Progesterone Receptor Membrane Component-1 is phosphorylated upon treatment with Progestins and binds to Estrogen Receptor α-coregulators PHB1 and PHB2 in breast cancer cells
09 May 2017 (online)
Combined menopausal hormone therapy, using estrogen and synthetic progestins, is associated with an increased risk of developing breast cancer. The effect of progestins on breast cells is complex and not fully understood yet. In previous in vitro and in vivo studies, we found different progestins to increase the proliferation of Progesterone Receptor Membrane Component-1 (PGRMC1)-overexpressing MCF7 breast cancer cells, revealing a potential role of PGRMC1 in forwarding membrane-initiated progestin signals into the cell. Therefore, the aim of this study was to further investigate the activation mechanism and downstream signaling of PGRMC1 upon progestin treatment. To identify posttranslational modifications and potential interaction partners of the receptor after progestin binding, co-immunoprecipitation experiments were performed using MCF-7/PGRMC1 cells, followed by mass spectrometry analysis. To further validate the results, proximity ligation assay and co-localization studies were conducted. Further, proliferation of MCF-7/PGRMC1 cells and MCF7/PGRMC1 cells, possessing point mutations at PGRMC1 phosphorylation sites, was investigated upon treatment with progestins. Here we show that treatment of MCF7/PGRMC1 cells with the progestin norethisterone (NET) induces phosphorylation of the receptor at Casein Kinase 2 (CK2) phosphorylation site Ser181. Point mutation of the Ser181 phosphorylation site in MCF7/PGRMC1 cells impairs proliferation upon NET treatment. Further, the Estrogen Receptor α (ERα)-coregulators Prohibitin 1 (PHB1) and Prohibitin 2 (PHB2) are identified as interaction partners of PGRMC1 after progestin treatment. This study gives further insight into the activation mechanism of the receptor upon progestin binding. It further suggests an important role of PGRMC1 in the progression of breast cancer in progestin-based hormone replacement therapy.