Intermolecular disulfide bond influences unphosphorylated STAT3 dimerization and function

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor activated by the phosphorylation of tyrosine 705 in response to many cytokines and growth factors. Recently, the roles for unphosphorylated STAT3 (U-STAT3) have been described in response to cytokine stimulation, in cancers, and in the maintenance of heterochromatin stability. It has been reported that U-STAT3 dimerizes, shuttles between the cytoplasm and nucleus, and binds to DNA, thereby driving genes transcription. Although many reports describe the active role of U-STAT3 in oncogenesis in addition to phosphorylated STAT3, the U-STAT3 functional pathway remains elusive. In this report, we describe the molecular mechanism of U-STAT3 dimerization, and we identify the presence of two intermolecular disulfide bridges between Cys367 and Cys542 and Cys418 and Cys426, respectively. Recently, we reported that the same cysteines contribute to the redox regulation of STAT3 signaling pathway both in vitro and in vivo. The presence of these disulfides is here demonstrated to largely contribute to the structure and the stability of U-STAT3 dimer as the dimeric form rapidly dissociates upon reduction in the S–S bonds. In particular, the Cys367–Cys542 disulfide bridge is shown to be critical for U-STAT3 DNA-binding activity. Mutation of the two Cys residues completely abolishes the DNA-binding capability of U-STAT3. Spectroscopic investigations confirm that the noncovalent interactions are sufficient for proper folding and dimer formation, but that the interchain disulfide bonds are crucial to preserve the functional dimer. Finally, we propose a reaction scheme of U-STAT3 dimerization with a first common step followed by stabilization through the formation of interchain disulfide bonds.

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

Molecular and Cellular Biology ◽

10.1128/mcb.23.12.4257-4266.2003 ◽

2003 ◽

Vol 23 (12) ◽

pp. 4257-4266 ◽

Cited By ~ 49

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.

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Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽

10.1182/blood-2007-12-128900 ◽

2008 ◽

Vol 112 (4) ◽

pp. 1056-1067 ◽

Cited By ~ 36

Author(s):

Mira T. Kassouf ◽

Hedia Chagraoui ◽

Paresh Vyas ◽

Catherine Porcher

Keyword(s):

Dna Binding ◽

Molecular Mechanisms ◽

Binding Activity ◽

Hematopoietic Stem ◽

Helix Loop Helix ◽

Experimental Systems ◽

Dna Binding Activity ◽

Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

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The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes

Development ◽

10.1242/dev.113.1.245 ◽

1991 ◽

Vol 113 (1) ◽

pp. 245-255 ◽

Cited By ~ 32

Author(s):

M. Van Doren ◽

H.M. Ellis ◽

J.W. Posakony

Keyword(s):

Dna Binding ◽

Cell Fate ◽

Protein Complexes ◽

Competitive Inhibitor ◽

Binding Activity ◽

Regulatory Function ◽

Biochemical Basis ◽

Dna Binding Activity

In Drosophila, a group of regulatory proteins of the helix-loop-helix (HLH) class play an essential role in conferring upon cells in the developing adult epidermis the competence to give rise to sensory organs. Proteins encoded by the daughterless (da) gene and three genes of the achaete-scute complex (AS-C) act positively in the determination of the sensory organ precursor cell fate, while the extramacrochaetae (emc) and hairy (h) gene products act as negative regulators. In the region upstream of the achaete gene of the AS-C, we have identified three ‘E box’ consensus sequences that are bound specifically in vitro by hetero-oligomeric complexes consisting of the da protein and an AS-C protein. We have used this DNA-binding activity to investigate the biochemical basis of the negative regulatory function of emc. Under the conditions of our experiments, the emc protein, but not the h protein, is able to antagonize specifically the in vitro DNA-binding activity of da/AS-C and putative da/da protein complexes. We interpret these results as follows: the heterodimerization capacity of the emc protein (conferred by its HLH domain) allows it to act in vivo as a competitive inhibitor of the formation of functional DNA-binding protein complexes by the da and AS-C proteins, thereby reducing the effective level of their transcriptional regulatory activity within the cell.

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Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2464-2476.1989 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2464-2476

Author(s):

M Cockell ◽

B J Stevenson ◽

M Strubin ◽

O Hagenbüchle ◽

P K Wellauer

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Dna Interaction ◽

Binding Activity ◽

Alpha Amylase ◽

Dna Binding Activity ◽

A Cell ◽

Flanking Regions

Footprint analysis of the 5'-flanking regions of the alpha-amylase 2, elastase 2, and trypsina genes, which are expressed in the acinar pancreas, showed multiple sites of protein-DNA interaction for each gene. Competition experiments demonstrated that a region from each 5'-flanking region interacted with the same cell-specific DNA-binding activity. We show by in vitro binding assays that this DNA-binding activity also recognizes a sequence within the 5'-flanking regions of elastase 1, chymotrypsinogen B, carboxypeptidase A, and trypsind genes. Methylation interference and protection studies showed that the DNA-binding activity recognized a bipartite motif, the subelements of which were separated by integral helical turns of DNA. The alpha-amylase 2 cognate sequence was found to enhance in vivo transcription of its own promoter in a cell-specific manner, which identified the DNA-binding activity as a transcription factor (PTF 1). The observation that PTF 1 bound to DNA sequences that have been defined as transcriptional enhancers by others suggests that this factor is involved in the coordinate expression of genes transcribed in the acinar pancreas.

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Characterization of CprK1, a CRP/FNR-Type Transcriptional Regulator of Halorespiration from Desulfitobacterium hafniense

Journal of Bacteriology ◽

10.1128/jb.188.7.2604-2613.2006 ◽

2006 ◽

Vol 188 (7) ◽

pp. 2604-2613 ◽

Cited By ~ 32

Author(s):

Krisztina Gábor ◽

Carla S. Veríssimo ◽

Barbara C. Cyran ◽

Paul ter Horst ◽

Nienke P. Meijer ◽

...

Keyword(s):

Dna Binding ◽

Redox Regulation ◽

Physiological Role ◽

Disulfide Bridge ◽

Reductive Dehalogenation ◽

Receptor Protein ◽

Oxidative Inactivation ◽

Desulfitobacterium Hafniense

ABSTRACT The recently identified CprK branch of the CRP (cyclic AMP receptor protein)-FNR (fumarate and nitrate reduction regulator) family of transcriptional regulators includes proteins that activate the transcription of genes encoding proteins involved in reductive dehalogenation of chlorinated aromatic compounds. Here we report the characterization of the CprK1 protein from Desulfitobacterium hafniense, an anaerobic low-G+C gram-positive bacterium that is capable of reductive dechlorination of 3-chloro-4-hydroxyphenylacetic acid (Cl-OHPA). The gene encoding CprK1 was cloned and functionally overexpressed in Escherichia coli, and the protein was subsequently purified to homogeneity. To investigate the interaction of CprK1 with three of its predicted binding sequences (dehaloboxes), we performed in vitro DNA-binding assays (electrophoretic mobility shift assays) as well as in vivo promoter probe assays. Our results show that CprK1 binds its target dehaloboxes with high affinity (dissociation constant, 90 nM) in the presence of Cl-OHPA and that transcriptional initiation by CprK1 is influenced by deviations in the dehaloboxes from the consensus TTAAT----ATTAA sequence. A mutant CprK1 protein was created by a Val→Glu substitution at a conserved position in the recognition α-helix that gained FNR-type DNA-binding specificity, recognizing the TTGAT----ATCAA sequence (FNR box) instead of the dehaloboxes. CprK1 was subject to oxidative inactivation in vitro, most likely caused by the formation of an intermolecular disulfide bridge between Cys11 and Cys200. The possibility of redox regulation of CprK1 by a thiol-disulfide exchange reaction was investigated by using two Cys→Ser mutants. Our results indicate that a Cys11-Cys200 disulfide bridge does not appear to play a physiological role in the regulation of CprK1.

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The rat cytomegalovirus homologue of parvoviral rep genes, r127, encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication

Journal of General Virology ◽

10.1099/vir.0.79864-0 ◽

2004 ◽

Vol 85 (7) ◽

pp. 2001-2013 ◽

Cited By ~ 6

Author(s):

Koen W. R. van Cleef ◽

Wendy M. A. Scaf ◽

Karen Maes ◽

Suzanne J. F. Kaptein ◽

Erik Beuken ◽

...

Keyword(s):

Dna Binding ◽

Virus Replication ◽

Deletion Mutant ◽

Nuclear Protein ◽

Human Herpesvirus ◽

Binding Activity ◽

Dna Binding Activity ◽

Double Stranded Dna

An intriguing feature of the rat cytomegalovirus (RCMV) genome is open reading frame (ORF) r127, which shows similarity to the rep genes of parvoviruses as well as the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B). Counterparts of these genes have not been found in other herpesviruses. Here, it is shown that the r127 gene is transcribed during the early and late phases of virus replication in vitro as an unspliced 1·1 kb transcript containing the complete r127 ORF. Transcripts of r127 were also detected in various organs of RCMV-infected rats at 1 week post-infection (p.i.), but only in the salivary gland at 4 months p.i. Using rabbit polyclonal antibodies raised against the r127-encoded protein (pr127), pr127 was found to be expressed as early as 12 h p.i. within the nuclei of RCMV-infected cells in vitro. Expression of pr127 was also observed within the nuclei of cells in various organs of RCMV-infected rats at 3 weeks p.i. Moreover, pr127 was demonstrated to bind single- as well as double-stranded DNA. Finally, an RCMV r127 deletion mutant (RCMVΔr127) was generated, in which the r127 ORF was disrupted. This deletion mutant, however, was shown to replicate with a similar efficiency as wild-type RCMV (wt RCMV), both in vitro and in vivo. Taken together, it is concluded that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo.

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CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

Journal of Biological Chemistry ◽

10.1074/jbc.m401854200 ◽

2004 ◽

Vol 279 (44) ◽

pp. 45887-45896 ◽

Cited By ~ 45

Author(s):

Mark J. Demma ◽

Serena Wong ◽

Eugene Maxwell ◽

Bimalendu Dasmahapatra

Keyword(s):

Dna Binding ◽

Mammalian Cells ◽

P53 Protein ◽

Binding Activity ◽

Mutant P53 ◽

Dependent Manner ◽

Wild Type ◽

Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

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Identification of a cell-specific DNA-binding activity that interacts with a transcriptional activator of genes expressed in the acinar pancreas.

Molecular and Cellular Biology ◽

10.1128/mcb.9.6.2464 ◽

1989 ◽

Vol 9 (6) ◽

pp. 2464-2476 ◽

Cited By ~ 83

Author(s):

M Cockell ◽

B J Stevenson ◽

M Strubin ◽

O Hagenbüchle ◽

P K Wellauer

Keyword(s):

Dna Binding ◽

Dna Sequences ◽

Dna Interaction ◽

Binding Activity ◽

Alpha Amylase ◽

Dna Binding Activity ◽

A Cell ◽

Flanking Regions

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

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5552 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5552-5562 ◽

Cited By ~ 24

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.

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Reduced sulphydryl groups are required for DNA binding of Ku protein

Biochemical Journal ◽

10.1042/bj2930769 ◽

1993 ◽

Vol 293 (3) ◽

pp. 769-774 ◽

Cited By ~ 14

Author(s):

W W Zhang ◽

M Yaneva

Keyword(s):

Dna Binding ◽

Protein A ◽

Binding Activity ◽

Heat Inactivation ◽

Cysteine Residues ◽

Dna Binding Activity ◽

Sulphydryl Groups ◽

Ku Protein

The Ku protein, a DNA-binding complex that is composed of two subunits of 70 kDa and of 86 kDa, has been suggested to play a role in gene transcription. The dependence of the in vitro DNA-binding activity of affinity-purified Ku protein on reduced cysteine residues has been studied using sulphydryl-modifying agents. Inhibition of the DNA-binding activity was caused by alkylation with N-ethylmaleimide and by crosslinking with azadicarboxylic acid bis(dimethylamide). Treatment of the protein with a large excess of N-ethylmaleimide after it had bound to DNA did not completely dissociate the complex from the DNA, suggesting that some cysteines may be in direct contact with DNA. Pre-incubation of the protein at 37 degrees C or above caused rapid inactivation of DNA binding. The elevated temperature azadicarboxylic acid bis(dimethylamide) treatments resulted in the formation of a crosslinked product, which was detected by Western blotting. The effects of azadicarboxylic acid bis(dimethylmaleimide) and heat were completely reversible by treatment with a reducing agent, such as dithiothreitol. These results demonstrate that in vitro DNA-binding activity of the Ku protein requires reduced sulphydryl groups. Interestingly, the DNA-binding activity of Ku protein was protected from heat inactivation by the presence of a HeLa cell nuclear extract, suggesting that a nuclear factor or factors may be responsible for the maintenance of the reduced cysteines of the Ku protein in vivo. Thus, the biochemical function of the Ku protein may be regulated through oxidation-reduction of its cysteine residues.

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