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Query: UNIPROT:P06889 (Mol)
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The promoter of the human major histocompatibility complex class II-associated invariant-chain gene (Ii) contains two NF-kappa B/Rel binding sites located at -109 to -118 (Ii kappa B-1) and -163 to -172 (Ii kappa B-2) from the transcription start site. We report here that the differential function of each of these NF-kappa B/Rel sites in several distinct cell types depends on cell-specific binding of NF-kappa B/Rel transcription factors. Ii kappa B-1 is a positive regulatory element in B-cell lines and in the Ii-expressing T-cell line, H9, but acts as a negative regulatory element in myelomonocytic and glia cell lines. In vivo protein-DNA contacts are detectable at Ii kappa B-1 in cell lines in which this site is functional as either a positive or negative regulator. Electrophoretic mobility supershift assays determine that members of the NF-kappa B/Rel family of transcription factors can bind to this site in vitro and that DNA-binding complexes that contain p50, p52, p65, and cRel correlate with positive regulation whereas the presence of p50 correlates with negative regulation. Ii kappa B-2 is a site of positive regulation in B-cell lines and a site of negative regulation in H9 T cells, myelomonocytic, and glial cell lines. In vivo occupancy of this site is observed only in the H9 T-cell line. Again, in vitro supershift studies indicate that the presence of p50, p52, p65, and cRel correlates with positive function whereas the presence of only p50 and p52 correlates with negative function. This differential binding of specific NF-kappa B/Rel subunits is likely to mediate the disparate functions of these two NF-kappa B/Rel binding sites.
Mol Cell Biol 1994 May
PMID:Function of NF-kappa B/Rel binding sites in the major histocompatibility complex class II invariant chain promoter is dependent on cell-specific binding of different NF-kappa B/Rel subunits. 816 52

Exposure of monocytic cells to bacterial lipopolysaccharide (LPS) activates the NF-kappa B/Rel family of proteins and leads to the rapid induction of inflammatory gene products, including tissue factor (TF). TF is the primary cellular initiator of the coagulation protease cascades. Here we report the characterization of a nuclear complex from human monocytic cells that bound to a kappa B-like site, 5'-CGGAGTTTCC-3', in the 5'-flanking region of the human TF gene. This nuclear complex was activated by LPS with kinetics that preceded induction of the TF gene. In vitro binding studies demonstrated that the TF site bound translated c-Rel and p65 homodimers but not p50/p65 heterodimers or p50 homodimers. Base-pair substitutions in the TF site indicated that the presence of a cytosine at position 1 precluded binding of NF-kappa B. In fact, under low-ionic-strength conditions, the TF complex did not migrate with translated p50/p65 dimers but instead comigrated with c-Rel/p65 dimers. Antibodies against the NF-kappa B and Rel proteins and UV cross-linking studies revealed the presence of c-Rel and p65 and the absence of p50 in the TF complex and further showed that c-Rel/p65 heterodimers selectively bound to the TF kappa B-like site. Functional studies indicated that the TF site conferred LPS inducibility on a heterologous promoter and was transactivated by c-Rel or p65. Taken together, our results demonstrated that binding of c-Rel/p65 heterodimers to a novel kappa B-like site mediated LPS induction of TF gene expression in monocytic cells.
Mol Cell Biol 1994 Jun
PMID:Lipopolysaccharide induction of tissue factor gene expression in monocytic cells is mediated by binding of c-Rel/p65 heterodimers to a kappa B-like site. 819 20

NF-kappa B is inducible transcription factor present in many cell types in a latent cytoplasmic form. So far, only immune cells including mature B cells, thymocytes, and adherent macrophages have been reported to contain constitutively active forms of NF-kappa B in the nucleus. A recent study showed that the human immunodeficiency virus type 1 (HIV-1) promoter is highly active in several brain regions of transgenic mice (J. R. Corboy, J. M. Buzy, M. C. Zink, and J. E. Clements, Science 258:1804-1807, 1992). Since the activity of this viral enhancer is governed mainly by two binding sites for NF-kappa B, we were prompted to investigate the state of NF-kappa B activity in neurons. Primary neuronal cultures derived from rat hippocampus and cerebral cortex showed a high constitutive expression of an HIV-1 long terminal repeat-driven luciferase reporter gene, which was primarily dependent on intact NF-kappa B binding sites and was abolished upon coexpression of the NF-kappa B-specific inhibitor I kappa B-alpha. Indirect immunofluorescence and confocal laser microscopy showed that the activity of NF-kappa B correlated with the presence of the NF-kappa B subunits p50 and RelA (p65) in nuclei of cultured neurons. NF-kappa B was also constitutively active in neurons in vivo. As investigated by electrophoretic mobility shift assays, constitutive NF-kappa B DNA-binding activity was highly enriched in fractions containing neuronal nuclei prepared from rat cerebral cortex. Nuclear NF-kappa B-specific immunostaining was also seen in cryosections from mouse cerebral cortex and hippocampus. Only a subset of neurons was stained. Activated NF-kappa B in the brain is likely to participate in normal brain function and to reflect a distinct state of neuronal activity or differentiation. Furthermore, it may explain the high level of activity of the HIV-1 enhancer in neurons, an observation potentially relevant for the etiology of the AIDS dementia complex caused by HIV infection of the central nervous system.
Mol Cell Biol 1994 Jun
PMID:Constitutive NF-kappa B activity in neurons. 819 37

The predominant inducible form of the NF-kappa B transcription factor is a heteromeric complex containing two Rel-related DNA-binding subunits, termed p65 and p50. Prior transfection studies have shown that when these p65 and p50 subunits are expressed independently as stable homodimers, p65 stimulates kappa B-directed transcription, whereas p50 functions as a kappa B-specific repressor. While authentic p50 homodimers (previously termed KBF1) have been detected in nuclear extracts from nontransfected cells, experimental evidence supporting the existence of p65 homodimers in vivo was lacking. We now provide direct biochemical evidence for the presence of an endogenous pool of inducible p65 homodimers in intact human T cells. As with the prototypical NF-kappa B p50-p65 heterodimer, this novel p65 homodimeric form of NF-kappa B is functionally sequestered in the cytoplasm but rapidly appears in the nuclear compartment following cellular stimulation. Site-directed mutagenesis studies indicate that the homodimerization function of p65 is dependent upon the presence of cysteine 216 and a conserved recognition motif for protein kinase A (RRPS; amino acids 273 to 276), both of which reside within a 91-amino-acid segment of the Rel homology domain that mediates self-association. In contrast, mutations at these two sites do not affect heterodimerization of p65 with p50 or its functional interaction with I kappa B alpha. These later findings indicate that neither homo- nor heterodimer formation is an absolute prerequisite for I kappa B alpha recognition of p65. Taken together with prior in vivo transcription studies, these results suggest that the biological activities of p65 and p50 homodimers are independently regulated, thereby providing an integrated and flexible control mechanism for the rapid activation and repression of NF-kappa B/Rel-directed gene expression.
Mol Cell Biol 1993 Dec
PMID:A novel NF-kappa B complex containing p65 homodimers: implications for transcriptional control at the level of subunit dimerization. 824 97

NF-kappa B and related factors are important transducers of external signals to the cell nucleus. They are abundant in the brain, where they may be significant for the regulation of gene transcription in plasticity-related processes for instance, via activation of protein kinase C. The subunit composition and levels of these factors in the mouse and rat brain and other tissues, using an assay based on gel retardation of the oligonucleotides corresponding to the kappa B DNA-element, are reported here. Three major kappa B-binding factors were observed. Factors I and II were activated by the dissociating agent deoxycholate. DNA protein cross-linking and antibody neutralization experiments suggest that factor I is a heterodimer of c-Rel and p65; factor II is a heterodimer of p50 and p65 (authentic NF-kappa B), and of p50 and c-Rel; factor III is the p50 homodimer (KBF1). All three factors were generally expressed in the 17-day-old rat embryo and 5-day-old pup, whereas in the adult rat, expression was more limited and showed certain tissue specificity. Factor II was the most generally expressed and the only factor observed in adult brain. Factor I was only detected in the adult testis whereas factor III was observed in the adult spleen and, in small amounts, in the liver and lung. Two minor kappa B-specific factors (A and B), distinctive to the brain and spleen, respectively, showed very slow gel mobility. Their estimated molecular weights were about 125 kDa and 95 kDa, respectively. Expression of factor A was stable in the rat brain during development. Factor A may be identical to a previously described brain-specific factor, BETA (Korner et al., Neuron, 3 (1989) 563-572). Thus, the expression pattern of kappa B-binding activities is apparently developmentally regulated and tissue-specific particularly in the adult. In the adult mouse and rat brain, only factors II (probably NF-kappa B and p50/c-Rel heterodimer) and A (probably BETA) could be observed.
Brain Res Mol Brain Res 1993 Oct
PMID:NF-kappa B-like factors in the murine brain. Developmentally-regulated and tissue-specific expression. 825 75

We have characterized constitutive and cytokine-regulated MGSA/GRO alpha, -beta, and -gamma gene expression in normal retinal pigment epithelial (RPE) cells and a malignant melanoma cell line (Hs294T) to discern the mechanism for MGSA/GRO constitutive expression in melanoma. In RPE cells, constitutive MGSA/GRO alpha, -beta, and -gamma mRNAs are not detected by Northern (RNA) blot analysis although nuclear runoff experiments show that all three genes are transcribed. In Hs294T cells, constitutive MGSA/GRO alpha expression is detectable by Northern blot analysis, and the level of basal MGSA/GRO alpha transcription is 8- to 30-fold higher than in RPE cells. In contrast, in Hs294T cells, basal MGSA/GRO beta and -gamma transcription is only twofold higher than in RPE cells and no beta or gamma mRNA is detected by Northern blot. These data suggest that the constitutive MGSA/GRO alpha mRNA in Hs294T cells is due to increased basal MGSA/GRO alpha gene transcription. The cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) significantly increase the mRNA levels for all three MGSA/GRO isoforms in Hs294T and RPE cells, and both transcriptional and posttranscriptional mechanisms are operational. Nuclear runoff assays indicate that in RPE cells, a 1-h IL-1 treatment induces a 10- to 20-fold increase in transcription of MGSA/GRO alpha, -beta and -gamma but only a 2-fold increase in Hs294T cells. Similarly, chloramphenicol acetyltransferase (CAT) reporter gene analysis using the MGSA/GRO alpha, -beta, and -gamma promoter regions demonstrates that IL-1 treatment induces an 8- to 14-fold increase in CAT activity in RPE cells but only a 2-fold increase in Hs294T cells. The effect of deletion or mutation of the MGSA/GRO alpha NF-kappa B element, combined with data from gel mobility shift analyses, indicates that the NF-kappa B p50/p65 heterodimer in RPE cells plays an important role in IL-1- and TNF alpha-enhanced gene transcription. In Hs294T cells, gel shift analyses indicate that IL-1 and TNF alpha induce NF-kappa B complex formation; however, transactivation does not occur, suggesting that subtle differences in the NF-kappa B complexes may result in the inability of the cytokines IL-1 and TNF alpha to activate transcription of the MGSA/GRO genes. IL-1 and TNF alpha posttranscriptionally regulate MGSA/GRO mRNA levels in both cell types. In Hs294T cells, IL-1 increases the half-life of MGSA/GRO alpha from 15 min to 6 h (a 24-fold increase in half-life). These data indicate that IL-1 and TNF alpha transcriptionally and posttranscriptionally regulate MGSA/GRO alpha, -beta, and -gamma mRNA levels in RPE cells, while in Hs294T cells, the major effect of IL-1 and TNF alpha is on mRNA stability.
Mol Cell Biol 1994 Jan
PMID:MGSA/GRO transcription is differentially regulated in normal retinal pigment epithelial and melanoma cells. 826 46

Endogenous proteins phosphorylated in vitro with nuclear extracts prepared from skin fibroblasts of familial and sporadic AD subjects and age/sex-matched controls show qualitative/quantitative changes which were dependent upon the addition of natural and synthetic double-stranded DNA (dsDNA) to assay mixtures. Control and AD cell-free homogenates showed a pronounced increase in four proteins migrating with molecular weights of 180- (p180), 75-(p75), 65- (p65), and 34-kD (p34). Optimal stimulation of each protein required a different concentration of dsDNA. In AD samples, an additional dsDNA-stimulated protein, p40, was observed. In extracts prepared from sporadic AD individuals, synthetic dsDNA stimulated the phosphorylation of two additional proteins, with molecular weights of 73- and 37-kD, which were not found in familial AD or control samples. These results suggest that dsDNA-stimulated phosphoprotein changes may be used to distinguish between familial and sporadic forms of AD.
Biochem Mol Biol Int 1993 Oct
PMID:Studies of biochemical changes in cultured skin fibroblasts derived from sporadic and familial Alzheimer's disease individuals: qualitative and quantitative changes in double-stranded DNA-stimulated phosphorylation of endogenous nucleoproteins. 827 15

Transcription factor NF-kappa B represents a family of closely related homo- and heterodimeric factors. The most abundant form of NF-kappa B is the p50/p65 heterodimer. We determined the complete genomic structure of the human gene and a partial structure of the mouse gene encoding p65. The human gene consists of ten exons and spans about 8.1 kbp of DNA. The exon-intron organization in the rel homology domain (exons 2 to 7) is conserved when compared to human and turkey c-rel, strengthening the evolutionary relationship between p65 and c-rel. The lengths of the corresponding introns 5 and 6 in the human and mouse p65 genes are not conserved. However, a surprisingly high degree of conservation of intron sequences was observed between both species. We show that the naturally occurring shorter variant of p65 (p65 delta) can be generated by alternative splicing of intron 6, not only in humans but also in mouse. In addition, the existence of another, as yet unknown splice variant of p65 is predicted.
Hum Mol Genet 1993 Nov
PMID:Genomic organization of the gene encoding the p65 subunit of NF-kappa B: multiple variants of the p65 protein may be generated by alternative splicing. 828 Nov 53

The murine c-myc gene contains two elements responsive to the rel-oncogene-related family of NF-kappa B factors. Previously we have shown that factor binding to these two NF-kappa B elements mediates induction of transcription of the c-myc promoter upon interleukin-1 treatment of human dermal fibroblasts and human T-cell leukemia virus type I tax gene expression in T cells (D. J. Kessler, M. P. Duyao, D. B. Spicer, and G. E. Sonenshein, J. Exp. Med. 176:787-792, 1992; M. P. Duyao, D. J. Kessler, D. B. Spicer, C. Bartholomew, J. L. Cleveland, M. Siekevitz, and G. E. Sonenshein, J. Biol. Chem. 267:16288-16291, 1992). To begin to delineate the specific roles of the individual members of the NF-kappa B family, here we have tested their effects on activation of a c-myc promoter/exon 1-CAT construct in NIH 3T3 cells. Classical NF-kappa B (p65/p50) was a potent transcriptional activator of the c-myc promoter. Cotransfection with either p65 alone or p65 in combination with p50 mediated significant induction. In contrast, expression of either v-rel or chicken c-rel failed to transactivate, while murine c-rel induced c-myc promoter activity only slightly. Furthermore, induction by classical NF-kappa B was inhibited by coexpression of either v-rel or chicken c-rel. Thus, individual members of the rel family have differential effects of the c-myc promoter, which can modulate overall transcriptional activity and allow for precise regulation of this oncogene under diverse physiologic conditions.
Mol Cell Biol 1994 Feb
PMID:Differential regulation of the c-myc oncogene promoter by the NF-kappa B rel family of transcription factors. 828 84

The subunits of NF-kappa B, NFKB1 (formerly p50) and RelA (formerly p65), belong to a growing family of transcription factors that share extensive similarity to the c-rel proto-oncogene product. The homology extends over a highly conserved stretch of approximately 300 amino acids termed the Rel homology domain (RHD). This region has been shown to be involved in both multimerization (homo- and heterodimerization) and DNA binding. It is now generally accepted that homodimers of either subunit are capable of binding DNA that contains a kappa B site originally identified in the immunoglobulin enhancer. Recent studies have demonstrated that the individual subunits of the NF-kappa B transcription factor complex can be distinguished by their ability to bind distinct DNA sequence motifs. By using NFKB1 and RelA subunit fusion proteins, different regions within the RHD were found to confer DNA-binding and multimerization functions. A fusion protein that contains 34 N-terminal amino acids of NFKB1 and 264 amino acids of RelA displayed preferential binding to an NFKB1-selective DNA motif while dimerizing with the characteristics of RelA. Within the NFKB1 portion of this fusion protein, a single amino acid change of His to Arg altered the DNA-binding specificity to favor interaction with the RelA-selective DNA motif. Furthermore, substitution of four amino acids from NFKB1 into RelA was able to alter the DNA-binding specificity of the RelA protein to favor interaction with the NFKB1-selective site. Taken together, these findings demonstrate the presence of a distinct subdomain within the RHD involved in conferring the DNA-binding specificity of the Rel family of proteins.
Mol Cell Biol 1993 Jul
PMID:Acquisition of NFKB1-selective DNA binding by substitution of four amino acid residues from NFKB1 into RelA. 832 Nov 92


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