Phosphorylation of
estrogen receptor-α (ERα) is critical for its
transcription factor activity and may determine its predictive and therapeutic value as a
biomarker for ERα-positive breast
cancers. Recent attention has turned to the poorly understood ERα 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 ERα immunoprecipitates from human
breast cancer cells is robustly phosphorylated exclusively by
ligand (
estradiol and
tamoxifen) activation of ERα and not by
growth factor stimulation (
EGF,
insulin,
heregulin-β). In a reciprocal fashion, Ser305 phosphorylation is induced by
growth factors but not
ligand activation of ERα. 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 ERα 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 ERα 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 ERα activation mechanisms thought to underlie the biologic and clinical diversity of
hormone-dependent breast
cancers.