scholarly journals NF-kappa B p100 (Lyt-10) is a component of H2TF1 and can function as an I kappa B-like molecule.

1993 ◽  
Vol 13 (10) ◽  
pp. 6089-6101 ◽  
Author(s):  
R I Scheinman ◽  
A A Beg ◽  
A S Baldwin
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Phorbol Esters ◽  
Interleukin 1 ◽  
Binding Activity ◽  
Proteolytic Processing ◽  
Cellular Growth ◽  
Nf Kappa B ◽  
Dna Binding Activity ◽  
Multiple Copies

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.

scholarly journals NF-kappa B p100 (Lyt-10) is a component of H2TF1 and can function as an I kappa B-like molecule

1993 ◽  
Vol 13 (10) ◽  
pp. 6089-6101
Author(s):  
R I Scheinman ◽  
A A Beg ◽  
A S Baldwin
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Phorbol Esters ◽  
Interleukin 1 ◽  
Binding Activity ◽  
Proteolytic Processing ◽  
Cellular Growth ◽  
Nf Kappa B ◽  
Dna Binding Activity ◽  
Multiple Copies

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.

scholarly journals Constitutive nuclear NFκB/rel DNA-binding activity of rat thymocytes is increased by stimuli that promote apoptosis, but not inhibited by pyrrolidine dithiocarbamate

1995 ◽  
Vol 312 (3) ◽  
pp. 833-838 ◽  
Author(s):  
A F G Slater ◽  
M Kimland ◽  
S A Jiang ◽  
S Orrenius
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Gradient Centrifugation ◽  
Binding Activity ◽  
Pyrrolidine Dithiocarbamate ◽  
Nf Kappa B ◽  
Dna Binding Activity ◽  
Apoptotic Activity

Rat thymocytes spontaneously undergo apoptotic death in cell culture, and are also sensitive to the induction of apoptosis by various stimuli. We show that unstimulated thymocytes constitutively express a p50-containing nuclear factor kappa B (NF kappa B)/rel DNA-binding activity in their nuclei. When the cells were fractionated by density-gradient centrifugation this activity was found to be most pronounced in immature CD4+8+ thymocytes, the cell population that undergoes selection by apoptosis in vivo and that is most sensitive to external inducers of apoptosis in vitro. The intensity of the NF kappa B/rel protein-DNA complex was significantly enhanced 30 min after exposing thymocytes to methylprednisolone or etoposide, two agents well known to induce apoptosis in these cells. Expression of this DNA-binding activity therefore correlates with the subsequent occurrence of apoptosis. By analogy to other systems, it has been suggested that antioxidants such as pyrrolidine dithiocarbamate (PDTC) inhibit thymocyte apoptosis by preventing the activation of an NF kappa B/rel transcription factor. However, we have found that etoposide induces a very similar enhancement of the NF kappa B/rel DNA-binding activity in the presence or absence of PDTC, despite a pronounced inhibition of apoptotic DNA fragmentation in the former situation. Dithiocarbamates therefore do not exert their anti-apoptotic activity in thymocytes by inhibiting the activation of this transcription factor.

scholarly journals I kappa B/MAD-3 masks the nuclear localization signal of NF-kappa B p65 and requires the transactivation domain to inhibit NF-kappa B p65 DNA binding.

1992 ◽  
Vol 3 (12) ◽  
pp. 1339-1352 ◽  
Author(s):  
P A Ganchi ◽  
S C Sun ◽  
W C Greene ◽  
D W Ballard
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Binding Activity ◽  
Transactivation Domain ◽  
Nf Kappa B ◽  
Dna Binding Activity ◽  
Localization Signal

The active nuclear form of the NF-kappa B transcription factor complex is composed of two DNA binding subunits, NF-kappa B p65 and NF-kappa B p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. The NF-kappa B p65 subunit provides the transactivation activity in this complex and serves as an intracellular receptor for a cytoplasmic inhibitor of NF-kappa B, termed I kappa B. In contrast, NF-kappa B p50 alone fails to stimulate kappa B-directed transcription, and based on prior in vitro studies, is not directly regulated by I kappa B. To investigate the molecular basis for the critical regulatory interaction between NF-kappa B and I kappa B/MAD-3, a series of human NF-kappa B p65 mutants was identified that functionally segregated DNA binding, I kappa B-mediated inhibition, and I kappa B-induced nuclear exclusion of this transcription factor. Results from in vivo expression studies performed with these NF-kappa B p65 mutants revealed the following: 1) I kappa B/MAD-3 completely inhibits NF-kappa B p65-dependent transcriptional activation mediated through the human immunodeficiency virus type 1 kappa B enhancer in human T lymphocytes, 2) the binding of I kappa B/MAD-3 to NF-kappa B p65 is sufficient to retarget NF-kappa B p65 from the nucleus to the cytoplasm, 3) selective deletion of the functional nuclear localization signal present in the Rel homology domain of NF-kappa B p65 disrupts its ability to engage I kappa B/MAD-3, and 4) the unique C-terminus of NF-kappa B p65 attenuates its own nuclear localization and contains sequences that are required for I kappa B-mediated inhibition of NF-kappa B p65 DNA binding activity. Together, these findings suggest that the nuclear localization signal and transactivation domain of NF-kappa B p65 constitute a bipartite system that is critically involved in the inhibitory function of I kappa B/MAD-3. Unexpectedly, our in vivo studies also demonstrate that I kappa B/MAD-3 binds directly to NF-kappa B p50. This interaction is functional as it leads to retargeting of NF-kappa B p50 from the nucleus to the cytoplasm. However, no loss of DNA binding activity is observed, presumably reflecting the unique C-terminal domain that is distinct from that present in NF-kappa B p65.

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor

1986 ◽  
Vol 6 (12) ◽  
pp. 4723-4733
Author(s):  
L A Chodosh ◽  
R W Carthew ◽  
P A Sharp
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Binding Site ◽  
Rna Polymerase Ii ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Affinity Column ◽  
Dna Binding Activity ◽  
Dna Fragment

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

Cloning and characterization of E2F-2, a novel protein with the biochemical properties of transcription factor E2F

1993 ◽  
Vol 13 (12) ◽  
pp. 7802-7812
Author(s):  
M Ivey-Hoyle ◽  
R Conroy ◽  
H E Huber ◽  
P J Goodhart ◽  
A Oliff ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Hela Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Identity ◽  
Biochemical Properties ◽  
Binding Activity ◽  
Dna Binding Activity ◽  
Novel Protein

E2F is a mammalian transcription factor that appears to play an important role in cell cycle regulation. While at least two proteins (E2F-1 and DP-1) with E2F-like activity have been cloned, studies from several laboratories suggest that additional homologs may exist. A novel protein with E2F-like properties, designated E2F-2, was cloned by screening a HeLa cDNA library with a DNA probe derived from the DNA binding domain of E2F-1 (K. Helin, J. A. Lees, M. Vidal, N. Dyson, E. Harlow, and A. Fattaey, Cell 70:337-350, 1992). E2F-2 exhibits overall 46% amino acid identity to E2F-1. Both the sequence and the function of the DNA and retinoblastoma gene product binding domains of E2F-1 are conserved in E2F-2. The DNA binding activity of E2F-2 is dramatically enhanced by complementation with particular sodium dodecyl sulfate-polyacrylamide gel electrophoresis-purified components of HeLa cell E2F, and anti-E2F-2 antibodies cross-react with components of purified HeLa cell E2F. These observations are consistent with a model in which E2F binds DNA as a heterodimer of two distinct proteins, and E2F-2 is functionally and immunologically related to one of these proteins.

Treatment of cultured myotubes with the proteasome inhibitor β-lactone increases the expression of the transcription factor C/EBPβ

2007 ◽  
Vol 292 (1) ◽  
pp. C216-C226 ◽  
Author(s):  
Wei Wei ◽  
Hongmei Yang ◽  
Michael Menconi ◽  
Peirang Cao ◽  
Chester E. Chamberlain ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transcription Factor ◽  
Dna Binding ◽  
Proteasome Inhibitor ◽  
Binding Activity ◽  
Dominant Negative ◽  
Dna Binding Activity ◽  
Cultured Myotubes ◽  
Nuclear Levels ◽  
Factor C

The role of the proteasome in the regulation of cellular levels of the transcription factor CCAAT/enhancer-binding protein β (C/EBPβ) is poorly understood. We tested the hypothesis that C/EBPβ levels in cultured myotubes are regulated, at least in part, by proteasome activity. Treatment of cultured L6 myotubes, a rat skeletal muscle cell line, with the specific proteasome inhibitor β-lactone resulted in increased nuclear levels of C/EBPβ as determined by Western blotting and immunofluorescent detection. This effect of β-lactone reflected inhibited degradation of C/EBPβ. Surprisingly, the increased C/EBPβ levels in β-lactone-treated myotubes did not result in increased DNA-binding activity. In additional experiments, treatment of the myotubes with β-lactone resulted in increased nuclear levels of growth arrest DNA damage/C/EBP homologous protein (Gadd153/CHOP), a dominant-negative member of the C/EBP family that can form heterodimers with other members of the C/EBP family and block DNA binding. Coimmunoprecipitation and immunofluorescent detection provided evidence that C/EBPβ and Gadd153/CHOP interacted and colocalized in the nuclei of the β-lactone-treated myotubes. When Gadd153/CHOP expression was downregulated by transfection of myotubes with siRNA targeting Gadd153/CHOP, C/EBPβ DNA-binding activity was restored in β-lactone-treated myotubes. The results suggest that C/EBPβ is degraded by a proteasome-dependent mechanism in skeletal muscle cells and that Gadd153/CHOP can interact with C/EBPβ and block its DNA-binding activity. The observations are important because they increase the understanding of the complex regulation of the expression and activity of C/EBPβ in skeletal muscle.

scholarly journals Two monomers of yeast transcription factor ADR1 bind a palindromic sequence symmetrically to activate ADH2 expression.

1991 ◽  
Vol 11 (3) ◽  
pp. 1566-1577 ◽  
Author(s):  
S K Thukral ◽  
A Eisen ◽  
E T Young
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Dna Binding ◽  
Dimethyl Sulfate ◽  
Binding Activity ◽  
Single Amino Acid ◽  
Minor Effect ◽  
Palindromic Sequence ◽  
Amino Terminal ◽  
Dna Binding Activity

ADR1 is a transcription factor from Saccharomyces cerevisiae that regulates ADH2 expression through a 22-bp palindromic sequence (UAS1). Size fractionation studies revealed that full-length ADR1 and a truncated ADR1 protein containing the first 229 amino acids, which has the complete DNA-binding domain, ADR1:17-229, exist as monomers in solution. However, two complexes were formed with target DNA-binding sites. UV-cross-linking studies suggested that these two complexes represent one and two molecules of ADR1 bound to DNA. Studies of ADR1 complexes formed with wild-type UAS1, asymmetrically altered UAS1, and one half of UAS1 showed that ADR1 can bind to one half of UAS1 and gives rise to a complex containing one molecule of ADR1. Dimethyl sulfate interference studies were consistent with this interpretation and in addition indicated that purine contact sites in each half of UAS1 were identical. Increasing the distance between the two halves of UAS1 had at most a minor effect of the thermodynamics of formation of the two complexes. These data are more consistent with ADR1 binding as two independent monomers, one to each half of UAS1. However, binding of two ADR1 monomers at UAS1 is apparently essential for transactivation in vivo. Further, we have identified a stretch of 18 amino acid residues amino terminal to the zinc two-finger domains of ADR1 which is essential for DNA-binding activity. Single amino acid substitutions of residues in this region resulted in severely reduced DNA-binding activity.

scholarly journals I(kappa)B(gamma) inhibits DNA binding of NF-kappaB p50 homodimers by interacting with residues that contact DNA.

1996 ◽  
Vol 16 (11) ◽  
pp. 6477-6485 ◽  
Author(s):  
S Bell ◽  
J R Matthews ◽  
E Jaffray ◽  
R T Hay
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Nf Kappa B ◽  
Protein Footprinting ◽  
I Kappa B Alpha ◽  
Dna Binding Activity ◽  
Cellular Genes ◽  
Phosphate Backbone ◽  
Nf Kappab ◽  
Contact Dna

NF-(kappa)B is an inducible transcription factor that activates many cellular genes involved in stress and immune response and whose DNA binding activity and cellular distribution are regulated by I(kappa)B inhibitor proteins. The interaction between NF-(kappa)B p50 and DNA was investigated by protein footprinting using chemical modification and partial proteolysis. Both methods confirmed lysine-DNA contacts already found in the crystal structure (K-147, K-149, K-244, K-275, and K-278) but also revealed an additional contact in the lysine cluster K-77-K-78-K-80 which was made on an extended DNA. Molecular modelling of such a DNA-protein complex revealed that lysine 80 is ideally placed to make phosphate backbone contacts in the extended DNA. Thus, it seems likely that the entire AB loop, containing lysines 77, 78, and 80, forms a C-shaped clamp that closes around the DNA recognition site. The same protein footprinting approaches were used to probe the interaction of p50 with the ankyrin repeat containing proteins I(kappa)B(gamma) and I(kappa)B(alpha). Lysine residues in p50 that were protected from modification by DNA were also protected from modification by I(kappa)B(gamma) but not I(kappa)B(alpha). Similarly, proteolytic cleavage at p50 residues which contact DNA was inhibited by bound I(kappa)B(gamma) but was enhanced by the presence of I(kappa)B(alpha). Thus, I(kappa)B(gamma) inhibits the DNA binding activity of p50 by direct interactions with residues contacting DNA, whereas the same residues remain exposed in the presence of I(kappa)B(alpha), which binds to p50 but does not block DNA binding.

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