Comprehensive mapping of post-translational modifications of progesterone receptor (PR) and functional analysis of novel methylation and acetylation of PR.

There is increasing evidence that progesterone is involved in breast cancer progression. The progesterone receptor (PR) is a member of the nuclear receptor superfamily. Its activity is regulated at multiple levels of the receptor activation pathway including receptor maturation, ligand-bind...

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Main Author: Chung, Hwa Hwa.
Other Authors: Lin Chun Ling Valerie
Format: Theses and Dissertations
Language:English
Published: 2013
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Online Access:http://hdl.handle.net/10356/54720
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Institution: Nanyang Technological University
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spelling sg-ntu-dr.10356-547202023-02-28T18:31:00Z Comprehensive mapping of post-translational modifications of progesterone receptor (PR) and functional analysis of novel methylation and acetylation of PR. Chung, Hwa Hwa. Lin Chun Ling Valerie School of Biological Sciences DRNTU::Science::Biological sciences There is increasing evidence that progesterone is involved in breast cancer progression. The progesterone receptor (PR) is a member of the nuclear receptor superfamily. Its activity is regulated at multiple levels of the receptor activation pathway including receptor maturation, ligand-binding, co-regulator recruitment and interaction with gene promoters. Post-translational modifications (PTMs) can regulate each of the steps through alteration of local properties of the protein. To gain in-depth understanding of the mechanisms that regulate PR activity, this study conducted a comprehensive mapping of PR PTMs using the highly sensitive liquid chromatography tandem Mass Spectrometry (LC-MS/MS) and identified 4 novel phosphorylation sites and 8 novel 'methylation and 1 acetylation sites on PR. The MS study was further extended to the next 2 parts of this project which focused on validating K464 methylation and K183 acetylation and determining the biological relevance of the modifications in regulating activity of PR. The autonomous transcriptional activation function 1, AF-1, of PR is responsible for its ligand-independent activation and synergize with AF-2 (activation function 2) to mediate maximal ligand-induced PR activity. One of the novel methylation sites is located at K464 of AF-l. Mutational analysis revealed that K464 and its methylation play a key role in suppressing ligand-independent PR phosphorylation and AF-l activation. A single mutation K464Q or K464A exhibited ligand-independent PR gel upshift that is akin to the ligand-induced PR gel upshift on SDS-PAGE because of phosphorylation. Both K464Q and K464A mutants displayed heightened ligand-independent and ligand-dependent activity that is due to increased activity of AF-l. The study also suggests that the heightened ligand-induced activity of PRB-K464Q is mediated through increased functional interaction with transcription co-regulators NCoR-l(Nuclear receptor corepressor 1) and SRC-l (Steroid receptor coactivator 1). Contrastingly, a mutation of K464 to the bulkier arginine or phenylalanine, mimicking methylation effects, had no effect or inhibited PR activity which suggests K464 methylation inhibits PR activity. In conclusion, this study demonstrated that K464 is a key element that represses ligand-independent and ligandinduced activation of AF -1. K183 is identified as a novel acetylation site of PR by LC/MS/MS. Mutagenesis studies verified that K183 is the primary site of acetylation. Overexpression and gene silencing approaches confirmed that the acetyltransferase p300 was the primary enzyme for K183 acetylation. In contrast to the repressive effect of methylation described earlier, the consequence of K183 acetylation was to promote PR phosphorylation and enhance PR activity). More interestingly, the effect of K183 acetylation seemed to be exerted specifically on' selective promoters. In summary, the results presented herein indicate that acetylation serves as a finely tuned regulatory mechanism to control activation magnitude and promoter selectivity of PR. ​Doctor of Philosophy (SBS) 2013-07-25T07:08:26Z 2013-07-25T07:08:26Z 2013 2013 Thesis http://hdl.handle.net/10356/54720 en 246 p. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic DRNTU::Science::Biological sciences
spellingShingle DRNTU::Science::Biological sciences
Chung, Hwa Hwa.
Comprehensive mapping of post-translational modifications of progesterone receptor (PR) and functional analysis of novel methylation and acetylation of PR.
description There is increasing evidence that progesterone is involved in breast cancer progression. The progesterone receptor (PR) is a member of the nuclear receptor superfamily. Its activity is regulated at multiple levels of the receptor activation pathway including receptor maturation, ligand-binding, co-regulator recruitment and interaction with gene promoters. Post-translational modifications (PTMs) can regulate each of the steps through alteration of local properties of the protein. To gain in-depth understanding of the mechanisms that regulate PR activity, this study conducted a comprehensive mapping of PR PTMs using the highly sensitive liquid chromatography tandem Mass Spectrometry (LC-MS/MS) and identified 4 novel phosphorylation sites and 8 novel 'methylation and 1 acetylation sites on PR. The MS study was further extended to the next 2 parts of this project which focused on validating K464 methylation and K183 acetylation and determining the biological relevance of the modifications in regulating activity of PR. The autonomous transcriptional activation function 1, AF-1, of PR is responsible for its ligand-independent activation and synergize with AF-2 (activation function 2) to mediate maximal ligand-induced PR activity. One of the novel methylation sites is located at K464 of AF-l. Mutational analysis revealed that K464 and its methylation play a key role in suppressing ligand-independent PR phosphorylation and AF-l activation. A single mutation K464Q or K464A exhibited ligand-independent PR gel upshift that is akin to the ligand-induced PR gel upshift on SDS-PAGE because of phosphorylation. Both K464Q and K464A mutants displayed heightened ligand-independent and ligand-dependent activity that is due to increased activity of AF-l. The study also suggests that the heightened ligand-induced activity of PRB-K464Q is mediated through increased functional interaction with transcription co-regulators NCoR-l(Nuclear receptor corepressor 1) and SRC-l (Steroid receptor coactivator 1). Contrastingly, a mutation of K464 to the bulkier arginine or phenylalanine, mimicking methylation effects, had no effect or inhibited PR activity which suggests K464 methylation inhibits PR activity. In conclusion, this study demonstrated that K464 is a key element that represses ligand-independent and ligandinduced activation of AF -1. K183 is identified as a novel acetylation site of PR by LC/MS/MS. Mutagenesis studies verified that K183 is the primary site of acetylation. Overexpression and gene silencing approaches confirmed that the acetyltransferase p300 was the primary enzyme for K183 acetylation. In contrast to the repressive effect of methylation described earlier, the consequence of K183 acetylation was to promote PR phosphorylation and enhance PR activity). More interestingly, the effect of K183 acetylation seemed to be exerted specifically on' selective promoters. In summary, the results presented herein indicate that acetylation serves as a finely tuned regulatory mechanism to control activation magnitude and promoter selectivity of PR.
author2 Lin Chun Ling Valerie
author_facet Lin Chun Ling Valerie
Chung, Hwa Hwa.
format Theses and Dissertations
author Chung, Hwa Hwa.
author_sort Chung, Hwa Hwa.
title Comprehensive mapping of post-translational modifications of progesterone receptor (PR) and functional analysis of novel methylation and acetylation of PR.
title_short Comprehensive mapping of post-translational modifications of progesterone receptor (PR) and functional analysis of novel methylation and acetylation of PR.
title_full Comprehensive mapping of post-translational modifications of progesterone receptor (PR) and functional analysis of novel methylation and acetylation of PR.
title_fullStr Comprehensive mapping of post-translational modifications of progesterone receptor (PR) and functional analysis of novel methylation and acetylation of PR.
title_full_unstemmed Comprehensive mapping of post-translational modifications of progesterone receptor (PR) and functional analysis of novel methylation and acetylation of PR.
title_sort comprehensive mapping of post-translational modifications of progesterone receptor (pr) and functional analysis of novel methylation and acetylation of pr.
publishDate 2013
url http://hdl.handle.net/10356/54720
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