Physical and functional interaction of filamin (actin-binding protein-280) and tumor necrosis factor receptor-associated factor 2

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is an intracellular protein involved in signal transduction from TNF receptor I and II and related receptors. TRAF2 is required for TNF-induced activation of c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and TRAF2 can also mediate activation of NF-kappaB. Here we have identified the actin-binding protein Filamin (actin-binding protein-280) as a TRAF2-interacting protein. Filamin binds to the Ring zinc finger domain of TRAF2. Overexpressed Filamin inhibits TRAF2-induced activation of JNK/SAPK and of NF-kappaB. Furthermore, ectopically expressed Filamin inhibits NF-kappaB activation induced via TNF, interleukin-1, Toll receptors, and TRAF6 but not activation induced via overexpression of NIK, a downstream effector in these pathways. Importantly, TNF fails to activate SAPK or NF-kappaB in a human melanoma cell line deficient in Filamin. Reintroduction of Filamin into these cells restores the TNF response. The data imply a role for Filamin in inflammatory signal transduction pathways.     

Receptor activator of NF-kappaB recruits multiple TRAF family adaptors and activates c-Jun N-terminal kinase

Receptor activator of NF-kappaB (RANK) is a recently cloned member of the tumor necrosis factor receptor (TNFR) superfamily, and its function has been implicated in osteoclast differentiation and dendritic cell survival. Many of the TNFR family receptors recruit various members of the TNF receptor-associated factor (TRAF) family for transduction of their signals to NF-kappaB and c-Jun N-terminal kinase. In this study, the involvement of TRAF family members and the activation of the JNK pathway in signal transduction by RANK were investigated. TRAF1, 2, 3, 5, and 6 were found to bind RANK in vitro. Association of RANK with each of these TRAF proteins was also detected in vivo. Expression of RANK in cultured cells also induced the activation of JNK, which was blocked by a dominant-negative form of JNK. Furthermore, by employing various C-terminal deletion mutants of RANK, the regions responsible for TRAF interaction and JNK activation were identified. TRAF5 was determined to bind to the C-terminal 11 amino acids and the other TRAF members to a region N-terminal to the TRAF5 binding site. The domain responsible for JNK activation was localized to the same region where TRAF1, 2, 3, and 6 bound, which suggests that these TRAF molecules might mediate the RANK-induced JNK activation.     

The role of TNF-receptor family members and other TRAF-dependent receptors in bone resorption

The contribution of osteoclasts to the process of bone loss in inflammatory arthritis has recently been demonstrated. Studies in osteoclast biology have led to the identification of factors responsible for the differentiation and activation of osteoclasts, the most important of which is the receptor activator of NF-kappa B ligand/osteoclast differentiation factor (RANKL/ODF), a tumor necrosis factor (TNF)-like protein. The RANKL/ODF receptor, receptor activator of NF-kappa B (RANK), is a TNF-receptor family member present on both osteoclast precursors and mature osteoclasts. Like other TNF-family receptors and the IL-1 receptor, RANK mediates its signal transduction via TNF receptor-associated factor (TRAF) proteins, suggesting that the signaling pathways activated by RANK and other inflammatory cytokines involved in osteoclast differentiation and activation are interconnected.     

TIRP,a novel Toll/interleukin-1 receptor (TIR) domain-containing adapter protein involved in TIR signaling

The Toll/interleukin-1 receptor (TIR) family members play important roles in host defense. These receptors signal through TIR domain-containing adapter proteins. In this report, we identified a novel TIR domain-containing adapter protein designated as TIRP. Co-immunoprecipitation experiments suggest that TIRP is associated with IL-1 receptors. TIRP also interacts with kinase-inactive mutants of IRAK and IRAK-4, IRAK-2, IRAK-M, and TRAF6. Overexpression of TIRP activates NF-kappaB and potentiates IL-1 receptor-mediated NF-kappaB activation. A dominant negative mutant of TIRP inhibits IL-1- but not tumor necrosis factor-triggered NF-kappaB activation. Moreover, TIRP-mediated NF-kappaB activation is inhibited by dominant negative mutants of IRAK, IRAK-2, TRAF6, and IKKbeta. Our findings suggest that TIRP is involved in IL-1-triggered NF-kappaB activation and functions upstream of IRAK, IRAK-2, TRAF6, and IKKbeta     

The role of TNF-receptor family members and other TRAF-dependent receptors in bone resorption

The contribution of osteoclasts to the process of bone loss in inflammatory arthritis has recently been demonstrated. Studies in osteoclast biology have led to the identification of factors responsible for the differentiation and activation of osteoclasts, the most important of which is the receptor activator of NF-κB ligand/osteoclast differentiation factor (RANKL/ODF), a tumor necrosis factor (TNF)-like protein. The RANKL/ODF receptor, receptor activator of NF-κB (RANK), is a TNF-receptor family member present on both osteoclast precursors and mature osteoclasts. Like other TNF-family receptors and the IL-1 receptor, RANK mediates its signal transduction via TNF receptor-associated factor (TRAF) proteins, suggesting that the signaling pathways activated by RANK and other inflammatory cytokines involved in osteoclast differentiation and activation are interconnected.     

TRAF6 is required for TRAF2-dependent CD40 signal transduction in nonhemopoietic cells

The emerging role of CD40, a tumor necrosis factor (TNF) receptor family member, in immune regulation, disease pathogenesis, and cancer therapy necessitates the analysis of CD40 signal transduction in a wide range of tissue types. In this study we present evidence that the CD40-interacting proteins TRAF2 and TRAF6 play an important physiological role in CD40 signaling in nonhemopoietic cells. Using mutational analysis of the CD40 cytoplasmic tail, we demonstrate that the specific binding of TRAF2 to CD40 is required for efficient signaling on the NF-kappaB, Jun N-terminal protein kinase (JNK), and p38 axis. In fibroblasts lacking TRAF2 or in carcinoma cells in which TRAF2 has been depleted by RNA interference, the CD40-mediated activation of NF-kappaB and JNK is significantly reduced, and the activation of p38 and Akt is severely impaired. Interestingly, whereas the TRAF6-interacting membrane-proximal domain of CD40 has a minor role in signal transduction, studies utilizing TRAF6 knockout fibroblasts and RNA interference in epithelial cells reveal that the CD40-induced activation of NF-kappaB, JNK, p38, and Akt requires the integrity of TRAF6. Furthermore, we provide evidence that TRAF6 regulates CD40 signal transduction not only through its direct binding to CD40 but also indirectly via its association with TRAF2. These observations provide novel insight into the mechanisms of CD40 signaling and the multiple roles played by TRAF6 in signal transduction.     

The ectodermal dysplasia receptor activates the nuclear factor-kappaB, JNK, and cell death pathways and binds to ectodysplasin A

The ectodermal dysplasia receptor (EDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to play a key role in the process of ectodermal differentiation. We present evidence that EDAR is capable of activating the nuclear factor-kappaB, JNK, and caspase-independent cell death pathways and that these activities are impaired in mutants lacking its death domain or those associated with anhidrotic ectodermal dysplasia and the downless phenotype. Although EDAR possesses a death domain, it did not interact with the death domain-containing adaptor proteins TRADD and FADD. EDAR successfully interacted with various TRAF family members; however, a dominant-negative mutant of TRAF2 was incapable of blocking EDAR-induced nuclear factor-kappaB or JNK activation. Collectively, the above results suggest that EDAR utilizes a novel signal transduction pathway. Finally, ectodysplasin A can physically interact with the extracellular domain of EDAR and thus represents its biological ligand.     

Tumor necrosis factor alpha-induced activation of c-jun N-terminal kinase is mediated by TRAF2.

Tumor necrosis factor alpha (TNF alpha) a pro-inflammatory cytokine is an endogenous mediator of septic shock, inflammation, anti-viral responses and apoptotic cell death. TNF alpha elicits its complex biological responses through the individual or cooperative action of two TNF receptors of mol. wt 55 kDa (TNF-RI) and mol. wt 75 kDa (TNF-RII). To determine signaling events specific for TNF-RII we fused the extracellular domain of the mouse CD4 antigen to the intracellular domain of TNF-RII. Crosslinking of the chimeric receptor using anti-CD4 antibodies initiates exclusively TNF-RII-mediated signals. Our findings show that: (i) TNF-RII is able to activate two members of the MAP kinase family: extracellular regulated kinase (ERK) and c-jun N-terminal kinase (JNK); (ii) TRAF2, a molecule that binds TNF-RII and associates indirectly with TNF-RI, is sufficient to activate JNK upon overexpression; (iii) dominant-negative TRAF2 blocks TNF alpha-mediated JNK activation and (iv) TRAF2 signals the activation of JNK and NF-kappaB through different pathways. Our findings suggest that TNF alpha-mediated JNK activation in fibroblasts is independent of the cell death pathway and that TRAF2 occupies a key role in TNF receptor signaling to JNK.     

肿瘤坏死因子受体作用因子3(TRAF3)结构及生物学功能

目前在哺乳动物中发现6个肿瘤坏死因子受体作用因子(TNF receptor associated factors,TRAFs)家族成员,它们主要参与TNF受体家族信号通路.这些TRAF成员在C末端有螺旋卷曲结构和保守的TRAF结构域.TRAF3是TRAF家族中功能最为多样化的成员之一.1996年对TRAF3基因敲除小鼠进行研究发现,小鼠在出生早期死亡,这阻碍了TRAF3的生物学功能进一步研究,另一方面也证实了TRAF3在出生后发育以及维持正常的免疫系统功能方面有着重要生物学功能.10年后研究发现,TRAF3的缺失能够导致非经典NF-κB信号通路激活,这使得TRAF3在该信号通路中的功能得到了进一步的阐述.最近研究表明,TRAF3不仅能够负向调节NF-κB和MAPK信号通路,还能够正向调节Ⅰ型干扰素的产生.通过研究还发现,TRAF3可能存在着负向调节钙调蛋白磷酸酶活性的新功能.因此,研究TRAF3在免疫信号通路中的作用以及与之相关的病毒疾病具有重要的意义.     

Tumor necrosis factor receptor 2 (TNFR2) signaling is negatively regulated by a novel, carboxyl-terminal TNFR-associated factor 2 (TRAF2)-binding site

Tumor necrosis factor (TNF) superfamily receptors typically induce both NF-kappaB and JNK activation by recruiting the TRAF2 signal transduction protein to their cytoplasmic domain. The type 2 TNF receptor (TNFR2), however, is a poor activator of these signaling pathways despite its high TRAF2 binding capability. This apparent paradox is resolved here by the demonstration that TNFR2 carries a novel carboxyl-terminal TRAF2-binding site (T2bs-C) that prevents the delivery of activation signals from its conventional TRAF2-binding site (T2bs-N). T2bs-C does not conform to canonical TRAF2 binding motifs and appears to bind TRAF2 indirectly via an as yet unidentified intermediary. Specific inactivation of T2bs-N by site-directed mutagenesis eliminated most of the TRAF2 recruited to the TNFR2 cytoplasmic domain but had no effect on ligand-dependent activation of the NF-kappaB or JNK pathways. By contrast, inactivation of T2bs-C had little effect on the amount of TRAF2 recruited but greatly enhanced ligand-dependent NF-kappaB and JNK activation. In wild-type TNFR2 therefore, T2bs-C acts in a dominant fashion to attenuate signaling by the intrinsically more active T2bs-N but not by preventing TRAF2 recruitment. This unique uncoupling of TRAF2 recruitment and signaling at T2bs-N may be important in the modulation by TNFR2 of signaling through coexpressed TNFR1.     

TNF signaling: early events and phosphorylation

Tumor necrosis factor-alpha (TNF) is a major mediator of apoptosis as well as immunity and inflammation. Inappropriate production of TNF or sustained activation of TNF signaling has been implicated in the pathogenesis of a wide spectrum of human diseases, including cancer, osteoporosis, sepsis, diabetes, and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. TNF binds to two specific receptors, TNF-receptor type I (TNF-R1, CD120a, p55/60) and TNF-receptor type II (TNF-R2, CD120b, p75/80). Signaling through TNF-R1 is extremely complex, leading to both cell death and survival signals. Many findings suggest an important role of phosphorylation of the TNF-R1 by number of protein kinases. Role of TNF-R2 phosphorylation on its signaling properties is understood less than TNF-R1. Other cellular substrates as TRADD adaptor protein, TRAF protein family and RIP kinases are reviewed in relation to TNF receptor-mediated apoptosis or survival pathways and regulation of their actions by phosphorylation.     

Modulation of tumor necrosis factor and interleukin-1-dependent NF-kappaB activity by mPLK/IRAK

The innate immune response is an important defense against pathogenic agents. A component of this response is the NF-kappaB-dependent activation of genes encoding inflammatory cytokines such as interleukin-8 (IL-8) and cell adhesion molecules like E-selectin. Members of the serine/threonine innate immune kinase family of proteins have been proposed to mediate the innate immune response. One serine/threonine innate immune kinase family member, the mouse Pelle-like kinase/human interleukin-1 receptor-associated kinase (mPLK/IRAK), has been proposed to play an obligate role in promoting IL-1-mediated inflammation. However, it is currently unknown whether mPLK/IRAK catalytic activity is required for IL-1-dependent NF-kappaB activation. The present study demonstrates that mPLK/IRAK catalytic activity is not required for IL-1-mediated activation of an NF-kappaB-dependent signal. Intriguingly, catalytically inactive mPLK/IRAK inhibits type 1 tumor necrosis factor (TNF) receptor-dependent NF-kappaB activation. The pathway through which mPLK/IRAK mediates this TNF response is TRADD- and TRAF2-independent. Our data suggest that in addition to its role in IL-1 signaling, mPLK/IRAK is a component of a novel signal transduction pathway through which TNF R1 activates NF-kappaB-dependent gene expression.