The RxxRxRxxC motif conserved in all Rel/kappa B proteins is essential for the DNA-binding activity and redox regulation of the v-Rel oncoprotein

1992 ◽  
Vol 12 (7) ◽  
pp. 3094-3106 ◽  
Author(s):  
S Kumar ◽  
A B Rabson ◽  
C Gélinas
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Redox Regulation ◽  
Biological Activities ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Lymphoid Cells ◽  
Redox Control ◽  
Dna Binding Activity

The v- and c-Rel oncoproteins bind to oligonucleotides containing kappa B motifs, form heterodimers with other members of the Rel family, and modulate expression of genes linked to kappa B motifs. Here, we report that the RxxRxRxxC motif conserved in all Rel/kappa B family proteins is absolutely required for v-Rel protein-DNA contact and its resulting transforming activity. We also demonstrate that serine substitution of the cysteine residue conserved within this motif enables v-Rel to escape redox control, thereby promoting overall DNA binding. These mutant proteins retained the ability to competitively inhibit kappa B-mediated transcriptional activation of the human immunodeficiency virus long terminal repeat but failed to efficiently transform chicken lymphoid cells both in vitro and in vivo. Our data indicate that reduction of the conserved cysteine residue in the RxxRxRxxC motif may be required for optimal DNA-protein interactions. These results provide direct biochemical evidence that the DNA-binding activity of v-Rel is subject to redox control and that the conserved cysteine residue in the RxxRxRxxC motif is critical for this regulation. These studies suggest that the DNA-binding, transcriptional, and biological activities of Rel family proteins may also be subject to redox control in vivo.

scholarly journals The RxxRxRxxC motif conserved in all Rel/kappa B proteins is essential for the DNA-binding activity and redox regulation of the v-Rel oncoprotein.

1992 ◽  
Vol 12 (7) ◽  
pp. 3094-3106 ◽  
Author(s):  
S Kumar ◽  
A B Rabson ◽  
C Gélinas
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Redox Regulation ◽  
Biological Activities ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Lymphoid Cells ◽  
Redox Control ◽  
Dna Binding Activity

The v- and c-Rel oncoproteins bind to oligonucleotides containing kappa B motifs, form heterodimers with other members of the Rel family, and modulate expression of genes linked to kappa B motifs. Here, we report that the RxxRxRxxC motif conserved in all Rel/kappa B family proteins is absolutely required for v-Rel protein-DNA contact and its resulting transforming activity. We also demonstrate that serine substitution of the cysteine residue conserved within this motif enables v-Rel to escape redox control, thereby promoting overall DNA binding. These mutant proteins retained the ability to competitively inhibit kappa B-mediated transcriptional activation of the human immunodeficiency virus long terminal repeat but failed to efficiently transform chicken lymphoid cells both in vitro and in vivo. Our data indicate that reduction of the conserved cysteine residue in the RxxRxRxxC motif may be required for optimal DNA-protein interactions. These results provide direct biochemical evidence that the DNA-binding activity of v-Rel is subject to redox control and that the conserved cysteine residue in the RxxRxRxxC motif is critical for this regulation. These studies suggest that the DNA-binding, transcriptional, and biological activities of Rel family proteins may also be subject to redox control in vivo.

scholarly journals The conserved redox-sensitive cysteine residue of the DNA-binding region in the c-Rel protein is involved in the regulation of the phosphorylation of the protein

2000 ◽  
Vol 352 (2) ◽  
pp. 583-591 ◽  
Author(s):  
Corine GLINEUR ◽  
Elisabeth DAVIOUD-CHARVET ◽  
Bernard VANDENBUNDER
Keyword(s):  
Dna Binding ◽  
Redox Regulation ◽  
Phosphorylation Site ◽  
Nuclear Factor Κb ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Redox Control ◽  
Aspartic Acid Residue ◽  
Dna Binding Activity ◽  
Thioredoxin Reductases

The DNA-binding activity of the transcription nuclear factor κB (NF-κB) is regulated by a redox-control mechanism involving the reduction of a disulphide bond from a specific cysteine residue conserved in all members of the NF-κB family. Thioredoxin is involved in this redox control. DNA binding and transactivating capacity of NF-κB are up-regulated by inducible phosphorylation. Here we demonstrate that the conserved redox cysteine in the c-Rel protein is involved in the phosphorylation regulation of the protein. When this cysteine residue is mutated to an aspartic acid residue, the mutant protein loses its capacity to be phosphorylated and its DNA-binding activity. In addition, our results suggest that, when the conserved redox cysteine is chemically modified by N-ethylmaleimide and 2-chloro-1,3-dinitrobenzene, the protein c-Rel cannot be phosphorylated. In contrast, the protein in which the cysteine residue was replaced by a serine residue, creating a potential phosphorylation site, is highly phosphorylated and binds κB sequences. The protein could loose the redox regulation of the phosphorylation when the residue replacing the cysteine can be itself phosphorylated. We also show that specific inhibitors of thioredoxin reductases impair the phosphorylation of the c-Rel protein, suggesting that the redox regulation of the protein controls its phosphorylation.

scholarly journals Hypoxia-inducible factor 1 levels vary exponentially over a physiologically relevant range of O2 tension

1996 ◽  
Vol 271 (4) ◽  
pp. C1172-C1180 ◽  
Author(s):  
B. H. Jiang ◽  
G. L. Semenza ◽  
C. Bauer ◽  
H. H. Marti
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Functional Characterization ◽  
Hypoxia Inducible Factor ◽  
Binding Activity ◽  
Maximal Response ◽  
Hypoxia Inducible Factor 1 ◽  
Dna Binding Activity

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric basic helix-loop-helix protein implicated in the transcriptional activation of genes encoding erythropoietin, glycolytic enzymes, and vascular endothelial growth factor in hypoxic mammalian cells. In this study, we have quantitated HIF-1 DNA-binding activity and protein levels of the HIF-1 alpha and HIF-1 beta subunits in human HeLa cells exposed to O2 concentrations ranging from 0 to 20% in the absence or presence of 1 mM KCN to inhibit oxidative phosphorylation and cellular O2 consumption. HIF-1 DNA-binding activity, HIF-1 alpha protein and HIF-1 beta protein each increased exponentially as cells were subjected to decreasing O2 concentrations, with a half maximal response between 1.5 and 2% O2 and a maximal response at 0.5% O2, both in the presence and absence of KCN. The HIF-1 response was greatest over O2 concentrations associated with ischemic/hypoxic events in vivo. These results provide evidence for the involvement of HIF-1 in O2 homeostasis and represent a functional characterization of the putative O2 sensor that initiates hypoxia signal transduction leading to HIF-1 expression.

scholarly journals Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

1995 ◽  
Vol 15 (10) ◽  
pp. 5552-5562 ◽  
Author(s):  
E Roulet ◽  
M T Armentero ◽  
G Krey ◽  
B Corthésy ◽  
C Dreyer ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Xenopus Laevis ◽  
Transcriptional Activation ◽  
Binding Activity ◽  
New Members ◽  
Binding Domains ◽  
Dna Binding Activity

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

Regulation of c-jun expression during hypoxic and low-glucose stress

1994 ◽  
Vol 14 (8) ◽  
pp. 5032-5042
Author(s):  
W A Ausserer ◽  
B Bourrat-Floeck ◽  
C J Green ◽  
K R Laderoute ◽  
R M Sutherland
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Squamous Carcinoma ◽  
Glucose Deprivation ◽  
Binding Activity ◽  
Hypoxic Exposure ◽  
Redox Control ◽  
Dna Binding Activity ◽  
Low Glucose ◽  
Squamous Carcinoma Cell

Hypoxic stress in tumor cells has been implicated in malignant progression and in the development of therapeutic resistance. We have investigated the effects of acute hypoxic exposure on regulation of the proto-oncogene c-jun in SiHa cells, a human squamous carcinoma cell line. Hypoxic exposure produced increased levels of c-jun mRNA resulting from both message stabilization and transcriptional activation. A superinduction of c-jun message resulted during simultaneous oxygen and glucose deprivation, with several characteristics of an induction mediated by oxidative-stress pathways. This superinduction was blocked by preincubation of cells with the glutathione precursor N-acetyl cysteine or with phorbol 12-myristate 13-acetate, which indicates redox control of c-jun expression and probable involvement of protein kinase C. By gel retardation assay, no increase in AP-1 DNA binding activity was found to be concomitant with the transcriptional activation of c-jun. A lack of increased DNA binding was observed for the consensus AP-1 sequence and for the two AP-1 sequence variants found within the c-Jun promoter. Additionally, hypoxic and low-glucose stress produced no activation of stably transfected AP-1 reporter sequences. Taken together, these results indicate that the transcriptional activation of c-jun during hypoxic and low-glucose stress involves redox control and is unlikely to be mediated by AP-1 recognition elements within the c-jun promoter.

Cooperative DNA binding of the human HoxB5 (Hox-2.1) protein is under redox regulation in vitro

1993 ◽  
Vol 13 (8) ◽  
pp. 4609-4617
Author(s):  
C K Galang ◽  
C A Hauser
Keyword(s):  
Dna Binding ◽  
Redox Regulation ◽  
Mutational Analysis ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Nf Kappa B ◽  
Amino Terminal ◽  
Dna Binding Activity ◽  
Specific Oxidation

The human HoxB5 (Hox-2.1) gene product is a sequence-specific DNA binding protein. Cooperative interactions stabilize in vitro DNA binding of the HoxB5 protein to tandem binding sites by at least 100-fold relative to binding to a single site. The HoxB5 homeodomain is sufficient for sequence-specific DNA binding but not for cooperative DNA binding. Here we report that the additional protein sequence required for cooperativity is a small domain adjacent to the homeodomain on the amino-terminal side. We further show that cooperative DNA binding is under redox regulation. The HoxB5 protein binds to DNA in vitro both when oxidized or reduced but binds cooperatively only when oxidized. Mutational analysis has revealed that the cysteine residue in the turn between homeodomain helices 2 and 3 is necessary for cooperative binding and redox regulation. The enhanced DNA binding of oxidized HoxB5 protein is the opposite of the redox regulation reported for other mammalian transcription factors such as Fos, Jun, USF, NF-kappa B, c-Myb, and v-Rel, in which oxidation of cysteine residues inhibits DNA binding. Thus, specific oxidation of nuclear proteins is a potential regulatory mechanism that can act to either decrease or increase their DNA binding activity.

scholarly journals Cooperative DNA binding of the human HoxB5 (Hox-2.1) protein is under redox regulation in vitro.

1993 ◽  
Vol 13 (8) ◽  
pp. 4609-4617 ◽  
Author(s):  
C K Galang ◽  
C A Hauser
Keyword(s):  
Dna Binding ◽  
Redox Regulation ◽  
Mutational Analysis ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Nf Kappa B ◽  
Amino Terminal ◽  
Dna Binding Activity ◽  
Specific Oxidation

The human HoxB5 (Hox-2.1) gene product is a sequence-specific DNA binding protein. Cooperative interactions stabilize in vitro DNA binding of the HoxB5 protein to tandem binding sites by at least 100-fold relative to binding to a single site. The HoxB5 homeodomain is sufficient for sequence-specific DNA binding but not for cooperative DNA binding. Here we report that the additional protein sequence required for cooperativity is a small domain adjacent to the homeodomain on the amino-terminal side. We further show that cooperative DNA binding is under redox regulation. The HoxB5 protein binds to DNA in vitro both when oxidized or reduced but binds cooperatively only when oxidized. Mutational analysis has revealed that the cysteine residue in the turn between homeodomain helices 2 and 3 is necessary for cooperative binding and redox regulation. The enhanced DNA binding of oxidized HoxB5 protein is the opposite of the redox regulation reported for other mammalian transcription factors such as Fos, Jun, USF, NF-kappa B, c-Myb, and v-Rel, in which oxidation of cysteine residues inhibits DNA binding. Thus, specific oxidation of nuclear proteins is a potential regulatory mechanism that can act to either decrease or increase their DNA binding activity.

scholarly journals Cysteine 64 of Ref-1 Is Not Essential for Redox Regulation of AP-1 DNA Binding

2003 ◽  
Vol 23 (12) ◽  
pp. 4257-4266 ◽  
Author(s):  
Jared M. Ordway ◽  
Derek Eberhart ◽  
Tom Curran
Keyword(s):  
Transcription Factors ◽  
Dna Binding ◽  
Redox Regulation ◽  
Binding Activity ◽  
Regulation Of Transcription ◽  
Binding Domains ◽  
Dna Binding Activity ◽  
Reducing Activity

ABSTRACT Ref-1 participates in DNA repair as well as in redox regulation of transcription factor function. The redox function of Ref-1 involves reduction of oxidized cysteine residues within the DNA binding domains of several transcription factors, including Fos and Jun. Reduction of these residues is required for DNA binding, providing a redox-dependent mechanism for regulation of target gene expression. Previous in vitro studies implicated cysteine 65 of human Ref-1 (cysteine 64 of mouse Ref-1) as the redox catalytic site. We analyzed the in vivo role of cysteine 64 in redox regulation of AP-1 activity by introducing a cysteine-to-alanine point mutation into the endogenous mouse Ref-1 gene (ref-1 C64A). Unlike Ref-1 null mice, which die very early in embryonic development, homozygous ref-1 C64A mice are viable, they survive to normal life expectancy, and they display no overt abnormal phenotype. Although Ref-1 provides the major AP-1-reducing activity in murine cells, ref-1 C64A cells retain normal levels of endogenous AP-1 DNA binding activity in vivo as well as normal Fos- and Jun-reducing activity in vitro. These results demonstrate that Ref-1 cysteine 64/65 is not required for redox regulation of AP-1 DNA binding in vivo, and they challenge previous hypotheses regarding the mechanism by which Ref-1 regulates the redox-dependent activity of specific transcription factors.

scholarly journals Regulation of c-jun expression during hypoxic and low-glucose stress.

1994 ◽  
Vol 14 (8) ◽  
pp. 5032-5042 ◽  
Author(s):  
W A Ausserer ◽  
B Bourrat-Floeck ◽  
C J Green ◽  
K R Laderoute ◽  
R M Sutherland
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Squamous Carcinoma ◽  
Glucose Deprivation ◽  
Binding Activity ◽  
Hypoxic Exposure ◽  
Redox Control ◽  
Dna Binding Activity ◽  
Low Glucose ◽  
Squamous Carcinoma Cell

Hypoxic stress in tumor cells has been implicated in malignant progression and in the development of therapeutic resistance. We have investigated the effects of acute hypoxic exposure on regulation of the proto-oncogene c-jun in SiHa cells, a human squamous carcinoma cell line. Hypoxic exposure produced increased levels of c-jun mRNA resulting from both message stabilization and transcriptional activation. A superinduction of c-jun message resulted during simultaneous oxygen and glucose deprivation, with several characteristics of an induction mediated by oxidative-stress pathways. This superinduction was blocked by preincubation of cells with the glutathione precursor N-acetyl cysteine or with phorbol 12-myristate 13-acetate, which indicates redox control of c-jun expression and probable involvement of protein kinase C. By gel retardation assay, no increase in AP-1 DNA binding activity was found to be concomitant with the transcriptional activation of c-jun. A lack of increased DNA binding was observed for the consensus AP-1 sequence and for the two AP-1 sequence variants found within the c-Jun promoter. Additionally, hypoxic and low-glucose stress produced no activation of stably transfected AP-1 reporter sequences. Taken together, these results indicate that the transcriptional activation of c-jun during hypoxic and low-glucose stress involves redox control and is unlikely to be mediated by AP-1 recognition elements within the c-jun promoter.

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