IKKgamma /NEMO facilitates the recruitment of the IkappaB proteins into the IkappaB kinase complex

IKKgamma/NEMO is an essential regulatory component of the IkappaB kinase complex that is required for NF-kappaB activation in response to various stimuli including tumor necrosis factor-alpha and interleukin-1beta. To investigate the mechanism by which IKKgamma/NEMO regulates the IKK complex, we examined the ability of IKKgamma/NEMO to recruit the IkappaB proteins into this complex. IKKgamma/NEMO binding to wild-type, but not to a kinase-deficient IKKbeta protein, facilitated the association of IkappaBalpha and IkappaBbeta with the high molecular weight IKK complex. Following tumor necrosis factor-alpha treatment of HeLa cells, the majority of the phosphorylated form of endogenous IkappaBalpha was associated with the high molecular weight IKK complex in HeLa cells and parental mouse embryo fibroblasts but not in IKKgamma/NEMO-deficient cells. Finally, we demonstrate that IKKgamma/NEMO facilitates the association of the IkappaB proteins and IKKbeta and leads to increases in IKKbeta kinase activity. These results suggest that an important function of IKKgamma/NEMO is to facilitate the association of both IKKbeta and IkappaB in the high molecular weight IKK complex to increase IkappaB phosphorylation.     

TLR8-mediated NF-kappaB and JNK activation are TAK1-independent and MEKK3-dependent

TLR8-mediated NF-kappaB and IRF7 activation are abolished in human IRAK-deficient 293 cells and IRAK4-deficient fibroblast cells. Both wild-type and kinase-inactive mutants of IRAK and IRAK4, respectively, restored TLR8-mediated NF-kappaB and IRF7 activation in the IRAK- and IRAK4-deficient cells, indicating that the kinase activity of IRAK and IRAK4 is probably redundant for TLR8-mediated signaling. We recently found that TLR8 mediates a unique NF-kappaB activation pathway in human 293 cells and mouse embryonic fibroblasts, accompanied only by IkappaBalpha phosphorylation and not IkappaBalpha degradation, whereas interleukin (IL)-1 stimulation causes both IkappaBalpha phosphorylation and degradation. The intermediate signaling events mediated by IL-1 (including IRAK modifications and degradation and TAK1 activation) were not detected in cells stimulated by TLR8 ligands. TLR8 ligands trigger similar levels of IkappaBalpha phosphorylation and NF-kappaB and JNK activation in TAK1(-/-) mouse embryo fibroblasts (MEFs) as compared with wild-type MEFs, whereas lack of TAK1 results in reduced IL-1-mediated NF-kappaB activation and abolished IL-1-induced JNK activation. The above results indicate that although TLR8-mediated NF-kappaB and JNK activation are IRAK-dependent, they do not require IRAK modification and are TAK1-independent. On the other hand, TLR8-mediated IkappaBalpha phosphorylation, NF-kappaB, and JNK activation are completely abolished in MEKK3(-/-) MEFs, whereas IL-1-mediated signaling was only moderately reduced in these deficient MEFs as compared with wild-type cells. The differences between IL-1R- and TLR8-mediated NF-kappaB activation are also reflected at the level of IkappaB kinase (IKK) complex. TLR8 ligands induced IKKgamma phosphorylation, whereas IKKalpha/beta phosphorylation and IKKgamma ubiquitination that can be induced by IL-1 were not detected in cells treated with TLR8 ligands. We postulate that TLR8-mediated MEKK3-dependent IKKgamma phosphorylation might play an important role in the activation of IKK complex, leading to IkappaBalpha phosphorylation.     

The human herpes virus 8-encoded viral FLICE inhibitory protein physically associates with and persistently activates the Ikappa B kinase complex

The human herpesvirus 8 (HHV8, also called Kaposi's sarcoma-associated herpesvirus) has been linked to Kaposi's sarcoma and primary effusion lymphoma (PEL) in immunocompromised individuals. We demonstrate that PEL cell lines have a constitutively active NF-kappaB pathway, which is associated with persistent phosphorylation of IkappaBalpha. To elucidate the mechanism of NF-kappaB activation in PEL cell lines, we have investigated the role of viral FLICE inhibitory protein (vFLIP) in this process. We report that stable expression of HHV8 vFLIP in a variety of cell lines is associated with persistent NF-kappaB activation caused by constitutive phosphorylation of IkappaBalpha. HHV8 vFLIP gets recruited to a approximately 700-kDa IkappaB kinase (IKK) complex and physically associates with IKKalpha, IKKbeta, NEMO/IKKgamma, and RIP. HHV8 vFLIP is incapable of activating NF-kappaB in cells deficient in NEMO/IKKgamma, thereby suggesting an essential role of an intact IKK complex in this process. Our results suggest that HHV8 vFLIP might contribute to the persistent NF-kappaB activation observed in PEL cells by associating with and stimulating the activity of the cellular IKK complex.     

IKKgamma inhibits activation of NF-kappaB by NIK

IKKgamma is a component of the IKK complex, which regulates NF-kappaB activity. To investigate the role of IKKgamma, we expressed wild type IKKgamma containing 412 amino acids, and deletion mutants containing residues 1-312 and 101-412, using murine IKKgamma cDNA. In a co-transfection assay with a CAT reporter plasmid, NIK activated NF-kappaB-dependent gene expression approximately two fold and this expression was inhibited by co-transfection of a wild type IKKgamma expression plasmid. In binding assays IKKgamma inhibited the association of IkappaBalpha with IKKbeta and the subsequent phosphorylation of IkappaBalpha that is activated by NIK. Inhibition by IKKgamma also occurred in an assay with a dominant negative mutant of NIK but not with a C-terminal deletion mutant of IKKgamma, indicating that the C-terminal 100 amino acids of IKKgamma are important for negative regulation of NF-kappaB activation. In addition, the interaction of IKKbeta with IKKgamma was inhibited by co-transfection with a NIK expression plasmid. Our results suggest that overexpression of IKKgamma inhibits activation of NF-kappaB by NIK by competing with NIK for interaction with IKKbeta.     

Lymphotoxin beta receptor induces sequential activation of distinct NF-kappa B factors via separate signaling pathways

Lymphotoxin beta receptor (LTbetaR)-induced activation of NF-kappaB in mouse embryo fibroblasts was mediated by the classical pathway and by an alternative or second pathway. The classical pathway involved the IkappaB kinase (IKK)beta- and IKKgamma-dependent degradation of IkappaBalpha and resulted in the rapid but transient activation of primarily RelA-containing NF-kappaB dimers. The alternative or second pathway proceeded via NF-kappaB-inducing kinase (NIK)-, IKKalpha-, and protein synthesis-dependent processing of the inhibitory NF-kappaB2 p100 precursor protein to the p52 form and resulted in a delayed but sustained activation of primarily RelB-containing NF-kappaB dimers. This second pathway was independent of the classical IKK complex, which is governed by its central IKKgamma regulatory subunit. The sequential engagement of two distinct pathways, coupled with the negative feedback inhibition of RelA complexes by NF-kappaB-induced resynthesis of IkappaBalpha, resulted in a pronounced temporal change in the nature of the NF-kappaB activity during the course of stimulation. Initially dominant RelA complexes were replaced with time by RelB complexes. Therefore, the alternative activation path mediated by processing of p100 was necessary for sustained NF-kappaB activity in mouse embryo fibroblasts in response to LTbetaR stimulation. Based on the phenotype of mice deficient in various components of the LTbetaR-induced activation of p100 processing, we conclude that this pathway is critically involved in the function of stromal cells during the generation of secondary lymphoid organ microarchitectures.     

Characterization of the bovine IkappaB kinases (IKK)alpha and IKKbeta,the regulatory subunit NEMO and their substrate IkappaBalpha

The Nuclear factor (NF)-kappaB signalling pathway plays a critical role in the regulation and coordination of a wide range of cellular events such as cell growth, apoptosis and cell differentiation. Activation of the IKK (inhibitor of NF-kappaB kinase) complex is a crucial step and a point of convergence of all known NF-kappaB signalling pathways. To analyse bovine IKKalpha (IKK1), IKKbeta (IKK2) and IKKgamma (or NF-kappaB Essential MOdulator, NEMO) and their substrate IkappaBalpha (Inhibitor of NF-kappaB), the corresponding cDNAs of these molecules were isolated, sequenced and characterized. A comparison of the amino acid sequences with those of their orthologues in other species showed a very high degree of identity, suggesting that the IKK complex and its substrate IkappaBalpha are evolutionarily highly conserved components of the NF-kappaB pathway. Bovine IKKalpha and IKKbeta are related protein kinases showing 50% identity which is especially prominent in the kinase and leucine zipper domains. Co-immunoprecipitation assays and GST-pull-down experiments were carried out to determine the composition of bovine IKK complexes compared to that in human Jurkat T cells. Using these approaches, the presence of bovine IKK complexes harbouring IKKalpha, IKKbeta, NEMO and the interaction of IKK with its substrate IkappaBalpha could be demonstrated. Parallel experiments using human Jurkat T cells confirmed the high degree of conservation also at the level of protein-protein interactions. Finally, a yeast two-hybrid analysis showed that bovine NEMO molecules, in addition to the binding to IKKalpha and IKKbeta, also strongly interact with each other.     

Complete reconstitution of human IkappaB kinase (IKK) complex in yeast. Assessment of its stoichiometry and the role of IKKgamma on the complex activity in the absence of stimulation

The IkappaB kinase (IKK) complex, composed of two catalytic subunits (IKKalpha and IKKbeta) and a regulatory subunit (IKKgamma), is the key enzyme in activation of nuclear factor kappaB (NF-kappaB). To study the mechanism and structure of the complex, we wanted to recombinantly express IKK in a model organism that lacks IKK. For this purpose, we have recombinantly reconstituted all three subunits together in yeast and have found that it is biochemically similar to IKK isolated from human cells. We show that there is one regulatory subunit per kinase subunit. Thus, the core subunit composition of IKKalpha.beta.gamma complex is alpha(1)beta(1)gamma(2), and the core subunit composition of IKKbeta.gamma is beta(2)gamma(2). The activity of the IKK complex (alpha+beta+gamma or beta+gamma) expressed in yeast (which lack NF-kappaB and IKK) is 4-5-fold higher than an equivalent amount of IKK from nonstimulated HeLa cells. In the absence of IKKgamma, IKKbeta shows a level of activity similar to that of IKK from nonstimulated HeLa cells. Thus, IKKgamma activates IKK complex in the absence of upstream stimuli. Deleting the gamma binding domain of IKKbeta or IKKalpha prevented IKKgamma induced activation of IKK complex in yeast, but it did not prevent the incorporation of IKKgamma into IKK and large complex formation. The possibility of IKK complex being under negative control in mammalian cells is discussed.     

Detection of IKKbeta-IKKgamma subcomplexes in monocytic cells and characterization of associated signaling

The IkappaB kinase (IKK) complex is one major step in the regulation of the NF-kappaB/Rel system that is involved in inflammatory and immune responses as well as in proliferation and apoptosis. At present it is not clear whether besides the "classical" IKKalpha-IKKbeta-IKKgamma configuration additional complexes exist in vivo that solely contain IKKbeta and IKKgamma (without IKKalpha). In the current study we were able to demonstrate in monocytic cells that endogenous complexes, which only include IKKbeta as the kinase-active molecule do indeed exist in vivo and that these complexes contain IKKgamma as an additional component. Furthermore, we showed that these IKKbeta-IKKgamma complexes are involved in mainstream NF-kappaB activation cascades because they can be activated by tumor necrosis factor. In contrast, these subcomplexes appear not to participate in NIK-dependent pathways. As a next step we showed that exogenous IKKbeta-IKKgamma complexes can be formed in an intact cell by overexpression and that these artificial complexes fulfill the requirement for participation in regular signaling. Finally, in the absence of IKKalpha we found a retarded proteolysis of IkappaBalpha, but not of IkappaB in, which is associated with a reduced IKK activity. Differential pathways represented by various IKK subcomplexes may open attractive possibilities in treatment of inflammation or cancer allowing specific therapeutic intervention.     

Regulation of I(kappa)B kinase complex by phosphorylation of (gamma)-binding domain of I(kappa)B kinase (beta) by Polo-like kinase 1

IkappaB kinase (IKK) complex is a key regulator of NF-kappaB pathways. Signal-induced interaction of the IKKgamma (NEMO) subunit with the C-terminal IKKgamma/NEMO-binding domain (gammaBD) of IKKbeta is an essential interaction for IKK regulation. Underlying regulatory mechanism(s) of this interaction are not known. Phosphorylation of gammaBD has been suggested to play a regulatory role for IKK activation. However, a kinase that phosphorylates gammaBD has not been identified. In this study, we used a C-terminal fragment of IKKbeta as substrate and purified Polo-like kinase 1 (Plk1) from HeLa cell extracts by standard chromatography as a gammaBD kinase. Plk1 phosphorylates serines 733, 740, and 750 in the gammaBD of IKKbeta in vitro. Phosphorylating gammaBD with Plk1 decreased its affinity for IKKgamma in pulldown assay. We generated phosphoantibodies against serine 740 and showed that gammaBD is phosphorylated in vivo. Expressing a constitutively active Plk1 in mammalian cells reduced tumor necrosis factor (TNF)-induced IKK activation, resulting in decreased phosphorylation of endogenous IkappaBalpha and reduced NF-kappaB activation. To activate endogenous Plk1, cells were treated with nocodazole, which reduced TNF-induced IKK activation, and increased the phosphorylation of gammaBD. Knocking down Plk1 in mammalian cells restored TNF-induced IKK activation in nocodazole-treated cells. Activation of Plk1 inhibited TNF-induced expression of cyclin D1. In cells in which Plk1 was knocked down, TNFalpha increased expression of cyclin D1 and the proportion of cells in the S phase of the cell cycle. Taken together, this study shows that phosphorylation regulates the interaction of gammaBD of IKKbeta with IKKgamma and therefore plays a critical role for IKK activation. Moreover, we identify Plk1 as a gammaBD kinase, which negatively regulates TNF-induced IKK activation and cyclin D1 expression, thereby affecting cell cycle regulation. Untimely activation of cyclin D1 by TNFalpha can provide a potential mechanism for an involvement of TNFalpha in inflammation-induced cancer.     

vCLAP, a caspase-recruitment domain-containing protein of equine Herpesvirus-2, persistently activates the Ikappa B kinases through oligomerization of IKKgamma

vCLAP, the E10 gene product of equine herpesvirus-2, is a caspase-recruitment domain (CARD)-containing protein that has been shown to induce both apoptosis and NF-kappaB activation in mammalian cells. vCLAP has a cellular counterpart, Bcl10/cCLAP, which is also an activator of apoptosis and NF-kappaB. Recent studies demonstrated that vCLAP activates NF-kappaB through an IkappaB kinase (IKK)-dependent pathway, but the underlying mechanism remains unknown. In this report, we demonstrate that vCLAP associates stably with the IKK complex through direct binding to the C-terminal region of IKKgamma. Consistent with this finding, IKKgamma was found to be essential for vCLAP-induced NF-kappaB activation, and the association between vCLAP and the IKK complex induced persistent activation of the IKKs. Moreover, enforced oligomerization of the isolated C-terminal region of vCLAP, which interacts with IKKgamma, can trigger NF-kappaB activation. Finally, substitution of the C-terminal region of IKKgamma, which interacts with vCLAP, with the CARD of vCLAP or Bcl10 produced a molecule that was able to activate NF-kappaB when ectopically expressed in IKKgamma-deficient cells. These data suggest that vCLAP-induced oligomerization of IKKgamma, which is mediated by the CARD of vCLAP, could be the mechanism by which vCLAP induces activation of NF-kappaB.     

MUC1 oncoprotein activates the IkappaB kinase beta complex and constitutive NF-kappaB signalling

Nuclear factor-kappaB (NF-kappaB) is constitutively activated in diverse human malignancies by mechanisms that are not understood. The MUC1 oncoprotein is aberrantly overexpressed by most human carcinomas and, similarly to NF-kappaB, blocks apoptosis and induces transformation. This study demonstrates that overexpression of MUC1 in human carcinoma cells is associated with constitutive activation of NF-kappaB p65. We show that MUC1 interacts with the high-molecular-weight IkappaB kinase (IKK) complex in vivo and that the MUC1 cytoplasmic domain binds directly to IKKbeta and IKKgamma. Interaction of MUC1 with both IKKbeta and IKKgamma is necessary for IKKbeta activation, resulting in phosphorylation and degradation of IkappaBalpha. Studies in non-malignant epithelial cells show that MUC1 is recruited to the TNF-R1 complex and interacts with IKKbeta-IKKgamma in response to TNFalpha stimulation. TNFalpha-induced recruitment of MUC1 is dependent on TRADD and TRAF2, but not the death-domain kinase RIP1. In addition, MUC1-mediated activation of IKKbeta is dependent on TAK1 and TAB2. These findings indicate that MUC1 is important for physiological activation of IKKbeta and that overexpression of MUC1, as found in human cancers, confers sustained induction of the IKKbeta-NF-kappaB p65 pathway.     

ABIN-1 binds to NEMO/IKKgamma and co-operates with A20 in inhibiting NF-kappaB

Nuclear factor kappaB (NF-kappaB) plays a pivotal role in inflammation, immunity, stress responses, and protection from apoptosis. Canonical activation of NF-kappaB is dependent on the phosphorylation of the inhibitory subunit IkappaBalpha that is mediated by a multimeric, high molecular weight complex, called IkappaB kinase (IKK) complex. This is composed of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, NEMO/IKKgamma. The latter protein is essential for the activation of IKKs and NF-kappaB, but its mechanism of action is not well understood. Here we identified ABIN-1 (A20 binding inhibitor of NF-kappaB) as a NEMO/IKKgamma-interacting protein. ABIN-1 has been previously identified as an A20-binding protein and it has been proposed to mediate the NF-kappaB inhibiting effects of A20. We find that both ABIN-1 and A20 inhibit NF-kappaB at the level of the IKK complex and that A20 inhibits activation of NF-kappaB by de-ubiquitination of NEMO/IKKgamma. Importantly, small interfering RNA targeting ABIN-1 abrogates A20-dependent de-ubiquitination of NEMO/IKKgamma and RNA interference of A20 impairs the ability of ABIN-1 to inhibit NF-kappaB activation. Altogether our data indicate that ABIN-1 physically links A20 to NEMO/IKKgamma and facilitates A20-mediated de-ubiquitination of NEMO/IKKgamma, thus resulting in inhibition of NF-kappaB.     

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.