Regulation of Ikappa B kinase (IKK)gamma /NEMO function by IKKbeta -mediated phosphorylation

The IkappaB kinase (IKK) complex includes the catalytic components IKKalpha and IKKbeta in addition to the scaffold protein IKKgamma/NEMO. Increases in the activity of the IKK complex result in the phosphorylation and subsequent degradation of IkappaB and the activation of the NF-kappaB pathway. Recent data indicate that the constitutive activation of the NF-kappaB pathway by the human T-cell lymphotrophic virus, type I, Tax protein leads to enhanced phosphorylation of IKKgamma/NEMO by IKKbeta. To address further the significance of IKKbeta-mediated phosphorylation of IKKgamma/NEMO, we determined the sites in IKKgamma/NEMO that were phosphorylated by IKKbeta, and we assayed whether IKKgamma/NEMO phosphorylation was involved in modulating IKKbeta activity. IKKgamma/NEMO is rapidly phosphorylated following treatment of cells with stimuli such as tumor necrosis factor-alpha and interleukin-1 that activate the NF-kappaB pathway. By using both in vitro and in vivo assays, IKKbeta was found to phosphorylate IKKgamma/NEMO predominantly in its carboxyl terminus on serine residue 369 in addition to sites in the central region of this protein. Surprisingly, mutation of these carboxyl-terminal serine residues increased the ability of IKKgamma/NEMO to stimulate IKKbeta kinase activity. These results indicate that the differential phosphorylation of IKKgamma/NEMO by IKKbeta and perhaps other kinases may be important in regulating IKK activity.     

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.     

FAF1 suppresses IkappaB kinase (IKK) activation by disrupting the IKK complex assembly

This study presents a molecular inhibitory mechanism by Fas-associated factor 1 (FAF1) on IkappaB kinase (IKK) activation, where divergent NF-kappaB-activating stimuli converge. FAF1 interacts with IKKbeta in response to proinflammatory stimuli (such as tumor necrosis factor-alpha, interleukin-1beta, and lipopolysaccharide) and suppresses IKK activation. Interaction of the leucine-zipper domain of IKKbeta with FAF1 affected the IKK heterocomplex (IKKalpha/beta) and homocomplex (IKKalpha/alpha, IKKbeta/beta) formations and attenuated IKKgamma recruitment to IKKbeta. Overexpression of FAF1 reduced the level of IKKbeta activity, whereas FAF1 depletion increased the activity. These results indicate that FAF1 inhibits IKK activation and its downstream signaling by interrupting the IKK complex assembly through physical interaction with IKKbeta. Taken together, FAF1 robustly suppresses NF-kappaB activation through the inhibition of IKK activation in combination with previously reported cytoplasmic retention of NF-kappaB p65 (Park, M. Y., Jang, H. D., Lee, S. Y., Lee, K. J., and Kim, E. (2004) J. Biol. Chem. 279, 2544-2549). Such redundant suppression would prevent inadvertent activation of the NF-kappaB pathway.     

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.     

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.     

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.     

Essential role for IkappaB kinase beta in remodeling Carma1-Bcl10-Malt1 complexes upon T cell activation

T cell receptor (TCR) signaling to IkappaB kinase (IKK)/NF-kappaB is controlled by PKCtheta-dependent activation of the Carma1, Bcl10, and Malt1 (CBM) complex. Antigen-induced phosphorylation of Bcl10 has been reported, but its physiological function is unknown. Here we show that the putative downstream kinase IKKbeta is required for initial CBM complex formation. Further, upon engagement of IKKbeta/Malt1/Bcl10 with Carma1, IKKbeta phosphorylates Bcl10 in the C terminus and thereby interferes with Bcl10/Malt1 association and Bcl10-mediated IKKgamma ubiquitination. Mutation of the IKKbeta phosphorylation sites on Bcl10 enhances expression of NF-kappaB target genes IL-2 and TNFalpha after activation of primary T cells. Thus, our data provide evidence that IKKbeta serves a dual role upstream of its classical substrates, the IkappaB proteins. While being essential for triggering initial CBM complex formation, IKKbeta-dependent phosphorylation of Bcl10 exhibits a negative regulatory role in T cell activation.     

An induced proximity model for NF-kappa B activation in the Nod1/RICK and RIP signaling pathways

Nod1 is an Apaf-1-like molecule composed of a caspase-recruitment domain (CARD), nucleotide-binding domain, and leucine-rich repeats that associates with the CARD-containing kinase RICK and activates nuclear factor kappaB (NF-kappaB). We show that self-association of Nod1 mediates proximity of RICK and the interaction of RICK with the gamma subunit of the IkappaB kinase (IKKgamma). Similarly, the RICK-related kinase RIP associated via its intermediate region with IKKgamma. A mutant form of IKKgamma deficient in binding to IKKalpha and IKKbeta inhibited NF-kappaB activation induced by RICK or RIP. Enforced oligomerization of RICK or RIP as well as of IKKgamma, IKKalpha, or IKKbeta was sufficient for induction of NF-kappaB activation. Thus, the proximity of RICK, RIP, and IKK complexes may play an important role for NF-kappaB activation during Nod1 oligomerization or trimerization of the tumor necrosis factor alpha receptor.     

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.     

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.     

IKK/NF-kappaB regulates skeletal myogenesis via a signaling switch to inhibit differentiation and promote mitochondrial biogenesis

Nuclear factor kappaB (NF-kappaB) is involved in multiple skeletal muscle disorders, but how it functions in differentiation remains elusive given that both anti- and promyogenic activities have been described. In this study, we resolve this by showing that myogenesis is controlled by opposing NF-kappaB signaling pathways. We find that myogenesis is enhanced in MyoD-expressing fibroblasts deficient in classical pathway components RelA/p65, inhibitor of kappaB kinase beta (IKKbeta), or IKKgamma. Similar increases occur in myoblasts lacking RelA/p65 or IKKbeta, and muscles from RelA/p65 or IKKbeta mutant mice also contain higher fiber numbers. Moreover, we show that during differentiation, classical NF-kappaB signaling decreases, whereas the induction of alternative members IKKalpha, RelB, and p52 occurs late in myogenesis. Myotube formation does not require alternative signaling, but it is important for myotube maintenance in response to metabolic stress. Furthermore, overexpression or knockdown of IKKalpha regulates mitochondrial content and function, suggesting that alternative signaling stimulates mitochondrial biogenesis. Together, these data reveal a unique IKK/NF-kappaB signaling switch that functions to both inhibit differentiation and promote myotube homeostasis.