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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 |
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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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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.</description><identifier>ISSN: 0039-128X</identifier><identifier>EISSN: 1878-5867</identifier><identifier>DOI: 10.1016/j.steroids.2010.03.001</identifier><identifier>PMID: 20223255</identifier><identifier>CODEN: STEDAM</identifier><language>eng</language><publisher>Kidlington: Elsevier Inc</publisher><subject>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</subject><ispartof>Steroids, 2010-06, Vol.75 (6), p.457-465</ispartof><rights>2010</rights><rights>2015 INIST-CNRS</rights><rights>Crown Copyright 2010. Published by Elsevier Inc. All rights reserved.</rights><rights>2010 Elsevier Inc. 2010</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c500t-bec233ef0d18f8d27d5f65c81d44d83b2c0d7ba288aedb6e3a86d9be27e0b8f13</citedby><cites>FETCH-LOGICAL-c500t-bec233ef0d18f8d27d5f65c81d44d83b2c0d7ba288aedb6e3a86d9be27e0b8f13</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,314,780,784,885,27924,27925</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=22650686$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/20223255$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Popovic, Natasa</creatorcontrib><creatorcontrib>Ruzdijic, Sabera</creatorcontrib><creatorcontrib>Kanazir, Dusan T.</creatorcontrib><creatorcontrib>Niciforovic, Ana</creatorcontrib><creatorcontrib>Adzic, Miroslav</creatorcontrib><creatorcontrib>Paraskevopoulou, Elissavet</creatorcontrib><creatorcontrib>Pantelidou, Constantia</creatorcontrib><creatorcontrib>Radojcic, Marija</creatorcontrib><creatorcontrib>Demonacos, Constantinos</creatorcontrib><creatorcontrib>Krstic-Demonacos, Marija</creatorcontrib><title>Site-specific and dose-dependent effects of glucocorticoid receptor phosphorylation in yeast Saccharomyces cerevisiae</title><title>Steroids</title><addtitle>Steroids</addtitle><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.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Glucocorticoid receptor</subject><subject>Humans</subject><subject>Molecular Sequence Data</subject><subject>Mutant Proteins - metabolism</subject><subject>Mutation</subject><subject>Peptide Mapping</subject><subject>Phosphopeptides - genetics</subject><subject>Phosphopeptides - metabolism</subject><subject>Phosphorylation</subject><subject>Protein Isoforms - genetics</subject><subject>Protein Isoforms - metabolism</subject><subject>Rats</subject><subject>Receptors, Glucocorticoid - chemistry</subject><subject>Receptors, Glucocorticoid - genetics</subject><subject>Receptors, Glucocorticoid - metabolism</subject><subject>Saccharomyces cerevisiae - genetics</subject><subject>Saccharomyces cerevisiae - metabolism</subject><subject>Saccharomyces cerevisiae Proteins - chemistry</subject><subject>Saccharomyces cerevisiae Proteins - genetics</subject><subject>Saccharomyces cerevisiae Proteins - metabolism</subject><subject>Signal Transduction - physiology</subject><subject>Transcription</subject><subject>Vertebrates: endocrinology</subject><subject>Yeast</subject><issn>0039-128X</issn><issn>1878-5867</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2010</creationdate><recordtype>article</recordtype><recordid>eNqFkcFuEzEQhi0EomnhFSpfEKcNY7u761wQqAKKVIlDQeJmecfjxtFmvdhOpLw9rpIWOHGwLNnf_DP2x9ilgKUA0b3bLHOhFIPLSwn1ENQSQDxjC6F73bS665-zBYBaNULqn2fsPOcNAHRqJV-yMwlSKtm2C7a7C4WaPBMGH5DbyXEXMzWOZpocTYWT94Ql8-j5_bjDiDGVgLUzT4Q0l5j4vI65rnQYbQlx4mHiB7K58DuLuLYpbg9ImSMl2occLL1iL7wdM70-7Rfsx-dP369vmttvX75ef7xtsAUozUAolSIPTmivnexd67sWtXBXV06rQSK4frBSa0tu6EhZ3bnVQLInGLQX6oK9P-bOu2FLDut7kh3NnMLWpoOJNph_b6awNvdxb2ok9CtVA96eAlL8taNczDZkpHG0E8VdNr1SquoQUMnuSGKKOSfyT10EmAdlZmMelZkHZQaUqcpq4eXfMz6VPTqqwJsTYDPa0Sc7Ych_ONm10Omuch-OHNUf3QdKJmOgCcmFaqoYF8P_ZvkNYfa9_A</recordid><startdate>20100601</startdate><enddate>20100601</enddate><creator>Popovic, Natasa</creator><creator>Ruzdijic, Sabera</creator><creator>Kanazir, Dusan T.</creator><creator>Niciforovic, Ana</creator><creator>Adzic, Miroslav</creator><creator>Paraskevopoulou, Elissavet</creator><creator>Pantelidou, Constantia</creator><creator>Radojcic, Marija</creator><creator>Demonacos, Constantinos</creator><creator>Krstic-Demonacos, Marija</creator><general>Elsevier Inc</general><general>Elsevier</general><scope>6I.</scope><scope>AAFTH</scope><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20100601</creationdate><title>Site-specific and dose-dependent effects of glucocorticoid receptor phosphorylation in yeast Saccharomyces cerevisiae</title><author>Popovic, Natasa ; Ruzdijic, Sabera ; Kanazir, Dusan T. ; Niciforovic, Ana ; Adzic, Miroslav ; Paraskevopoulou, Elissavet ; Pantelidou, Constantia ; Radojcic, Marija ; Demonacos, Constantinos ; Krstic-Demonacos, Marija</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c500t-bec233ef0d18f8d27d5f65c81d44d83b2c0d7ba288aedb6e3a86d9be27e0b8f13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2010</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Glucocorticoid receptor</topic><topic>Humans</topic><topic>Molecular Sequence Data</topic><topic>Mutant Proteins - metabolism</topic><topic>Mutation</topic><topic>Peptide Mapping</topic><topic>Phosphopeptides - genetics</topic><topic>Phosphopeptides - metabolism</topic><topic>Phosphorylation</topic><topic>Protein Isoforms - genetics</topic><topic>Protein Isoforms - metabolism</topic><topic>Rats</topic><topic>Receptors, Glucocorticoid - chemistry</topic><topic>Receptors, Glucocorticoid - genetics</topic><topic>Receptors, Glucocorticoid - metabolism</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Saccharomyces cerevisiae - metabolism</topic><topic>Saccharomyces cerevisiae Proteins - chemistry</topic><topic>Saccharomyces cerevisiae Proteins - genetics</topic><topic>Saccharomyces cerevisiae Proteins - metabolism</topic><topic>Signal Transduction - physiology</topic><topic>Transcription</topic><topic>Vertebrates: endocrinology</topic><topic>Yeast</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Popovic, Natasa</creatorcontrib><creatorcontrib>Ruzdijic, Sabera</creatorcontrib><creatorcontrib>Kanazir, Dusan T.</creatorcontrib><creatorcontrib>Niciforovic, Ana</creatorcontrib><creatorcontrib>Adzic, Miroslav</creatorcontrib><creatorcontrib>Paraskevopoulou, Elissavet</creatorcontrib><creatorcontrib>Pantelidou, Constantia</creatorcontrib><creatorcontrib>Radojcic, Marija</creatorcontrib><creatorcontrib>Demonacos, Constantinos</creatorcontrib><creatorcontrib>Krstic-Demonacos, Marija</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Steroids</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Popovic, Natasa</au><au>Ruzdijic, Sabera</au><au>Kanazir, Dusan T.</au><au>Niciforovic, Ana</au><au>Adzic, Miroslav</au><au>Paraskevopoulou, Elissavet</au><au>Pantelidou, Constantia</au><au>Radojcic, Marija</au><au>Demonacos, Constantinos</au><au>Krstic-Demonacos, Marija</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Site-specific and dose-dependent effects of glucocorticoid receptor phosphorylation in yeast Saccharomyces cerevisiae</atitle><jtitle>Steroids</jtitle><addtitle>Steroids</addtitle><date>2010-06-01</date><risdate>2010</risdate><volume>75</volume><issue>6</issue><spage>457</spage><epage>465</epage><pages>457-465</pages><issn>0039-128X</issn><eissn>1878-5867</eissn><coden>STEDAM</coden><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.</abstract><cop>Kidlington</cop><pub>Elsevier Inc</pub><pmid>20223255</pmid><doi>10.1016/j.steroids.2010.03.001</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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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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