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 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.
Popovic
Natasa
N
VINCA Institute of Nuclear Sciences, P.O. Box 522, 11001 Belgrade, Serbia.
Ruzdijic
Sabera
S
Kanazir
Dusan T
DT
Niciforovic
Ana
A
Adzic
Miroslav
M
Paraskevopoulou
Elissavet
E
Pantelidou
Constantia
C
Radojcic
Marija
M
Demonacos
Constantinos
C
Krstic-Demonacos
Marija
M
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
D050505:Mutant Proteins; D010748:Phosphopeptides; D020033:Protein Isoforms; D011965:Receptors, Glucocorticoid; D029701:Saccharomyces cerevisiae Proteins