Phosphorylation of serine 212 confers novel activity to human estrogen receptor α

Estrogen receptor alpha (ER alpha) can be phosphorylated at various residues, one of which is serine 212 in the DNA binding domain. The majority of human nuclear receptors conserves, as a motif, this serine residue within their DNA binding domain. Among these nuclear receptors, phosphorylation of the corresponding threonine 38 in the nuclear receptor CAR is essential for determining its activity [9]. Here, we have investigated the role of phosphorylated serine 212 in the regulation of ER alpha activity by comparing it with serine 236, another potential phosphorylation site within the DNA binding domain, and demonstrated that phosphorylation of serine 212 confers upon ER alpha a distinct activity regulating gene expression in Huh-7 cells. In Western blot analysis, wild type ER alpha and mutants ER alpha S212A, ER alpha S212D, ER alpha S236A and ER alpha S236D were equally expressed in the nucleus, thus indicating that phosphorylation does not determine nuclear localization of ER alpha. ER alpha S212D, but not ER alpha S236D, retained its capability of activating an ERE-reporter gene in luciferase assays. Similar results were also obtained for human ER beta: the ER beta S176D mutant retained its trans-activation activity, but the ER beta S200D mutant did not. cDNA microarray and Ingenuity Pathway Analysis, employed on Huh-7 cells ectopically expressing either ER alpha S212A or ER alpha S212D, revealed that phosphorylation of serine 212 enabled ER alpha to regulate a unique set of genes and cellular functions. Published by Elsevier Inc.