Identification of phosphorylation sites in the mouse oestrogen receptor

Abstract Phosphorylation sites in the mouse oestrogen receptor, expressed in COS-1 cells in the presence of 17β-oestradiol, have been mapped by solid phase microsequencing. The receptor was first radiolabelled with [ 32 P]orthophosphate and a number of 3 H- or 14 C-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 α-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.