Using yeast to study glucocorticoid receptor phosphorylation
Само за регистроване кориснике
1998
Аутори
Počuča, NatašaRuždijić, Sabera
Demonacos, Constantinos
Kanazir, Dušan T.
Krstić-Demonacos, Marija
Чланак у часопису (Објављена верзија)
Метаподаци
Приказ свих података о документуАпстракт
The glucocorticoid receptor (GR) is a phosphoprotein and a member of the steroid/thyroid receptor superfamily of ligand dependent transcription factors. When the glucocorticoid receptor is expressed in yeast (Saccharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation. We have studied the glucocorticoid receptor phosphorylation in yeast and demonstrated that the receptor is phosphorylated in both the absence and presence of hormone, on serine and threonine residues. This phosphorylation occurs within 15 min upon addition of radioactivity in both hormone treated and untreated cells. As reported for mammalian cells, additional phosphorylation occurs upon hormone binding and this phosphorylation is dependent on the type of the ligand. We have followed the hormone dependent receptor phosphorylation by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment. In addition, the appearance of horm...one dependent phosphoisoforms of the receptor depends 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 amino acids 130-247. We altered the phosphorylation sites within this domain that correspond to the amino acids phosphorylated in mouse hormone treated cells. Using phosphopeptide maps we show that hormone changes the peptide pattern of metabolically labelled receptor, and we identify peptides which are phosphorylated in hormone dependent manner. Then we determine that phosphorylation of residues S224 and S232 is increased 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 suitable system to study glucocorticoid receptor phosphorylation. The genetic manipulability of yeast cells, together with conservation of the phosphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of molecules involved in the glucocorticoid receptor phosphorylation pathway and further our understanding of this process. (C) 1998 Elsevier Science Ltd. All rights reserved.
Извор:
Journal of Steroid Biochemistry and Molecular Biology, 1998, 66, 5-6, 303-318
DOI: 10.1016/S0960-0760(98)00057-0
ISSN: 0960-0760
PubMed: 9749836
WoS: 000075747500005
Scopus: 2-s2.0-0031788142
Колекције
Институција/група
VinčaTY - JOUR AU - Počuča, Nataša AU - Ruždijić, Sabera AU - Demonacos, Constantinos AU - Kanazir, Dušan T. AU - Krstić-Demonacos, Marija PY - 1998 UR - https://vinar.vin.bg.ac.rs/handle/123456789/2188 AB - The glucocorticoid receptor (GR) is a phosphoprotein and a member of the steroid/thyroid receptor superfamily of ligand dependent transcription factors. When the glucocorticoid receptor is expressed in yeast (Saccharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation. We have studied the glucocorticoid receptor phosphorylation in yeast and demonstrated that the receptor is phosphorylated in both the absence and presence of hormone, on serine and threonine residues. This phosphorylation occurs within 15 min upon addition of radioactivity in both hormone treated and untreated cells. As reported for mammalian cells, additional phosphorylation occurs upon hormone binding and this phosphorylation is dependent on the type of the ligand. We have followed the hormone dependent receptor phosphorylation by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment. In addition, the appearance of hormone dependent phosphoisoforms of the receptor depends 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 amino acids 130-247. We altered the phosphorylation sites within this domain that correspond to the amino acids phosphorylated in mouse hormone treated cells. Using phosphopeptide maps we show that hormone changes the peptide pattern of metabolically labelled receptor, and we identify peptides which are phosphorylated in hormone dependent manner. Then we determine that phosphorylation of residues S224 and S232 is increased 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 suitable system to study glucocorticoid receptor phosphorylation. The genetic manipulability of yeast cells, together with conservation of the phosphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of molecules involved in the glucocorticoid receptor phosphorylation pathway and further our understanding of this process. (C) 1998 Elsevier Science Ltd. All rights reserved. T2 - Journal of Steroid Biochemistry and Molecular Biology T1 - Using yeast to study glucocorticoid receptor phosphorylation VL - 66 IS - 5-6 SP - 303 EP - 318 DO - 10.1016/S0960-0760(98)00057-0 ER -
@article{ author = "Počuča, Nataša and Ruždijić, Sabera and Demonacos, Constantinos and Kanazir, Dušan T. and Krstić-Demonacos, Marija", year = "1998", abstract = "The glucocorticoid receptor (GR) is a phosphoprotein and a member of the steroid/thyroid receptor superfamily of ligand dependent transcription factors. When the glucocorticoid receptor is expressed in yeast (Saccharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation. We have studied the glucocorticoid receptor phosphorylation in yeast and demonstrated that the receptor is phosphorylated in both the absence and presence of hormone, on serine and threonine residues. This phosphorylation occurs within 15 min upon addition of radioactivity in both hormone treated and untreated cells. As reported for mammalian cells, additional phosphorylation occurs upon hormone binding and this phosphorylation is dependent on the type of the ligand. We have followed the hormone dependent receptor phosphorylation by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment. In addition, the appearance of hormone dependent phosphoisoforms of the receptor depends 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 amino acids 130-247. We altered the phosphorylation sites within this domain that correspond to the amino acids phosphorylated in mouse hormone treated cells. Using phosphopeptide maps we show that hormone changes the peptide pattern of metabolically labelled receptor, and we identify peptides which are phosphorylated in hormone dependent manner. Then we determine that phosphorylation of residues S224 and S232 is increased 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 suitable system to study glucocorticoid receptor phosphorylation. The genetic manipulability of yeast cells, together with conservation of the phosphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of molecules involved in the glucocorticoid receptor phosphorylation pathway and further our understanding of this process. (C) 1998 Elsevier Science Ltd. All rights reserved.", journal = "Journal of Steroid Biochemistry and Molecular Biology", title = "Using yeast to study glucocorticoid receptor phosphorylation", volume = "66", number = "5-6", pages = "303-318", doi = "10.1016/S0960-0760(98)00057-0" }
Počuča, N., Ruždijić, S., Demonacos, C., Kanazir, D. T.,& Krstić-Demonacos, M.. (1998). Using yeast to study glucocorticoid receptor phosphorylation. in Journal of Steroid Biochemistry and Molecular Biology, 66(5-6), 303-318. https://doi.org/10.1016/S0960-0760(98)00057-0
Počuča N, Ruždijić S, Demonacos C, Kanazir DT, Krstić-Demonacos M. Using yeast to study glucocorticoid receptor phosphorylation. in Journal of Steroid Biochemistry and Molecular Biology. 1998;66(5-6):303-318. doi:10.1016/S0960-0760(98)00057-0 .
Počuča, Nataša, Ruždijić, Sabera, Demonacos, Constantinos, Kanazir, Dušan T., Krstić-Demonacos, Marija, "Using yeast to study glucocorticoid receptor phosphorylation" in Journal of Steroid Biochemistry and Molecular Biology, 66, no. 5-6 (1998):303-318, https://doi.org/10.1016/S0960-0760(98)00057-0 . .