IkappaBalpha Protein Research Articles

Yersinia enterocolitica impairs activation of transcription factor NF-kappaB: involvement in the induction of programmed cell death and in the suppression of the macrophage tumor necrosis factor alpha production.

In this study, we investigated the activity of transcription factor NF-kappaB in macrophages infected with Yersinia enterocolitica. Although triggering initially a weak NF-kappaB signal, Y. enterocolitica inhibited NF-kappaB activation in murine J774A.1 and peritoneal macrophages within 60 to 90 min. Simultaneously, Y. enterocolitica prevented prolonged degradation of the inhibitory proteins IkappaB-alpha and IkappaB-beta observed by treatment with lipopolysaccharide (LPS) or nonvirulent, plasmid-cured yersiniae. Analysis of different Y. enterocolitica mutants revealed a striking correlation between the abilities of these strains to inhibit NF-kappaB and to suppress the tumor necrosis factor alpha (TNF-alpha) production as well as to trigger macrophage apoptosis. When NF-kappaB activation was prevented by the proteasome inhibitor MG-132, nonvirulent yersiniae as well as LPS became able to trigger J774A.1 cell apoptosis and inhibition of the TNF-alpha secretion. Y. enterocolitica also impaired the activity of NF-kappaB in epithelial HeLa cells. Although neither Y. enterocolitica nor TNF-alpha could induce HeLa cell apoptosis alone, TNF-alpha provoked apoptosis when activation of NF-kappaB was inhibited by Yersinia infection or by the proteasome inhibitor MG-132. Together, these data demonstrate that Y. enterocolitica suppresses cellular activation of NF-kappaB, which inhibits TNF-alpha release and triggers apoptosis in macrophages. Our results also suggest that Yersinia infection confers susceptibility to programmed cell death to other cell types, provided that the appropriate death signal is delivered.

Role of Rel/NF-kappaB transcription factors during the outgrowth of the vertebrate limb.

The development of the vertebrate limb serves as an amenable system for studying signaling pathways that lead to tissue patterning and proliferation. Limbs originate as a consequence of a differential growth of cells from the lateral plate mesoderm at specific axial levels. At the tip of the limb primordia the progress zone, a proliferating group of mesenchymal cells, induces the overlying ectoderm to differentiate into a specialized structure termed the apical ectodermal ridge. Subsequent limb outgrowth requires reciprocal signalling between the ridge and the progress zone. The Rel/NF-kappaB family of transcription factors is induced in response to several signals that lead to cell growth, differentiation, inflammatory responses, apoptosis and neoplastic transformation. In unstimulated cells, NF-kappaB is associated in the cytoplasm with an inhibitory protein, I-kappaB. In response to an external signal, I-kappaB is phosphorylated, ubiquitinated and degraded, releasing NF-kappaB to enter the nucleus and activate transcription. Here we show that Rel/NF-kappaB genes are expressed in the progress zone of the developing chick limb bud. When the activity of Rel/NF-kappaB proteins is blocked by infection with viral vectors that produce transdominant-negative I-kappaBalpha proteins, limb outgrowth is arrested. Our results indicate that Rel/NF-kappaB transcription factors play a role in vertebrate limb development.

Activation of p38 mitogen-activated protein kinase by sodium salicylate leads to inhibition of tumor necrosis factor-induced IkappaB alpha phosphorylation and degradation.

Many actions of the proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) on gene expression are mediated by the transcription factor NF-kappaB. Activation of NF-kappaB by TNF and IL-1 is initiated by the phosphorylation of the inhibitory subunit, IkappaB, which targets IkappaB for degradation and leads to the release of active NF-kappaB. The nonsteroidal anti-inflammatory drug sodium salicylate (NaSal) interferes with TNF-induced NF-kappaB activation by inhibiting phosphorylation and subsequent degradation of the IkappaB alpha protein. Recent evidence indicated that NaSal activates the p38 mitogen-activated protein kinase (MAPK), raising the possibility that inhibition of NF-kappaB activation by NaSal is mediated by p38 MAPK. We now show that inhibition of TNF-induced IkappaB alpha phosphorylation and degradation by NaSal is prevented by treatment of cells with SB203580, a highly specific p38 MAPK inhibitor. Both p38 activation and inhibition of TNF-induced IkappaB alpha degradation were seen after only 30 s to 1 min of NaSal treatment. Induction of p38 MAPK activation and inhibition of TNF-induced IkappaB alpha degradation were demonstrated with pharmacologically achievable doses of NaSal. These findings provide evidence for a role of NaSal-induced p38 MAPK activation in the inhibition of TNF signaling and suggest a possible role for the p38 MAPK in the anti-inflammatory actions of salicylates. In addition, these results implicate the p38 MAPK as a possible negative regulator of TNF signaling that leads to NF-kappaB activation.

Serine Phosphorylation-regulated Ubiquitination and Degradation of β-Catenin

Several lines of evidence suggest that accumulation of cytoplasmic beta-catenin transduces an oncogenic signal. We show that beta-catenin is ubiquitinated and degraded by the proteosome and that beta-catenin stability is regulated by a diacylglycerol-independent protein kinase C-like kinase activity, which is required for beta-catenin ubiquitination. We also define a six-amino acid sequence found in both beta-catenin and the NF-kappaB regulatory protein IkappaBalpha, which, upon phosphorylation, targets both proteins for ubiquitination. Mutation of a single serine within the ubiquitination targeting sequence prevents ubiquitination of beta-catenin. Mutations within the ubiquitination targeting sequence of beta-catenin may be oncogenic.

Distinct functional properties of IkappaB alpha and IkappaB beta.

The biological activity of the transcription factor NF-kappaB is controlled mainly by the IkappaB alpha and IkappaB beta proteins, which restrict NF-kappaB to the cytoplasm and inhibit its DNA binding activity. Here, we carried out experiments to determine and compare the mechanisms by which IkappaB alpha and IkappaB beta inhibit NF-kappaB-dependent transcriptional activation. First, we found that in vivo IkappaB alpha is a stronger inhibitor of NF-kappaB than is IkappaB beta. This difference is directly correlated with their abilities to inhibit NF-kappaB binding to DNA in vitro and in vivo. Moreover, IkappaB alpha, but not IkappaB beta, can remove NF-kappaB from functional preinitiation complexes in in vitro transcription experiments. Second, we showed that both IkappaBs function in vivo not only in the cytoplasm but also in the nucleus, where they inhibit NF-kappaB binding to DNA. Third, the inhibitory activity of IkappaB beta, but not that of IkappaB alpha, is facilitated by phosphorylation of the C-terminal PEST sequence by casein kinase II and/or by the interaction of NF-kappaB with high-mobility group protein I (HMG I) on selected promoters. The unphosphorylated form of IkappaB beta forms stable ternary complexes with NF-kappaB on the DNA either in vitro or in vivo. These experiments suggest that IkappaB alpha works as a postinduction repressor of NF-kappaB independently of HMG I, whereas IkappaB beta functions preferentially in promoters regulated by the NF-kappaB/HMG I complexes.

Failure of a Second X-ray Dose to Activate Nuclear Factor κB in Normal Rat Astrocytes

Induced gene expression and subsequent cytokine production have been implicated in the normal tissue injury response to radiotherapy. However, studies of radiation-induced gene expression have used single radiation doses rather than the fractionated exposures typical of the clinical situation. To study the effects of multiple radiation doses on gene expression, we investigated nuclear factor kappaB (NFkappaB) DNA binding activity in primary astrocyte cultures after one and two exposures to x-rays. After a single dose of x-rays (3.8-15 gray (Gy)), NFkappaB binding activity in astrocytes increased in a dose-dependent manner, reaching a maximum by 2-4 h and returning to control levels by 8 h after irradiation. In split-dose experiments, when an interval of 24 h was used between two doses of 7.5 Gy, the second 7.5-Gy exposure failed to induce NFkappaB activation. The period of desensitization induced by the first radiation exposure was dose-dependent, persisting approximately 72 h after 7.5 Gy compared with 24 h after 1.5 Gy. No changes in IkappaBalpha protein levels were detected. However, the presence of a transcription inhibitor prevented the desensitizing effect of the initial irradiation. Irradiation also prevented NFkappaB activation in astrocytes by a subsequent exposure to H2O2, but it had no effect on the activation induced by tumor necrosis factor-alpha. These data indicate that an initial x-ray exposure can desensitize astrocytes to the NFkappaB-activating effects of a subsequent radiation exposure. Furthermore, they suggest that this desensitization depends on gene transcription and may have some specificity for NFkappaB activation mediated by reactive oxygen species.

The Interleukin-1 Receptor-associated Kinase Is Degraded by Proteasomes following Its Phosphorylation

Following interleukin (IL)-1 stimulation, the majority of the cellular interleukin-1 receptor-associated kinase (IRAK) translocates to a discrete subset of the Type I IL-1 receptor (IL-1R1) in MRC-5 human lung fibroblasts. As the IRAK becomes multiphosphorylated, it is degraded by proteasomes at a rate comparable to that of the degradation of the phosphorylated IkappaBalpha protein. Proteasome inhibitors block the degradation of phosphorylated IRAK and correspondingly increase the amount of IL-1R1 that can be coimmunoprecipitated with IRAK. The nonspecific kinase inhibitor K-252b blocks IRAK phosphorylation and degradation, but does not inhibit IRAK association with the IL-1R1 indicating that translocation of IRAK to the IL-1R1 and its phosphorylation are independent events. The IL-1 specificity of these effects is indicated by the lack of IRAK phosphorylation and degradation by IL-1 in the presence of the IL-1 receptor antagonist or by the activation of MRC-5 cells by tumor necrosis factor alpha. Long term exposure of MRC-5 cells to IL-1 desensitizes the resynthesized IkappaBalpha to IL-1, but not to tumor necrosis factor alpha stimulation, but no additional effects on IRAK are seen.

IkappaB alpha is a target for the mitogen-activated 90 kDa ribosomal S6 kinase.

The activity of transcription factor NFkappaB is regulated by its subcellular localization. In most cell types, NFkappaB is sequestered in the cytoplasm due to binding of the inhibitory protein IkappaB alpha. Stimulation of cells with a wide variety of agents results in degradation of IkappaB alpha which allows translocation of NFkappaB to the nucleus. Degradation of IkappaB alpha is triggered by phosphorylation of two serine residues, i.e. Ser32 and Ser36, by as yet unknown kinases. Here we report that the mitogen-activated 90 kDa ribosomal S6 kinase (p90rsk1) is an IkappaB alpha kinase. p90rsk1 phosphorylates IkappaB alpha at Ser32 and it physically associates with IkappaB alpha in vivo. Moreover, when the function of p90rsk1 is impaired by expression of a dominant-negative mutant, IkappaB alpha degradation in response to mitogenic stimuli, e.g. 12-O-tetradecanoylphorbol 13-acetate (TPA), is inhibited. Finally, NFkappaB cannot be activated by TPA in cell lines that have low levels of p90rsk1. We conclude that p90rsk1 is an essential kinase required for phosphorylation and subsequent degradation of IkappaB alpha in response to mitogens.

A threshold nuclear level of the v-Rel oncoprotein is required for transformation of avian lymphocytes.

The net distribution of eukaryotic transcription factors between the cytoplasm and the nucleus provides an effective mechanism for controlling gene expression. We have utilized cis-acting signals for both nuclear import and nuclear export to experimentally manipulate the distribution of the v-Rel oncoprotein between the nucleus and the cytoplasm. The respective abilities of the v-Rel oncoprotein to localize to the nucleus in chicken embryo fibroblasts, to activate kappaB-dependent transcription in yeast, and to transform avian lymphoid cells were each markedly reduced by the fusion of a cis-acting nuclear export signal onto v-Rel. Our results demonstrate that a threshold nuclear function of v-Rel is required for manifestation of its oncogenic properties. In contrast, while increased expression of the avian IkappaB-alpha protein was able to prevent nuclear localization of v-Rel in chicken embryo fibroblasts, coexpression of IkappaB-alpha with v-Rel in the target cell for v-Rel mediated transformation did not reduce the ability of v-Rel to transform avian lymphoid cells or alter the distribution of v-Rel between the nucleus and the cytoplasm in v-Rel-transformed cells. Our results suggest that the ability of IkappaB-alpha to inhibit nuclear localization of v-Rel is affected by cell-type specific differences between fibroblasts and lymphoid cells.

Stress response decreases NF-kappaB nuclear translocation and increases I-kappaBalpha expression in A549 cells.

The stress response and stress proteins confer protection against diverse forms of cellular and tissue injury, including acute lung injury. The stress response can inhibit nonstress protein gene expression, therefore transcriptional inhibition of proinflammatory responses could be a mechanism of protection against acute lung injury. To explore this possibility, we determined the effects of the stress response on nuclear translocation of the transcription factor NF-kappaB, an important regulator of proinflammatory gene expression. In A549 cells induction of the stress response decreased tumor necrosis factor-alpha (TNF-alpha)-mediated NF-kappaB nuclear translocation. TNF-alpha initiates NF-kappaB nuclear translocation by causing dissociation of the inhibitory protein I-kappaBalpha from NF-kappaB and rapid degradation of I-kappaBalpha. Prior induction of the stress response inhibited TNF-alpha-mediated dissociation of I-kappaBalpha from NF-kappaB and subsequent degradation of I-kappaBalpha. Induction of the stress response also increased expression of I-kappaBalpha. We conclude that the stress response affects NFkappaB-mediated gene regulation by two independent mechanisms. The stress response stabilizes I-kappaBalpha and induces expression of I-kappaBalpha. The composite result of these two effects is to decrease NF-kappaB nuclear translocation. We speculate that the protective effect of the stress response against acute lung injury involves a similar effect on the I-kappaB/NF-kappaB pathway.

The Serine/Threonine Phosphatase Inhibitor Calyculin A Induces Rapid Degradation of IκBβ: REQUIREMENT OF BOTH THE N- AND C-TERMINAL SEQUENCES

Signal-initiated activation of the transcription factor NF-kappaB is mediated through proteolysis of its cytoplasmic inhibitory proteins IkappaBalpha and IkappaBbeta. While most NF-kappaB inducers trigger the degradation of IkappaBalpha, only certain stimuli are able to induce the degradation of IkappaBbeta. The degradation of IkappaBalpha is targeted by its site-specific phosphorylations, although the mechanism underlying the degradation of IkappaBbeta remains elusive. In the present study, we have analyzed the effect of phosphatase inhibitors on the proteolysis of IkappaBbeta. We show that the serine/threonine phosphatase inhibitor calyculin A induces the hyperphosphorylation and subsequent degradation of IkappaBbeta in both human Jurkat T cells and the murine 70Z-3 preB cells, which is associated with the nuclear expression of active NF-kappaB. The calyculin A-mediated degradation of IkappaBbeta is further enhanced by the cytokine tumor necrosis factor-alpha (TNF-alpha), although TNF-alpha alone is unable to induce the degradation of IkappaBbeta. Mutational analyses have revealed that the inducible degradation of IkappaBbeta induced by calyculin A, and TNF-alpha requires two N-terminal serines (serines 19 and 23) that are homologous to the inducible phosphorylation sites present in IkappaBalpha. Furthermore, the C-terminal 51 amino acid residues, which are rich in serines and aspartic acids, are also required for the inducible degradation of IkappaBbeta. These results suggest that the degradation signal of IkappaBbeta may be controlled by the opposing actions of protein kinases and phosphatases and that both the N- and C-terminal sequences of IkappaBbeta are required for the inducible degradation of this NF-kappaB inhibitor.

IκBα Overexpression in Human Breast Carcinoma MCF7 Cells Inhibits Nuclear Factor-κB Activation but Not Tumor Necrosis Factor-α-induced Apoptosis

Nuclear factor-kappaB (NF-kappaB) is one of major component induced by tumor necrosis factor-alpha (TNF), and its role in the signaling of TNF-induced cell death remains controversial. In order to delineate whether the involvement of NF-kappaB activation is required for triggering of the apoptotic signal of TNF, we inhibited the nuclear translocation of this transcription factor in TNF-sensitive MCF7 cells by introducing a human MAD-3 mutant cDNA coding for a mutated IkappaB alpha that is resistant to both phosphorylation and proteolytic degradation and that behaves as a potent dominant negative IkappaB alpha protein. Our results demonstrated that the mutated IkappaB alpha was stably expressed in the transfected MCF7 cells and blocked the TNF-induced NF-kappaB nuclear translocation. Indeed, TNF treatment of these cells induced the proteolysis of only the endogenous IkappaB alpha but not the mutated IkappaB alpha. The nuclear NF-kappaB released from the endogenous IkappaB alpha within 30 min of TNF treatment was rapidly inhibited by the mutated IkappaB alpha. There was no significant difference either in cell viability or in the kinetics of cell death between control cells and the mutated IkappaB alpha transfected cells. Furthermore, electron microscopic analysis showed that the cell death induced by TNF in both control and mutated IkappaB alpha transfected cells was apoptotic. The inhibition of NF-kappaB translocation in mutated IkappaB alpha-transfected cells persisted throughout the same time course that apoptosis was occurring. Our data provide direct evidence that the inhibition of NF-kappaB did not alter TNF-induced apoptosis in MCF7 cells and support the view that TNF-mediated apoptosis is NF-kappaB independent.

Identification of a novel NF-kappaB p50-related protein in B lymphocytes.

In most cell types other than mature B lymphocytes and macrophages, the transcription factor NF-kappaB remains in an inactive form in the cytosol by being bound to the inhibitory proteins IkappaBalpha and IkappaBbeta. To investigate the regulation of constitutively active NF-kappaB in B lymphocytes, we have examined the composition of Rel protein complexes in different mouse B-cell lines. As reported previously, the constitutively active complex in mature B cells was predominantly p50:c-Rel. However, the kappaB binding complex in the plasmacytomas that were examined lacked c-Rel and instead contained only a p50-related protein. This p50-related protein (p55) cross-reacts with three different p50 antisera, exists in both the cytosol and the nucleus, and is the protein that binds to kappaB sites in plasma cells. Transfection of reporter constructs into plasma cells indicates that the p55 complex is also transcriptionally active. The p55 protein can be detected in splenocytes from mice lacking the p105/p50 gene, and therefore it appears to be the product of a distinct gene. The implications of the existence of a NF-kappaB p50-related protein in plasma cells that is capable of binding to kappaB sites and activating transcription are discussed.

Inhibition of NFκB Activity through Maintenance of IκBα Levels Contributes to Dihydrotestosterone-mediated Repression of the Interleukin-6 Promoter

Androgens repress expression of many genes, yet the mechanism of this activity has remained elusive. The cytokine, interleukin-6, is active in a variety of biological systems, and its expression is repressed by androgens. Accordingly we dissected the mechanism of androgen's ability to inhibit interleukin-6 expression at the molecular level. In a series of co-transfection assays, we found that 5alpha-dihydrotestosterone, through the androgen receptor, repressed activation of the interleukin-6 promoter, in part, by inhibiting NFkappaB activity. It did not appear that 5alpha-dihydrotestosterone inhibited NFkappaB by activating the androgen receptor to compete for the NFkappaB response element as we could not detect androgen receptor binding to the IL-6 promoter by DNase I footprinting assay. However, by electrophoretic mobility shift assay we found that 5alpha-dihydrotestosterone repressed formation of NFkappaB middle dotNFkappaB response element complex formation. In LNCaP prostate carcinoma cells, 5alpha-dihydrotestosterone achieved this effect through maintenance of IkappaBalpha protein levels in the face of phorbol ester, a stimulus that results in IkappaBalpha degradation. Finally, we confirmed that IkappaBalpha inhibits NFkappaB-mediated activation of the interleukin-6 promoter. These data suggest that maintenance of IkappaBalpha levels may represent the first identified mechanism for androgen-mediated repression of a natural androgen-regulated gene.

Glucocorticoid-mediated Repression of NFκB Activity in Endothelial Cells Does Not Involve Induction of IκBα Synthesis

Repression of NFkappaB-dependent gene expression is one of the major elements of immunosuppression by glucocorticoids. Protein-protein interactions between the glucocorticoid receptor and NFkappaB have been characterized and shown to be a possible mechanism of mutual inhibition of transactivation properties. More recently, glucocorticoid-mediated induction of IkappaBalpha, an inhibitor of NFkappaB, has been described in monocytes and lymphocytes; an increase in IkappaBalpha mRNA and protein resulted in inactivation and cytosolic retention of NFkappaB. Thus, rather than the physical interaction between the glucocorticoid receptor and NFkappaB, the up-regulation of IkappaBalpha was presented as the key element in immunosuppression by glucocorticoids. In contrast, we show that the IkappaBalpha pathway is not involved in glucocorticoid-mediated inhibition of NFkappaB activity in endothelial cells. Although transcriptional activation by NFkappaB was significantly reduced in the presence of glucocorticoids, we did not detect induction of IkappaBalpha protein that could prevent nuclear translocation of NFkappaB upon stimulation with lipopolysaccharide or tumor necrosis factor alpha. Furthermore, treatment with glucocorticoids did not seem to affect the transcription rate or mRNA stability of IkappaBalpha. We therefore conclude that, although induction of IkappaBalpha expression by glucocorticoids seems to be of importance in monocytes and lymphocytes, it cannot explain inhibition of NFkappaB-dependent gene expression in endothelial cells. Our results emphasize the relevance of physical interaction between the glucocorticoid receptor and NFkappaB in endothelial cells and thus in suppression of inflammation by glucocorticoids.

Chronic human immunodeficiency virus type 1 infection of myeloid cells disrupts the autoregulatory control of the NF-kappaB/Rel pathway via enhanced IkappaBalpha degradation.

Productive human immunodeficiency virus type 1 (HIV-1) infection causes sustained NF-kappaB DNA-binding activity in chronically infected monocytic cells. A direct temporal correlation exists between HIV infection and the appearance of NF-kappaB DNA-binding activity in myelomonoblastic PLB-985 cells. To examine the molecular basis of constitutive NF-kappaB DNA-binding activity in HIV1 -infected cells, we analyzed the phosphorylation and turnover of IkappaBalpha protein, the activity of the double-stranded RNA-dependent protein kinase (PKR) and the intracellular levels of NF-kappaB subunits in the PLB-985 and U937 myeloid cell models. HIV-1 infection resulted in constitutive, low-level expression of type 1 interferon (IFN) at the mRNA level. Constitutive PKR activity was also detected in HIV-1-infected cells as a result of low-level IFN production, since the addition of anti-IFN-alpha/beta antibody to the cells decreased PKR expression. Furthermore, the analysis of IkappaBalpha turnover demonstrated an increased degradation of IkappaBalpha in HIV-1-infected cells that may account for the constitutive DNA binding activity. A dramatic increase in the intracellular levels of NF-kappaB subunits c-Rel and NF-kappaB2 p100 and a moderate increase in NF-kappaB2 p52 and RelA(p65) were detected in HIV-1-infected cells, whereas NF-kappaB1 p105/p50 levels were not altered relative to the levels in uninfected cells. We suggest that HIV-1 infection of myeloid cells induces IFN production and PKR activity, which in turn contribute to enhanced IkappaBalpha phosphorylation and subsequent degradation. Nuclear translocation of NF-kappaB subunits may ultimately increase the intracellular pool of NF-kappaB/IkappaBalpha by an autoregulatory mechanism. Enhanced turnover of IkappaBalpha and the accumulation of NF-kappaB/Rel proteins may contribute to the chronically activated state of HIV-1-infected cells.

A Sustained Reduction in IκB-β May Contribute to Persistent NF-κB Activation in Human Endothelial Cells

The responses of vascular endothelial cells (EC) to tumor necrosis factor-alpha (TNF), interleukin-1alpha (IL-1), and phorbol myristate acetate (PMA) were compared with respect to the kinetics of (i) NF-kappaB activation, (ii) IkappaB-alpha and IkappaB-beta degradation, and (iii) NF-kappaB-dependent cell surface molecule expression. TNF rapidly (</=20 min) and persistently (>20 h) activates NF-kappaB; IL-1 rapidly activates NF-kappaB, but activity declines by 3 h and further by 20 h; PMA slowly and transiently activates NF-kappaB. Untreated EC contain the inhibitory proteins IkappaB-alpha and IkappaB-beta. The onset of NF-kappaB activation correlates with degradation of IkappaB-alpha, but IkappaB-alpha reappears by 4 h without resequestration of NF-kappaB. TNF causes a rapid but partial (50%) reduction in IkappaB-beta, which does not recover by 22 h; IL-1 and PMA cause slower and less sustained reductions in IkappaB-beta. All three agonists induce de novo expression of E-selectin (CD62E) and vascular cell adhesion molecule-1 (CD106) and increase expression of intercellular adhesion molecule-1 (CD54) at 4 h. TNF induces sustained increases in vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and increases human leukocyte antigen class I molecules at 24 h. We conclude that TNF causes persistent activation of NF-kappaB in human EC and that this may result from sustained reductions in IkappaB-beta levels.

MEK Kinase Is Involved in Tumor Necrosis Factor α-Induced NF-κB Activation and Degradation of IκB-α

Signal-dependent activation of the transcription factor NF-kappaB is dominantly regulated by degradation of IkappaB-alpha protein. However, the signaling pathways that lead to the degradation are not clear. Here we report that mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) kinase, an activator of stress-activated protein kinases/jun kinase-1 (SAPKs/JNK1), is involved in such signaling pathways. The transient overexpression of MEK kinase in NIH3T3 fibroblasts activates kappaB-CAT reporter expression in a synergistic manner with TNFalpha stimulation. In contrast, overexpression of kinase-negative MEK kinase suppresses TNFalpha-induced reporter expression. The overexpression of MEK kinase suppresses the inhibitory activity of co-transfected IkappaB-alpha on the kappaB-CAT or human immunodeficiency virus-long terminal repeat-luciferase reporter expression and causes the simultaneous disappearance of the overexpressed IkappaB-alpha. The disappearance of exogenous IkappaB-alpha by the overexpression of MEK kinase is prevented by calpain inhibitor-I, an inhibitor of IkappaB-alpha degradation. These results suggest that MEK kinase is a signal mediator involved in TNFalpha-induced NF-kappaB activation and that the activation of NF-kappaB by MEK kinase is regulated through the degradation of IkappaB-alpha.

PEST-dependent cytoplasmic retention of v-Rel by I(kappa)B-alpha: evidence that I(kappa)B-alpha regulates cellular localization of c-Rel and v-Rel by distinct mechanisms

Association of c-Rel with the inhibitor of kappaB-alpha (IkappaB-alpha) protein regulates both cellular localization and DNA binding. The ability of v-Rel, the oncogenic viral counterpart of avian c-Rel, to evade regulation by p40, the avian IkappaB-alpha protein, contributes to v-Rel-mediated oncogenesis. The yeast two-hybrid system was utilized to dissect Rel:IkappaB-alpha interactions in vivo. We find that distinct domains in c-Rel and v-Rel are required for association with p40. Furthermore, while the ankyrin repeat domain of p40 is sufficient for association with c-Rel, both the ankyrin repeat domain and the PEST domain are required for association with v-Rel. Two amino acid differences between c-Rel and v-Rel that are principally responsible for PEST-dependent association of v-Rel with p40 were identified. These same amino acids were principally responsible for PEST-dependent cytoplasmic retention of v-Rel by p40. The presence of mutations in c-Rel that were sufficient to confer PEST-dependent association of the mutant c-Rel protein with p40 did not increase the weak oncogenicity of c-Rel. However, the introduction of these two c-Rel-derived amino acids into v-Rel markedly reduced the oncogenicity of v-Rel. Deletion of the NLS of either c-Rel or v-Rel did not abolish association with p40, but did confer PEST-dependent association of c-Rel with p40. Surprisingly, deletion of the nuclear localization signal in v-Rel did not affect oncogenicity by v-Rel. Analysis of several mutant c-Rel and v-Rel proteins demonstrated that association of Rel proteins with p40 is necessary but not sufficient for cytoplasmic retention. These results are not consistent with the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by the same mechanism. Rather, these results support the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by distinct mechanisms.

Role of IkBα Ubiquitination in Signal-induced Activation of NF-kB in Vivo

In unstimulated cells, the transcription factor NF-kappaB is held in the cytoplasm in an inactive state by the inhibitor protein IkappaBalpha. Stimulation of cells results in rapid phosphorylation and degradation of IkappaBalpha, thus releasing NF-kappaB, which translocates to the nucleus and activates transcription of responsive genes. Here we demonstrate that in cells where proteasomal degradation is inhibited, signal induction by tumor necrosis factor alpha results in the rapid accumulation of higher molecular weight forms of IkappaBalpha that dissociate from NF-kappaB and are consistent with ubiquitin conjugation. Removal of the high molecular weight forms of IkappaBalpha by a recombinant ubiquitin carboxyl-terminal hydrolase and reactivity of the immunopurified material with a monoclonal antibody specific for ubiquitin indicated that IkappaBalpha was conjugated to multiple copies of ubiquitin. Western blot analysis of immunopurified IkappaBalpha from cells expressing epitope-tagged versions of IkappaBalpha and ubiquitin revealed the presence of multiple copies of covalently bound tagged ubiquitin. An S32A/S36A mutant of IkappaBalpha that is neither phosphorylated nor degraded in response to signal induction fails to undergo inducible ubiquitination in vivo. Thus signal-induced activation of NF-kappaB involves phosphorylation-dependent ubiquitination of IkappaBalpha, which targets the protein for rapid degradation by the proteasome and releases NF-kappaB for translocation to the nucleus.