TAK1-binding protein 2 (TAB2) and TAB3 are redundantly required for TLR-induced cytokine production in macrophages.

Transforming growth factor-ß-activated kinase 1 (TAK1) plays a pivotal role in innate and adaptive immunity. TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB pathways downstream of diverse immune receptors, including toll-like receptors (TLRs). Upon stimulation with TLR ligands, TAK1 is activated via recruitment to the lysine 63-linked polyubiquitin chain through TAK1-binding protein 2 (TAB2) and TAB3. However, the physiological importance of TAB2 and TAB3 in macrophages is still controversial. A previous study has shown that mouse bone marrow-derived macrophages (BMDMs) isolated from mice double deficient for TAB2 and TAB3 produced tumor necrosis factor (TNF)-α and interleukin (IL)-6 to the similar levels as control wild-type BMDMs in response to TLR ligands such as lipopolysaccharide (LPS) or Pam3CSK4, indicating that TAB2 and TAB3 are dispensable for TLR signaling. In this study, we revisited the role of TAB2 and TAB3 using an improved mouse model. We observed a significant impairment in the production of pro-inflammatory cytokines and chemokine in LPS- or Pam3CSK4-treated BMDMs deficient for both TAB2 and TAB3. Double deficiency of TAB2 and TAB3 resulted in the decreased activation of NF-κB and MAPK pathways as well as the slight decrease in TAK1 activation in response to LPS or Pam3CSK4. Notably, the TLR-mediated expression of inhibitor of NF-κB (IκB)ζ was severely compromised at the protein and messenger RNA (mRNA) levels in the TAB2/TAB3 double-deficient BMDMs, thereby impeding IL-6 production. Our results suggest that TAB2 and TAB3 play a redundant and indispensable role in the TLR signaling pathway.

GRIM-19 is a target of mycobacterial Zn2+ metalloprotease 1 and indispensable for NLRP3 inflammasome activation.

Tuberculosis is a communicable disease caused by Mycobacterium tuberculosis which primarily infects macrophages and establishes intracellular parasitism. A mycobacterial virulence factor Zn2+ metalloprotease 1 (Zmp1) is known to suppress interleukin (IL)-1ß production by inhibiting caspase-1 resulting in phagosome maturation arrest. However, the molecular mechanism of caspase-1 inhibition by Zmp1 is still elusive. Here, we identified GRIM-19 (also known as NDUFA13), an essential subunit of mitochondrial respiratory chain complex I, as a novel Zmp1-binding protein. Using the CRISPR/Cas9 system, we generated GRIM-19 knockout murine macrophage cell line J774.1 and found that GRIM-19 is essential for IL-1ß production during mycobacterial infection as well as in response to NLRP3 inflammasome-activating stimuli such as extracellular ATP or nigericin. We also found that GRIM-19 is required for the generation of mitochondrial reactive oxygen species and NLRP3-dependent activation of caspase-1. Loss of GRIM-19 or forced expression of Zmp1 resulted in a decrease in mitochondrial membrane potential. Our study revealed a previously unrecognized role of GRIM-19 as an essential regulator of NLRP3 inflammasome and a molecular mechanism underlying Zmp1-mediated suppression of IL-1ß production during mycobacterial infection.

TAK1 determines susceptibility to endoplasmic reticulum stress and leptin resistance in the hypothalamus.

Sustained endoplasmic reticulum (ER) stress disrupts normal cellular homeostasis and leads to the development of many types of human diseases, including metabolic disorders. TAK1 (also known as MAP3K7) is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family and is activated by a diverse set of inflammatory stimuli. Here, we demonstrate that TAK1 regulates ER stress and metabolic signaling through modulation of lipid biogenesis. We found that deletion of Tak1 increased ER volume and facilitated ER-stress tolerance in cultured cells, which was mediated by upregulation of sterol-regulatory-element-binding protein (SREBP)-dependent lipogenesis. In the in vivo setting, central nervous system (CNS)-specific Tak1 deletion upregulated SREBP-target lipogenic genes and blocked ER stress in the hypothalamus. Furthermore, CNS-specific Tak1 deletion prevented ER-stress-induced hypothalamic leptin resistance and hyperphagic obesity under a high-fat diet (HFD). Thus, TAK1 is a crucial regulator of ER stress in vivo, which could be a target for alleviation of ER stress and its associated disease conditions.

Cyclic adenosine monophosphate suppresses the transcription of proinflammatory cytokines via the phosphorylated c-Fos protein.

Intracellular cyclic adenosine monophosphate (cAMP) suppresses innate immunity by inhibiting proinflammatory cytokine production from monocytic cells. Enhanced expression of interleukin-10 (IL-10) has been suggested to be the mechanism of suppression. However, cAMP is still capable of suppressing production of the cytokines TNF-alpha and IL-12 in IL-10-deficient dendritic cells (DCs). Here, we demonstrated that the transcription factor c-Fos was responsible for the cAMP-mediated suppression of inflammatory cytokine production. c-Fos accumulated at high amounts in response to cAMP and lipopolysaccharide (LPS). Overexpression of c-Fos suppressed LPS-induced cytokine production, whereas cAMP-mediated suppression of TNF-alpha and IL-12 was impaired in Fos(-/-) DCs or in RAW264.7 cells treated with c-Fos siRNA. c-Fos physically interacted with p65 protein and reduced the recruitment of p65 to the Tnf promoter. Multiple sites of c-Fos were phosphorylated by the IKKbeta protein. Thus, we propose that c-Fos is a substrate of IKKbeta and is responsible for the immunosuppressive effect of cAMP.

Effects of Psidium guajava leaf extract on secretion systems of Gram-negative enteropathogenic bacteria.

We screened a total of 672 plant-tissue extracts to search for phytochemicals that inhibit the function of the type III secretion system (T3SS) of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among candidates examined, we found that an extract from the leaves of Psidium guajava (guava) inhibited the secretion of the EspB protein from EPEC and EHEC without affecting bacterial growth. The guava extract (GE) also inhibited EPEC and EHEC from adhering to and injecting EspB protein into HEp-2 cells. GE seemed to block the translocation of EspB from the bacterial cells to the culture medium. In addition to EPEC and EHEC, GE also inhibited the T3SS of Yersinia pseudotuberculosis and Salmonella enterica serovar Typhimurium. After exposure to GE, Y. pseudotuberculosis stopped the secretion of Yop proteins and lost its ability to induce the apoptosis of mouse bone marrow-derived macrophages. S. Typhimurium exposed to GE ceased the secretion of Sip proteins and lost its ability to invade HEp-2 cells. GE inhibited EspC secretion, the type V secretion protein of EPEC, but not Shiga toxin2 from EHEC. Thus, our results suggest that guava leaves contain a novel type of antimicrobial compound that could be used for the therapeutic treatment and prevention of gram-negative enteropathogenic bacterial infections.

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

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 ß-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.

Loss of SOCS3 in T helper cells resulted in reduced immune responses and hyperproduction of interleukin 10 and transforming growth factor-beta 1.

Suppressor of cytokine signaling (SOCS)3 is a major negative feedback regulator of signal transducer and activator of transcription (STAT)3-activating cytokines. Transgenic mouse studies indicate that high levels of SOCS3 in T cells result in type 2 T helper cell (Th2) skewing and lead to hypersensitivity to allergic diseases. To define the physiological roles of SOCS3 in T cells, we generated T cell-specific SOCS3 conditional knockout mice. We found that the mice lacking SOCS3 in T cells showed reduced immune responses not only to ovalbumin-induced airway hyperresponsiveness but also to Leishmania major infection. In vitro, SOCS3-deficient CD4+ T cells produced more transforming growth factor (TGF)-beta1 and interleukin (IL)-10, but less IL-4 than control T cells, suggesting preferential Th3-like differentiation. We found that STAT3 positively regulates TGF-beta1 promoter activity depending on the potential STAT3 binding sites. Furthermore, chromatin immunoprecipitation assay revealed that more STAT3 was recruited to the TGF-beta1 promoter in SOCS3-deficient T cells than in control T cells. The activated STAT3 enhanced TGF-beta1 and IL-10 expression in T cells, whereas the dominant-negative form of STAT3 suppressed these. From these findings, we propose that SOCS3 regulates the production of the immunoregulatory cytokines TGF-beta1 and IL-10 through modulating STAT3 activation.

Activation of p38alpha/beta MAPK in myogenesis via binding of the scaffold protein JLP to the cell surface protein Cdo.

The p38 mitogen-activated protein kinase (MAPK) pathway plays an important role in cell differentiation, but the signaling mechanisms by which it is activated during this process are largely unknown. Cdo is an immunoglobulin superfamily member that functions as a component of multiprotein cell surface complexes to promote myogenesis. In this study, we report that the Cdo intracellular region interacts with JLP, a scaffold protein for the p38alpha/beta MAPK pathway. Cdo, JLP, and p38alpha/beta form complexes in differentiating myoblasts, and Cdo and JLP cooperate to enhance levels of active p38alpha/beta in transfectants. Primary myoblasts from Cdo(-/-) mice, which display a defective differentiation program, are deficient in p38alpha/beta activity, and the expression of an activated form of MKK6 (an immediate upstream activator of p38) rescues the ability of Cdo(-/-) cells to differentiate. These results document a novel mechanism of signaling during cell differentiation: the interaction of a MAPK scaffold protein with a cell surface receptor.

A major lipid raft protein raftlin modulates T cell receptor signaling and enhances th17-mediated autoimmune responses.

The membrane microdomains known as lipid rafts have been shown to act as platforms for the initiation of various receptor signals. Through proteomic analysis, we have identified a novel protein termed Raftlin (raft-linking protein) as a major protein in lipid rafts. To determine the physiological and immunological functions of Raftlin in mammals, we generated Raftlin-deficient mice, as well as Raftlin-transgenic (Tg) mice. Although Raftlin was originally identified in B cells, we observe no severe abnormalities in the B cells of these mice, presumably due to a high expression of Raftlin-homologue (Raftlin-2). T cells, in contrast, expressed a substantial amount of Raftlin but no Raftlin-2. In Raftlin-deficient mice, T cell-dependent Ab production was reduced, and experimental autoimmune encephalomyelitis, a Th17-dependent autoimmune disease model, was ameliorated. In Raftlin-Tg mice, in contrast, Ab production was enhanced and experimental autoimmune encephalomyelitis was more severe. Cytokine production, especially that of IL-17, was reduced in Raftlin-deficient T cells, while it was enhanced in Raftlin-Tg T cells. We found that these changes were associated with the strength of the TCR-mediated signals. Importantly, localization of Lck protein in the lipid rafts was enhanced by Raftlin overexpression and reduced by Raftlin deficiency. These data indicate that Raftlin modulates TCR signals and is necessary for the fine-tuning of T cell-mediated immune responses.

miR-935 Inhibits Oral Squamous Cell Carcinoma and Targets Inositol Polyphosphate-4-phosphatase Type IA (INPP4A).

BACKGROUND/AIM: Oral squamous cell carcinoma (OSCC) is a common malignancy with poor prognosis. Therefore, novel therapeutic options are needed to improve prognosis of OSCC. Recently, microRNAs (miRs) have received increasing attention as a potential therapeutic tool for carcinomas. However, no definitive miR-based drugs for patients with OSCC have been reported to date. The aim of this study was to identify new miRs potentially involved in cellular processes associated with OSCC malignancy, which could lead to novel therapeutic strategies. MATERIALS AND METHODS: We identified miRs that are modulated in OSCC and possibly regulate OSCC malignancy, using miR microarray on OSCC cell lines. RESULTS: miR-935 and miR-509-3p were down-regulated in OSCC cell lines and patient tissues. When miR-935 was overexpressed in HSC-3-M3 cells, proliferation, migration, and invasion of the cell line was suppressed, whereas apoptosis was increased. Moreover, we showed that the gene inositol polyphosphate-4-phosphatase type I A (INPP4A) is a potential target whose expression is positively regulated by miR-935. CONCLUSION: miR-935 may function as a tumor suppressor by inhibiting OSCC malignancy via INPP4A induction. Therefore, miR-935 can be a new therapeutic candidate for OSCC treatment.

An F-box protein, FBXW5, negatively regulates TAK1 MAP3K in the IL-1beta signaling pathway.

TAK1, a member of the MAP3K family, plays an essential role in activation of JNK/p38 MAPKs and IKK in the IL-1beta and TNFalpha signaling pathway. Upon stimulation, TAK1 is rapidly and transiently activated. While the activation mechanism of TAK1 in these signaling pathways is well characterized, how its activity is terminated still remains unclear. To identify the molecule(s) involved in TAK1 regulation, we performed tandem affinity purification (TAP) in HeLa cells stably expressing TAP-tagged TAK1. FBXW5, an F-box family protein, was identified as a previously unknown component of the IL-1beta-induced TAK1 complex. FBXW5 associated with endogenous TAK1 in an IL-1beta-dependent manner. Overexpression of FBXW5 inhibited IL-1beta-induced activation of JNK/p38 MAPKs and NF-kappaB as well as phosphorylation of TAK1 on Thr187. Conversely, knockdown of FBXW5 resulted in the prolonged activation of TAK1 upon IL-1beta stimulation. These results suggest that FBXW5 negatively regulates TAK1 in the IL-1beta signaling pathway.

Peptidoglycan and lipopolysaccharide activate PLCgamma2, leading to enhanced cytokine production in macrophages and dendritic cells.

In macrophages and monocytes, microbial components trigger the production of pro-inflammatory cytokine through Toll-like receptors (TLRs). Although major TLR signaling pathways are mediated by serine/threonine kinases, including TAK1, IKK and MAP kinases, tyrosine phosphorylation of intracellular proteins by TLR ligands has been suggested in a number of reports. Here, we demonstrated that peptidoglycan (PGN) of a Gram-positive bacterial cell wall component, a TLR2 ligand and lipopoysaccharide (LPS) of a Gram-positive bacterial component, a TLR4 ligand induced tyrosine phosphorylation of phospholipase Cgamma-2 (PLCgamma2), leading to intracellular free Ca2+ mobilization in bone marrow-derived macrophages (BMMphi) and bone marrow-derived dendritic cells (BMDC). PGN- and LPS-induced Ca2+ mobilization was not observed in BMDC from PLCgamma2 knockout mice. Thus, PLCgamma2 is essential for TLR2 and TLR4-mediated Ca2+ flux. In PLCgamma2-knockdown cells, PGN-induced IkappaB-alpha phosphorylation and p38 activation were reduced. Moreover, PLCgamma2 was necessary for the full production of TNF-alpha and IL-6. These data indicate that the PLCgamma2 pathway plays an important role in bacterial ligands-induced activation of macrophages and dendritic cells.

Suppressor of cytokine signaling 1 protects mice against concanavalin A-induced hepatitis by inhibiting apoptosis.

UNLABELLED: Acute liver failure is associated with significant mortality. However, the underlying pathophysiological mechanism is not yet fully understood. Suppressor of cytokine signaling-1 (SOCS1), which is a negative-feedback molecule for cytokine signaling, has been shown to be rapidly induced during liver injury. Here, using liver-specific SOCS1-conditional-knockout mice, we demonstrated that SOCS1 deletion in hepatocytes enhanced concanavalin A (ConA)-induced hepatitis, which has been shown to be dependent on activated T and natural killer T (NKT) cells. Although serum cytokine level and NKT cell activation were similar in wild-type (WT) and SOCS1-deficient mice after ConA treatment, proapoptotic signals, including signal transducers and activators of transcription 1 (STAT1) and Jun-terminal kinase (JNK) activation, were enhanced in SOCS1-deficient livers compared with those in WT livers. SOCS1-deficient hepatocytes had higher expression of Fas antigen and were more sensitive to anti-Fas antibody-induced apoptosis than were WT hepatocytes. Furthermore, SOCS1-deficient hepatocytes were more sensitive to tumor necrosis factor (TNF)-alpha-induced JNK activation and apoptosis. These data indicate that SOCS1 is important to the prevention of hepatocyte apoptosis induced by Fas and TNF-alpha. In contrast, SOCS1 overexpression in the liver by adenoviral gene transfer prevented ConA-induced liver injury. CONCLUSION: These findings indicate that SOCS1 plays important negative roles in fulminant hepatitis and that forced expression of SOCS1 is therapeutic in preventing hepatitis.

Prostaglandin E2 is a major soluble factor produced by stromal cells for preventing inflammatory cytokine production from dendritic cells.

Dendritic cells (DCs) are specialized antigen-presenting cells that play pivotal roles in initiating immune responses. However, DC maturation is usually strongly restricted by the stromal microenvironment, especially in non-lymphoid tissues, such as skin and mucosa. Although suppression of DC maturation by stromal cells has been well documented, the molecular basis of this suppression has not been established. In this study, we examined the role of fibroblasts for DC maturation in vitro. The mouse embryonic fibroblasts (MEFs) strongly suppressed LPS-induced DC maturation. Although suppression of class II MHC and CD40 required DC-MEF contact, soluble factors in the culture supernatant of MEFs were sufficient for the suppression of IL-12 and tumor necrosis factor-alpha production. Using molecular-size selection and HPLC, we determined that prostaglandin E2 (PGE2) is a major soluble inhibitory factor secreted by MEFs. This was confirmed by the fact that cyclooxygenase inhibitors inhibited the production of the suppressive factor by MEFs. These results suggest that PGE2 is a major soluble factor produced by MEFs for the suppression of inflammatory cytokine production from DCs, while a contact mechanism between MEFs and DCs is required for the suppression to induce T cell-stimulating molecules.

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-kappaB 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-kappaB-activating TRAF family proteins are potential cellular E3 ubiquitin ligases toward Tax.

Adaptor protein SH2-B linking receptor-tyrosine kinase and Akt promotes adipocyte differentiation by regulating peroxisome proliferator-activated receptor gamma messenger ribonucleic acid levels.

Adipocyte differentiation is regulated by insulin and IGF-I, which transmit signals by activating their receptor tyrosine kinase. SH2-B is an adaptor protein containing pleckstrin homology and Src homology 2 (SH2) domains that have been implicated in insulin and IGF-I receptor signaling. In this study, we found a strong link between SH2-B levels and adipogenesis. The fat mass and expression of adipogenic genes including peroxisome proliferator-activated receptor gamma (PPARgamma) were reduced in white adipose tissue of SH2-B-/- mice. Reduced adipocyte differentiation of SH2-B-deficient mouse embryonic fibroblasts (MEFs) was observed in response to insulin and dexamethasone, whereas retroviral SH2-B overexpression enhanced differentiation of 3T3-L1 preadipocytes to adipocytes. SH2-B overexpression enhanced mRNA level of PPARgamma in 3T3-L1 cells, whereas PPARgamma levels were reduced in SH2-B-deficient MEFs in response to insulin. SH2-B-mediated up-regulation of PPARgamma mRNA was blocked by a phosphatidylinositol 3-kinase inhibitor, but not by a MAPK kinase inhibitor. Insulin-induced Akt activation and the phosphorylation of forkhead transcription factor (FKHR/Foxo1), a negative regulator of PPARgamma transcription, were up-regulated by SH2-B overexpression, but reduced in SH2-B-deficient MEFs. These data indicate that SH2-B is a key regulator of adipogenesis both in vivo and in vitro by regulating the insulin/IGF-I receptor-Akt-Foxo1-PPARgamma pathway.

Receptor activator of NF-kappaB ligand (RANKL) activates TAK1 mitogen-activated protein kinase kinase kinase through a signaling complex containing RANK, TAB2, and TRAF6.

The receptor activator of NF-kappaB (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-kappaB through IkappaB 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-kappaB 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-kappaB 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.

TAK1 regulates resident macrophages by protecting lysosomal integrity.

Hematopoietic cell survival and death is critical for development of a functional immune system. Here, we report that a protein kinase, TAK1, is selectively required for resident macrophage integrity during embryogenesis. Hematopoietic lineage-specific deletion of Tak1 gene (Tak1HKO) caused accumulation of cellular debris in the thymus in perinatal mice. Although no overt alteration in thymocytes and blood myeloid populations was observed in Tak1HKO mice, we found that thymic and lung macrophages were diminished. In the in vitro setting, Tak1 deficiency caused profound disruption of lysosomes and killed bone marrow-derived macrophages (BMDMs) without any exogenous stressors. Inhibition of the lysosomal protease, cathepsin B, partially blocked Tak1-deficient BMDM death, suggesting that leakage of the lysosomal contents is in part the cause of cell death. To identify the trigger of this cell death, we examined involvement of TNF and Toll-like receptor pathways. Among them, we found that deletion of Tnfr1 partially rescued cell death. Finally, we show that Tnfr1 deletion partially restored thymic and lung macrophages in vivo. These results suggest that autocrine and potentially paracrine TNF kills Tak1-deficient macrophages during development. Our results reveal that TAK1 signaling maintains proper macrophage populations through protecting lysosomal integrity.

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

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

Structural insights into E2-E3 interaction for LC3 lipidation.

The members of the LC3/Atg8 family of proteins are covalently attached to phagophore and autophagosomal membranes. At the last step of the LC3 lipidation cascade, LC3 is transferred from the E2 enzyme ATG3 to phosphatidylethanolamine (PE). This transfer is stimulated by the ATG12-ATG5-ATG16L1 E3 complex, but the mechanism is not fully understood. We recently found that ATG12 of the E3 binds to a short sequence in the flexible region (FR) of ATG3 with high affinity, and that this interaction is critical for E2-E3 complex formation. These findings, together with detailed structural analyses of this interaction, define the properties of ATG12 and provide new insights of how LC3 transfer begins with ATG3 recruitment by ATG12.