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Query: UNIPROT:P06889 (Mol)
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NF-kappa B and C/EBP represent distinct families of transcription factors that target unique DNA enhancer elements. The heterodimeric NF-kappa B complex is composed of two subunits, a 50- and a 65-kDa protein. All members of the NF-kappa B family, including the product of the proto-oncogene c-rel, are characterized by their highly homologous approximately 300-amino-acid N-terminal region. This Rel homology domain mediates DNA binding, dimerization, and nuclear targeting of these proteins. C/EBP contains the bZIP region, which is characterized by two motifs in the C-terminal half of the protein: a basic region involved in DNA binding and a leucine zipper motif involved in dimerization. The C/EBP family consist of several related proteins, C/EBP alpha, C/EBP beta, C/EBP gamma, and C/EBP delta, that form homodimers and that form heterodimers with each other. We now demonstrated the unexpected cross-coupling of members of the NF-kappa B family three members of the C/EBP family. NF-kappa B p65, p50, and Rel functionally synergize with C/EBP alpha, C/EBP beta, and C/EBP delta. This cross-coupling results in the inhibition of promoters with kappa B enhancer motifs and in the synergistic stimulation of promoters with C/EBP binding sites. These studies demonstrate that NF-kappa B augments gene expression mediated by a multimerized c-fos serum response element in the presence of C/EBP. We show a direct physical association of the bZIP region of C/EBP with the Rel homology domain of NF-kappa B. The cross-coupling of NF-kappa B with C/EBP highlights a mechanism of gene regulation involving an interaction between distinct transcription factor families.
Mol Cell Biol 1993 Jul
PMID:Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZIP interaction. 832 Dec 3

NF-kappa B is an important transcription factor regulating expression of genes involved in immune function, inflammation, and cellular growth control. NF-kappa B activity is induced by numerous stimuli, such as phorbol esters, B- and T-cell mitogens, the cytokines tumor necrosis factor and interleukin-1, and serum growth factors. The standard model for the induction of NF-kappa B activity involves the release of the transcription factor from a cytoplasmic inhibitor termed I kappa B, allowing translocation of NF-kappa B to the nucleus. I kappa B contains multiple copies of the so-called ankyrin repeat, which are apparently necessary for its function. Subunits comprising NF-kappa B and related binding activities are members of the Rel multigene family. Two such subunits, p50 and p52 (also called p50B), are proteolytically processed from precursors of 105 kDa (also called p105 and NFKB1) and 100 kDa (also called p100, NFKB2, and Lyt-10), respectively. Both contain N-terminal Rel-homologous domains as well as multiple copies of C-terminal ankyrin repeats. We show here that NF-kappa B p100 is a component of the previously identified DNA-binding activity H2TF1. In addition, we show that p100 is localized in the cytoplasm in HeLa cells, where it is associated with c-Rel, p50, or p65 (RelA). In transient-transfection assays, p100 represses the ability of NF-kappa B p65 to activate a kappa B-containing reporter construct. Transfection of p100 also results in a loss of nuclear p65 DNA binding to a kappa B probe, as measured by an electrophoretic mobility shift assay, and a loss of nuclear p65 immunoreactivity, as measured by immunoblotting. This loss of nuclear p65 is paralleled by a gain of p65 DNA-binding activity and immunoreactivity in the cytoplasm. We interpret these data as demonstrating that p100 functions as an I kappa B-like molecule to sequester Rel family members in the cytoplasm. Proteolytic processing of p100 to the activator p52 is predicted to generate several new forms of Rel family heterodimers and therefore represents a form of regulation of NF-kappa B activity distinct from the classic I kappa B pathway.
Mol Cell Biol 1993 Oct
PMID:NF-kappa B p100 (Lyt-10) is a component of H2TF1 and can function as an I kappa B-like molecule. 841 11

Interleukin-8 (IL-8), a chemotactic cytokine for T lymphocytes and neutrophils, is induced in several cell types by a variety of stimuli including the inflammatory cytokines IL-1 and tumor necrosis factor alpha TNF-alpha. Several cis elements, including a binding site for the inducible transcription factor NF-kappa B, have been identified in the regulatory region of the IL-8 gene. We have examined the ability of various NF-kappa B subunits to bind to, and activate transcription from, the IL-8 promoter. A nuclear complex was induced in phorbol myristate acetate-treated Jurkat T cells which bound specifically to the kappa B site of the IL-8 promoter and was inhibited by addition of purified I kappa B alpha to the reaction mixture. Only antibody to RelA (p65), but not to NFKB1 (p50), NFKB2 (p50B), c-Rel, or RelB was able to abolish binding, suggesting that RelA is a major component in these kappa B binding complexes. Gel mobility shift analysis with in vitro-translated and purified proteins indicated that whereas the kappa B element in the human immunodeficiency virus type 1 long terminal repeat bound to all members of the kappa B/Rel family examined, the IL-8 kappa B site bound only to RelA and to c-Rel and NFKB2 homodimers, but not to NFKB1 homodimers or heterodimers of NFKB1-RelA. Transient transfection analysis demonstrated a kappa B-dependent expression of the IL-8 promoter in a human fibrosarcoma cell line (8387) and in Jurkat T lymphocytes. Cotransfection with various NF-kappa B subunits indicated that RelA and c-Rel, but neither NFKB1 nor heterodimeric NFKB1-RelA, was able to activate transcription from the IL-8 promoter. Furthermore, cotransfection of NFKB1 and RelA, although able to support activation from the human immunodeficiency virus type 1 long terminal repeat, failed to activate expression from the IL-8 promoter. Antisense oligonucleotides to RelA, but not NFKB1, inhibited phorbol myristate acetate-induced IL-8 production in Jurkat T lymphocytes. These data demonstrate the differential ability of members of the kappa B/Rel family to bind to, and activate transcription from, the IL-8 promoter. Furthermore, while providing a novel example of a kappa B-regulated promoter in which the classical NF-kappa B complex is unable to activate transcription from the kappa B element, these data provide direct evidence for the role of RelA in regulation of IL-8 gene expression.
Mol Cell Biol 1993 Oct
PMID:NF-kappa B subunit-specific regulation of the interleukin-8 promoter. 841 15

Retinoic acid (RA) treatment of human embryonal carcinoma (EC) NTera-2 (NT2) cells induces expression of major histocompatibility complex (MHC) class I and beta-2 microglobulin surface molecules. We found that this induction was accompanied by increased levels of MHC class I mRNA, which was attributable to the activation of the two conserved upstream enhancers, region I (NF-kappa B like) and region II. This activation coincided with the induction of nuclear factor binding activities specific for the two enhancers. Region I binding activity was not present in undifferentiated NT2 cells, but binding of an NF-kappa B heterodimer, p50-p65, was induced following RA treatment. The p50-p65 heterodimer was produced as a result of de novo induction of p50 and p65 mRNAs. Region II binding activity was present in undifferentiated cells at low levels but was greatly augmented by RA treatment because of activation of a nuclear hormone receptor heterodimer composed of the retinoid X receptor (RXR beta) and the RA receptor (RAR beta). The RXR beta-RAR beta heterodimer also bound RA responsive elements present in other genes which are likely to be involved in RA triggering of EC cell differentiation. Furthermore, transfection of p50 and p65 into undifferentiated NT2 cells synergistically activated region I-dependent MHC class I reporter activity. A similar increase in MHC class I reporter activity was demonstrated by cotransfection of RXR beta and RAR beta. These data show that following RA treatment, heterodimers of two transcription factor families are induced to bind to the MHC enhancers, which at least partly accounts for RA induction of MHC class I expression in NT2 EC cells.
Mol Cell Biol 1993 Oct
PMID:Retinoic acid induction of major histocompatibility complex class I genes in NTera-2 embryonal carcinoma cells involves induction of NF-kappa B (p50-p65) and retinoic acid receptor beta-retinoid X receptor beta heterodimers. 841 17

The -300 region of the interleukin 1 beta (IL-1 beta) promoter contains a functional NF-kappa B binding site composed of the decamer sequence 5'-GGGAAAATCC-3'. Probes representing the -300 region or the NF-kappa B site alone interacted with NF-kappa B proteins present in phorbol myristate acetate-, lipopolysaccharide-, or Sendai virus-induced myeloid cell extracts as well as recombinant NFKB1 (p50) and RelA (p65); furthermore, NF-kappa B protein-DNA complex formation was dissociated in vitro by the addition of recombinant I kappa B alpha. Mutation of the NF-kappa B site in the context of the IL-1 beta promoter reduced the responsiveness of the IL-1 beta promoter to various inducers, including phorbol ester, Sendai virus, poly(rI-rC), and IL-1 beta. A 4.4-kb IL-1 beta promoter fragment linked to a chloramphenicol acetyltransferase reporter gene was also preferentially inducible by coexpression of individual NF-kappa B subunits compared with a mutated IL-1 beta promoter fragment. When multiple copies of the IL-1 beta NF-kappa B site were linked to an enhancerless simian virus 40 promoter, this element was able to mediate phorbol ester- or lipopolysaccharide-inducible gene expression. In cotransfection experiments, RelA (p65) and c-Rel (p85) were identified as the main subunits responsible for the activation of the IL-1 beta NF-kappa B site; also, combinations of NFKB1 (p50) and RelA (p65) or c-Rel and RelA were strong transcriptional activators of reporter gene activity. The presence of a functional NF-kappa B binding site in the IL-1 beta promoter suggests that IL-1 positively autoregulates its own synthesis, since IL-1 is a strong inducer of NF-kappa B binding activity. Thus, the IL-1 beta gene may be considered as an important additional member of the family of cytokine genes regulated in part by the NF-kappa B/rel family of transcription factors.
Mol Cell Biol 1993 Oct
PMID:Characterization of a functional NF-kappa B site in the human interleukin 1 beta promoter: evidence for a positive autoregulatory loop. 841 23

The interleukin-8 promoter is transcriptionally activated by interleukin-1, tumor necrosis factor alpha, phorbol myristate acetate, or hepatitis B virus X protein through a sequence located between positions -91 and -71. This region contains an NF-kappa B-like and a C/EBP-like binding site. We show here that several members of the NF-kappa B family, including p65, p50, p52, and c-Rel, can bind to this region, confirming an authentic NF-kappa B binding site in the interleukin-8 promoter. Further, C/EBP binds only weakly to the interleukin-8 promoter site. Electrophoretic mobility shift assays with proteins overexpressed in COS cells and with nuclear extracts from tumor necrosis factor alpha-stimulated HeLa cells demonstrated a strong cooperative binding of C/EBP to its site when NF-kappa B is bound to its adjacent binding site. Transfection studies lead to a model that suggests a highly complex regulation of interleukin-8 gene expression at multiple levels: independent binding of C/EBP and NF-kappa B to their respective sites, cooperative binding of C/EBP and NF-kappa B to DNA, and positive synergistic activation through the C/EBP binding site and inhibition through the NF-kappa B binding site by combinations of C/EBP and NF-kappa B. Thus, the ultimate regulation of interleukin-8 gene expression depends on the ratio of cellular C/EBP and NF-kappa B.
Mol Cell Biol 1993 Nov
PMID:Distinct mechanisms for regulation of the interleukin-8 gene involve synergism and cooperativity between C/EBP and NF-kappa B. 841 6

We previously reported that either oxidation or alkylation of NF-kappa B in vitro abrogates DNA binding. We used this phenomenon to help elucidate structural determinants of NF-kappa B binding. We now demonstrate that Cys-62 of NF-kappa B p50 mediates the redox effect and lies within an N-terminal region required for DNA binding but not for dimerization. Several point mutations in this region confer a transdominant negative binding phenotype to p50. The region is highly conserved in all Rel family proteins, and we have determined that it is also critical for DNA binding of NF-kappa B p65. Replacement of the N-terminal region of p65 with the corresponding region from p50 changes its DNA-binding specificity towards that of p50. These data suggest that the N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65. We have defined within the N-terminal region a sequence motif, R(F/G)(R/K)YXCE, which is present in Rel family proteins and also in zinc finger proteins capable of binding to kappa B sites. The potential significance of this finding is discussed.
Mol Cell Biol 1993 Feb
PMID:N-terminal DNA-binding domains contribute to differential DNA-binding specificities of NF-kappa B p50 and p65. 842 7

Inducible expression of human immunodeficiency virus (HIV) is regulated by a cellular transcription factor, nuclear factor kappa B (NF-kappa B). NF-kappa B is composed of distinct subunits; five independent genes, NFKB1(p105), NFKB2(p100), RelA(p65), c-rel and relB, that encode related proteins that bind to kappa B DNA elements have been isolated. We have previously found that NFKB2(p49/p52) acts in concert with RelA(p65) to stimulate the HIV enhancer in Jurkat T-leukemia cells. Here we examine the biochemical basis for the transcriptional regulation of HIV by NFKB2. Using Scatchard analysis, we have determined the dissociation constants of homodimeric p49 and heterodimeric p49/p65 for binding to the HIV kappa B site. p49 has a approximately 18-fold-lower affinity for the HIV kappa B site (KD = 69.1 pM) than does the approximately 50-kDa protein NFKB1(p50) derived from p105 (KD = 3.9 pM). In contrast, the affinity of heterodimeric NFKB2(p49)/RelA(p65) for this site is approximately 6-fold higher (KD = 11.8 pM) than that of p49 alone. Consistent with these findings, in vitro transcription was stimulated 18-fold by the addition of preformed, heterodimeric NFKB2(p49)/RelA(p65) protein. Transcriptional activation of the HIV enhancer was also subject to regulation by recently cloned I kappa B-alpha(MAD-3). Recombinant I kappa B-alpha(MAD-3) inhibited the DNA binding activity of p65, p49/p65, and p50/p65 but stimulated the binding of NFKB2(p49) or NFKB1(p50). Functional activation of an HIV reporter plasmid by p49/p65 in transiently transfected Jurkat T-leukemia cells was also inhibited by coexpression of MAD-3. These data suggest that binding of the NFKB2 subunit to the HIV enhancer is facilitated by RelA(p65) and that this NFKB2(p49)/p65 heterodimeric complex mediates transcriptional activation which is subject to regulation by MAD-3.
Mol Cell Biol 1993 Mar
PMID:Dimerization of NF-KB2 with RelA(p65) regulates DNA binding, transcriptional activation, and inhibition by an I kappa B-alpha (MAD-3). 844 77

The NF-kappa B transcription factor complex is composed of a 50-kDa (p50) and a 65-kDa (p65) subunit. Both subunits bind to similar DNA motifs and elicit transcriptional activation as either homo- or heterodimers. By using chimeric proteins that contain the DNA binding domain of the yeast transcriptional activator GAL4 and subdomains of p65, three distinct transcriptional activation domains were identified. One domain was localized to a region of 42 amino acids containing a potential leucin zipper structure, consistent with earlier reports. Two other domains, both acidic and rich in prolines, were also identified. Of perhaps more significance, the same minimal activation domains that were functional in mammalian cells were also functional in the yeast Saccharomyces cerevisiae. Coexpression of the NF-kappa B inhibitory molecule, I kappa B, reduced the transcriptional activity of p65 significantly, suggesting the ability of I kappa B to function in a similar manner in S. cerevisiae. Surprisingly, while the conserved rel homology domain of p65 demonstrated no transcriptional activity in either mammalian cells or S. cerevisiae, the corresponding domain in p50 was a strong transcriptional activator in S. cerevisiae. The observation that similar domains elicit transcriptional activation in mammalian cells and S. cerevisiae demonstrates strong conservation of the transcriptional machinery required for NF-kappa B function and provides a powerful genetic system to study the transcriptional mechanisms of these proteins.
Mol Cell Biol 1993 Mar
PMID:Conservation of transcriptional activation functions of the NF-kappa B p50 and p65 subunits in mammalian cells and Saccharomyces cerevisiae. 844 4

Nuclear factor kappa B (NF-kappa B) is a critical regulator of several genes which are involved in immune and inflammation responses. NF-kappa B, consisting of a 50-kDa protein (p50) and a 65-kDa protein (p65), is bound to a cytoplasmic retention protein called I kappa B. Stimulation of cells with a variety of inducers, including cytokines such as tumor necrosis factor and interleukin-1, leads to the activation and the translocation of p50/65 NF-kappa B into the nucleus. However, the in vivo mechanism of the activation process remains unknown. Here, we provide the first evidence that the in vivo mechanism of NF-kappa B activation is through the phosphorylation and subsequent loss of its inhibitor, I kappa B alpha. We also show that both I kappa B alpha loss and NF-kappa B activation are inhibited in the presence of antioxidants, demonstrating that the loss of I kappa B alpha is a prerequisite for NF-kappa B activation. Finally, we demonstrate that I kappa B alpha is rapidly resynthesized after loss, indicating that an autoregulatory mechanism is involved in the regulation of NF-kappa B function. We propose a mechanism for the activation of NF-kappa B through the modification and loss of I kappa B alpha, thereby establishing its role as a mediator of NF-kappa B activation.
Mol Cell Biol 1993 Jun
PMID:Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. 849 53


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