title: Site-specific and dose-dependent effects of glucocorticoid receptor phosphorylation in yeast Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: VINCA Institute of Nuclear Sciences, P.O. Box 522, 11001 Belgrade, Serbia.
authors.forename: Natasa
authors.initials: N
authors.lastname: Popovic
authors.forename: Sabera
authors.initials: S
authors.lastname: Ruzdijic
authors.forename: Dusan T
authors.initials: DT
authors.lastname: Kanazir
authors.forename: Ana
authors.initials: A
authors.lastname: Niciforovic
authors.forename: Miroslav
authors.initials: M
authors.lastname: Adzic
authors.forename: Elissavet
authors.initials: E
authors.lastname: Paraskevopoulou
authors.forename: Constantia
authors.initials: C
authors.lastname: Pantelidou
authors.forename: Marija
authors.initials: M
authors.lastname: Radojcic
authors.forename: Constantinos
authors.initials: C
authors.lastname: Demonacos
authors.forename: Marija
authors.initials: M
authors.lastname: Krstic-Demonacos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology
chemical_list: D050505:Mutant Proteins; D010748:Phosphopeptides; D020033:Protein Isoforms; D011965:Receptors, Glucocorticoid; D029701:Saccharomyces cerevisiae Proteins