Ligand dependent induction of phospho-serine 294 in the hinge region of estrogen receptor alpha within human breast cancer cells

5458 Activated estrogen receptor (ERα) plays a critical role in normal mammary gland physiology as well as in breast cancer development, where it also serves as a major target for breast cancer therapeutics. Serine (S) phosphorylation within the 67 kDa receptor protein is known to contribute significantly to ERα activation by orchestrating its nuclear translocation and the recruitment of transcriptional co-regulators to DNA-bound receptor. Since biochemical methods for detection of S phosphorylation are severely limited, we employed mass spectrometry (MS) to identify and semi-quantitate novel S phosphorylation sites within endogenously expressed ERα from control and growth stimulated human breast cancer cells (MCF7). ERα immunoprecipitated from cultured MCF7 cells was proteolytically digested, and the resulting peptides were MS analyzed using nano-LC-ESI-MS/MS (Q-STAR, MDS Sciex) and vMALDI-MSn (FinniganTM LTQTM, Thermo-Electron). S phosphorylation changes in ERα peptides were semi-quantitated using an ion trap instrument (4000 QTRAP, ABI/MDS Sciex) operated in multiple reaction monitoring (MRM) mode. Trypsin digests yielded 75% peptide coverage of the ERα hinge region (D domain residues Q280 to K309), leading to identification of a novel phospho-S site, pS294. Comparison of pS294 levels relative to induction of known ERα phospho-S sites revealed an 11-fold increase in pS294 relative to a 10-fold increase in pS167 and a 6-fold increase in pS154 following estradiol (10 nM, 30 min) stimulation. In contrast, EGF (50 ng/ml, 10 min) stimulation produced no detectable induction of pS294 but increased pS167 20-fold and pS154 3-fold. The novel observation of significant ERα pS294 induction following estradiol but not EGF stimulation of MCF7 cells suggests that the hinge region actively participates within the ERα activation function 2a (AF2a) domain to selectively recruit co-regulators in response to ligands like estradiol, but not in response to ligand-independent ERα activators like EGF.