Cobrotoxin inhibits NF-kappa B activation and target gene expression through reaction with NF-kappa B signal molecules

Cobrotoxin is known to bind with cysteine residues of biological molecules such as nicotine acetylcholine receptor. Cobrotoxin may modify IKKs and p50 through protein-protein interaction since cysteine residues are present in the kinase domains of IKKalpha and IKKbeta and in the p50 of NF-kappaB. Our surface plasmon resonance analysis showed that cobrotoxin directly binds to p50 (K(d) = 1.54 x 10(-)(5) M), IKKalpha (K(d) = 3.94 x 10(-)(9) M) and IKKbeta (K(d) = 3.4 x 10(-)(8) M) with high binding affinity. Moreover, these protein-protein interactions suppressed the lipopolysaccharide (LPS, 1 microg/mL)- and the sodium nitroprusside (SNP, 200 microM)-induced DNA binding activity of NF-kappaB and NF-kappaB-dependent luciferase activity in astrocytes and Raw 264.7 macrophages. These inhibitory effects were correlated with the inhibition of IkappaB release and p50 translocation. Inhibition of NF-kappaB by cobrotoxin resulted in reductions in the LPS-induced expressions of COX-2, iNOS, cPLA(2), IL-4, and TNF-alpha in astrocytes and in COX-2 expression induced by SNP, LPS, and TNF-alpha in astrocytes. Moreover, these inhibitory effects of cobrotoxin were reversed by adding reducing agents, dithiothreitol and glutathione. In addition, cobrotoxin did not have any inhibitory effect on NF-kappaB activity in cells carrying mutant p50 (C62S), IKKalpha (C178A), and IKKbeta (C179A), with the exception of IKKbeta (K44A) mutant plasmid. Confocal microscopic analysis showed that cobrotoxin is uptaken into the nucleus of cells. These results demonstrate that cobrotoxin directly binds to the sulfhydryl groups of p50 and IKKs, and that this results in reduced IkappaB release and the translocation of p50, thereby inhibiting the activation of NF-kappaB.     

Interleukin-1-induced NF-kappaB activation is NEMO-dependent but does not require IKKbeta

Activation of NF-kappaB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IkappaB kinase (IKK) complex, which contains two kinases named IKKalpha and IKKbeta and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKbeta is required for TNF-induced NF-kappaB activation. To determine whether NEMO and IKKalpha can form a functional IKK complex capable of activating the classical NF-kappaB pathway in the absence of IKKbeta, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IkappaBalpha degradation absolutely requires NEMO and IKKbeta. In contrast, we consistently observed intact IkappaBalpha degradation and NF-kappaB activation in response to IL-1 in two separate cell lines lacking IKKbeta. Furthermore, exogenously expressed, catalytically inactive IKKbeta blocked TNF- but not IL-1-induced IkappaBalpha degradation in wild-type MEFs, and reconstitution of IKKalpha/beta double knockout cells with IKKalpha rescued IL-1- but not TNF-induced NF-kappaB activation. Finally, we have shown that incubation of IKKbeta-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-kappaB activation. Our results therefore demonstrate that NEMO and IKKalpha can form a functional IKK complex that activates the classical NF-kappaB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways.     

Activation of IkappaB kinase beta by protein kinase C isoforms

The atypical protein kinase C (PKC) isotypes (lambda/iotaPKC and zetaPKC) have been shown to be critically involved in important cell functions such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-alpha) and are required for the activation of NF-kappaB by this cytokine through a mechanism that most probably involves the phosphorylation of IkappaB. The inability of these PKC isotypes to directly phosphorylate IkappaB led to the hypothesis that zetaPKC may use a putative IkappaB kinase to functionally inactivate IkappaB. Recently several groups have molecularly characterized and cloned two IkappaB kinases (IKKalpha and IKKbeta) which phosphorylate the residues in the IkappaB molecule that serve to target it for ubiquitination and degradation. In this study we have addressed the possibility that different PKCs may control NF-kappaB through the activation of the IKKs. We report here that alphaPKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of zetaPKC positively modulates IKKbeta activity but not that of IKKalpha, whereas the transfection of a zetaPKC dominant negative mutant severely impairs the activation of IKKbeta but not IKKalpha in TNF-alpha-stimulated cells. We also show that cell stimulation with phorbol 12-myristate 13-acetate activates IKKbeta, which is entirely dependent on the activity of alphaPKC but not that of the atypical isoforms. In contrast, the inhibition of alphaPKC does not affect the activation of IKKbeta by TNF-alpha. Interestingly, recombinant active zetaPKC and alphaPKC are able to stimulate in vitro the activity of IKKbeta but not that of IKKalpha. In addition, evidence is presented here that recombinant zetaPKC directly phosphorylates IKKbeta in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-kappaB pathway at the level of IKKbeta activation and IkappaB degradation.     

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.     

SIMPL is a tumor necrosis factor-specific regulator of nuclear factor-kappaB activity

The IL-1 receptor-associated kinase (IRAK/mPLK) is linked to the regulation of nuclear factor-kappaB (NF-kappaB)-dependent gene expression. Here we describe a novel binding partner of IRAK/mPLK that we term SIMPL (signaling molecule that associates with the mouse pelle-like kinase). Overexpression of SIMPL leads to the activation of NF-kappaB-dependent promoters, and inactivation of SIMPL inhibits IRAK/mPLK as well as tumor necrosis factor receptor type I-induced NF-kappaB activity. Dominant inhibitory alleles of IkappaB kinase (IKKalpha or IKKbeta) block the activation of NF-kappaB by IRAK/mPLK and SIMPL. Furthermore, SIMPL binds IRAK/mPLK and the IKKs in vitro and in vivo. In the presence of antisense mRNA to SIMPL, the physical association between IRAK/mPLK and IKKbeta but not IRAK/mPLK and IKKalpha is greatly diminished. Moreover, dominant-negative SIMPL blocks IKKalpha- or IKKbeta-induced NF-kappaB activity. These results lead us to propose a model in which SIMPL functions to regulate NF-kappaB activity by linking IRAK/mPLK to IKKbeta/alpha-containing complexes.     

Role of IKKgamma/nemo in assembly of the Ikappa B kinase complex

IKKgamma/NEMO is a protein that is critical for the assembly of the high molecular weight IkappaB kinase (IKK) complex. To investigate the role of IKKgamma/NEMO in the assembly of the IKK complex, we conducted a series of experiments in which the chromatographic distribution of extracts prepared from cells transiently expressing epitope-tagged IKKgamma/NEMO and the IKKs were examined. When expressed alone following transfection, IKKalpha and IKKbeta were present in low molecular weight complexes migrating between 200 and 400 kDa. However, when coexpressed with IKKgamma/NEMO, both IKKalpha and IKKbeta migrated at approximately 600 kDa which was similar to the previously described IKK complex that is activated by cytokines such as tumor necrosis factor-alpha. When either IKKalpha or IKKbeta was expressed alone with IKKgamma/NEMO, IKKbeta but not IKKalpha migrated in the higher molecular weight IKK complex. Constitutively active or inactive forms of IKKbeta were both incorporated into the high molecular weight IKK complex in the presence of IKKgamma/NEMO. The amino-terminal region of IKKgamma/NEMO, which interacts directly with IKKbeta, was required for formation of the high molecular weight IKK complex and for stimulation of IKKbeta kinase activity. These results suggest that recruitment of the IKKs into a high molecular complex by IKKgamma/NEMO is a crucial step involved in IKK function.     

Gene expression profiling in conjunction with physiological rescues of IKKalpha-null cells with wild type or mutant IKKalpha reveals distinct classes of IKKalpha/NF-kappaB-dependent genes

Cellular responses to stress-like stimuli require the IkappaB kinase (IKK) signalsome (IKKalpha, IKKbeta, and NEMO/IKKgamma) to activate NF-kappaB-dependent genes. IKKbeta and NEMO/IKKgamma are required to release NF-kappaB p65/p50 heterodimers from IkappaBalpha, resulting in their nuclear migration and sequence-specific DNA binding; but IKKalpha was found to be dispensable for this initial phase of canonical NF-kappaB activation. Nevertheless, IKKalpha-/- mouse embryonic fibroblasts (MEFs) fail to express NF-kappaB targets in response to proinflammatory stimuli, uncovering a nuclear role for IKKalpha in NF-kappaB activation. However, it remains unknown whether the global defect in NF-kappaB-dependent gene expression of IKKalpha-/- cells is caused by the absence of IKKalpha kinase activity. We show by gene expression profiling that rescue of near physiological levels of wild type IKKalpha in IKKalpha-/- MEFs globally restores expression of their canonical NF-kappaB target genes. To prove that the kinase activity of IKKalpha was required on a genomic scale, the same physiological rescue was performed with a kinase-dead, ATP binding domain IKKalpha mutant (IKKalpha(K44M)). Remarkably, the IKKalpha(K44M) protein rescued approximately 28% of these genes, albeit in a largely stimulus-independent manner with the notable exception of several genes that also acquired tumor necrosis factor-alpha responsiveness. Thus the IKKalpha-containing signalsome unexpectedly functions in the presence and absence of extracellular signals in both kinase-dependent and -independent modes to differentially modulate the expression of five distinct classes of IKKalpha/NF-kappaB-dependent genes.     

IkappaB kinase alpha regulates subcellular distribution and turnover of cyclin D1 by phosphorylation

IkappaB kinases (IKKs), IKKalpha and IKKbeta, with a regulatory subunit IKKgamma/NEMO constitute a high molecular weight IKK complex that regulates NF-kappaB activity. Although IKKalpha and IKKbeta share structural and biochemical similarities, IKKalpha has been shown to have distinct biological roles. Here we show that IKKalpha plays a critical role in regulating cyclin D1 during the cell cycle. Analysis of IKKalpha-/- mouse embryo fibroblast cells showed that cyclin D1 is overexpressed and localized in the nucleus compared with parental mouse embryo fibroblasts. IKKalpha associates with and phosphorylates cyclin D1. Analysis on cyclin D1 mutants demonstrated that IKKalpha phosphorylates cyclin D1 at Thr286. Reconstitution of IKKalpha in knockout cells leads to nuclear export and increased degradation of cyclin D1. Further, RNAi-mediated knockdown of IKKalpha results in similar changes as observed in IKKalpha-/- cells. These results suggest a novel role of IKKalpha in regulating subcellular localization and proteolysis of cyclin D1 by phosphorylation of cyclin D1 at Thr286, the same residue earlier found to be a target for glycogen synthase kinase-3beta-induced phosphorylation.     

IkappaB kinase alpha (IKKalpha) regulation of IKKbeta kinase activity

Two related kinases, IkappaB kinase alpha (IKKalpha) and IKKbeta, phosphorylate the IkappaB proteins, leading to their degradation and the subsequent activation of gene expression by NF-kappaB. IKKbeta has a much higher level of kinase activity for the IkappaB proteins than does IKKalpha and is more critical than IKKalpha in modulating tumor necrosis factor alpha activation of the NF-kappaB pathway. These results indicate an important role for IKKbeta in activating the NF-kappaB pathway but leave open the question of the role of IKKalpha in regulating this pathway. In the current study, we demonstrate that IKKalpha directly phosphorylates IKKbeta. Moreover, IKKalpha either directly or indirectly enhances IKKbeta kinase activity for IkappaBalpha. Finally, transfection studies to analyze NF-kappaB-directed gene expression suggest that IKKalpha is upstream of IKKbeta in activating the NF-kappaB pathway. These results indicate that IKKalpha, in addition to its previously described ability to phosphorylate IkappaBalpha, can increase the ability of IKKbeta to phosphorylate IkappaBalpha.     

The human T-cell leukemia virus type-1 Tax protein regulates the activity of the IkappaB kinase complex

Two cytokine-inducible kinases, IKKalpha and IKKbeta, are components of a 700-kDa kinase complex that specifically phosphorylates IkappaB. Phosphorylation of IkappaB by IKK leads to its ubiquitination and subsequent degradation, resulting in the nuclear translocation of NF-kappaB. The oncogenic protein Tax, encoded by human T-cell leukemia virus type-1 (HTLV-1), stimulates IKK activity to result in constitutive nuclear levels of NF-kappaB. In an attempt to gain insights into the mechanism by which Tax mediates constitutive activation of the NF-kappaB pathway, we analyzed the chromatographic distribution of IKK proteins using cellular extracts prepared from three T lymphocytes either lacking or containing Tax. IKK kinase activity and the distribution of proteins in the IKK complex were characterized. In extracts prepared from cells containing Tax, the activity of both IKKalpha and IKKbeta present in the 700-kDa IKK complex were increased. Surprisingly, cell lines expressing Tax also contained an additional peak of IKKbeta, but not IKKalpha activity, that migrated at 300 kDa rather than at 700 kDa. We noted that extracts containing Tax had extremely low levels of IkappaBbeta, but not IkappaBalpha, and contained predominantly a truncated form of the MAP3K MEKK1. These results suggest that Tax may target several components of the NF-kappaB pathway leading to constitutive activation of this important regulator of cellular gene expression.     

Caspase inhibition sensitizes inhibitor of NF-kappaB kinase beta-deficient fibroblasts to caspase-independent cell death via the generation of reactive oxygen species

Cells lacking functional NF-kappaB die after ligation of some tumor necrosis factor (TNF) receptor family members through failure to express NF-kappaB-dependent anti-apoptotic genes. NF-kappaB activation requires the IkappaB kinase (IKK) complex containing two catalytic subunits named IKKalpha and IKKbeta that regulate distinct NF-kappaB pathways. IKKbeta is critical for classical signaling that induces pro-inflammatory and anti-apoptotic gene profiles, whereas IKKalpha regulates the non-canonical pathway involved in lymphoid organogenesis and B-cell development. To determine whether IKKalpha and IKKbeta differentially function in rescuing cells from death induced by activators of the classical and non-canonical pathways, we analyzed death after ligation of the TNF and lymphotoxin-beta receptors, respectively. Using murine embryonic fibroblasts (MEFs) lacking each of the IKKs, the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, and dominant negative Fas-associated death domain protein, we found that deletion of these kinases sensitized MEFs to distinct cell death pathways. MEFs lacking IKKalpha were sensitized to death in response to both cytokines that was entirely caspase-dependent, demonstrating that IKKalpha functions in this process. Surprisingly, death of IKKbeta-/- MEFs was not blocked by caspase inhibition, demonstrating that IKKbeta negatively regulates caspase-independent cell death (CICD). CICD was strongly activated by both TNF and lymphotoxin-beta receptor ligation in IKKbeta-/- MEFs and was accompanied by loss of mitochondrial membrane potential and the generation of reactive oxygen species. CICD was inhibited by the anti-oxidant butylated hydroxyanosole and overexpression of Bcl-2, neither of which blocked caspase-dependent apoptosis. Our findings, therefore, demonstrate that both IKKalpha and IKKbeta regulate cytokine-induced apoptosis, and IKKbeta additionally represses reactive oxygen species- and mitochondrial-dependent CICD.     

Acute alcohol exposure exerts anti-inflammatory effects by inhibiting IkappaB kinase activity and p65 phosphorylation in human monocytes

Acute alcohol use is associated with impaired immune responses and decreased proinflammatory cytokine production. Our earlier studies have shown that acute alcohol intake inhibits NF-kappaB DNA binding in an IkappaBalpha-independent manner. We report using human peripheral blood monocytes and Chinese hamster ovary cells transfected with CD14 cells that acute alcohol treatment in vitro exerts NF-kappaB inhibition by disrupting phosphorylation of p65. Immunoprecipitation of p65 and IkappaBalpha revealed that acute alcohol exposure for 1 h decreased NF-kappaB-IkappaBalpha complexes in the cytoplasm. Phosphorylation of p65 at Ser(536) is mediated by IkappaB kinase (IKK)beta and is required for NF-kappaB-dependent cellular responses. We show that acute alcohol treatment decreased LPS-induced IKKalpha and IKKbeta activity resulting in decreased phosphorylation of p65 at Ser(536). Furthermore, nuclear expression of IKKalpha increased after alcohol treatment, which may contribute to inhibition of NF-kappaB. Decreased phosphorylation of nuclear p65 at Ser(276) was likely not due to alcohol-induced inhibition of protein kinase A and mitogen- and stress-activated protein kinase-1 activity. Although decreased IkappaBalpha phosphorylation after acute alcohol treatment was attributable to reduced IKKbeta activity, degradation of IkappaBalpha during alcohol exposure was IKKbeta-independent. Alcohol-induced degradation of IkappaBalpha in the presence of a 26S proteasome inhibitor suggested proteasome-independent IkappaBalpha degradation. Collectively, our studies suggest that acute alcohol exposure modulates IkappaBalpha-independent NF-kappaB activity primarily by affecting phosphorylation of p65. These findings further implicate an important role for IKKbeta in the acute effects of alcohol in immune cells.