USING YEAST TO STUDY GLUCOCORTICOID RECEPTOR PHOSPHORYLATION

The glucocorticoid receptor (GR) is a phosphoprotein and a member of t he steroid/thyroid receptor superfamily of ligand dependent transcript ion factors. When the glucocorticoid receptor is expressed in yeast (S accharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation. We have studied the glucocorticoid recepto r phosphorylation in yeast and demonstrated that the receptor is phosp horylated in both the absence and presence of hormone, on serine and t hreonine residues. This phosphorylation occurs within 15 min upon addi tion of radioactivity in both hormone treated and untreated cells. As reported for mammalian cells, additional phosphorylation occurs upon h ormone binding and this phosphorylation is dependent on the type of th e ligand. We have followed the hormone dependent receptor phosphorylat ion by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment. In addition, th e appearance of hormone dependent phosphoisoforms of the receptor depe nds on the potency of the agonist used. Using this method we show that the residues contributing to the hormone dependent mobility shift are localized in one of the transcriptional activation domains, between a mino acids 130-247. We altered the phosphorylation sites within this d omain that correspond to the amino acids phosphorylated in mouse hormo ne treated cells. Using phosphopeptide maps we show that hormone chang es the peptide pattern of metabolically labelled receptor, and we iden tify peptides which are phosphorylated in hormone dependent manner. Th en we determine that phosphorylation of residues S224 and S232 is incr eased in the presence of hormone, whereas phosphorylation of residues T171 and S246 is constitutive. Finally, we show that in both yeast and mammalian cells the same residues on the glucocorticoid receptor are phosphorylated. Our results suggest that yeast cells would be a suitab le system to study glucocorticoid receptor phosphorylation. The geneti c manipulability of yeast cells, together with conservation of the pho sphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of m olecules involved in the glucocorticoid receptor phosphorylation pathw ay and further our understanding of this process. (C) 1998 Elsevier Sc ience Ltd. All rights reserved.