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.

Altered expression and insulin-induced trafficking of Na+-K+-ATPase in rat skeletal muscle: effects of high-fat diet and exercise

2009 ◽  
Vol 297 (1) ◽  
pp. E38-E49 ◽  
Author(s):  
Dana Galuska ◽  
Olga Kotova ◽  
Romain Barrès ◽  
Daria Chibalina ◽  
Boubacar Benziane ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Transcription Factor ◽  
Dna Binding ◽  
Protein Expression ◽  
Atpase Activity ◽  
Binding Activity ◽  
Insulin Resistant ◽  
Dna Binding Activity ◽  
Skeletal Muscle Insulin Resistance ◽  
Subunit Expression

Skeletal muscle Na+-K+-ATPase plays a central role in the clearance of K+ from the extracellular fluid, therefore maintaining blood [K+]. Na+-K+-ATPase activity in peripheral tissue is impaired in insulin resistant states. We determined effects of high-fat diet (HFD) and exercise training (ET) on skeletal muscle Na+-K+-ATPase subunit expression and insulin-stimulated translocation. Skeletal muscle expression of Na+-K+-ATPase isoforms and transcription factor DNA binding was determined before or after 5 days of swim training in Wistar rats fed chow or HFD for 4 or 12 wk. Skeletal muscle insulin resistance was observed after 12 wk of HFD. Na+-K+-ATPase α1-subunit protein expression was increased 1.6-fold ( P < 0.05), whereas α2- and β1-subunits and protein expression were decreased twofold ( P < 0.01) in parallel with decrease in plasma membrane Na+-K+-ATPase activity after 4 wk of HFD. Exercise training restored α1-, α2-, and β1-subunit expression and Na+-K+-ATPase activity to control levels and reduced β2-subunit expression 2.2-fold ( P < 0.05). DNA binding activity of the α1-subunit-regulating transcription factor ZEB (AREB6) and α1 mRNA expression were increased after HFD and restored by ET. DNA binding activity of Sp-1, a transcription factor involved in the regulation of α2- and β1-subunit expression, was decreased after HFD. ET increased phosphorylation of the Na+-K+-ATPase regulatory protein phospholemman. Phospholemman mRNA and protein expression were increased after HFD and restored to control levels after ET. Insulin-stimulated translocation of the α2-subunit to plasma membrane was impaired by HFD, whereas α1-subunit translocation remained unchanged. Alterations in sodium pump function precede the development of skeletal muscle insulin resistance. Disturbances in skeletal muscle Na+-K+-ATPase regulation, particularly the α2-subunit, may contribute to impaired ion homeostasis in insulin-resistant states such as obesity and type 2 diabetes.

scholarly journals Mutations in the DNA-binding Domain of the Transcription Factor Bright Act as Dominant Negative Proteins and Interfere with Immunoglobulin Transactivation

2004 ◽  
Vol 279 (50) ◽  
pp. 52465-52472 ◽  
Author(s):  
Jamee C. Nixon ◽  
Jaya Rajaiya ◽  
Carol F. Webb
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
B Cell ◽  
Heavy Chain ◽  
Cell Development ◽  
Binding Activity ◽  
B Cell Development ◽  
Dominant Negative ◽  
Wild Type ◽  
Dna Binding Activity

Bright, for B cell regulator of immunoglobulin heavy chain transcription, binds A+T-rich sequences in the intronic enhancer regions of the murine heavy chain locus and 5′-flanking sequences of some variable heavy chain promoters. Most resting B cells do not express Bright; however, it is induced after stimulation with antigen or polyclonal mitogens. Bright activation results in up-regulation of μ transcription; however, it is not clear whether Bright function is critical for normal B cell development. To begin to address Bright function during B cell development, seven mutated forms of Bright were produced. Five of the seven mutants revealed little or no DNA binding activity. Furthermore, because Bright binds DNA as a dimer, two of the mutants formed complexes with wild type Bright and acted in a dominant negative fashion. Dominant negative Bright prevented the up-regulation of μ transcription in transfected Chinese hamster ovary cells transfected with wild type Bright. These data identify regions within Bright that are required for the DNA binding activity of Bright and for its function as a transcription factor.

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.

Characterization of a dominant negativeC. elegansTwist mutant protein with implications for human Saethre-Chotzen syndrome

Development ◽  
2002 ◽  
Vol 129 (11) ◽  
pp. 2761-2772
Author(s):  
Ann K. Corsi ◽  
Thomas M. Brodigan ◽  
Erik M. Jorgensen ◽  
Michael Krause
Keyword(s):  
Dna Binding ◽  
Binding Activity ◽  
Binding Domain ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Dna Binding Domain ◽  
Mesodermal Cell ◽  
C Elegans ◽  
Dna Binding Activity ◽  
A Charge

Twist is a transcription factor that is required for mesodermal cell fates in all animals studied to date. Mutations of this locus in humans have been identified as the cause of the craniofacial disorder Saethre-Chotzen syndrome. The Caenorhabditis elegans Twist homolog is required for the development of a subset of the mesoderm. A semidominant allele of the gene that codes for CeTwist, hlh-8, has defects that occur earlier in the mesodermal lineage than a previously studied null allele of the gene. The semidominant allele has a charge change (E29K) in the basic DNA-binding domain of CeTwist. Surprisingly, the mutant protein retains DNA-binding activity as both a homodimer and a heterodimer with its partner E/Daughterless (CeE/DA). However, the mutant protein blocks the activation of the promoter of a target gene. Therefore, the mutant CeTwist may cause cellular defects as a dominant negative protein by binding to target promoters as a homo- or heterodimer and then blocking transcription. Similar phenotypes as those caused by the E29K mutation were observed when amino acid substitutions in the DNA-binding domain that are associated with the human Saethre-Chotzen syndrome were engineered into the C. elegans protein. These data suggest that Saethre-Chotzen syndrome may be caused, in some cases, by dominant negative proteins, rather than by haploinsufficiency of the locus.

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.

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