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Query: UNIPROT:P06889 (Mol)
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Optimal activation of T cells requires at least two signals delivered by the T-cell receptor complex and costimulatory molecules such as CD28. The CD28 signaling participates in the transcription of the interleukin-2 gene through activation of an enhancer termed the CD28-responsive element (CD28RE). Stimulation of CD28 enhances mitogen-mediated induction of CD28RE-binding proteins including members of the NF-kappaB/Rel transcription factor family, although the underlying mechanism remains elusive. In this report, we show that CD28 costimulation leads to biphasic induction of NF-kappaB/Rel heterodimers, including early-phase induction of p50/RelA and c-Rel/RelA and late-phase induction of p50/c-Rel. Interestingly, activation of these NF-kappaB/Rel complexes by the CD28 signal is associated with the rapid degradation of both IkappaBalpha and IkappaBbeta, two major cytoplasmic inhibitors of NF-kappaB/Rel. Although IkappaBalpha degradation can be induced by phorbol ester alone, degradation of IkappaBbeta is largely dependent on the CD28 costimulatory signal. We further demonstrate that CD28-mediated transactivation of the CD28RE enhancer is potently inhibited by an N-terminal truncation mutant of IkappaBbeta that is incapable of responding to the degradation signals. Together, these results suggest that the CD28 costimulatory signal augments activation of NF-kappaB/Rel by promoting degradation of IkappaBbeta as well as enhancing degradation of IkappaBalpha and that induction of NF-kappaB/Rel serves as an essential step in the signal-mediated activation of the CD28RE enhancer.
Mol Cell Biol 1996 Dec
PMID:CD28 mediates a potent costimulatory signal for rapid degradation of IkappaBbeta which is associated with accelerated activation of various NF-kappaB/Rel heterodimers. 894 28

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.
Mol Cell Biol 1996 Dec
PMID:Identification of a novel NF-kappaB p50-related protein in B lymphocytes. 894 64

The accumulation of lipofuscin to cardiomyocytes is a classical parameter of aging and is believed to reflect oxidative stress. NF-kB transcription factor complex is one of the cellular sensors which responds to oxidative stress and regulates gene expression. Our purpose was to study whether aging affects the level and distribution of DNA binding activities of NF-kB transcription factors both in cardiac sarcoplasm and nuclear extracts. We used electrophoretic mobility shift assays (EMSA) to characterize the DNA binding activities of NF-kB and two other transcription factors. AP-1 and Sp-1, in the myocardium of 4 months and 24 months old male and female NMRI-mice. The protein levels of p50, p52, and p65 components of NF-kB-complex and an inhibitory IkB-alpha/MAD-3 were assayed with Western blots. Surprisingly, aging upregulated by 123% the nuclear NF-kB binding activity in the male and female mice. The sarcoplasmic NF-kB activity, activated by deoxycholate, did not show any change during aging. Aging-induced increase in nuclear NF-kB protein-DNA binding activity was observed both by gel retardation and UV-crosslinking assays. In immunoblotting, the level of p52 component but not those of p50 and p65 components of NF-kB-complex was slightly increased in nuclear fractions. Aging did not affect the sarcoplasmic levels of p50, p52, and p65 proteins. Supershift EMSA assays showed that the nuclear NF-kB complex contained p50, p52, and p65 components. The level of inhibitory IkB-alpha/MAD-3 protein was unaffected by aging both in nuclear and sarcoplasmic fractions. Aging down-regulated the nuclear Sp-1 binding activities but did not affect AP-1 binding activities. Statistically significant sex-related differences did not appear in the aging responses of transcription factors. These results indicate that NF-kB transcription factor pathway is activated during aging in cardiac muscle and could be the signaling route regulating gene expression. However, the activation mechanism of NF-kB during aging whether oxidative stress responsive or not in vivo needs further studies.
J Mol Cell Cardiol 1996 Mar
PMID:Aging-induced up-regulation of nuclear binding activities of oxidative stress responsive NF-kB transcription factor in mouse cardiac muscle. 901 32

Numerous genes required during the immune or inflammation response as well as the adhesion process are regulated by nuclear factor kappaB (NF-kappaB). Associated with its inhibitor, I kappaB, NF-kappaB resides as an inactive form in the cytoplasm. Upon stimulation by various agents, I kappaB is proteolyzed and NF-kappaB translocates to the nucleus, where it activates its target genes. The transduction pathways that lead to I kappaB inactivation remain poorly understood. In this study, we have characterized a cellular mutant, the 70/Z3-derived 1.3E2 murine pre-B cell line, that does not activate NF-kappaB in response to several stimuli. We demonstrate that upon stimulation by lipopolysaccharide, Taxol, phorbol myristate acetate, interleukin-1, or double-stranded RNA, I kappaB alpha is not degraded, as a result of an absence of induced phosphorylation on serines 32 and 36. Neither a mutation in I kappaB alpha nor a mutation in p50 or relA, the two major subunits of NF-kappaB in this cell line, accounts for this phosphorylation defect. As well as culminating in the inducible phosphorylation of I kappaB alpha on serines 32 and 36, all the stimuli that are inactive on 1.3E2 cells exhibit a sensitivity to the antioxidant pyrrolidine dithiocarbamate (PDTC). In contrast, stimuli such as hyperosmotic shock or phosphatase inhibitors, which use PDTC-insensitive pathways, induce I kappaB alpha degradation in 1.3E2. Analysis of the redox status of 1.3E2 does not reveal any difference from wild-type 70Z/3. We also report that the human T-cell leukemia virus type 1 (HTLV-1)-derived Tax trans-activator induces NF-kappaB activity in 1.3E2, suggesting that this viral protein does not operate via the defective pathway. Finally, we show that two other I kappaB molecules, I kappaB beta and the recently identified I kappaB epsilon, are not degraded in the 1.3E2 cell line following stimulation. Our results demonstrate that 1.3E2 is a cellular transduction mutant exhibiting a defect in a step that is required by several different stimuli to activate NF-kappaB. In addition, this analysis suggests a common step in the signaling pathways that trigger I kappaB alpha, I kappaB beta, and I kappaB epsilon degradation.
Mol Cell Biol 1997 Mar
PMID:Characterization of a mutant cell line that does not activate NF-kappaB in response to multiple stimuli. 903 71

The antitumor antibiotic mitomycin C is activated by several bioreductive enzymes, including DT-diaphorase. In HT29 cells, mitomycin C treatment results in the induction of DT-diaphorase as reflected in elevated steady state DT-diaphorase mRNA levels. An increase in the transcriptional rate was demonstrated by nuclear run-on assay. To investigate the molecular basis of the change in transcriptional activity caused by mitomycin C treatment, electrophoretic mobility shift assays were used to demonstrate the induction of nuclear factor binding to elements in the 5' flanking region of the DT-diaphorase gene. Treatment of HT29 cells with mitomycin C resulted in the dose-dependent induction of binding activity directed to the activator protein-1 (AP-1) binding element with a time course similar to that of mRNA elevation. Supershift assays using specific antibodies to Jun and Fos demonstrated the participation of both proteins in the binding activities generated. A binding activity for the nuclear factor-kappaB (NF-kappaB) site was induced with a similar time course. Both competitor and supershift experiments indicated that a heterodimer of the NF-kappaB proteins p50 and p65 was contained in the bound complex. To further investigate the functional consequences of such binding, we transfected HT29 cells with a plasmid containing 3 kb of the DT-diaphorase 5' region upstream of a reporter gene, chloramphenicol acetyltransferase. Treatment with mitomycin C resulted in a 5.5-fold increase in the expression of a chloramphenicol acetyltransferase construct containing 3 kb of DT-diaphorase promoter sequence. Using a series of deletion mutations based on this full-length construct, we found that two regions of the DT-diaphorase promoter region, positions -346 to -588 (containing the AP-1 element) and positions -785 to -890 (containing the NF-kappaB element) are required for the full expression of the mitomycin C response. The specific involvement of these binding elements was confirmed using mutational analysis. The results demonstrate that mutation of either element alone or of both diminishes the response, indicating an additive interaction between the elements at a minimum. However, inducibility characterizes a promoter fragment as small as 78 base-pairs from the transcription start site. Treatment of cells with mitomycin C induced binding to a 38-base-pair region (-40 to -78) devoid of known transcription factor binding elements. These data suggest that mitomycin C induces the overexpression of DT-diaphorase through a mechanism involving both the AP-1 and NF-kappaB response elements and that inducibility depends on a novel factor binding element.
Mol Pharmacol 1997 Mar
PMID:Involvement of activator protein-1 and nuclear factor-kappaB transcription factors in the control of the DT-diaphorase expression induced by mitomycin C treatment. 905 97

Multiple endocrine neoplasia type 1 (MEN 1) is inherited as an autosomal dominant disorder, characterized by neoplasia and hyperplasia in specific endocrine organs. The MEN 1 gene, which is most probably a tumor suppressor gene, has been localized to a region of approximately 900 kb on chromosome 11q13. The nuclear factor-kappa B (NF-kappa B) is a transcription factor with pleiotropic expression, which is involved in the regulation of expression of many cellular genes. The p50/p65 heterodimer is the most abundant form of NF-kappa B. The gene encoding the p65 subunit (NF-kappa B3/REL A) was recently localized in the 900-kb MEN 1 region and was considered a good candidate gene for MEN 1. The structure and nucleotide sequence of the NF-kappa B3 coding region in MEN 1 patients were compared with those of non-MEN 1 subjects, to determine the potential role of this gene in MEN 1 tumorigenesis. Southern blot analysis with constitutional DNA from probands of 14 independent MEN 1 families and DNA from four MEN 1 tumor specimens did not reveal any structural abnormality of the NF-kappa B3 gene. Direct sequencing of cDNAs from two affected subjects from 2 different MEN 1 families, as well as nucleotide sequence analysis of exon/intron boundaries in these patients, did not reveal MEN 1-specific point mutations or other small structural aberrations in the NF-kappa B3 gene. These results make it very unlikely that the NF-kappa B3 gene is the gene responsible for the development of MEN 1.
Biochem Mol Med 1997 Feb
PMID:Exclusion of the nuclear factor-kappa B3 (REL A) gene as candidate for the multiple endocrine neoplasia type 1 (MEN 1) gene. 906 84

Two polypeptides of 50 and 45 kDa were adenylated by incubation of a mitochondrial extract from Leishmania tarentolae with [alpha-32P]ATP. These proteins were components of a complex that sedimented at 20S in glycerol gradients and migrated as a single band of approximately 1800 kDa in a native gel. The facts that RNA ligase activity cosedimented at 20S and that the ATP-labeled p45 and p50 polypeptides were deadenylated upon incubation with a ligatable RNA substrate suggested that these proteins may represent charged intermediates of a mitochondrial RNA ligase. Hybridization of native gel blots with guide RNA (gRNA) probes showed the presence of gRNA in the previously identified T-IV complexes that sedimented in glycerol at 10S and contained terminal uridylyl transferase (TUTase) activity, and also in a previously unidentified class of heterodisperse complexes that sedimented throughout the gradient. gRNAs were not detected in the p45 + p50-containing 1800 kDa complex. The heterodisperse gRNA-containing complexes were sensitive to incubation at 27 degrees C and appear to represent complexes of T-IV subunits with mRNA. Polyclonal antiserum to a 70 kDa protein that purified with terminal uridylyl transferase activity was generated, and the antiserum was used to show that this p70 polypeptide was a component of both the T-IV and the heterodisperse gRNA-containing complexes. We propose that the p45 + p50-containing 1800 kDa complex and the p70 + gRNA-containing heterodisperse complexes interact in the editing process. Further characterization of these various complexes should increase our knowledge of the biochemical mechanisms involved in RNA editing.
Mol Biochem Parasitol 1997 Mar
PMID:Native gel analysis of ribonucleoprotein complexes from a Leishmania tarentolae mitochondrial extract. 910 45

The CD28 costimulatory signal enhances antigen-mediated induction of interleukin-2 (IL-2) gene transcription through activation of an enhancer termed the CD28-responsive element (CD28RE). Although various nuclear proteins have been shown to bind to CD28RE, their in vivo functions in the regulation of this enhancer remain elusive. In this report, we show that CD28RE binds distinct transcription factors in cells treated with different mitogenic stimuli. Stimulation of the T-cell receptor (TCR) complex in the absence of a CD28 costimulatory signal induces a member of the nuclear factor of the activated T cells, NF-ATp; however, this treatment fails to activate the CD28RE enhancer activity. Significant activation of CD28RE was detected when the cells were treated with both the TCR stimulators and an anti-CD28 monoclonal antibody (anti-CD28), which induces the NF-kappaB/Rel enhancer binding proteins in addition to NF-ATp. The costimulatory activity of anti-CD28 can be further enhanced by a phorbol ester. Kinetic analyses demonstrate that activation of endogenous IL-2 gene transcription is correlated with the binding of CD28RE by NF-ATp and different NF-kappaB/Rel species. Transient-transfection studies reveal that expression of either NF-ATp or the p50-RelA NF-kappaB heterodimer leads to the potent transactivation of both the CD28RE enhancer and the intact IL-2 promoter in mitogen-stimulated cells. Remarkably, coexpression of these two families of enhancer-binding proteins in Jurkat T cells results in the transactivation of the CD28RE enhancer even in the absence of any cellular stimuli. Together, these results suggest that activation of IL-2 gene transcription by the TCR- and CD28-mediated signals involves the interaction of CD28RE with NF-ATp and various NF-kappaB/Rel transcription factors.
Mol Cell Biol 1997 May
PMID:Regulation of the interleukin-2 CD28-responsive element by NF-ATp and various NF-kappaB/Rel transcription factors. 911 30

A novel member of the I kappaB family has been identified as a protein that associated with the p50 subunit of NF-kappaB in a yeast two-hybrid screen. Similar to previously known I kappaB proteins, this member, I kappaB epsilon, has six consecutive ankyrin repeats. I kappaB epsilon mRNA is widely expressed in different human tissues, with highest levels in spleen, testis, and lung. I kappaB epsilon interacts with different NF-kappaB proteins, including p65 (RelA), c-Rel, p50, and p52, in vitro and in vivo and inhibits the DNA-binding activity of both p50-p65 and p50-c-Rel complexes effectively. Endogenous and transfected NF-kappaB (RelA-dependent) transcriptional activation is inhibited by I kappaB epsilon. I kappaB epsilon mRNA is expressed at different levels in specific cell types and is synthesized constitutively in transformed B-cell lines. It also displays differential induction in response to tumor necrosis factor alpha, interleukin-1, or phorbol ester stimulation compared to I kappaB alpha in non-B-cell lines. Therefore, I kappaB epsilon represents a novel I kappaB family member which provides an alternative mechanism for regulation of NF-kappaB-dependent transcription.
Mol Cell Biol 1997 Oct
PMID:A new member of the I kappaB protein family, I kappaB epsilon, inhibits RelA (p65)-mediated NF-kappaB transcription. 931 79

The oxidative stress responsive transcription factor nuclear factor-kappa B (NF-kappa B) consists of a p50 (50 kDa) and p65/RelA (65 kDa) component and can be activated in vitro by TNF alpha, IL1 beta, hydrogen peroxide and oxygen radicals. All of the above factors are also known to be elevated at certain times after transient global ischemia. The present study was performed to determine if NF-kappa B was activated in vivo by transient global forebrain ischemia. Adult male rats were subjected to 30 min of 4-vessel occlusion (4-VO) and sacrificed at selected post-ischemic time points. Levels of NF-kappa B p50 and p65 subunits were determined by immunocytochemistry, Western blot and electrophoretic mobility-shift analysis. The enhancer complex was also confirmed by immuno-gel-shift analysis. Specific labeling of DNA strand breaks and DNA fragmentation was examined in situ by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Western blot analysis of hippocampus showed induction of p50 and p65. A time course of NF-kappa B induction in hippocampus showed a p50-specific band at 6 h that increased in intensity over 12, 48 h and then decreased by 96 h post-ischemia. Immunocytochemistry revealed at 24 h post-ischemia that p65 and p50 immunoreactivity was present in neuronal nuclei of hippocampal CA1 neurons as well as all other hippocampal regions and several other forebrain regions which were not vulnerable to transient forebrain ischemia. At 72 h post-ischemia, nuclear NF-kappa B immunoreactivity had disappeared in all brain areas except in hippocampal CA1 neurons which were degenerating. No evidence for DNA fragmentation as revealed by TUNEL staining could be observed at 24 h. However, at 72 h, hippocampal CA1 neurons were heavily labeled. The results of this study demonstrate that global forebrain ischemia causes a transient activation of NF-kappa B in many forebrain regions. NF-kappa B remains persistently activated in the vulnerable hippocampal CA1 sector. Because of the persistent activation of NF-kappa B in these neurons, the possibility exists that NF-kappa B has a role in programmed cell death in hippocampal CA1 neurons.
Brain Res Mol Brain Res 1997 Sep
PMID:Global cerebral ischemia activates nuclear factor-kappa B prior to evidence of DNA fragmentation. 933 15


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