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Site-specific and dose-dependent effects of glucocorticoid receptor phosphorylation in yeast Saccharomyces cerevisiae

The glucocorticoid receptor (GR) signal transduction and transcriptional regulation are efficiently recapitulated when GR is expressed in Saccharomyces cerevisiae. In this report we demonstrate that the in vivo GR phosphorylation pattern, hormone dependency and interdependency of phosphorylation eve...

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Published in:Steroids 2010-06, Vol.75 (6), p.457-465
Main Authors: Popovic, Natasa, Ruzdijic, Sabera, Kanazir, Dusan T., Niciforovic, Ana, Adzic, Miroslav, Paraskevopoulou, Elissavet, Pantelidou, Constantia, Radojcic, Marija, Demonacos, Constantinos, Krstic-Demonacos, Marija
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container_title Steroids
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creator Popovic, Natasa
Ruzdijic, Sabera
Kanazir, Dusan T.
Niciforovic, Ana
Adzic, Miroslav
Paraskevopoulou, Elissavet
Pantelidou, Constantia
Radojcic, Marija
Demonacos, Constantinos
Krstic-Demonacos, Marija
description The glucocorticoid receptor (GR) signal transduction and transcriptional regulation are efficiently recapitulated when GR is expressed in Saccharomyces cerevisiae. In this report we demonstrate that the in vivo GR phosphorylation pattern, hormone dependency and interdependency of phosphorylation events were similar in yeast and mammalian cells. GR phosphorylation at S246 exhibited inhibitory effect on S224 and S232 phosphorylation, suggesting the conservation of molecular mechanisms that control this interdependence between yeast and mammalian cells. To assess the effects of GR phosphorylation the mutated GR derivatives T171A, S224A, S232A, S246A were overexpressed and their transcriptional activity was analysed. These receptor derivatives displayed significant hormone inducible transcription when overexpressed in S. cerevisiae. We have established an inducible methionine expression system, which allows the close regulation of the receptor protein levels to analyse the dependence of GR function on its phosphorylation and protein abundance. Using this system we observed that GR S246A mutation increased its activity across all of the GR concentrations tested. The activity of the S224A and S246A mutants was mostly independent of GR protein levels, whereas the WT, T171A and S232A mediated transcription diminished with declining GR protein levels. Our results suggest that GR phosphorylation at specific residues affects its transcriptional functions in a site selective manner and these effects were directly linked to GR dosage.
doi_str_mv 10.1016/j.steroids.2010.03.001
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identifier ISSN: 0039-128X
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source Elsevier
subjects Amino Acid Sequence
Animals
Biological and medical sciences
Fundamental and applied biological sciences. Psychology
Glucocorticoid receptor
Humans
Molecular Sequence Data
Mutant Proteins - metabolism
Mutation
Peptide Mapping
Phosphopeptides - genetics
Phosphopeptides - metabolism
Phosphorylation
Protein Isoforms - genetics
Protein Isoforms - metabolism
Rats
Receptors, Glucocorticoid - chemistry
Receptors, Glucocorticoid - genetics
Receptors, Glucocorticoid - metabolism
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - chemistry
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
Signal Transduction - physiology
Transcription
Vertebrates: endocrinology
Yeast
title Site-specific and dose-dependent effects of glucocorticoid receptor phosphorylation in yeast Saccharomyces cerevisiae
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