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.     

Identification of a novel 300-kDa factor termed IkappaB alphaE3-F1 that is required for ubiquitinylation of IkappaB alpha

Intracellular superoxide (O(2)*- was manipulated in M14 melanoma cells by overexpression or repression of Cu/Zn SOD using a tetracycline-inducible expression system. Scavenging intracellular O(2)*- increased tumor cell sensitivity to daunorubicin, etoposide, and pMC540, whereas expression of the antisense SOD mRNA significantly decreased cell sensitivity to drug treatment. Whereas Cu/Zn SOD overexpressing cells exhibited higher activation of the executioner caspase 3 upon drug exposure, caspase 3 activation was significantly lower when Cu/Zn SOD was repressed by antisense expression. These data show that intracellular O(2)*- regulates tumor cell response to drug-induced cell death via a direct or indirect effect on the caspase activation pathway.     

IkappaB kinases: key regulators of the NF-kappaB pathway

The nuclear factor (NF)-kappaB pathway is important for the expression of a wide variety of genes that are involved in the control of the host immune and inflammatory response, and in the regulation of cellular proliferation and survival. The constitutive activation of this pathway is associated with inflammatory and autoimmune diseases, such as asthma, rheumatoid arthritis and inflammatory bowel disease, in addition to atherosclerosis, Alzheimer's disease, cancer and diabetes. One of the key steps in activating the NF-kappaB pathway is the stimulation of the IkappaB (inhibitor of kappaB) kinases. Recent data indicate that these kinases activate the NF-kappaB pathway through distinct steps that are operative in both the cytoplasm and the nucleus. A better understanding of the mechanisms that activate this pathway provides the potential for defining new therapeutic targets that might prevent the aberrant activation of NF-kappaB in a variety of human diseases.     

Binding of manumycin A inhibits IkappaB kinase beta activity

IkappaB kinase (IKK) catalytic subunits play a key role in cytokinemediated nuclear factor (NF)-kappaB signaling, and a loss of NF-kappaB function appears to inhibit inflammation and oncogenesis. Manumycin A is a potent and selective farnesyltransferase inhibitor with antitumor activity. We found that manumycin A caused a rapid and potent inhibition of IKK activity induced by tumor necrosis factor alpha in a number of cell types. Most unexpectedly, other classes of farnesyltransferase inhibitors had no inhibitory effect. To identify the molecular mechanisms of manumycin A action, cultured human HepG2 hepatoma cells were transiently transfected with various IKKalpha and IKKbeta constructs, and a striking difference in manumycin A sensitivity was observed. Furthermore, cells expressing wild-type IKKbeta and IKKbeta mutated in the activation loop at Cys-179 exhibited covalent homotypic dimerization of IKKbeta in response to manumycin A, whereas substitution of Cys-662 and -716 conferred protection against dimer formation. Direct inhibition of IKK activity and formation of stable IKKbeta dimers were observed in the presence of manumycin A that could be blocked by dithiothreitol. IKK interaction with the adaptor protein IKKgamma/NEMO was disrupted in manumycin A-treated cells. Most importantly, administration of manumycin A to mice xenografted with murine B16F10 tumors caused potent IKK-suppressive effects. Thus, manumycin A with its epoxyquinoid moieties plays an important regulatory function in IKK signaling through pathways distinct from its role as a protein farnesylation inhibitor.     

A fourth IkappaB protein within the NF-kappaB signaling module

Inflammatory NF-kappaB/RelA activation is mediated by the three canonical inhibitors, IkappaBalpha, -beta, and -varepsilon. We report here the characterization of a fourth inhibitor, nfkappab2/p100, that forms two distinct inhibitory complexes with RelA, one of which mediates developmental NF-kappaB activation. Our genetic evidence confirms that p100 is required and sufficient as a fourth IkappaB protein for noncanonical NF-kappaB signaling downstream of NIK and IKK1. We develop a mathematical model of the four-IkappaB-containing NF-kappaB signaling module to account for NF-kappaB/RelA:p50 activation in response to inflammatory and developmental stimuli and find signaling crosstalk between them that determines gene-expression programs. Further combined computational and experimental studies reveal that mutant cells with altered balances between canonical and noncanonical IkappaB proteins may exhibit inappropriate inflammatory gene expression in response to developmental signals. Our results have important implications for physiological and pathological scenarios in which inflammatory and developmental signals converge.     

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.     

Proteolytic signaling by TNFalpha: caspase activation and IkappaB degradation

Following binding its death receptor on the plasma membrane, tumor necrosis factor (TNF) induces the receptor trimerization and recruits a number of death domain-containing molecules to form the receptor complex. The complex promotes activation of downstream caspase cascade and induces degradation of IkappaBalpha. Caspases are activated using mechanisms of oligomeration and 'self-controlled proteolysis'. According to their structures and functions, apoptosis related caspases can be divided into upstream and downstream caspases. In general, upstream caspases cleave and activate downstream caspases by proteolysis of the Asp-X site. Activated caspases then cleaved target substrates. To date, more than 70 proteins have been identified to be substrates of caspases in mammalian cells. Caspases can alter the function of their target proteins by destroying structural components of the cytoskeleton and nuclear scaffold or by removing their regulatory domains. Activation of NF-kappaB is dependent on the degradation of IkappaBalpha. IkappaB kinase (IKK) phosphorylates IkappaBalpha at the residues 32 and 36 followed by polyubiquitination at lysine 21 and 22 and subsequent degradation of the molecules by 26S proteasome. There is extensive crosstalk between the apoptotic and NF-kappaB signaling pathways that emanate from TNF-R1. On the one hand, activation of NF-kappaB can inactivate caspases; on the other hand, activated caspases can inhibit the activation of NF-kappaB. Both processes involve in proteolysis. This crosstalk may be important for maintaining the balance between the two pathways and for determining whether a cell should live or die.     

IKKepsilon is part of a novel PMA-inducible IkappaB kinase complex

Here we report the identification of a novel PMA-inducible IkappaB kinase complex, distinct from the well-characterized high-molecular weight IkappaB kinase complex containing IKKalpha, IKKbeta, and IKKgamma. We have characterized one kinase from this complex, which we designate IKKepsilon. Although recombinant IKKepsilon directly phosphorylates only serine 36 of IKBalpha, the PMA-activated endogenous IKKepsilon complex phosphorylates both critical serine residues. Remarkably, this activity is due to the presence of a distinct kinase in this complex. A dominant-negative mutant of IKKepsilon blocks induction of NF-kappaB by both PMA and activation of the T cell receptor but has no effect on the activation of NF-KB by TNFalpha or IL-1. These observations indicate that the activation of NF-kappaB requires multiple distinct IkappaB kinase complexes, which respond to both overlapping and discrete signaling pathways.     

Phosphorylation and stabilization of TAp63gamma by IkappaB kinase-beta

Post-translational modification of the p53 family members is key to their regulation. Here we report the phosphorylation of TAp63gamma, but not DeltaNp63gamma, by IkappaB kinase beta (IKKbeta). Activation of IKKbeta by gamma radiation or tumor necrosis factor-alpha led to increased TAp63gamma protein levels in cells. IKKbeta, but not its kinase-defective mutant IKKbeta-K44A, led to this observed stabilization of TAp63gamma. This stabilization of TAp63gamma in response to gamma radiation was significantly decreased in the absence of IKKbeta. Phosphorylation of TAp63gamma blocks ubiquitylation and possible degradation of this protein. We postulate that phosphorylation of TAp63gamma by IKKbeta stabilizes the TAp63gamma protein by blocking ubiquitylation-dependent degradation of this protein.