Phosphorylation by the protein kinase CK2 promotes calpain-mediated degradation of IkappaBalpha.

Rapid IkappaBalpha turnover has been implicated in the high basal NF-kappaB activity in WEHI 231 B immature IgM(+) B cells. Here we show that treatment of WEHI 231 cells with apigenin, a selective inhibitor of the protein kinase CK2, decreased the rate of IkappaBalpha turnover and nuclear levels of NF-kappaB. Turnover of IkappaBalpha in these cells is mediated in part by the protease calpain. Since both CK2 and calpain target the proline-glutamic acid-serine-threonine (PEST) domain, we investigated the role of CK2 in the degradation of IkappaBalpha by calpain using an in vitro phosphorylation/degradation assay. CK2 phosphorylation enhanced mu-calpain-mediated degradation of wild-type IkappaBalpha, but not of mutant 3CIkappaBalpha, with S283A, T291A, and T299A mutations in phosphorylation sites within the PEST domain. Roles for CK2 and calpain in IkappaBalpha turnover were similarly shown in CH31 immature and CH12 mature IgM(+) B cells, but not in A20 and M12 IgG(+) B cells. These findings demonstrate for the first time that CK2 phosphorylation of serine/threonine residues in the PEST domain promotes calpain-mediated degradation of IkappaBalpha and thereby increases basal NF-kappaB levels in IgM(+) B cells.     

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.     

Transient nuclear factor kappaB (NF-kappaB) activation stimulated by interleukin-1beta may be partly dependent on proteasome activity, but not phosphorylation and ubiquitination of the IkappaBalpha molecule, in C6 glioma cells. Regulation of NF-kappaB linked to chemokine production.

We previously reported that several stresses can induce cytokine-induced neutrophil chemoattractant expression in a nuclear factor kappaB (NF-kappaB)-dependent manner. In this study, we focused further on the regulation of NF-kappaB. The activation of NF-kappaB and the subsequent cytokine-induced neutrophil chemoattractant induction in response to interleukin-1beta (IL-1beta) were inhibited by proteasome inhibitors, MG132 and proteasome inhibitor I. Translocation of NF-kappaB into nuclei occurs by the phosphorylation, multi-ubiquitination, and degradation of IkappaBalpha, a regulatory protein of NF-kappaB. Nascent IkappaBalpha began to degrade 5 min after treatment with IL-1beta and disappeared completely after 15 min. However, IkappaBalpha returned to basal levels after 45-60 min. Interestingly, resynthesized IkappaBalpha was already phosphorylated at Ser-32. These results suggest that 1) the upstream signals are still activated, although the translocation of NF-kappaB peaks at 15 min; and 2) the regulated protein(s) acts downstream of IkappaBalpha phosphorylation. Western blotting showed that the resynthesized and phosphorylated IkappaB molecules were also upward-shifted by multi-ubiquitination in response to IL-1beta treatment. On the other hand, ATP-dependent Leu-Leu-Val-Tyr cleaving activity transiently increased, peaked at 15 min, and then decreased to basal levels at 60 min. Furthermore, the cytosolic fraction that was stimulated by IL-1beta for 15 min, but not for 0 and 60 min, could degrade phosphorylated and multi-ubiquitinated IkappaBalpha. These results indicate that the transient translocation of NF-kappaB in response to IL-1beta may be partly dependent on transient proteasome activation.     

Low intracellular zinc impairs the translocation of activated NF-kappa B to the nuclei in human neuroblastoma IMR-32 cells

In the current work, we studied how variations in extracellular zinc concentrations modulate different steps involved in nuclear factor kappaB (NF-kappaB) activation in human neuroblastoma IMR-32 cells. Cells were incubated in media containing varying concentrations of zinc (1.5, 5, 15, and 50 microm). Within 3 h, the intracellular zinc content was lower in cells exposed to 1.5 and 5 microm, compared with the other groups. Low intracellular zinc concentrations were associated with the activation of NF-kappaB, based on high levels of IkappaBalpha phosphorylation, low IkappaBalpha concentrations, and high NF-kappaB binding activity in total cell fractions. However, the active dimer accumulated in the cytosol, as shown by a low ratio of nuclear/cytosolic NF-kappaB binding activity. This altered nuclear translocation was accompanied by a decreased transactivation of an endogenous NF-kappaB-driven gene (ikba) and of a reporter gene (pNF-kappaB-luc). In cells with low intracellular zinc concentrations, a low rate of in vitro tubulin polymerization was measured compared with the other groups. We conclude that low intracellular zinc concentrations induce tubulin depolymerization, which may be one signal for NF-kappaB activation. However, NF-kappaB nuclear translocation is impaired, which inhibits the transactivation of NF-kappaB-driven genes. This could affect cell survival, and be an important factor in certain zinc deficiency-associated pathologies.     

Interference effect of epigallocatechin-3-gallate on targets of nuclear factor kappaB signal transduction pathways activated by EB virus encoded latent membrane protein 1

AIM: To elucidate the interference effect of epigallocatechin-3-gallate (EGCG) on targets of nuclear factor kappaB (NF-kappaB) signal transduction pathway activated by EB virus encoded latent membrane protein 1 (LMP1) in nasopharyngeal carcinoma (NPC) cell lines. METHODS: The survival rates of CNE1 and CNE-LMP1 cell lines after the EGCG treatment were determined by MTT assay. NF-kappaB activation in CNE1 and CNE-LMP1 cells after EGCG treatment was analyzed by promoter luciferase reporter system. And then nuclear translocation of NF-kappaB (p65) after the EGCG treatment was analyzed by immunofluorescence and western blotting. Meanwhile, the changes of IkappaBalpha phosphorylation were observed after the EGCG treatment. EGFR promoter activity was analyzed by promoter luciferase reporter system and EGFR phosphorylation was observed by western blotting after the EGCG treatment. RESULTS: EGCG inhibited the survival rates of CNE1 and CNE-LMP1 cells and NF-kappaB activation caused by LMP1 in CNE-LMP1 cells. EGCG also suppressed the nuclear translocation of NF-kappaB (p65) and IkappaBalpha phosphorylation. Meanwhile, EGCG inhibited EGFR promoter activity and EGFR phosphorylation. CONCLUSIONS: EGCG inhibited not only the dose-dependent survival rate of NPC cells, but also the dose-dependent activation of NF-kappaB. This inhibition of LMP1-caused NF-kappaB activation was mediated via the phosphorylative degradation of its inhibitory protein IkappaBalpha, and then EGCG inhibited EGFR activity which was a downstream gene from NF-kappaB. This study suggests that interference effect of EGCG on targets of signal transduction pathway plays an important role in the anticancer function.     

IkappaBalpha is essential for maintaining basal c-Jun N-terminal kinase (JNK) activation and regulating JNK-mediated resistance to tumor necrosis factor cytotoxicity in L929 cells.

Early activation of c-Jun N-terminal kinase (JNK) is believed to block apoptosis in response to death signals such as tumor necrosis factor (TNF). Brief exposure of murine L929 fibroblasts to anisomycin for 1 hr to activate JNK resulted in resistance to TNF killing. TNF rapidly induced cytoplasmic shrinkage in control cells, but not in the anisomycin-pretreated L929 cells. However, the induced TNF resistance was suppressed in the L929 cells which were engineered to stably inhibit IkappaBalpha protein expression by antisense mRNA ( approximately 80% reduction in protein expression). No constitutive NF-kappaB nuclear translocation and increased TNF resistance were found in these IkappaBalpha antisense cells. Notably, these cells had a significantly reduced basal level of JNK activation (50-70%), compared to vector control cells. Furthermore, brief exposure of L929 cells to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), resulted in resistance to TNF killing, probably due to preconsumption of caspases by wortmannin. Nonetheless, wortmannin-induced TNF resistance was suppressed in the IkappaBalpha antisense cells. Thus, these observations indicate that IkappaBalpha is essential for maintaining the basal level of JNK activation and regulating the JNK-induced TNF resistance.     

Polyglutamine-expanded ataxin-7 decreases nuclear translocation of NF-kappaB p65 and impairs NF-kappaB activity by inhibiting proteasome activity of cerebellar neurons

Our recent study indicated that polyglutamine-expanded ataxin-7-Q75 induced apoptotic death of cultured cerebellar neurons by downregulating Bcl-x(L) expression and activating mitochondrial apoptotic cascade. Mutant polyglutamine-expanded proteins are believed to impair the proteolytic function of ubiquitin-proteasome system by sequestering components of proteasomes. Proteasome degradation of IkappaBalpha permits nuclear translocation of NF-kappaB and is required for continuous NF-kappaB activity, which supports the survival of cultured cerebellar neurons by inducing Bcl-x(L) expression. Thus, we tested the hypothesis that mutant ataxin-7-Q75 causes proteasome dysfunction and impairs NF-kappaB activity, leading to reduced Bcl-x(L) expression, caspase activation and cerebellar neuronal death. EMSA assays indicate that DNA-binding activity of NF-kappaB was significantly decreased in cerebellar neurons expressing ataxin-7-Q75. Similar to mutant ataxin-7-Q75, NF-kappaB inhibitor APEQ induced cerebellar neuronal death by decreasing Bcl-x(L) expression and activating caspase-9. Mutant ataxin-7-Q75 inhibited the proteolytic activity of proteasomes in cerebellar neurons. Proteasome inhibitor MG132 also caused cerebellar neuronal death by decreasing Bcl-x(L) expression and activating caspase-9. Both ataxin-7-Q75 and MG132 caused the cytosolic accumulation of IkappaBalpha in cerebellar neurons. Mutant ataxin-7-Q75 or MG132 increased the cytosolic level of NF-kappaB p65 and decreased the nuclear NF-kappaB p65 level. Our study provides the evidence that polyglutamine-expanded ataxin-7-Q75 decreases nuclear translocation of NF-kappaB p65 and impairs NF-kappaB activity by inhibiting proteasome activity of cerebellar neurons.