Phosphorylation-dependent sumoylation of estrogen-related receptor α1

We previously showed that estrogen-related receptor alpha 1 (ERR alpha 1) can compete with estrogen receptor alpha (ER alpha) for binding to estrogen response elements (EREs), repressing transcription in the mammary carcinoma cell line MCF-7. Given that ERR alpha 1 can function in the absence of ligands and exists as a phosphoprotein in vivo, we wished to determine sites of phosphorylation involved in regulating its transcriptional activity. Using a combination of electrophoretic mobility shift analysis, phospho-specific fluorescent dye staining, and site-directed mutagenesis, we identified two novel in vivo sites of phosphorylation in the A/B ligand-independent activation domain of ERR alpha 1 at Ser(19) and Ser(22). Inhibition of phosphorylation at amino acid residue 22 did not have a significant effect on ERR alpha 1's transcriptional activity. However, mutation of amino acid residue 19 from serine to alanine enhanced two-fold ERR alpha 1's response to the coactivator GRIP-1. We also identified two sites of sumoylation at Lys(14) and Lys(403). We found that inhibition of sumoylation at Lys(14) could enhance five-fold ERR alpha 1's response to coactivator GRIP-1. Furthermore, phosphorylation of Ser(19) enhanced the sumoylation at Lys(14). Taken together, we conclude that phosphorylation at Ser(19) and sumoylation at Lys(14) within the A/B domain play roles in regulating ERR alpha 1's transcriptional activities via affecting its response to coactivators.