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.
Vu
Elizabeth H
EH
McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706-1599, USA.
Kraus
Richard J
RJ
Mertz
Janet E
JE
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