TNAP, a novel repressor of NF-kappaB-inducing kinase, suppresses NF-kappaB activation

NF-kappaB-inducing kinase (NIK) has been implicated as an essential component of NF-kappaB activation. However, the regulatory mechanism of NIK signaling remains elusive. We have identified a novel NIK interacting protein, TNAP (for TRAFs and NIK-associated protein). In mammalian cells, TNAP physically interacts with NIK, TRAF2, and TRAF3 but not IKK1 or IKK2. TNAP specifically inhibits NF-kappaB activation induced by tumor necrosis factor (TNF)-alpha, TNF receptor 1, TRADD, RIP, TRAF2, and NIK but does not affect IKK1- and IKK2-mediated NF-kappaB activation. Knockdown of TNAP by lentiviral-mediated small interference RNA potentiates TNF-alpha-induced NF-kappaB activation. TNAP suppresses NIK kinase activity and subsequently reduces p100 processing, p65 phosphorylation, and IkappaBalpha degradation. These data suggest that TNAP is a repressor of NIK activity and regulates both the classical and alternative NF-kappaB signaling pathways.     

NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase

The activation of NF-kappaB by receptors in the tumor necrosis factor (TNF) receptor and Toll/interleukin-1 (IL-1) receptor families requires the TRAF family of adaptor proteins. Receptor oligomerization causes the recruitment of TRAFs to the receptor complex, followed by the activation of a kinase cascade that results in the phosphorylation of IkappaB. TANK is a TRAF-binding protein that can inhibit the binding of TRAFs to receptor tails and can also inhibit NF-kappaB activation by these receptors. However, TANK also displays the ability to stimulate TRAF-mediated NF-kappaB activation. In this report, we investigate the mechanism of the stimulatory activity of TANK. We find that TANK interacts with TBK1 (TANK-binding kinase 1), a novel IKK-related kinase that can activate NF-kappaB in a kinase-dependent manner. TBK1, TANK and TRAF2 can form a ternary complex, and complex formation appears to be required for TBK1 activity. Kinase-inactive TBK1 inhibits TANK-mediated NF-kappaB activation but does not block the activation mediated by TNF-alpha, IL-1 or CD40. The TBK1-TANK-TRAF2 signaling complex functions upstream of NIK and the IKK complex and represents an alternative to the receptor signaling complex for TRAF-mediated activation of NF-kappaB.     

Tumor necrosis factor-induced nuclear factor kappaB activation is impaired in focal adhesion kinase-deficient fibroblasts

Focal adhesion kinase (FAK) is widely involved in important cellular functions such as proliferation, migration, and survival, although its roles in immune and inflammatory responses have yet to be explored. We demonstrate a critical role for FAK in the tumor necrosis factor (TNF)-induced activation of nuclear factor (NF)-kappaB, using FAK-deficient (FAK-/-) embryonic fibroblasts. Interestingly, TNF-induced interleukin (IL)-6 production was nearly abolished in FAK-/- fibroblasts, whereas a normal level of production was obtained in FAK+/- or FAK+/+ fibroblasts. FAK deficiency did not affect the three types of mitogen-activated protein kinases, ERK, JNK, and p38. Similarly, TNF-induced activation of activator protein 1 or NF-IL-6 was not impaired in FAK-/- cells. Of note, TNF-induced NF-kappaB DNA binding activity and activation of IkappaB kinases (IKKs) were markedly impaired in FAK-/- cells, whereas the expression of TNF receptor I or other signaling molecules such as receptor-interacting protein (RIP), tumor necrosis factor receptor-associated factor 2 (TRAF2), IKKalpha, IKKbeta, and IKKgamma was unchanged. Also, TNF-induced association of FAK with RIP and subsequent association of RIP with TRAF2 were not observed, resulting in a failure of RIP to recruit the IKK complex in FAK-/- cells. The reintroduction of wild type FAK into FAK-/- cells restored the interaction of RIP with TRAF2 and the IKK complex and allowed recovery of NF-kappaB activation and subsequent IL-6 production. Thus, we propose a novel role for FAK in the NF-kappaB activation pathway leading to the production of cytokines.     

Caspase-mediated cleavage converts the tumor necrosis factor (TNF) receptor-associated factor (TRAF)-1 from a selective modulator of TNF receptor signaling to a general inhibitor of NF-kappaB activation

The role of tumor necrosis factor (TNF) receptor-associated factor (TRAF)-1 in NF-kappaB activation by various members of the TNF receptor family is not well understood, and conflicting data have been published. Here, we show that TRAF1 differentially affects TRAF2 recruitment and activation of NF-kappaB by members of the TNF receptor family. Interestingly, a naturally occurring caspase-derived cleavage product of TRAF1 solely comprising its TRAF domain (TRAF1-(164-416)) acted as a general inhibitor of NF-kappaB activation. In contrast, a corresponding fragment generated by cleavage of TRAF3 showed no effect in this regard. In accordance with these functional data, TRAF1, but not TRAF3, interacted with the IKK complex via its N-TRAF domain. Endogenous TRAF1 and the overexpressed TRAF domain of TRAF1 were found to be constitutively associated with the IKK complex, whereas endogenous receptor interacting protein was only transiently associated with the IKK complex upon TNF stimulation. Importantly, the caspase-generated TRAF1-fragment, but not TRAF1 itself inhibited IKK activation. Our results suggest that TRAF1 and TRAF1-(164-416) exert their regulatory effects on receptor-induced NF-kappaB activation not only by modulation of TRAF2 receptor interaction but especially TRAF1-(164-416) also by directly targeting the IKK complex.     

Activation of the Ikappa B kinases by RIP via IKKgamma /NEMO-mediated oligomerization

To understand the mechanism of activation of the IkappaB kinase (IKK) complex in the tumor necrosis factor (TNF) receptor 1 pathway, we examined the possibility that oligomerization of the IKK complex triggered by ligand-induced trimerization of the TNF receptor 1 complex is responsible for activation of the IKKs. Gel filtration analysis of the IKK complex revealed that TNFalpha stimulation induces a large increase in the size of this complex, suggesting oligomerization. Substitution of the C-terminal region of IKKgamma, which interacts with RIP, with a truncated DR4 lacking its cytoplasmic death domain, produced a molecule that could induce IKK and NF-kappaB activation in cells in response to TRAIL. Enforced oligomerization of the N terminus of IKKgamma or truncated IKKalpha or IKKbeta lacking their serine-cluster domains can also induce IKK and NF-kappaB activation. These data suggest that IKKgamma functions as a signaling adaptor between the upstream regulators such as RIP and the IKKs and that oligomerization of the IKK complex by upstream regulators is a critical step in activation of this complex.     

The carboxyl-terminal region of IkappaB kinase gamma (IKKgamma) is required for full IKK activation

IkappaB kinase gamma (IKKgamma) (also known as NEMO, Fip-3, and IKKAP-1) is the essential regulatory component of the IKK complex; it is required for NF-kappaB activation by various stimuli, including tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), phorbol esters, lipopolysaccharides, and double-stranded RNA. IKKgamma is encoded by an X-linked gene, deficiencies in which may result in two human genetic disorders, incontinentia pigmenti (IP) and hypohidrotic ectodermal dysplasia with severe immunodeficiency. Subsequent to the linkage of IKKgamma deficiency to IP, we biochemically characterized the effects of a mutation occurring in an IP-affected family on IKK activity and NF-kappaB signaling. This particular mutation results in premature termination, such that the variant IKKgamma protein lacks its putative C-terminal Zn finger and, due to decreased mRNA stability, is underexpressed. Correspondingly, IKK and NF-kappaB activation by TNF-alpha and, to a lesser extent, IL-1 are reduced. Mutagenesis of the C-terminal region of IKKgamma was performed in an attempt to define the role of the putative Zn finger and other potential functional motifs in this region. The mutants were expressed in IKKgamma-deficient murine embryonic fibroblasts (MEFs) at levels comparable to those of endogenous IKKgamma in wild-type MEFs and were able to associate with IKKalpha and IKKbeta. Substitution of two leucines within a C-terminal leucine zipper motif markedly reduced IKK activation by TNF-alpha and IL-1. Another point mutation resulting in a cysteine-to-serine substitution within the putative Zn finger motif affected IKK activation by TNF-alpha but not by IL-1. These results may explain why cells that express these or similar mutant alleles are sensitive to TNF-alpha-induced apoptosis despite being able to activate NF-kappaB in response to other stimuli.     

Nonstructural 5A protein of hepatitis C virus modulates tumor necrosis factor alpha-stimulated nuclear factor kappa B activation

The hepatitis C virus nonstructural protein 5A (NS5A) is a multifunctional phosphoprotein that leads to pleiotropic responses, in part by regulating cell growth and cellular signaling pathways. Here we show that overexpression of NS5A inhibits tumor necrosis factor (TNF)-alpha-induced nuclear factor kappaB (NF-kappaB) activation in HEK293 cells, as determined by luciferase reporter gene expression and by electrophoretic mobility shift assay. When overexpressed, NS5A cannot inhibit the recruitment of TNF receptor-associated factor 2 (TRAF2) and IkappaB kinase (IKK)beta into the TNF receptor 1-TNF receptor-associated death domain complex. In contrast, NS5A is a part of the TNF receptor 1 signaling complex. NF-kappaB activation by TNF receptor-associated death domain and TRAF2 was inhibited by NS5A, whereas MEKK1 and IKKbeta-dependent NF-kappaB activation was not affected, suggesting that NS5A may inhibit NF-kappaB activation signaled by TRAF2. Coimmunoprecipitation and colocalization of NS5A and TRAF2 expressed in vivo provide compelling evidence that NS5A directly interacts with TRAF2. This interaction was mapped to the middle one-third (amino acids 148-301) of NS5A and the TRAF domain of TRAF2. Our findings suggest a possible molecular mechanism that could explain the ability of NS5A to negatively regulate TNF-alpha-induced NF-kappaB activation.     

TNFR-associated factor-3 is associated with BAFF-R and negatively regulates BAFF-R-mediated NF-kappa B activation and IL-10 production

TALL-1 is a member of the TNF family that is critically involved in B cell survival, maturation, and progression of lupus-like autoimmune diseases. TALL-1 has three receptors, including BCMA, TACI, and BAFF-R, which are mostly expressed by B lymphocytes. Gene knockout studies have indicated that BAFF-R is the major stimulatory receptor for TALL-1 signaling and is required for normal B cell development. The intracellular signaling mechanisms of BAFF-R are not known. In this report, we attempted to identify BAFF-R-associated downstream proteins by yeast two-hybrid screening. This effort identified TNFR-associated factor (TRAF)3 as a protein specifically interacting with BAFF-R in yeast two-hybrid assays. Coimmunoprecipitation experiments indicated that BAFF-R interacts with TRAF3 in B lymphoma cells and this interaction is stimulated by TALL-1 treatment. Domain mapping experiments indicated that both a 6-aa membrane proximal region and the C-terminal 35 aa of BAFF-R are required for its interaction with TRAF3. Moreover, overexpression of TRAF3 inhibits BAFF-R-mediated NF-kappaB activation and IL-10 production. Taken together, our findings suggest that TRAF3 is a negative regulator of BAFF-R-mediated NF-kappaB activation and IL-10 production.     

The LIM protein Ajuba influences interleukin-1-induced NF-kappaB activation by affecting the assembly and activity of the protein kinase Czeta/p62/TRAF6 signaling complex

The Zyxin/Ajuba family of cytosolic LIM domain-containing proteins has the potential to shuttle from sites of cell adhesion into the nucleus and thus can be candidate transducers of environmental signals. To understand Ajuba's role in signal transduction pathways, we performed a yeast two-hybrid screen with the LIM domain region of Ajuba. We identified the atypical protein kinase C (aPKC) scaffold protein p62 as an Ajuba binding partner. A prominent function of p62 is the regulation of NF-kappaB activation in response to interleukin-1 (IL-1) and tumor necrosis factor signaling through the formation of an aPKC/p62/TRAF6 multiprotein signaling complex. In addition to p62, we found that Ajuba also interacted with tumor necrosis factor receptor-associated factor 6 (TRAF6) and PKCzeta. Ajuba recruits TRAF6 to p62 and in vitro activates PKCzeta activity and is a substrate of PKCzeta. Ajuba null mouse embryonic fibroblasts (MEFs) and lungs were defective in NF-kappaB activation following IL-1 stimulation, and in lung IKK activity was inhibited. Overexpression of Ajuba in primary MEFs enhances NF-kappaB activity following IL-1 stimulation. We propose that Ajuba is a new cytosolic component of the IL-1 signaling pathway modulating IL-1-induced NF-kappaB activation by influencing the assembly and activity of the aPKC/p62/TRAF6 multiprotein signaling complex.     

TRAF2 Suppresses Basal IKK Activity in Resting Cells and TNFα Can Activate IKK in TRAF2 and TRAF5 Double Knockout Cells

Tumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) and TRAF5 are adapter proteins involved in TNFα-induced activation of the c-Jun N-terminal kinase and nuclear factor κB (NF-κB) pathways. Currently, TNFα-induced NF-κB activation is believed to be impaired in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells. Here, we report instead that T2/5 DKO cells exhibit high basal IκB kinase (IKK) activity and elevated expression of NF-κB-dependent genes in unstimulated conditions. Although TNFα-induced receptor-interacting protein 1 ubiquitination is indeed impaired in T2/5 DKO cells, TNFα stimulation further increases IKK activity in these cells, resulting in significantly elevated expression of NF-κB target genes to a level higher than that in wild-type cells. Inhibition of NIK in T2/5 DKO cells attenuates basal IKK activity and restores robust TNFα-induced IKK activation to a level comparable with that seen in wild-type cells. This suggests that TNFα can activate IKK in the absence of TRAF2 and TRAF5 expression and receptor-interacting protein 1 ubiquitination. In addition, both the basal and TNFα-induced expression of anti-apoptotic proteins are normal in T2/5 DKO cells, yet these DKO cells remain sensitive to TNFα-induced cell death, due to the impaired recruitment of anti-apoptotic proteins to the TNFR1 complex in the absence of TRAF2. Thus, our data demonstrate that TRAF2 negatively regulates basal IKK activity in resting cells and inhibits TNFα-induced cell death by recruiting anti-apoptotic proteins to the TNFR1 complex rather than by activating the NF-κB pathway.     

Role of TRAF3 and -6 in the activation of the NF-kappa B and JNK pathways by X-linked ectodermal dysplasia receptor

X-linked ectodermal dysplasia receptor (XEDAR) is a recently isolated member of the tumor necrosis factor receptor family that has been shown to be highly expressed in ectodermal derivatives during embryonic development and binds to ectodysplasin-A2 (EDA-A2). By using a subclone of 293F cells with stable expression of XEDAR, we report that XEDAR activates the NF-kappaB and JNK pathways in an EDA-A2-dependent fashion. Treatment with EDA-A2 leads to the recruitment of TRAF3 and -6 to the aggregated XEDAR complex, suggesting a central role of these adaptors in the proximal aspect of XEDAR signaling. Whereas TRAF3 and -6, IKK1/IKKalpha, IKK2/IKKbeta, and NEMO/IKKgamma are involved in XEDAR-induced NF-kappaB activation, XEDAR-induced JNK activation seems to be mediated via a pathway dependent on TRAF3, TRAF6, and ASK1. Deletion and point mutagenesis studies delineate two distinct regions in the cytoplasmic domain of XEDAR, which are involved in binding to TRAF3 and -6, respectively, and play a major role in the activation of the NF-kappaB and JNK pathways. Taken together, our results establish a major role of TRAF3 and -6 in XEDAR signaling and in the process of ectodermal differentiation.     

Interleukin-8 induces nuclear transcription factor-kappaB through a TRAF6-dependent pathway

Considering the potential role of interleukin-8 (IL-8) in inflammation, angiogenesis, tumorigenesis, and metastasis, we investigated the molecular mechanism involved in IL-8-mediated signaling. In this report we provide evidence that like TNF, an inducer of NF-kappaB and also a NF-kappaB-dependent gene product, IL-8 induces NF-kappaB in a unique pathway. IL-8 induces NF-kappaB activation in a dose-dependent manner in different cell types as detected by a DNA-protein binding assay. IL-8 induces NF-kappaB-dependent reporter gene expression as well as ICAM-1, VCAM-1, and Cox-2 expression. IL-8 also induces IkappaBalpha phosphorylation followed by degradation and p65 translocation. IL-8 induces c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase (MAPK) in a dose- and time-dependent manner. IL-8-induced NF-kappaB activation is for the most part unaltered when cells are transfected with dominant-negative TRADD, FADD, or TRAF2, but is inhibited with dominant-negative TRAF6-, NIK-, IKK-, or IkappaBalpha-transfected cells. The data suggest that IL-8-induced NF-kappaB activation proceeds through a TRAF2-independent but TRAF6-dependent pathway, followed by recruitment of IRAK and activation of IKK. IL-8-induced NF-kappaB activation is not observed in a cell-permeable peptide that has TRAF6 binding motif-treated cells or IRAK-deficient cells. IL-8-induced NF-kappaB activation proceeds mostly through interaction with TRAF6 and partially through the Rho-GTPase pathways. This is the first report that IL-8 induces NF-kappaB in a distinct pathway, and activation of NF-kappaB and its dependent genes may be one of the pathways of IL-8-induced inflammation and angiogenesis.