scholarly journals Phosphorylation of serine-167 on the human oestrogen receptor is important for oestrogen response element binding and transcriptional activation

1997 ◽  
Vol 326 (1) ◽  
pp. 149-157 ◽  
Author(s):  
Enrique CASTAÑO ◽  
Daria P. VOROJEIKINA ◽  
Angelo C. NOTIDES
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Functional Significance ◽  
Oestrogen Receptor ◽  
Phosphorylation Site ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Hormone Binding ◽  
Dna Binding Affinity

We have studied the role of phosphorylation of the human oestrogen receptor (hOR; otherwise known as hER) at serine-167, which has been identified previously as the major oestrogen-induced phosphorylation site. We have tested transactivation by the hOR in yeast and cell-free transcription assays, and shown that mutation of serine-167 results in a 70% decrease in hOR-dependent transcription. Furthermore we explored the functional significance of phosphorylation at this site by hormone binding and DNA binding. DNA binding affinity was 10-fold lower when serine-167 was changed to alanine in the hOR. Cell-free transcription experiments showed that casein kinase II is the enzyme responsible for oestradiol-dependent phosphorylation of the hOR at serine-167. This suggests that a conformational change of the hOR must occur upon hormone binding that exposes serine-167 to casein kinase II, resulting in transactivation of oestrogen-responsive genes.

scholarly journals Casein kinase II phosphorylation site mutations in c-Myb affect DNA binding and transcriptional cooperativity with NF-M.

1995 ◽  
Vol 15 (11) ◽  
pp. 5966-5974 ◽  
Author(s):  
M Oelgeschläger ◽  
J Krieg ◽  
J M Lüscher-Firzlaff ◽  
B Lüscher
Keyword(s):  
Dna Binding ◽  
Phosphorylation Site ◽  
Direct Interaction ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Wild Type ◽  
Type C ◽  
A Site

Phosphorylation of c-Myb has been implicated in the regulation of the binding of c-Myb to DNA. We show that murine c-Myb is phosphorylated at Ser-11 and -12 in vivo and that these sites can be phosphorylated in vitro by casein kinase II (CKII), analogous to chicken c-Myb. An efficient method to study DNA binding properties of full-length c-Myb and Myb mutants under nondenaturing conditions was developed. It was found that a Myb mutant in which Ser-11 and -12 were replaced with Ala (Myb Ala-11/12), wild-type c-Myb, and Myb Asp-11/12 bound to the A site of the mim-1 promoter with decreasing affinities. In agreement with this finding, Myb Ala-11/12 transactivated better than wild-type c-Myb and Myb Asp-11/12 on the mim-1 promoter or a synthetic Myb-responsive promoter. Similar observations were made for the myeloid-specific neutrophil elastase promoter. The presence of NF-M or an NF-M-like activity abolished partially the differences seen with the Ser-11/12 mutants, suggesting that the reduced DNA binding due to negative charge at positions 11 and 12 can be compensated for by NF-M. Since no direct interaction of c-Myb and NF-M was observed, we propose that the cooperativity is mediated by a third factor. Our data offer two possibilities for how casein kinase II phosphorylation can influence c-Myb function: first, by reducing c-Myb DNA binding and thereby influencing transactivation, and second, by enhancing the apparent cooperativity between c-Myb and NF-M or an NF-M-like activity.

scholarly journals Genetic dissection of thyroid hormone receptor beta: identification of mutations that separate hormone binding and transcriptional activation.

1995 ◽  
Vol 15 (3) ◽  
pp. 1499-1512 ◽  
Author(s):  
R Uppaluri ◽  
H C Towle
Keyword(s):  
Thyroid Hormone ◽  
Dna Binding ◽  
Ligand Binding ◽  
Reporter Gene ◽  
Transcriptional Activation ◽  
Thyroid Hormone Receptor ◽  
Receptor Activation ◽  
Hormone Binding ◽  
D Region

The thyroid hormone receptors (TR) are members of the nuclear receptor family of ligand-mediated transcription factors. The large region of TR that lies C-terminal to its DNA-binding domain subserves functions of ligand binding, dimerization, and transactivation. Little is known regarding the structural or functional determinants of these processes. We have utilized genetic screening in the yeast Saccharomyces cerevisiae to identify residues involved in these functions. Random mutations of the rat TR beta 1 isoform between amino acid residues 179 and 456 were screened, and mutants with reduced hormone-dependent activation of reporter gene activity were isolated. In this paper we describe the characterization of a class of mutants that exhibit a dissociation between hormone binding and transcriptional activation. These mutants retained hormone binding (> 15% of the wild-type level) yet failed to transactivate a reporter gene. A number of these mutations occurred within the D region, which links the DNA-binding and ligand-binding domains of the receptor. One subset of these mutations abrogated DNA binding, supporting a role of the D region in this process. The remainder retain DNA binding and thus highlight residues critical for receptor activation. In addition, an unexpected group of "superactivator" mutations that led to enhanced hormone-dependent activation in S. cerevisiae were found. These mutations localized to the carboxy-terminal portion of the receptor in a region which contains elements conserved across the superfamily of nuclear receptors. The hormone-dependent phenotype of these superactivator mutations suggests an important role of this segment in ligand-mediated transcriptional activation.

scholarly journals Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Krystyna Ślaska-Kiss ◽  
Nikolett Zsibrita ◽  
Mihály Koncz ◽  
Pál Albert ◽  
Ákos Csábrádi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Binding ◽  
Binding Affinity ◽  
Dna Methyltransferase ◽  
Restriction Enzymes ◽  
Dna Binding Protein ◽  
E Coli ◽  
Dna Binding Affinity ◽  
Higher Eukaryotes

AbstractTargeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

scholarly journals Role of GCR2 in transcriptional activation of yeast glycolytic genes.

1992 ◽  
Vol 12 (9) ◽  
pp. 3834-3842 ◽  
Author(s):  
H Uemura ◽  
Y Jigami
Keyword(s):  
Dna Binding ◽  
Fusion Protein ◽  
Transcriptional Activation ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Gene Products ◽  
Lacz Expression ◽  
Genetic Method ◽  
Potential Interactions

The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.

scholarly journals Varicella-Zoster Virus Fc Receptor Component gI Is Phosphorylated on Its Endodomain by a Cyclin-Dependent Kinase

1999 ◽  
Vol 73 (2) ◽  
pp. 1320-1330 ◽  
Author(s):  
Ming Ye ◽  
Karen M. Duus ◽  
Junmin Peng ◽  
David H. Price ◽  
Charles Grose
Keyword(s):  
Fusion Protein ◽  
Varicella Zoster Virus ◽  
Phosphorylation Site ◽  
Cytoplasmic Tail ◽  
Casein Kinase Ii ◽  
Fc Receptor ◽  
Casein Kinase ◽  
Cyclin Dependent Kinase ◽  
Varicella Zoster

Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.

Nuclear import of serum response factor (SRF) requires a short amino-terminal nuclear localization sequence and is independent of the casein kinase II phosphorylation site

1994 ◽  
Vol 107 (11) ◽  
pp. 3029-3036
Author(s):  
J. Rech ◽  
I. Barlat ◽  
J.L. Veyrune ◽  
A. Vie ◽  
J.M. Blanchard
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Serum Response Factor ◽  
Phosphorylation Site ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Resting Cells ◽  
Response Factor ◽  
Amino Terminal ◽  
Serum Response

Serum stimulation of resting cells is mediated at least in part at the transcriptional level by the activation of numerous genes among which c-fos constitutes a model. Serum response factor (SRF) forms a ternary complex at the c-fos serum response element (SRE) with an accessory protein p62TCF/Elk-1. Both proteins are the targets of multiple phosphorylation events and their role is still unknown in the amino terminus of SRF. While the transcriptional activation domain has been mapped between amino acids 339 and 508, the DNA-binding and the dimerization domains have been mapped to between amino acids 133–235 and 168–235, respectively, no role has been proposed for the amino-terminal portion of the molecule. We demonstrate in the present work that amino acids 95 to 100 contain a stretch of basic amino acids that are sufficient to target a reporter protein to the nucleus. Moreover, this sequence appears to be the only nuclear localization signal operating in SRF. Finally, whereas the global structure around this putative nuclear location signal is reminiscent of what is found in the SV40 T antigen, the casein kinase II phosphorylation site does not determine the rate of cyto-nuclear protein transport of this protein.

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