Abstract 3933: ERα phosphorylation patterns at hinge domain Ser294 and Ser305 differentiate ligand-dependent from ligand-independent receptor activation
Authors: Scott Gary K. , Held Jason , Britton David J. , Lee Elbert , Schilling Birgit , Baldwin Michael , Gibson Brad W. , Benz Christopher C. 
Published: August 2012

Abstract
Domain-specific phosphorylation of estrogen receptor alpha (ERα) is critical for its transcription factor activity and may determine its predictive and therapeutic biomarker value. Recent attention has turned to the poorly understood ERα hinge domain in light of preliminary evidence that phosphorylation at serine 305 (pSer305) associates with poor clinical outcome and endocrine resistance. Using multiple reaction monitoring (MRM) mass spectrometry (MS), we have studied a number of ERα overexpressing human breast cancer cells lines (MCF-7, BT474, T47D) and found that robust ERα phosphorylation occurs on a neighboring hinge domain site at Ser294, but only upon ligand (estradiol, tamoxifen) stimulation of the receptor since growth factor cell stimulation (EGF, insulin, heregulin-α, forskolin) fails to induce pSer294. As well, pSer294 formation appears to be essential for full ligand induction of ERα gene transcription in experimental models. Selective inhibition of cyclin-dependent kinases (CDKs) by roscovitine or SNS-032 in the breast cancer cell lines blocks ligand induced pSer294 formation, indicating that this hinge domain phosphorylation is mediated by CDKs; likewise, cell-free studies with recombinant ERα and specific cyclin-CDK complexes reveal that Ser294 phosphorylation is most effectively induced by the transcription-regulating and cell cycle-independent kinases, CDK7 and CDK9. In contrast to this ligand specific induction of pSer294, western analysis of immunopurified ERα from MCF-7 probed with a newly available pSer305 monoclonal demonstrates that pSer305 formation occurs in response to growth factor stimulation (EGF, insulin, forskolin) but not in response to cell stimulation by ligand alone. Interestingly, co-treatment with ligand and growth factor reduces pSer294 formation relative to ligand stimulation alone while co-treatment reduces pSer305 formation relative to growth factor stimulation alone, suggesting that these two hinge phosphorylation sites not only respond reciprocally to specify different aspects of ERα function but are also antagonistically responsive to crosstalk. Unlike the better studied N-terminal domain phosphorylation sites at Ser118 and Ser167, which are phosphorylated jointly and additively upon combined ligand-dependent and ligand-independent ERα activation, the two hinge domain sites at Ser294 and Ser305 appear to respond in a reciprocal fashion differentiating ligand-dependent from ligand-independent ERα activation. Pending current development of a pSer294 monoclonal, future immunohistochemical interrogation of the ERα hinge domain phosphorylation status at Ser294 and Ser305 is expected to improve the biomarker value of this receptor by better predicting ERα overexpressing breast cancers most likely to respond to endocrine agents.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3933. doi:1538-7445.AM2012-3933