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Identification of phosphorylation sites in the mouse oestrogen receptor
H Lahooti1, R White, S A Hoare
1Molecular Endocrinology, Laboratory Imperial Cancer Research Fund, London, U.K.
Abstract:
Phosphorylation sites in the mouse oestrogen receptor, expressed in COS-1 cells in the presence of 17 beta-oestradiol, have been mapped by solid phase microsequencing. The receptor was first radio-labelled with [32P]orthophosphate and a number of 3H- or 14C-labelled amino acids, immunopurified and then tryptic peptides were separated by thin layer chromatography or high performance liquid chromatography. Amino acid sequence analysis indicated that Ser-122, Ser-156, Ser-158 and Ser-298 were phosphorylated. The substitution of Ser-122 and Ser-298 with alanine had a negligible effect on the transcriptional activity of the receptor in transfected cells. However, a reduction of transcriptional activity was observed when Ser-122 was mutated in the context of mutations in a putative amphipathic alpha-helix involved in AF-2 activity. Thus a region of AF-1 that encompasses Ser-122 appears to interact with AF-2 in the full-length receptor.
Insights
Researchers identified key phosphorylation sites on the mouse oestrogen receptor, revealing how Ser-122 interacts with AF-2 to influence transcriptional activity.
Area of Science:
- Molecular Biology
- Endocrinology
- Protein Chemistry
Background:
- The oestrogen receptor (ER) is a crucial nuclear receptor involved in various physiological processes.
- Understanding the post-translational modifications, such as phosphorylation, of ER is vital for elucidating its regulatory mechanisms.
- Specific phosphorylation sites can modulate ER's transcriptional activity and interactions with co-regulators.
Purpose of the Study:
- To map the phosphorylation sites on the mouse oestrogen receptor.
- To investigate the functional impact of specific phosphorylation sites on ER transcriptional activity.
- To explore the interplay between different functional domains of the oestrogen receptor.
Main Methods:
- Expression of mouse oestrogen receptor in COS-1 cells with 17 beta-oestradiol.
- Radio-labelling of the receptor with [32P]orthophosphate and amino acids.
- Immunopurification and tryptic peptide mapping using thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC).
- Amino acid sequencing and site-directed mutagenesis (Serine to Alanine substitutions).
Main Results:
- Identified four phosphorylation sites: Ser-122, Ser-156, Ser-158, and Ser-298.
- Mutating Ser-122 and Ser-298 to alanine showed negligible impact on transcriptional activity.
- Mutating Ser-122 within a specific helical region (involved in AF-2 activity) reduced transcriptional activity.
- A region in AF-1, including Ser-122, appears to interact with AF-2 in the full-length receptor.
Conclusions:
- Ser-122 is a key phosphorylation site influencing oestrogen receptor transcriptional activity.
- The N-terminal AF-1 domain, specifically Ser-122, plays a role in interacting with the AF-2 domain.
- These findings provide insights into the complex regulation of oestrogen receptor function through phosphorylation and domain interactions.