Phosphorylation-dependent sumoylation of estrogen-related receptor alpha1.
We previously showed that estrogen-related receptor alpha1 (ERRalpha1) can compete with estrogen receptor alpha (ERalpha) for binding to estrogen response elements (EREs), repressing transcription in the mammary carcinoma cell line MCF-7. Given that ERRalpha1 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 ERRalpha1 at Ser19 and Ser22. Inhibition of phosphorylation at amino acid residue 22 did not have a significant effect on ERRalpha1's transcriptional activity. However, mutation of amino acid residue 19 from serine to alanine enhanced two-fold ERRalpha1's response to the coactivator GRIP-1. We also identified two sites of sumoylation at Lys14 and Lys403. We found that inhibition of sumoylation at Lys14 could enhance five-fold ERRalpha1's response to coactivator GRIP-1. Furthermore, phosphorylation of Ser19 enhanced the sumoylation at Lys14. Taken together, we conclude that phosphorylation at Ser19 and sumoylation at Lys14 within the A/B domain play roles in regulating ERRalpha1's transcriptional activities via affecting its response to coactivators.
McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706-1599, USA.
Elizabeth H
EH
Vu
Richard J
RJ
Kraus
Janet E
JE
Mertz
D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D025842:SUMO-1 Protein / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D000097983:ERRalpha Estrogen-Related Receptor
D010744:Phosphoric Monoester Hydrolases; D011960:Receptors, Estrogen; D025842:SUMO-1 Protein; D000097983:ERRalpha Estrogen-Related Receptor