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

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