1beta is involved in regulating p38alpha activity in physiological conditions.     

Prostaglandin E2 enhances osteoclastic differentiation of precursor cells through protein kinase A-dependent phosphorylation of TAK1

Prostaglandin E2 (PGE2) synergistically enhances the receptor activator for NF-kappa B ligand (RANKL)-induced osteoclastic differentiation of the precursor cells. Here we investigated the mechanisms of the stimulatory effect of PGE2 on osteoclast differentiation. PGE2 enhanced osteoclastic differentiation of RAW264.7 cells in the presence of RANKL through EP2 and EP4 prostanoid receptors. RANKL-induced degradation of I kappa B alpha and phosphorylation of p38 MAPK and c-Jun N-terminal kinase in RAW264.7 cells were up-regulated by PGE2 in a cAMP-dependent protein kinase A (PKA)-dependent manner, suggesting that EP2 and EP4 signals cross-talk with RANK signals. Transforming growth factor beta-activated kinase 1 (TAK1), an important MAPK kinase kinase in several cytokine signals, possesses a PKA recognition site at amino acids 409-412. PKA directly phosphorylated TAK1 in RAW264.7 cells transfected with wild-type TAK1 but not with the Ser412 --> Ala mutant TAK1. Ser412 --> Ala TAK1 served as a dominant-negative mutant in PKA-enhanced degradation of I kappa B alpha, phosphorylation of p38 MAPK, and PGE2-enhanced osteoclastic differentiation in RAW264.7 cells. Furthermore, forskolin enhanced tumor necrosis factor alpha-induced I kappa B alpha degradation, p38 MAPK phosphorylation, and osteoclastic differentiation in RAW264.7 cells. Ser412 --> Ala TAK1 abolished the stimulatory effects of forskolin on those cellular events induced by tumor necrosis factor alpha. Ser412 --> Ala TAK1 also inhibited the forskolin-induced up-regulation of interleukin 6 production in RAW264.7 cells treated with lipopolysaccharide. These results suggest that the phosphorylation of the Ser412 residue in TAK1 by PKA is essential for cAMP/PKA-induced up-regulation of osteoclastic differentiation and cytokine production in the precursor cells.     

Determinants that control the specific interactions between TAB1 and p38alpha

Previous studies have revealed that transforming growth factor-beta-activated protein kinase 1 (TAB1) interacts with p38alpha and induces p38alpha autophosphorylation. Here, we examine the sequence requirements in TAB1 and p38alpha that drive their interaction. Deletion and point mutations in TAB1 reveal that a proline residue in the C terminus of TAB1 (Pro412) is necessary for its interaction with p38alpha. Furthermore, a cryptic D-domain-like docking site was identified adjacent to the N terminus of Pro412, putting Pro412 in the phi(B)+3 position of the docking site. Through mutational analysis, we found that the previously identified hydrophobic docking groove in p38alpha is involved in this interaction, whereas the CD domain and ED domain are not. Furthermore, chimeric analysis with p38beta (which does not bind to TAB1) revealed a previously unidentified locus of p38alpha comprising Thr218 and Ile275 that is essential for specific binding of p38alpha to TAB1. Converting either of these residues to the corresponding amino acid of p38beta abolishes p38alpha interaction with TAB1. These p38alpha mutants still can be fully activated by p38alpha upstream activating kinase mitogen-activated protein kinase kinase 6, but their basal activity and activation in response to some extracellular stimuli are reduced. Adjacent to Thr218 and Ile275 is a site where large conformational changes occur in the presence of docking-site peptides derived from p38alpha substrates and activators. This suggests that TAB1-induced autophosphorylation of p38alpha results from conformational changes that are similar but unique to those seen in p38alpha interactions with its substrates and activating kinases.     

Regulation of the JNK pathway by TGF-beta activated kinase 1 in rheumatoid arthritis synoviocytes

c-Jun N-terminal kinase (JNK) contributes to metalloproteinase (MMP) gene expression and joint destruction in inflammatory arthritis. It is phosphorylated by at least two upstream kinases, the mitogen-activated protein kinase kinases (MEK) MKK4 and MKK7, which are, in turn, phosphorylated by MEK kinases (MEKKs). However, the MEKKs that are most relevant to JNK activation in synoviocytes have not been determined. These studies were designed to assess the hierarchy of upstream MEKKs, MEKK1, MEKK2, MEKK3, and transforming growth factor-β activated kinase (TAK)1, in rheumatoid arthritis (RA). Using either small interfering RNA (siRNA) knockdown or knockout fibroblast-like synoviocytes (FLSs), MEKK1, MEKK2, or MEKK3 deficiency (either alone or in combination) had no effect on IL-1β-stimulated phospho-JNK (P-JNK) induction or MMP expression. However, TAK1 deficiency significantly decreased P-JNK, P-MKK4 and P-MKK7 induction compared with scrambled control. TAK1 knockdown did not affect p38 activation. Kinase assays showed that TAK1 siRNA significantly suppressed JNK kinase function. In addition, MKK4 and MKK7 kinase activity were significantly decreased in TAK1 deficient FLSs. Electrophoretic mobility shift assays demonstrated a significant decrease in IL-1β induced AP-1 activation due to TAK1 knockdown. Quantitative PCR showed that TAK1 deficiency significantly decreased IL-1β-induced MMP3 gene expression and IL-6 protein expression. These results show that TAK1 is a critical pathway for IL-1β-induced activation of JNK and JNK-regulated gene expression in FLSs. In contrast to other cell lineages, MEKK1, MEKK2, and MEKK3 did not contribute to JNK phosphorylation in FLSs. The data identify TAK1 as a pivotal upstream kinase and potential therapeutic target to modulate synoviocyte activation in RA.     

Regulation of the maintenance of peripheral T-cell anergy by TAB1-mediated p38 alpha activation

In anergic T cells, T-cell receptor (TCR)-mediated responses are functionally inactivated by negative regulatory signals whose mechanisms are poorly understood. Here, we show that CD4(+) T cells anergized in vivo by superantigen Mls-1(a) express a scaffolding protein, transforming growth factor beta-activated protein kinase 1-binding protein 1 (TAB1), that negatively regulates TCR signaling through the activation of mitogen-activated protein kinase p38 alpha. TAB1 was not expressed in naive and activated CD4(+) T cells. Inhibition of p38 activity in anergic T cells by a chemical inhibitor resulted in the recovery of interleukin 2 (IL-2) and the inhibition of IL-10 secretion. T-cell hybridoma 2B4 cells transduced with TAB1-containing retrovirus (TAB1-2B4 cells) showed activated p38 alpha, inhibited extracellular signal-regulated kinase (ERK) activity, culminating in reduced IL-2 levels and increased IL-10 production. The use of a p38 inhibitor or cotransfection of a dominant-negative form of p38 in TAB1-2B4 cells resulted in the recovery of ERK activity and IL-2 production. These results imply that TAB1-mediated activation of p38 alpha in anergic T cells regulates the maintenance of T-cell unresponsiveness both by inhibiting IL-2 production and by promoting IL-10 production.     

Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling

The cytokines IL-1 and TNF induce expression of a series of genes that regulate inflammation through activation of NF-kappaB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL-1- and TNF-induced activation of the NF-kappaB pathway. TAB2, a TAK1-binding protein, is involved in IL-1-induced NF-kappaB activation by physically linking TAK1 to TRAF6. However, IL-1-induced activation of NF-kappaB is not impaired in TAB2-deficient embryonic fibroblasts. Here we report the identification and characterization of a novel protein designated TAB3, a TAB2-like molecule that associates with TAK1 and can activate NF-kappaB similar to TAB2. Endogenous TAB3 interacts with TRAF6 and TRAF2 in an IL-1- and a TNF-dependent manner, respectively. Further more, IL-1 signaling leads to the ubiquitination of TAB2 and TAB3 through TRAF6. Cotransfection of siRNAs directed against both TAB2 and TAB3 inhibit both IL-1- and TNF-induced activation of TAK1 and NF-kappaB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL-1 and TNF signal transduction.     

Regulation of c-Jun N-terminal kinase by MEKK-2 and mitogen-activated protein kinase kinase kinases in rheumatoid arthritis

The mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK) is a critical regulator of collagenase-1 production in rheumatoid arthritis (RA). The MAPKs are regulated by upstream kinases, including MAPK kinases (MAPKKs) and MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in arthritis. RT-PCR studies of MAP3K gene expression in RA and osteoarthritis synovial tissue demonstrated mitogen-activated protein kinase/ERK kinase kinase (MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1, TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3 mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro kinase assays using MEKK2 immunoprecipitates demonstrated that IL-1 increased MEKK2-mediated phosphorylation of the key MAPKKs that activate JNK (MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.     

IRAK-mediated translocation of TRAF6 and TAB2 in the interleukin-1-induced activation of NFkappa B

The interleukin-1 (IL-1) receptor-associated kinase (IRAK) is required for the IL-1-induced activation of nuclear factor kappaB and c-Jun N-terminal kinase. The goal of this study was to understand how IRAK activates the intermediate proteins TRAF6, TAK1, TAB1, and TAB2. When IRAK is phosphorylated in response to IL-1, it binds to the membrane where it forms a complex with TRAF6; TRAF6 then dissociates and translocates to the cytosol. The membrane-bound IRAK similarly mediates the IL-1-induced translocation of TAB2 from the membrane to the cytosol. Different regions of IRAK are required for the translocation of TAB2 and TRAF6, suggesting that IRAK mediates the translocation of each protein separately. The translocation of TAB2 and TRAF6 is needed to form a TRAF6-TAK1-TAB1-TAB2 complex in the cytosol and thus activate TAK1. Our results show that IRAK is required for the IL-1-induced phosphorylation of TAK1, TAB1, and TAB2. The phosphorylation of these three proteins correlates strongly with the activation of nuclear factor kappaB but is not necessary to activate c-Jun N-terminal kinase.     

MEKK3 regulates IFN-gamma production in T cells through the Rac1/2-dependent MAPK cascades

MEKK3 is a conserved Ser/Thr protein kinase belonging to the MAPK kinase kinase (MAP3K) family. MEKK3 is constitutively expressed in T cells, but its function in T cell immunity has not been fully elucidated. Using Mekk3 T cell conditional knockout (T-cKO) mice, we show that MEKK3 is required for T cell immunity in vivo. Mekk3 T-cKO mice had reduced T cell response to bacterial infection and were defective in clearing bacterial infections. The Ag-induced cytokine production, especially IFN-γ production, was impaired in Mekk3-deficient CD4 T cells. The TCR-induced ERK1/2, JNK, and p38 MAPKs activation was also defective in Mekk3-deficient CD4 T cells. In vitro, MEKK3 is not required for Th1 and Th2 cell differentiation. Notably, under a nonpolarizing condition (Th0), Mekk3 deficiency led to a significant reduction of IFN-γ production in CD4 T cells. Furthermore, the IL-12/IL-18-driven IFN-γ production and MAPK activation in Mekk3-deficient T cells was not affected suggesting that MEKK3 may selectively mediate the TCR-induced MAPK signals for IFN-γ production. Finally, we found that MEKK3 activation by TCR stimulation requires Rac1/2. Taken together, our study reveals a specific role of MEKK3 in mediating the TCR signals for IFN-γ production.     

Interleukin-1 (IL-1) receptor-associated kinase leads to activation of TAK1 by inducing TAB2 translocation in the IL-1 signaling pathway

Interleukin-1 (IL-1) is a proinflammatory cytokine that recognizes a surface receptor complex and generates multiple cellular responses. IL-1 stimulation activates the mitogen-activated protein kinase kinase kinase TAK1, which in turn mediates activation of c-Jun N-terminal kinase and NF-kappaB. TAB2 has previously been shown to interact with both TAK1 and TRAF6 and promote their association, thereby triggering subsequent IL-1 signaling events. The serine/threonine kinase IL-1 receptor-associated kinase (IRAK) also plays a role in IL-1 signaling, being recruited to the IL-1 receptor complex early in the signal cascade. In this report, we investigate the role of IRAK in the activation of TAK1. Genetic analysis reveals that IRAK is required for IL-1-induced activation of TAK1. We show that IL-1 stimulation induces the rapid but transient association of IRAK, TRAF6, TAB2, and TAK1. TAB2 is recruited to this complex following translocation from the membrane to the cytosol upon IL-1 stimulation. In IRAK-deficient cells, TAB2 translocation and its association with TRAF6 are abolished. These results suggest that IRAK regulates the redistribution of TAB2 upon IL-1 stimulation and facilitates the formation of a TRAF6-TAB2-TAK1 complex. Formation of this complex is an essential step in the activation of TAK1 in the IL-1 signaling pathway.