Giichi Takaesu

TAB2, a novel adaptor protein, mediates activation of TAK1 MAPKKK by linking TAK1 to TRAF6 in the IL-1 signal transduction pathway.

The TAK1 MAPKKK mediates activation of JNK and NF-KB in the IL-1-activated signaling pathway. Here we report the identification of TAB2, a novel intermediate in the IL-1 pathway that functionally links TAK1 to TRAF6. Expression of TAB2 induces JNK and NF-kappaB activation, whereas a dominant-negative mutant TAB2 impairs their activation by IL-1. IL-1 stimulates translocation of TAB2 from the membrane to the cytosol where it mediates the IL-1-dependent association of TAK1 with TRAF6. These results define TAB2 as an adaptor linking TAK1 and TRAF6 and as a mediator of TAK1 activation in the IL-1 signaling pathway.

TAK1 is Critical for IκB Kinase-mediated Activation of the NF-κB Pathway

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

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‐κB signal transduction pathways. TAK1, a MAPKKK, is critical for both IL‐1‐ and TNF‐induced activation of the NF‐κB pathway. TAB2, a TAK1‐binding protein, is involved in IL‐1‐induced NF‐κB activation by physically linking TAK1 to TRAF6. However, IL‐1‐induced activation of NF‐κB 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‐κB 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‐κB. These results suggest that TAB2 and TAB3 function redundantly as mediators of TAK1 activation in IL‐1 and TNF signal transduction.

Receptor Activator of NF-κB Ligand (RANKL) Activates TAK1 Mitogen-Activated Protein Kinase Kinase Kinase through a Signaling Complex Containing RANK, TAB2, and TRAF6

ABSTRACT The receptor activator of NF-κB (RANK) and its ligand RANKL are key molecules for differentiation and activation of osteoclasts. RANKL stimulates transcription factors AP-1 through mitogen-activated protein kinase (MAPK) activation, and NF-κB through IκB kinase (IKK) activation. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is essential for activation of these kinases. In the interleukin-1 signaling pathway, TAK1 MAPK kinase kinase (MAPKKK) mediates MAPK and IKK activation via interaction with TRAF6, and TAB2 acts as an adapter linking TAK1 and TRAF6. Here, we demonstrate that TAK1 and TAB2 participate in the RANK signaling pathway. Dominant negative forms of TAK1 and TAB2 inhibit NF-κB activation induced by overexpression of RANK. In 293 cells stably transfected with full-length RANK, RANKL stimulation facilitates the formation of a complex containing RANK, TRAF6, TAB2, and TAK1, leading to the activation of TAK1. Furthermore, in murine monocyte RAW 264.7 cells, dominant negative forms of TAK1 and TAB2 inhibit NF-κB activation induced by RANKL and endogenous TAK1 is activated in response to RANKL stimulation. These results suggest that the formation of the TRAF6-TAB2-TAK1 complex is involved in the RANK signaling pathway and may regulate the development and function of osteoclasts.

Characterization of a serum response factor-like protein in Saccharomyces cerevisiae, Rlm1, which has transcriptional activity regulated by the Mpk1 (Slt2) mitogen-activated protein kinase pathway.

The Mpk1 (Slt2) mitogen-activated protein (MAP) kinase has been implicated in several biological processes in Saccharomyces cerevisiae. The Rlm1 protein, a member of the MADS box family of transcription factors, functions downstream of Mpk1 in the pathway. To characterize the role of Rlm1 in mediating the transcriptional activation by the Mpk1 pathway, we constructed a LexA-Rlm1 deltaN chimera in which sequences, including the MADS box domain of the Rlm1 protein, were replaced by the LexA DNA binding domain and tested the ability of this chimera to activate a LexA operator-controlled reporter gene. In this assay, the Rlm1 protein was found to activate transcription in a manner regulated by the Mpk1 pathway. The Mpk1 protein kinase phosphorylated Rlm1 deltaN in vitro and the LexA-Rlm1 deltaN chimera protein was phosphorylated in vivo in a Mpk1-dependent manner. These results suggest that Mpk1 regulates the transcriptional activity of Rlm1 by directly phosphorylating it. We identified a Mpk1-like protein kinase, Mlp1, as an Rlm1-associated protein by using the yeast two-hybrid system. Overexpression of MLP1 suppresses the caffeine-sensitive phenotype of the bck1 delta mutation. The additivity of the mlp1 delta defect with the Mpk1 delta defect with regard to the caffeine sensitivity, combined with the results of genetic epistasis experiments, suggested that the activity of Rlm1 is regulated independently by Mpk1 MAP kinase and the Mlp1 MAP kinase-like kinase.

Interleukin-1 (IL-1) Receptor-Associated Kinase Leads to Activation of TAK1 by Inducing TAB2 Translocation in the IL-1 Signaling Pathway

ABSTRACT 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-κB. 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.

Structure of the human ATG12~ATG5 conjugate required for LC3 lipidation in autophagy.

The autophagy factor ATG12~ATG5 conjugate exhibits E3 ligase–like activity which facilitates the lipidation of members of the LC3 family. The crystal structure of the human ATG12~ATG5 conjugate bound to the N-terminal region of ATG16L1, the factor that recruits the conjugate to autophagosomal membranes, reveals an integrated architecture in which ATG12 docks onto ATG5 through conserved residues. ATG12 and ATG5 are oriented such that other conserved residues on each molecule, including the conjugation junction, form a continuous surface patch. Mutagenesis data support the importance of both the interface between ATG12 and ATG5 and the continuous patch for E3 activity. The ATG12~ATG5 conjugate interacts with the E2 enzyme ATG3 with high affinity through another surface location that is exclusive to ATG12, suggesting a different role of the continuous patch in E3 activity. These findings provide a foundation for understanding the mechanism of LC3 lipidation.

A Drosophila MAPKKK, D‐MEKK1, mediates stress responses through activation of p38 MAPK

In cultured mammalian cells, the p38 mitogen‐activated protein kinase (MAPK) pathway is activated in response to a variety of environmental stresses. How ever, there is little evidence from in vivo studies to demonstrate a role for this pathway in the stress response. We identified a Drosophila MAPK kinase kinase (MAPKKK), D‐MEKK1, which can activate p38 MAPK. D‐MEKK1 is structurally similar to the mammalian MEKK4/MTK1 MAPKKK. D‐MEKK1 kinase activity was activated in animals under conditions of high osmolarity. Drosophila mutants lacking D‐MEKK1 were hypersensitive to environmental stresses, including elevated temperature and increased osmolarity. In these D‐MEKK1 mutants, activation of Drosophila p38 MAPK in response to stress was poor compared with activation in wild‐type animals. These results suggest that D‐MEKK1 regulation of the p38 MAPK pathway is critical for the response to environmental stresses in Drosophila.

TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation

Activation of the TAK1 kinase drives RIPK3-dependent necrosis and inhibits apoptosis downstream of TNF-α stimulation.

Structural basis of ATG3 recognition by the autophagic ubiquitin-like protein ATG12

Significance Autophagy-related (ATG)12 is a ubiquitin-like protein essential to autophagy. It has been known for years that ATG12 is conjugated to the structural protein ATG5 and that the resulting protein–protein conjugate acts like an E3 enzyme that facilitates the attachment of the LC3 ubiquitin-like protein to a lipid molecule, phosphatidylethanolamine. However, the exact role of ATG12 in the E3 complex and the significance of ATG12 being a ubiquitin-like protein have remained elusive. We show that ATG12 binds to a short peptide region of the E2 enzyme ATG3 and describe the structural details of this interaction. This work establishes ATG12 as the ATG3 recruitment factor and explains how ATG12 uses its ubiquitin-like fold for binding to the ATG3 peptide. The autophagic ubiquitin-like protein (ublp) autophagy-related (ATG)12 is a component of the ATG12∼ATG5–ATG16L1 E3 complex that promotes lipid conjugation of members of the LC3 ublp family. A role of ATG12 in the E3 complex is to recruit the E2 enzyme ATG3. Here we report the identification of the ATG12 binding sequence in the flexible region of human ATG3 and the crystal structure of the minimal E3 complexed with the identified binding fragment of ATG3. The structure shows that 13 residues of the ATG3 fragment form a short β-strand followed by an α-helix on a surface area that is exclusive to ATG12. Mutational analyses of ATG3 confirm that four residues whose side chains make contacts with ATG12 are important for E3 interaction as well as LC3 lipidation. Conservation of these four critical residues is high in metazoan organisms and plants but lower in fungi. A structural comparison reveals that the ATG3 binding surface on ATG12 contains a hydrophobic pocket corresponding to the binding pocket of LC3 that accommodates the leucine of the LC3-interacting region motif. These findings establish the mechanism of ATG3 recruitment by ATG12 in higher eukaryotes and place ATG12 among the members of signaling ublps that bind liner sequences.