Phosphorylation of activation function-1 regulates proteasome-dependent nuclear mobility and E6-associated protein ubiquitin ligase recruitment to the estrogen receptor β

The ubiquitin-proteasome pathway has been recognized as an important regulator in the hormonal response by estrogen receptor (ER) alpha, but its impact on ER beta function is poorly characterized. In the current study, we investigated the role of the ubiquitin-proteasome pathway in regulating ER beta activity and identified regulatory sites within the activation function (AF)-1 domain that modulate ER beta ubiquitination and nuclear dynamics in a hormone-independent manner. Although both ER alpha and ER beta were dependent on proteasome function for their maximal response to estrogen, they were regulated differently by proteasome inhibition in the absence of hormone, an effect shown to be dependent on their respective AF-1 domain. Given the role of AF-1 phosphorylation to regulate ERactivity, we found that sequential substitutions of specific serine residues contained in MAPK consensus sites conferred transcriptional activation of ER beta in a proteasome-dependent mannERthrough reduced ubiquitination and enhanced accumulation of mutant receptors. Specifically, serines 94 and 106 within ER beta AF-1 domain were found to modulate subnuclear mobility of the receptor to transit between inactive clusters and a more mobile state in a proteasome-dependent manner. In addition, cellular levels of ER beta were regulated through these sites by facilitating the recruitment of the ubiquitin ligase E6-associated protein in a phosphorylation-dependent manner. These findings suggest a role for ER beta AF-1 in contributing to the activation-degradation cycling of the receptor through a functional clustering of phosphorylated serine residues that cooperate in generating signals to the ubiquitin-proteasome pathway.