The ubiquitin-proteasome pathway has been recognized as an important regulator in the hormonal response by estrogen receptor ER, but its impact on ER function is poorly characterized. In the current study, we investigated the role of the ubiquitin-proteasome pathway in regulating ER activity and identified regulatory sites within the activation function AF-1 domain that modulate ER ubiquitination and nuclear dynamics in a hormone-independent manner. Whereas both ER and ER 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 ER activity, we found that sequential substitutions of specific serine residues contained in MAPK consensus sites conferred transcriptional activation of ER in a proteasome-dependent manner through reduced ubiquitination and enhanced accumulation of mutant receptors. Specifically, serines 94 and 106 within ER AF-1 domain were found to modulate sub-nuclear 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 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 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.