Ligand Binding Promotes CDK-Dependent Phosphorylation of ER-Alpha on Hinge Serine 294 but Inhibits Ligand-Independent Phosphorylation of Serine 305

Phosphorylation of estrogen receptor-alpha (ER alpha) is critical for its transcription factor activity and may determine its predictive and therapeutic value as a biomarker for ER alpha-positive breast cancers. Recent attention has turned to the poorly understood ERa hinge domain, as phosphorylation at serine 305 (Ser305) associates with poor clinical outcome and endocrine resistance. We show that phosphorylation of a neighboring hinge domain site, Ser294, analyzed by multiple reaction monitoring mass spectrometry of ER alpha immunoprecipitates from human breast cancer cells is robustly phosphorylated exclusively by ligand (estradiol and tamoxifen) activation of ER alpha and not by growth factor stimulation (EGF, insulin, heregulin-beta). In a reciprocal fashion, Ser305 phosphorylation is induced by growth factors but not ligand activation of ER alpha. Phosphorylation at Ser294 and Ser305 is suppressed upon co-stimulation by EGF and ligand, respectively, unlike the N-terminal (AF-1) domain Ser118 and Ser167 sites of ER alpha where phosphorylation is enhanced by ligand and growth factor co-stimulation. Inhibition of cyclin-dependent kinases (CDK) by roscovitine or SNS-032 suppresses ligand-activated Ser294 phosphorylation without affecting Ser118 or Ser104/Ser106 phosphorylation. Likewise, cell-free studies using recombinant ER alpha and specific cyclin-CDK complexes suggest that Ser294 phosphorylation is primarily induced by the transcription-regulating and cell-cycle-independent kinase CDK7. Thus, CDK-dependent phosphorylation at Ser294 differentiates ligand-dependent from ligand-independent activation of Ser305 phosphorylation, showing that hinge domain phosphorylation patterns uniquely inform on the various ER alpha activation mechanisms thought to underlie the biologic and clinical diversity of hormone-dependent breast cancers. Mol Cancer Res; 10(8); 1120-32. (C) 2012 AACR.