Identification of four novel phosphorylation sites in estrogen receptor α: IImpact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

Abstract

Background. Estrogen receptor (ER) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ER phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ER phosphorylation sites exist, COS-1 cells expressing human ER were labeled with [32P]H 3PO4in vivo and ER tryptic phosphopeptides were isolated to identify phosphorylation sites. Results. Previously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ER in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ER as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERα at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor. Conclusion. These novel ERα phosphorylation sites represent new means for modulation of ER activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor. © 2009 Williams et al; licensee BioMed Central Ltd.