Identification of phosphorylation sites in the mouse oestrogen receptor

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 radiolabelled with [P-32] orthophosphate and a number of H-3- or C-14-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 a-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.