Phosphorylation of human estrogen receptor α by protein kinase A regulates dimerization

Phosphorylation provides an important mechanism by which transcription factor activity is regulated. Estrogen receptor or (ER alpha) is phosphorylated on multiple sites, and stimulation of a number of growth factor receptors and/or protein kinases leads to ligand-independent and/or synergistic increase in transcriptional activation by ER alpha in the presence of estrogen, Here we show that ER alpha is phosphorylated by protein kinase A (PKA) on serine-236 within the DNA binding domain. Mutation of serine-236 to glutamic acid prevents DNA binding by inhibiting dimerization by ER alpha, whereas mutation to alanine has little effect on DNA binding or dimerization. Furthermore, PKA overexpression or activation of endogenous PKA inhibits dimerization in the absence of ligand, This inhibition is overcome by the addition of 17 beta-estradiol or the partial agonist 4-hydroxy tamoxifen. Interestingly, treatment with the complete antagonist ICI 182,780 does not overcome the inhibitory effect of PKA activation. Our results indicate that in the absence of ligand ER alpha forms dimers through interaction between DNA binding domains and that dimerization mediated by the ligand binding domain only occurs upon ligand binding but that the complete antagonist ICI 182,780 prevents dimerization through the ligand-binding domain. Heterodimer formation between ER alpha and ER beta is similarly affected by PKA phosphorylation of serine 236 of ER alpha, However, 4-hydroxytamoxifen is unable to overcome inhibition of dimerization by PKA. Thus, phosphorylation of ER alpha in the DNA binding domain provides a mechanism by which dimerization and thereby DNA binding by the estrogen receptor is regulated.