TAK1-binding Protein Research Articles

Transforming Growth Factor β-activated Kinase 1 (TAK1) Kinase Adaptor, TAK1-binding Protein 2, Plays Dual Roles in TAK1 Signaling by Recruiting Both an Activator and an Inhibitor of TAK1 Kinase in Tumor Necrosis Factor Signaling Pathway

Transforming growth factor beta-activated kinase 1 (TAK1) kinase is an indispensable signaling intermediate in tumor necrosis factor (TNF), interleukin 1, and Toll-like receptor signaling pathways. TAK1-binding protein 2 (TAB2) and its closely related protein, TAB3, are binding partners of TAK1 and have previously been identified as adaptors of TAK1 that recruit TAK1 to a TNF receptor signaling complex. TAB2 and TAB3 redundantly mediate activation of TAK1. In this study, we investigated the role of TAB2 by analyzing fibroblasts having targeted deletion of the tab2 gene. In TAB2-deficient fibroblasts, TAK1 was associated with TAB3 and was activated following TNF stimulation. However, TAB2-deficient fibroblasts displayed a significantly prolonged activation of TAK1 compared with wild type control cells. This suggests that TAB2 mediates deactivation of TAK1. We found that a TAK1-negative regulator, protein phosphatase 6 (PP6), was recruited to the TAK1 complex in wild type but not in TAB2-deficient fibroblasts. Furthermore, we demonstrated that both PP6 and TAB2 interacted with the polyubiquitin chains and this interaction mediated the assembly with TAK1. Our results indicate that TAB2 not only activates TAK1 but also plays an essential role in the deactivation of TAK1 by recruiting PP6 through a polyubiquitin chain-dependent mechanism.

MAPKAPK-2 Signaling Is Critical for Cutaneous Wound Healing

Cutaneous wound healing is a complex process, which is heavily dependent on successful inflammatory action. Mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2 or MK2), a major substrate of p38 MAPK, has been shown to be a major player in multiple inflammatory diseases, but its role in cutaneous wound healing has not yet been explored. In this study, by comparing excisional wounds made on the backs of MK2 knockout (KO) and MK2 wild-type (WT) mice, we found that the kinetics of wound healing are significantly affected by the absence of MK2 (P=0.010 to P<0.001). Histological examination showed a higher level of acanthosis of the migrating wound keratinocyte layer as well as a higher level of collagen deposition in the granulation tissue of the wounds from MK2 WT mice compared with those from MK2 KO mice. Interestingly, although MK2 did not influence macrophage and neutrophil infiltration of the wounds, the expression of many cytokines and chemokines was significantly affected at different days post wounding. Furthermore, the delayed healing rate of wounds in MK2 KO mice can be significantly improved by passive transfer of macrophages with intact MK2. Overall, these results show a critical role for MK2 gene expression in macrophages participating in the process of cutaneous wound healing.

X-linked Inhibitor of Apoptosis Protein and Its E3 Ligase Activity Promote Transforming Growth Factor-Β-mediated Nuclear Factor-κB Activation during Breast Cancer Progression

The precise sequence of events that enable mammary tumorigenesis to convert transforming growth factor-beta (TGF-beta) from a tumor suppressor to a tumor promoter remains incompletely understood. We show here that X-linked inhibitor of apoptosis protein (xIAP) is essential for the ability of TGF-beta to stimulate nuclear factor-kappaB (NF-kappaB) in metastatic 4T1 breast cancer cells. Indeed whereas TGF-beta suppressed NF-kappaB activity in normal mammary epithelial cells, those engineered to overexpress xIAP demonstrated activation of NF-kappaB when stimulated with TGF-beta. Additionally up-regulated xIAP expression also potentiated the basal and TGF-beta-stimulated transcriptional activities of Smad2/3 and NF-kappaB. Mechanistically xIAP (i) interacted physically with the TGF-beta type I receptor, (ii) mediated the ubiquitination of TGF-beta-activated kinase 1 (TAK1), and (iii) facilitated the formation of complexes between TAK1-binding protein 1 (TAB1) and IkappaB kinase beta that enabled TGF-beta to activate p65/RelA and to induce the expression of prometastatic (i.e. cyclooxygenase-2 and plasminogen activator inhibitor-1) and prosurvival (i.e. survivin) genes. We further observed that inhibiting the E3 ubiquitin ligase function of xIAP or expressing a mutant ubiquitin protein (i.e. K63R-ubiquitin) was capable of blocking xIAP- and TGF-beta-mediated activation of NF-kappaB. Functionally xIAP deficiency dramatically reduced the coupling of TGF-beta to Smad2/3 in NMuMG cells as well as inhibited their expression of mesenchymal markers in response to TGF-beta. More importantly, xIAP deficiency also abrogated the formation of TAB1.IkappaB kinase beta complexes in 4T1 breast cancer cells, thereby diminishing their activation of NF-kappaB, their expression of prosurvival/metastatic genes, their invasion through synthetic basement membranes, and their growth in soft agar. Collectively our findings have defined a novel role for xIAP in mediating oncogenic signaling by TGF-beta in breast cancer cells.

WDR34 is a novel TAK1-associated suppressor of the IL-1R/TLR3/TLR4-induced NF-kappaB activation pathway.

Toll-like receptors (TLRs) act as sensors of microbial components and elicit innate immune responses. All TLR signaling pathways activate the nuclear factor-kappaB (NF-kappaB), which controls the expression of inflammatory cytokine genes. Transforming growth factor-beta-activated kinase 1 (TAK1) is a serine/threonine protein kinase that is critically involved in the activation of NF-kappaB by tumor necrosis factor (TNFalpha), interleukin-1beta (IL-1beta) and TLR ligands. In this study, we identified a novel protein, WD40 domain repeat protein 34 (WDR34) as a TAK1-interacting protein in yeast two-hybrid screens. WDR34 interacted with TAK1, TAK1-binding protein 2 (TAB2), TAK1-binding protein 3 (TAB3) and tumor necrosis factor receptor-associated factor 6 (TRAF6) in overexpression and under physiological conditions. Overexpression of WDR34 inhibited IL-1beta-, polyI:C- and lipopolysaccharide (LPS)-induced but not TNFalpha-induced NF-kappaB activation, whereas knockdown of WDR34 by a RNA-interference construct potentiated NF-kappaB activation by these ligands. Our findings suggest that WDR34 is a TAK1-associated inhibitor of the IL-1R/TLR3/TLR4-induced NF-kappaB activation pathway.

Cross interference with TNF-α-induced TAK1 activation via EGFR-mediated p38 phosphorylation of TAK1-binding protein 1

Transforming growth factor-α-activated kinase 1 (TAK1) has been widely recognized as a kinase that regulates multiple intracellular signaling pathways evoked by cytokines and immune receptor activation. We have recently reported that tumor necrosis factor-α (TNF-α) triggers internalization of epidermal growth factor receptor (EGFR) through a TAK1-p38α signaling pathway, which results in a transient suppression of the EGFR. In the present study, we investigated the pathway of intracellular signaling in the opposite direction. Ligand-induced activation of EGFR caused phosphorylation of the TAK1-binding proteins TAB1 and TAB2 in a TAK1-independent manner. EGFR-mediated phosphorylation of TAB1 was completely inhibited by a chemical inhibitor and siRNA of p38α. The phosphorylation of TAB1 was occurred at Ser-423 and Thr-431, the residues underlying the p38-mediated feedback inhibition of TAK1. In contrast, phosphorylation of TAB2 was sustained, and largely resistant to p38 inhibition. The inducible phosphorylation of TAB1 interfered with a response of EGF-treated cells to TNF-α-induced TAK1 activation, which led to the reduction of NF-κB activation. Collectively, these results demonstrated that EGFR activation interfered with TNF-α-induced TAK1 activation via p38-mediated phosphorylation of TAB1.

Molecular discrimination of structurally equivalent Lys 63‐linked and linear polyubiquitin chains

At least eight types of ubiquitin chain exist, and individual linkages affect distinct cellular processes. The only distinguishing feature of differently linked ubiquitin chains is their structure, as polymers of the same unit are chemically identical. Here, we have crystallized Lys 63-linked and linear ubiquitin dimers, revealing that both adopt equivalent open conformations, forming no contacts between ubiquitin molecules and thereby differing significantly from Lys 48-linked ubiquitin chains. We also examined the specificity of various deubiquitinases (DUBs) and ubiquitin-binding domains (UBDs). All analysed DUBs, except CYLD, cleave linear chains less efficiently compared with other chain types, or not at all. Likewise, UBDs can show chain specificity, and are able to select distinct linkages from a ubiquitin chain mixture. We found that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO (NF-kappaB essential modifier) binds to linear chains exclusively, whereas the NZF (Npl4 zinc finger) domain of TAB2 (TAK1 binding protein 2) is Lys 63 specific. Our results highlight remarkable specificity determinants within the ubiquitin system.

Interaction between TAK1-TAB1-TAB2 and RCAN1-calcineurin defines a signalling nodal control point.

The calcium-activated protein phosphatase calcineurin is controlled by regulator of calcineurin (RCAN) in organisms ranging from yeast to mammals. Here we performed a yeast two-hybrid screen with RCAN1 as bait, identifying TAK1 binding protein 2 (TAB2) as an interacting partner. TAB2 interacted directly with RCAN1 in vitro and in vivo, recruiting TAK1, TAB1 and calcineurin, forming a macromolecular signalling complex. Overexpression of TAK1 and TAB1, or active TAK1(DeltaN), promoted direct phosphorylation of RCAN1 in vitro and in vivo. TAK1 phosphorylated RCAN1 at Ser 94 and Ser 136, converting RCAN1 from an inhibitor to a facilitator of calcineurin-NFAT signalling, and enhancing NFATc1 nuclear translocation, NFAT transcriptional activation and the hypertrophic growth of cultured cardiomyocytes. The TAK1-TAB1-TAB2 and the calcineurin-NFAT signalling modules did not interact in Rcan1/2- or Tab2-deficient mouse embryonic fibroblast (MEF) cultures. Calcineurin activation also dephosphorylated and inhibited TAK1 and TAB1, an effect that was absent in Rcan1/2 deficient MEFs. Functionally, TAK1 was indispensable for the cardiomyocyte growth response induced by pro-hypertrophic stimuli through calcineurin. These results describe a signalling relationship between two central regulatory pathways in which TAK1-TAB1-TAB2 selectively induces calcineurin-NFAT signalling through direct phosphorylation of RCAN1, while calcineurin activation diminishes TAK1 signalling by dephosphorylation of TAK1 and TAB1.

HSP90 is required for TAK1 stability but not for its activation in the pro-inflammatory signaling pathway

The protein kinase transforming-growth-factor-β-activated kinase-1 (TAK1) is a key regulator in the pro-inflammatory signaling pathway and is activated by tumor necrosis factor-α, interleukin-1 (IL-1) and lipopolysaccharide (LPS). We describe the identification of TAK1 as a client protein of the 90 kDa heat-shock protein (Hsp90)/cell division cycle protein 37 (Cdc37) chaperones. However, Hsp90 is not required for the activation of TAK1 as short exposure to the Hsp90 inhibitor, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) did not affect its activation by LPS or IL-1. Prolonged treatment of cells with 17-AAG inhibits Hsp90 and downregulates TAK1. Our results suggest that Hsp90 is required for the folding and stability of TAK1 but is displaced and no longer required when TAK1 is complexed to TAK1-binding protein-1 (TAB1). Structured summary MINT- 6797182: TAK1 (uniprotkb: O43318-2) physically interacts (MI: 0218) with CDC37 (uniprotkb: Q16543) and HSP90 (uniprotkb: P07900) by anti bait coimmunoprecipitation (MI: 0006) MINT- 6797194: TAK1 (uniprotkb: O43318-2) physically interacts (MI: 0218) with TAB1 (uniprotkb: Q15750), HSP90 (uniprotkb: P07900) and CDC37 (uniprotkb: Q16543) by anti bait coimmunoprecipitation (MI: 0006) MINT- 6797248: TAK1 (uniprotkb: Q62073) physically interacts (MI: 0218) with HSP90 (uniprotkb: P07901), CDC37 (uniprotkb: Q61081), TAB2 (uniprotkb: Q99K90) and TAB1 (uniprotkb: Q8CF89) by anti bait coimmunoprecipitation (MI: 0006) MINT- 6797232: TAK1 (uniprotkb: O43318-2) physically interacts (MI: 0218) with HSP90 (uniprotkb: P07900) and CDC37 (uniprotkb: Q16543) by pull down (MI: 0096) MINT- 6797216: TAK1 (uniprotkb: O43318-2) physically interacts (MI: 0218) with TAB2 (uniprotkb: Q9NYJ8), CDC37 (uniprotkb: Q16543), HSP90 (uniprotkb: P07900) and TAB1 (uniprotkb: Q15750) by anti bait coimmunoprecipitation (MI: 0006)

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.

Generation of a conditional mutant allele for Tab1 in mouse

TAK1 binding protein 1 (TAB1) binds and induces autophosphorylation of TGF-beta activating kinase (TAK1). TAK1, a mitogen-activated kinase kinase kinase, is involved in several distinct signaling pathways including non-Smad pathways for TGF-beta superfamily members and inflammatory responses caused by cytokines. Conventional disruption of the murine Tab1 gene results in late gestational lethality showing intraventricular septum defects and underdeveloped lung alveoli. To gain a better understanding of the roles of TAB1 in different tissues, at different stages of development, and in pathological conditions, we generated Tab1 floxed mice in which the loxP sites flank Exons 9 and 10 to remove the C-terminal region of TAB1 protein necessary for activation of TAK1. We demonstrate that Cre-mediated recombination using Sox2-Cre, a Cre line expressed in the epiblast during early embryogenesis, results in deletion of the gene and protein. These homozygous Cre-recombined null embryos display an identical phenotype to conventional null embryos. This animal model will be useful in revealing distinct roles of TAB1 in different tissues at different stages.

TAB4 Stimulates TAK1-TAB1 Phosphorylation and Binds Polyubiquitin to Direct Signaling to NF-κB

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.

Pinitol targets nuclear factor-κB activation pathway leading to inhibition of gene products associated with proliferation, apoptosis, invasion, and angiogenesis

Pinitol (3-O-methyl-chiroinositol), a component of traditional Ayurvedic medicine (talisapatra), has been shown to exhibit anti-inflammatory and antidiabetic activities through undefined mechanisms. Because the transcription factor nuclear factor-kappaB (NF-kappaB) has been linked with inflammatory diseases, including insulin resistance, we hypothesized that pinitol must mediate its effects through modulation of NF-kappaB activation pathway. We found that pinitol suppressed NF-kappaB activation induced by inflammatory stimuli and carcinogens. This suppression was not specific to cell type. Besides inducible, pinitol also abrogated constitutive NF-kappaB activation noted in most tumor cells. The suppression of NF-kappaB activation by pinitol occurred through inhibition of the activation of IkappaBalpha kinase, leading to sequential suppression of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Pinitol also suppressed the NF-kappaB reporter activity induced by tumor necrosis factor receptor (TNFR)-1, TNFR-associated death domain, TNFR-associated factor-2, transforming growth factor-beta-activated kinase-1 (TAK-1)/TAK1-binding protein-1, and IkappaBalpha kinase but not that induced by p65. The inhibition of NF-kappaB activation thereby led to down-regulation of gene products involved in inflammation (cyclooxygenase-2), proliferation (cyclin D1 and c-myc), invasion (matrix metalloproteinase-9), angiogenesis (vascular endothelial growth factor), and cell survival (cIAP1, cIAP2, X-linked inhibitor apoptosis protein, Bcl-2, and Bcl-xL). Suppression of these gene products by pinitol enhanced the apoptosis induced by TNF and chemotherapeutic agents and suppressed TNF-induced cellular invasion. Our results show that pinitol inhibits the NF-kappaB activation pathway, which may explain its ability to suppress inflammatory cellular responses.

Pellino 3b Negatively Regulates Interleukin-1-induced TAK1-dependent NFκB Activation

Pellino 3b Negatively Regulates Interleukin-1-induced TAK1-dependent NFκB Activation

HTLV-1 Tax-mediated TAK1 activation involves TAB2 adapter protein

Human T cell leukemia virus type 1 (HTLV-1) Tax is an oncoprotein that plays a crucial role in the proliferation and transformation of HTLV-1-infected T lymphocytes. It has recently been reported that Tax activates a MAPKKK family, TAK1. However, the molecular mechanism of Tax-mediated TAK1 activation is not well understood. In this report, we investigated the role of TAK1-binding protein 2 (TAB2) in Tax-mediated TAK1 activation. We found that TAB2 physically interacts with Tax and augments Tax-induced NF-κB activity. Tax and TAB2 cooperatively activate TAK1 when they are coexpressed. Furthermore, TAK1 activation by Tax requires TAB2 binding as well as ubiquitination of Tax. We also found that the overexpression of TRAF2, 5, or 6 strongly induces Tax ubiquitination. These results suggest that TAB2 may be critically involved in Tax-mediated activation of TAK1 and that NF-κB-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax.

Constitutive Activation of TAK1 by HTLV-1 Tax-dependent Overexpression of TAB2 Induces Activation of JNK-ATF2 but Not IKK-NF-κB

HTLV-1 Tax oncoprotein induces persistent activation of the transcription factor NF-kappaB and CREB (cAMP-response element-binding protein)/ATF. Transforming growth factor-beta-activated kinase 1 (TAK1) has been shown to play a critical role in these transcription factors. Here, we found that TAK1 was constitutively activated in Tax-positive HTLV-1-transformed T cells. Tax induced persistent overexpression of TAK1-binding protein 2 (TAB2), but not TAB3, which is essential for TAK1 activation. Surprisingly, TAK1 was not involved in the activation of NF-kappaB. On the other hand, JNK and p38 mitogen-activated protein kinases were activated by TAK1. In addition, ATF2, but not CREB, was a target for the TAK1-JNK pathway, and p38 negatively regulated TAK1 activity through TAB1 phosphorylation. These results indicate that Tax-mediated TAK1 activation is important for the activation of ATF2 rather than NF-kappaB.

Expression analysis of the TAB2 protein in adult mouse tissues

.Background:The Interleukin-1 (IL-1) signaling component TAK1 binding protein 2 (TAB2) plays a role in activating the NFκB and JNK signaling pathways. Additionally, TAB2 functions in the nucleus as a repressor of NFκB-mediated gene regulation.Objective:To obtain insight into the function of TAB2 in the adult mouse, we analyzed the in vivo TAB2 expression pattern.Materials and methods:Cell lines and adult mouse tissues were analyzed for TAB2 protein expression and localization.Results:Immunohistochemical staining for TAB2 protein revealed expression in the vascular endothelium of most tissues, hematopoietic cells and brain cells. While TAB2 is localized in both the nucleus and the cytoplasm in cell lines, cytoplasmic localization predominates in hematopoietic tissues in vivo.Conclusions:The TAB2 expression pattern shows striking similarities with previously reported IL-1 receptor expression and NFκB activation patterns, suggesting that TAB2 in vivo is playing a role in these signaling pathways.

TAK1 MAPK Kinase Kinase Mediates Transforming Growth Factor-β Signaling by Targeting SnoN Oncoprotein for Degradation

Transforming growth factor-beta (TGF-beta) regulates a variety of physiologic processes through essential intracellular mediators Smads. The SnoN oncoprotein is an inhibitor of TGF-beta signaling. SnoN recruits transcriptional repressor complex to block Smad-dependent transcriptional activation of TGF-beta-responsive genes. Following TGF-beta stimulation, SnoN is rapidly degraded, thereby allowing the activation of TGF-beta target genes. Here, we report the role of TAK1 as a SnoN protein kinase. TAK1 interacted with and phosphorylated SnoN, and this phosphorylation regulated the stability of SnoN. Inactivation of TAK1 prevented TGF-beta-induced SnoN degradation and impaired induction of the TGF-beta-responsive genes. These data suggest that TAK1 modulates TGF-beta-dependent cellular responses by targeting SnoN for degradation.

TGF-β-activated kinase 1 and TAK1-binding protein 1 cooperate to mediate TGF-β1-induced MKK3-p38 MAPK activation and stimulation of type I collagen

We have previously demonstrated that transforming growth factor-beta(1) (TGF-beta(1)) rapidly activates the mitogen-activated protein kinase kinase 3 (MKK3)-p38 MAPK signaling cascade, leading to the induction of type I collagen synthesis in mouse glomerular mesangial cells (Wang L, Ma R, Flavell RA, Choi ME. J Biol Chem 277: 47257-47262, 2002). In the present study, we investigated the functional role of upstream TGF-beta-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1) in the TGF-beta(1) signaling cascade. Rapid activation of endogenous TAK1 activity by TGF-beta(1) was observed in mouse mesangial cells. Transient overexpression of TAK1 with TAB1 enhanced the activation of MKK3 and p38 MAPK with or without TGF-beta(1) stimulation, whereas a dominant-negative mutant of TAK1 (TAK1DN) suppressed TGF-beta(1)-induced activation of MKK3 and p38 MAPK. Moreover, constitutive expression of TAK1DN reduced steady-state protein levels of MKK3 and p38 MAPK as well as MKK3 phosphorylation. Increased p38alpha MAPK activity by ectopic expression of either TAB1 or wild-type p38alpha MAPK resulted in enhanced TGF-beta(1)-induced type I collagen expression. In contrast, constitutive expression of TAK1DN inhibited collagen induction. Taken together, our data indicate that TAK1 and TAB1 play a pivotal role as upstream signal transducers activating the MKK3-p38 MAPK signaling cascade that leads to the induction of type I collagen expression by TGF-beta(1). In addition, our findings also suggest that TAK1 has a novel function in regulation of the steady-state protein levels of MKK3 and p38 MAPK.

Iap2 is required for a sustained response in the Drosophila Imd pathway

Fruit flies have effective immune response against Gram-negative bacteria. Upon infection, early JNK-signaling pathway mediated response is followed by the action of the Immune deficiency (Imd) signaling cascade, a Drosophila equivalent of mammalian TNF-receptor pathway, leading to the release of antimicrobial peptides. Recently, Tak1-binding protein 2 (Tab2) and Inhibitor of apoptosis 2 (Iap2) were identified as components of the Imd pathway. In this study, we carried out a genome-wide kinetic analysis of the role of Tab2 and Iap2 for immune response in Drosophila S2 cells using oligonucleotide microarrays. Tab2 RNAi abolished the induction of all immune response genes in S2 cells indicating its requirement for signaling both via the Imd and the JNK pathway. The role of Iap2 was more specific. Kinetic analysis indicated that Iap2 is required to sustain antimicrobial peptide gene expression in S2 cells. Furthermore, inactivation of Iap2 by RNAi resulted in impaired microbial resistance in Drosophila in vivo.

Protein Phosphatase 6 Down-regulates TAK1 Kinase Activation in the IL-1 Signaling Pathway

TAK1 (transforming growth factor beta-activated kinase 1) is a serine/threonine kinase that is a mitogen-activated protein kinase kinase kinase and an essential intracellular signaling component in inflammatory signaling pathways. Upon stimulation of cells with inflammatory cytokines, TAK1 binds proteins that stimulate autophosphorylation within its activation loop and is thereby catalytically activated. This activation is transient; it peaks within a couple of minutes and is subsequently down-regulated rapidly to basal levels. The mechanism of down-regulation of TAK1 has not yet been elucidated. In this study, we found that toxin inhibition of type 2A protein phosphatases greatly enhances interleukin 1 (IL-1)-dependent phosphorylation of Thr-187 in the TAK1 activation loop as well as the catalytic activity of TAK1. From proteomic analysis of TAK1-binding proteins, we identified protein phosphatase 6 (PP6), a type-2A phosphatase, and demonstrated that PP6 associated with and inactivated TAK1 by dephosphorylation of Thr-187. Ectopic and endogenous PP6 co-precipitated with TAK1, and expression of PP6 reduced IL-1 activation of TAK1 but did not affect osmotic activation of MLK3, another MAPKKK. Reduction of PP6 expression by small interfering RNA enhances IL-1-induced phosphorylation of Thr-187 in TAK1. Enhancement occurred without change in levels of PP2A showing specificity for PP6. Our results demonstrate that PP6 specifically down-regulates TAK1 through dephosphorylation of Thr-187 in the activation loop, which is likely important for suppressing inflammatory responses via TAK1 signaling pathways.