TAK1-binding Protein Research Articles

Minocycline targets the NF-κB Nexus through suppression of TGF-β1-TAK1-IκB signaling in ovarian cancer.

Substantial evidence supports the critical role of NF-κB in ovarian cancer. Minocycline, a tetracycline, has been shown to exhibit beneficial effects in this malignancy through regulation of a cohort of genes that overlap significantly with the NF-κB transcriptome. Here, it was examined whether or not the molecular mechanism could be attributed to modulation of NF-κB signaling using a combination of in vitro and in vivo models. Minocycline suppressed constitutive NF-κB activation in OVCAR-3 and SKOV-3 ovarian carcinoma cells and was correlated with attenuation of IκBα kinase (IKK) activation, IκBα phosphorylation and degradation, and p65 phosphorylation and nuclear translocation. The inhibition of IKK was found to be associated with suppression of TGF-β-activated-kinase-1 (TAK1) activation and its dissociation from TAK1-binding-protein-1 (TAB1), an indispensable functional mediator between TGF-β and TAK1. Further studies demonstrated that minocycline downregulated TGF-β1 expression. Enforced TGF-β1 expression induced NF-κB activity, and minocycline rescued this effect. Consistent with this finding, TGF-β1 knockdown suppressed NF-κB activation and abrogated the inhibitory effect of minocycline on this transcription factor. These results suggest that the minocycline-induced suppression of NF-κB activity is mediated, in part, through inhibition of TGF-β1. Furthermore, the influence of minocycline on NF-κB pathway activation was examined in female nude mice harboring intraperitoneal OVCAR-3 tumors. Both acute and chronic administration of minocycline led to suppression of p65 phosphorylation and nuclear translocation accompanied by downregulation of NF-κB activity and endogenous protein levels of its target gene products. These data reveal the therapeutic potential of minocycline as an agent targeting the pro-oncogenic TGF-β-NF-κB axis in ovarian cancer. This preclinical study lends support to the notion that ovarian cancer management would benefit from administration of minocycline.

Crystal structure of the p38α MAP kinase in complex with a docking peptide from TAB1

The mitogen-activated protein kinase (MAPK) p38α is a key regulator in many cellular processes, whose activity is tightly regulated by upstream kinases, phosphatases and other regulators. Transforming growth factor-β activated kinase 1 (TAK1) is an upstream kinase in p38α signaling, and its full activation requires a specific activator, the TAK1-binding protein (TAB1). TAB1 was also shown to be an inducer of p38α's autophosphorylation and/or a substrate driving the feedback control of p38α signaling. Here we determined the complex structure of the unphosphorylated p38α and a docking peptide of TAB1, which shows that the TAB1 peptide binds to the classical MAPK docking groove and induces long-range conformational changes on p38α. Our structural and biochemical analyses suggest that TAB1 is a reasonable substrate of p38α, yet the interaction between the docking peptide and p38α may not be sufficient to trigger trans-autophosphorylation of p38α.

MiR-155 Regulates Immune Modulatory Properties of Mesenchymal Stem Cells by Targeting TAK1-binding Protein 2

MSCs possess potent immunosuppressive capacity. We have reported that mouse MSCs inhibit T cell proliferation and function via nitric oxide. This immune regulatory capacity of MSCs is induced by the inflammatory cytokines IFNγ together with either TNFα or IL-1β. This effect of inflammatory cytokines on MSCs is extraordinary; logarithmic increases in the expression of iNOS and chemokines are often observed. To investigate the molecular mechanisms underlying this robust effect of cytokines, we examined the expression of microRNAs in MSCs before and after cytokine treatment. We found that miR-155 is most significantly up-regulated. Furthermore, our results showed that miR-155 inhibits the immunosuppressive capacity of MSCs by reducing iNOS expression. We further demonstrated that miR-155 targets TAK1-binding protein 2 (TAB2) to regulate iNOS expression. Additionally, knockdown of TAB2 reduced iNOS expression. In summary, our study demonstrated that miR-155 inhibits the immunosuppressive capacity of MSCs by reducing iNOS expression by targeting TAB2. Our data revealed a novel role of miR-155 in regulating the immune modulatory activities of MSCs.

Functional studies for theTRAF6mutation associated with hypohidrotic ectodermal dysplasia

Hypohidrotic ectodermal dysplasia (HED) is a rare condition characterized by hypotrichosis, hypohidrosis and hypodontia. A de novo heterozygous mutation in the tumour necrosis factor receptor-associated factor 6 gene (TRAF6) was recently identified in a patient with HED, while functional consequences resulting from the mutation remained unknown. To determine the mechanism by which the TRAF6 mutation results in HED. We performed coimmunoprecipitation (co-IP) studies to determine whether the mutation would affect the interaction of TRAF6 with transforming growth factor β-activated kinase 1 (TAK1), TAK1-binding protein 2 (TAB 2) and ectodysplasin-A receptor-associated death domain protein (EDARADD). We then performed co-IP and glutathione S-transferase-pulldown assays to determine the TRAF6 binding sequences in EDARADD. In addition, we analysed the effect of the mutant TRAF6 protein on the affinity between wild-type TRAF6 and EDARADD, as well as on EDARADD-mediated nuclear factor (NF)-κB activation. The mutant TRAF6 protein was capable of forming a complex with TAK1 and TAB 2 in a similar way to wild-type TRAF6. However, the mutant TRAF6 protein completely lost the affinity to EDARADD, while the wild-type TRAF6 bound to the N-terminal domain of EDARADD. Furthermore, the mutant TRAF6 inhibited the interaction between the wild-type TRAF6 and EDARADD, and also potentially reduced the EDARADD-mediated NF-κB activity. We conclude that the mutant TRAF6 protein shows a dominant negative effect against the wild-type TRAF6 protein, which is predicted to affect the EDARADD-mediated activation of NF-κB during the development of ectoderm-derived organs, and to lead to the HED phenotype.

TAK1 (MAP3K7) Signaling Regulates Hematopoietic Stem Cells through TNF-Dependent and -Independent Mechanisms

A cytokine/stress signaling kinase Tak1 (Map3k7) deficiency is known to impair hematopoietic progenitor cells. However, the role of TAK1 signaling in the stem cell function of the hematopoietic system is not yet well defined. Here we characterized hematopoietic stem cells (HSCs) harboring deletion of Tak1 and its activators, Tak1 binding proteins 1 and 2 (Tab1 and Tab2) using a competitive transplantation assay in a mouse model. Tak1 single or Tab1/Tab2 double deletions completely eliminated the reconstitution activity of HSCs, whereas Tab1 or Tab2 single deletion did not cause any abnormality. Tak1 single or Tab1/Tab2 double deficient lineage-negative, Sca-1+, c-Kit+ (LSK) cells did not proliferate and underwent cell death. We found that Tnfr1 deficiency restored the reconstitution activity of Tak1 deficient bone marrow cells for 6–18 weeks. However, the reconstitution activity of Tak1- and Tnfr1-double deficient bone marrow cells declined over the long term, and the number of phenotypically identified long-term hematopoietic stem cells were diminished. Our results indicate that TAB1- or TAB2-dependent activation of TAK1 is required for maintenance of the hematopoietic system through two mechanisms: one is prevention of TNF-dependent cell death and the other is TNF-independent maintenance of long-term HSC.

TAK1 kinase signaling regulates embryonic angiogenesis by modulating endothelial cell survival and migration

AbstractTGF-β activated kinase 1 (TAK1) is a mediator of various cytokine signaling pathways. Germline deficiency of Tak1 causes multiple abnormalities, including dilated blood vessels at midgestation. However, the mechanisms by which TAK1 regulates vessel formation have not been elucidated. TAK1 binding proteins 1 and 2 (TAB1 and TAB2) are activators of TAK1, but their roles in embryonic TAK1 signaling have not been determined. In the present study, we characterized mouse embryos harboring endothelial-specific deletions of Tak1, Tab1, or Tab2 and found that endothelial TAK1 and TAB2, but not TAB1, were critically involved in vascular formation. TAK1 deficiency in endothelial cells caused increased cell death and vessel regression at embryonic day 10.5 (E10.5). Deletion of TNF signaling largely rescued endothelial cell death in TAK1-deficient embryos at E10.5. However, embryos deficient in both TAK1 and TNF signaling still exhibited dilated capillary networks at E12.5. TAB2 deficiency caused reduced TAK1 activity, resulting in abnormal capillary blood vessels, similar to the compound deficiency of TAK1 and TNF signaling. Ablation of either TAK1 or TAB2 impaired cell migration and tube formation. Our results show that endothelial TAK1 signaling is important for 2 biologic processes in angiogenesis: inhibiting TNF-dependent endothelial cell death and promoting TNF-independent angiogenic cell migration.

Abstract 336: TAB1 Initiates p38α Autophosphorylation During Myocardial Ischemia

The p38 mitogen-activated protein kinase (MAPK) family has been implicated in many aspects of cell biology including inflammation, gene expression, cell cycle progression, differentiation and apoptosis. The canonical pathway of p38 activation results in dual phosphorylation of p38 induced via TGF-beta activated kinase 1 (TAK1) which is an upstream Ser/Thr kinase of the MAPK kinases (MKK) of p38; MKK3/6. However, a MKK-independent mechanism of activation is proposed as TAK 1 binding protein 1 (TAB1) appears to induce autophosphorylation of p38 through a direct interaction during myocardial ischaemia, worsening injury. When human embryonic kidney cells are transfected with wild-type TAB1, there is a marked increase in activating dual phosphorylation of p38 compared to untransfected cells (5.3 ± 1.9 normalised to untransfected). The ATP-competitive p38 inhibitor SB203580 (1µM) markedly reduced the phosphorylation of transfected p38 (20.4 ± 9.7 vs 5.4 ± 1.5). An SB203580-resistant form of p38 (T106M) was also co-transfected with TAB1, and the resulting p38 phosphorylation was no longer reduced by the presence of SB203580 (12.4 ± 7.5 vs 12.0 ± 6.7) but was reduced by another pharmacological inhibitor, BIRB796 (10µM) that does not depend on T106 for binding (5.6 ± 1.1 vs 1.6 ± 0.4, P<0.05). In conclusion, these findings suggest that p38 activation by autophosphorylation is TAB1-mediated.

Analysis of Nuclear Factor-κB (NF-κB) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-κB

Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli.

Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation

Oligomerization of tau is a key process contributing to the progressive death of neurons in Alzheimer's disease. Tau is modified by O-linked N-acetylglucosamine (O-GlcNAc), and O-GlcNAc can influence tau phosphorylation in certain cases. We therefore speculated that increasing tau O-GlcNAc could be a strategy to hinder pathological tau-induced neurodegeneration. Here we found that treatment of hemizygous JNPL3 tau transgenic mice with an O-GlcNAcase inhibitor increased tau O-GlcNAc, hindered formation of tau aggregates and decreased neuronal cell loss. Notably, increases in tau O-GlcNAc did not alter tau phosphorylation in vivo. Using in vitro biochemical aggregation studies, we found that O-GlcNAc modification, on its own, hinders tau oligomerization. O-GlcNAc also inhibits thermally induced aggregation of an unrelated protein, TAK-1 binding protein, suggesting that a basic biochemical function of O-GlcNAc may be to prevent protein aggregation. These results also suggest O-GlcNAcase as a potential therapeutic target that could hinder progression of Alzheimer's disease.

Epithelial transforming growth factor β-activated kinase 1 (TAK1) is activated through two independent mechanisms and regulates reactive oxygen species

Dysregulation in cellular redox systems results in accumulation of reactive oxygen species (ROS), which are causally associated with a number of disease conditions. Transforming growth factor β-activated kinase 1 (TAK1) is a signaling intermediate of innate immune signaling pathways and is critically involved in the redox regulation in vivo. Ablation of TAK1 causes accumulation of ROS, resulting in epithelial cell death and inflammation. Here we determine the mechanism by which TAK1 kinase is activated in epithelial tissues. TAB1 and TAB2 are structurally unrelated TAK1 binding protein partners. TAB2 is known to mediate polyubiquitin chain-dependent TAK1 activation in innate immune signaling pathways, whereas the role of TAB1 is not defined. We found that epithelial-specific TAB1 and TAB2 double- but not TAB1 or TAB2 single-knockout mice phenocopied epithelial-specific TAK1 knockout mice. We demonstrate that phosphorylation-dependent basal activity of TAK1 is dependent on TAB1. Ablation of both TAB1 and TAB2 diminished the activity of TAK1 in vivo and causes accumulation of ROS in the epithelial tissues. These results demonstrate that epithelial TAK1 activity is regulated through two unique, TAB1-dependent basal and TAB2-mediated stimuli-dependent mechanisms.

Reciprocal Inhibition between the Transforming Growth Factor-β-activated Kinase 1 (TAK1) and Apoptosis Signal-regulating Kinase 1 (ASK1) Mitogen-activated Protein Kinase Kinase Kinases and Its Suppression by TAK1-binding Protein 2 (TAB2), an Adapter Protein for TAK1

Mitogen-activated protein kinase kinase kinases (MAP3Ks) are activated by a wide spectrum of extracellular stimuli and are involved in various cellular events including proinflammatory and oxidative damage response through activations of two specific transcription factors, nuclear factor κB (NF-κB) and activator protein-1 (AP-1). Although members of the MAP3K family have both overlapping and distinct functions, the inter-regulatory mechanism of MAP3Ks remains largely unknown. In this study we demonstrated that transforming growth factor-β-activated kinase 1 (TAK1)-TAK1-binding protein 1 (TAB1) complex negatively regulates ASK1-mediated signaling, and TAB2 reciprocally regulates TAK1-induced NF-κB and apoptosis signal-regulating kinase 1 (ASK1)-mediated AP-1 activations through the TAK1-TAB2 interaction and the interferences of TAK1-ASK1 interaction. TAK1 interacted with the N or C terminus of ASK1 through the C-terminal TAB2 binding domain of TAK1, with resultant inhibition of ASK1-induced AP-1 activation. Interestingly, the interaction between TAK1 and TAB2 significantly attenuated the ASK1-TAK1 interaction through the competitive interaction with ASK1 to TAK1 and resulted in the activations of TAK1-induced activations of NF-κB and AP-1. More interestingly, H(2)O(2)- and TNF-α-induced apoptosis in TAK1-deficient mouse embryo fibroblast cells were dramatically enhanced by overexpression of ASK1, whereas the apoptosis was markedly inhibited by the overexpression of TAK1. Overall, these results demonstrate that TAK1 and its adapter protein, TAB2, reciprocally regulate both TAK1- and ASK1-mediated signaling pathways to direct the activations of NF-κB and AP-1.

Identification and Functional Characterization of Novel Phosphorylation Sites in TAK1-Binding Protein (TAB) 1

TAB1 was defined as a regulatory subunit of the protein kinase TAK1, which functions upstream in the pathways activated by interleukin (IL)-1, tumor necrosis factor (TNF), toll-like receptors (TLRs) and stressors. However, TAB1 also functions in the p38 MAPK pathway downstream of TAK1. We identified amino acids (aa) 452/453 and 456/457 of TAB1 as novel sites phosphorylated by TAK1 as well as by p38 MAPK in intact cells as well as in vitro. Serines 452/453 and 456/457 were phosphorylated upon phosphatase blockade by calyculin A, or in response to IL-1 or translational stressors such as anisomycin and sorbitol. Deletion or phospho-mimetic mutations of aa 452–457 of TAB1 retain TAB1 and p38 MAPK in the cytoplasm. The TAB1 mutant lacking aa 452–457 decreases TAB1-dependent phosphorylation of p38 MAPK. It also enhances TAB1-dependent CCL5 secretion in response to IL-1 and increases activity of a post-transcriptional reporter gene, which contains the CCL5 3′ untranslated region. These data suggest a complex role of aa 452–457 of TAB1 in controlling p38 MAPK activity and subcellular localization and implicate these residues in TAK1- or p38 MAPK-dependent post-transcriptional control of gene expression.

IKKepsilon involvement in Tax-mediated activation of INF pathway

HTLV-1 Tax de-regulates several cellular signaling pathways leading to cell transformation by altering gene expression, intracellular protein distribution and cell proliferation. Tax-1 induces persistent activation of several transcriptional factors and signal transduction pathways, including NF-κB and CREB/ATF. It is known that Tax-1 constitutively activates TAK1 (transforming growth factor-β-activated kinase 1) and modifies the interferon (INF) regulatory signals by controlling the expression of INF transcription factors 3 (INF3) and INF4. We have recently reported that HTLV-1 and HTLV-2 Tax proteins interact with TAK1-binding protein 2 (TAB2) of the NF-κB pathway and that both Tax proteins transactivate NF-κB promoters [1]. TAB2 functions as an adaptor protein to recruit TAK1 to TRAF2 (TNF-α receptor-associated factor) in TNF-α signaling pathways. In the present study we have investigated Tax-1 and Tax-2 role in modifying INF and NF-κB activation through the recruitment of IKKepsilon, an IκB kinase homologue involved in NF-κB and INF3 signaling pathways. By co-immunoprecipitation experiments, we have found that both IKKepsilon and Tax-1, but not Tax-2, are present in protein complexes in transfected cells. IKKepsilon and Tax-1 or Tax-2 role in the activation of INF responsive elements or NF-κB containing promoters have been analyzed after transfecting the protein genes in 293T cells and measuring the effect by luciferase assay. Co-expression of Tax-1 and IKKepsilon resulted in an increased IRF activation mediated by IKKepsilon. Interaction of IKKepsilon with Tax-1 and Tax-2 and their possible effects in the de-regulation of the IRF3 pathways will be discussed.

Shigella flexneri type III secreted effector OspF reveals new crosstalks of proinflammatory signaling pathways during bacterial infection

Shigella flexneri type III secreted effector OspF harbors a phosphothreonine lyase activity that irreversibly dephosphorylates MAP kinases (MAPKs) p38 and ERK in infected epithelial cells and thereby, dampens innate immunity. Whereas this activity has been well characterized, the impact of OspF on other host signaling pathways that control inflammation was unknown. Here we report that OspF potentiates the activation of the MAPK JNK and the transcription factor NF-κB during S. flexneri infection. This unexpected effect of OspF was dependent on the phosphothreonine lyase activity of OspF on p38, and resulted from the disruption of a negative feedback loop regulation between p38 and TGF-beta activated kinase 1 (TAK1), mediated via the phosphorylation of TAK1-binding protein 1. Interestingly, potentiated JNK activation was not associated with enhanced c-Jun signaling as OspF also inhibits c-Jun expression at the transcriptional level. Altogether, our data reveal the impact of OspF on the activation of NF-κB, JNK and c-Jun, and demonstrate the existence of a negative feedback loop regulation between p38 and TAK1 during S. flexneri infection. Furthermore, this study validates the use of bacterial effectors as molecular tools to identify the crosstalks that connect important host signaling pathways induced upon bacterial infection.

Association of HTLV Tax proteins with TAK1-binding protein 2 and RelA in calreticulin-containing cytoplasmic structures participates in Tax-mediated NF-κB activation

HTLV-1 is more pathogenic than HTLV-2 despite having a similar genome and closely related transactivating oncoproteins. Both Tax-1 protein from HTLV-1 and Tax-2 from HTLV-2 activate the NF-κB pathway. The mechanisms involved in Tax-1 deregulation of this signalling pathway have been thoroughly investigated, but little is known about regulation by Tax-2. We have compared the interaction of Tax-1 and Tax-2 with two key NF-κB signalling factors: TAK1-binding protein 2 (TAB2), an adaptor involved in the activation of TAK1 kinase, and RelA, the active subunit of the canonical RelA/p50 NF-κB transcription factor. Tax-2 formed stable complexes with both RelA and TAB2. These two NF-κB factors colocalized with Tax proteins in dotted cytoplasmic structures targeted by calreticulin, a multi-process calcium-buffering chaperone. Co-expression of RelA and/or TAB2 markedly increased Tax-mediated NF-κB activation. These findings provide new insights into the role of RelA, TAB2 and Tax in the deregulation of the NF-κB pathway.

Immunolocalization of TAK1, TAB1, and p38 in the developing rat molar

In tooth development, transforming growth factor beta (TGF-β) and bone morphogenetic protein (BMP) are involved in cell differentiation and matrix protein production. TGF-β and BMP have two signaling pathways: the Smad pathway and the non-Smad pathway. However, only a few studies have focused on the non-Smad pathway in tooth development. TGF-β-activated kinase 1 (TAK1) is activated by TGF-β or BMP and binds to TAK1-binding protein (TAB1), activating p38 or c-Jun N-terminal kinase (JNK), forming the non-Smad signaling pathway. In this study, we examined the distribution of these kinases, TGF-β receptor 1 (TGF-β-R1), BMP receptor-1B (BMPR-1B) and Smad4 in cells of the rat molar germ histochemically, in order to investigate the signaling pathway in each type of cell. Immunostaining for TGF-β-R1, BMPR-1B, Smad4, TAK1, TAB1 and phosphorylated-p38 (p-p38) showed similar reactions. In the cervical loop, reactions were clearer than in other enamel epithelium. In the inner enamel epithelium, signal increased with differentiation into ameloblasts, became strongest in the secretory stage, and decreased rapidly in the maturation stage. Signal also increased upon differentiation from preodontoblasts to odontoblasts. In Hertwig's epithelial sheath, with the exception of BMPR-1B, reactions were stronger in the later stage, showing more enamel protein secretion than in the early stage. However, no clear reaction corresponding to phosphorylated-JNK was observed in any type of cell. These results suggest that TGF-β or BMP is involved in the induction of differentiation of inner enamel epithelium cells into ameloblasts, and preodontoblast differentiation into odontoblasts, the regulation of cervical loop cell proliferation, the elongation or regulation of the epithelial sheath, and the secretion of enamel protein and dentin matrix protein through the non-Smad signaling pathway via TAK1, TAB1 and p38 as well as Smad signaling pathways in the rat molar germ.

Autoactivation of Transforming Growth Factor β-activated Kinase 1 Is a Sequential Bimolecular Process

Transforming growth factor-beta-activated kinase 1 (TAK1), an MAP3K, is a key player in processing a multitude of inflammatory stimuli. TAK1 autoactivation involves the interplay with TAK1-binding proteins (TAB), e.g. TAB1 and TAB2, and phosphorylation of several activation segment residues. However, the TAK1 autoactivation is not yet fully understood on the molecular level due to the static nature of available x-ray structural data and the complexity of cellular systems applied for investigation. Here, we established a bacterial expression system to generate recombinant mammalian TAK1 complexes. Co-expression of TAK1 and TAB1, but not TAB2, resulted in a functional and active TAK1-TAB1 complex capable of directly activating full-length heterotrimeric mammalian AMP-activated protein kinase (AMPK) in vitro. TAK1-dependent AMPK activation was mediated via hydrophobic residues of the AMPK kinase domain alphaG-helix as observed in vitro and in transfected cell culture. Co-immunoprecipitation of differently epitope-tagged TAK1 from transfected cells and mutation of hydrophobic alphaG-helix residues in TAK1 point to an intermolecular mechanism of TAB1-induced TAK1 autoactivation, as TAK1 autophosphorylation of the activation segment was impaired in these mutants. TAB1 phosphorylation was enhanced in a subset of these mutants, indicating a critical role of alphaG-helix residues in this process. Analyses of phosphorylation site mutants of the activation segment indicate that autophosphorylation of Ser-192 precedes TAB1 phosphorylation and is followed by sequential phosphorylation of Thr-178, Thr-187, and finally Thr-184. Finally, we present a model for the chronological order of events governing TAB1-induced TAK1 autoactivation.

TAB2 Scaffolds TAK1 and NLK in Repressing Canonical Wnt Signaling

The TAK1-NLK cascade is a mitogen-activated protein kinase-related pathway that plays an inhibitory role in canonical Wnt/beta-catenin signaling through regulating the LEF1/TCF family transcriptional factors. TAB2 (TAK1-binding protein 2) is a putative TAK1 interacting protein that is involved in the regulation of TAK1. Here, we found that TAB2 could directly interact with NLK and function as a scaffold protein to facilitate the interaction between TAK1 and NLK. Knocking down TAB2 using small interfering RNA abolished the interaction of TAK1 with NLK in mammalian cells. The intermediate region (residues 292-417) of TAB2 was mapped for its binding to NLK. TAB2-DeltaM, a TAB2 mutant lacking this region, showed a lower affinity for NLK and became defective in its scaffolding function. In addition, TAB2, but not TAB2-DeltaM, mediated TAK1-dependent activation of NLK and LEF1 polyubiquitylation, resulting in the inhibition of canonical Wnt signaling. Moreover, Wnt3a stimulation led to an increase in the interaction of TAB2 with NLK and the formation of a TAK1.TAB2.NLK complex, suggesting that this TAK1-TAB2-NLK pathway may constitute a negative feedback mechanism for canonical Wnt signaling.

Constitutive Association of TGF-β–Activated Kinase 1 with the IκB Kinase Complex in the Nucleus and Cytoplasm of Human Neutrophils and Its Impact on Downstream Processes

Neutrophils influence innate and adaptative immunity by generating numerous mediators whose regulation largely depends on the IkappaB kinase (IKK)/IkappaB/NF-kappaB signaling cascade. A singular feature of neutrophils is that they express several components of this pathway (namely, NF-kappaB/Rel proteins and IkappaB-alpha) in both the nucleus and cytoplasm. We recently reported that the IKK complex of neutrophils is similarly expressed and activated in both cellular compartments. However, the upstream IKK kinase has not yet been identified. In this study, we report that neutrophils express the mitogen-activated protein 3 kinase, TGF-beta-activated kinase 1 (TAK1), as well as its associated partners, TAK1-binding protein (TAB) 1, TAB2, and TAB4, in both the cytoplasm and nucleus. Following cell stimulation by TNF-alpha or LPS, TAK1 becomes rapidly and transiently activated. Blocking TAK1 kinase activity with a highly selective inhibitor (5z-7-oxozeaenol) attenuated the phosphorylation of nuclear and cytoplasmic IKKalpha/beta, IkappaB-alpha, and RelA, and also impaired IkappaB-alpha degradation and NF-kappaB DNA binding in activated neutrophils. Moreover, TAK1 was found to be involved in the activation of p38 MAPK and ERK, which also influence cytokine generation in neutrophils. As a result, inflammatory cytokine expression and release were profoundly impaired following TAK1 inhibition. Similarly, the delayed apoptosis observed in response to LPS or TNF-alpha was reversed by TAK1 inhibition. By contrast, IKKgamma phosphorylation and STAT1 activation were unaffected by TAK1 inhibition. Our data establish the central role of TAK1 in controlling nuclear and cytoplasmic signaling cascades in primary neutrophils, making it a promising target for therapeutic intervention in view of the foremost role of neutrophils in several chronic inflammatory conditions.

Specific Regulation of Noncanonical p38α Activation by Hsp90-Cdc37 Chaperone Complex in Cardiomyocyte

p38 is an important stress activated protein kinase involved in gene regulation, proliferation, differentiation, and cell death regulation in heart. p38 kinase activity can be induced through canonical pathway via upstream kinases or by noncanonical autophosphorylation. The intracellular p38 kinase activity is tightly regulated and maintained at low level under basal condition. The underlying regulatory mechanism for canonical p38 kinase activation is well-studied, but the regulation of noncanonical p38 autophosphorylation remains poorly understood. We investigated the molecular basis for the regulation of noncanonical p38 autophosphorylation and its potential functional impact in cardiomyocytes. Using both proteomic and biochemical tools, we established that heat shock protein (Hsp)90-Cdc37 chaperones are part of the p38alpha signaling complex in mammalian cells both in vitro and in vivo. The Hsp90-Cdc37 chaperone complex interacts with p38 via direct binding between p38 and Cdc37. Cdc37 expression is both sufficient and necessary to suppress noncanonical p38 activation via autophosphorylation at either basal state or under TAB1 (TAK1 binding protein-1) induction. In contrast, Cdc37 expression has no impact on p38 activation by canonical upstream kinase MKK3 or oxidative stress. Furthermore, Hsp90 inhibition results in p38 activation via autophosphorylation, and p38 activity contribute to apoptotic cell death induced by Hsp90 inhibition. Our study has revealed a so far uncharacterized function of Hsp90-Cdc37 as an endogenous regulator of noncanonical p38 activity.