Tightly bound nuclear progesterone receptor is not phosphorylated in primary chick oviduct cultures.
Oviduct cells from estradiol-treated chicks were grown in primary culture. After 3-5 days of culture in medium containing estradiol, 90% of the cellular progesterone binding sites were detected in the cytosol. After exposure to [3H]progesterone at 37 degrees C, 80% of the progesterone binding sites were found in nuclear fractions. Progesterone receptor phosphorylation was assessed after incubating the cells with [32P]orthophosphate. Receptor components were immunoprecipitated with a specific polyclonal antibody (IgG-G3) and analyzed by NaDodSO4/PAGE and autoradiography. In the cytosol, constant amounts of 32P-labeled 110-kDa subunit (the B subunit, one of the progesterone-binding components of the receptor) and of the non-steroid-binding heat shock protein hsp90 were found, whether cells had been exposed to progesterone or not. No 32P-labeled 79-kDa subunit (the A subunit, another progesterone-binding subunit) was detected. Various procedures were used to solubilize nuclear progesterone receptor (0.5 M KCl, micrococcal nuclease, NaDodSO4), and in no case was 32P-labeled B subunit detected in the extracts. However, nonradioactive B subunit was detected by immunoblot in a nuclear KCl extract of progesterone-treated cells. These results suggest that the fraction of the B subunit that becomes strongly attached to nuclear structures is not phosphorylated upon exposure of cells to progesterone.
Garcia
T
T
Jung-Testas
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I
Baulieu
E E
EE
D000818:Animals; D002467:Cell Nucleus / Q000032:analysis / Q000378:metabolism*; D002478:Cells, Cultured; D002645:Chickens; D003600:Cytosol / Q000032:analysis; D005260:Female; D007074:Immunoglobulin G / Q000276:immunology; D010057:Oviducts / Q000378:metabolism*; D010766:Phosphorylation; D011980:Receptors, Progesterone / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
D007074:Immunoglobulin G; D011980:Receptors, Progesterone