Phosphorylation of transfected wild type and mutated progesterone receptors.
An expression vector encoding wild type or mutated forms of the rabbit progesterone receptor was transfected into COS-7 cells and phosphorylation was studied by incubation with 32Pi followed by specific immunoprecipitation. The features of phosphorylation of the wild type receptor were identical to those previously observed in uterine cells: there was a basal level of phosphorylation which was increased approximately 7-fold by incubation with the hormone. The hyperphosphorylated receptor had decreased electrophoretic mobility ("upshift"). These experiments thus showed that the presence of the receptor specific kinase is not restricted to the target cells. Cleavage of the receptor by hydroxylamine and cyanogen bromide, and use of receptor mutants deleted in the N-terminal region, showed the absence of any detectable phosphorylation downstream from amino acid 520 (thus in the DNA and steroid binding domains). The majority of the phosphorylation sites were localized between amino acids 166 and 520. This localization was similar for basal and hormone-induced phosphorylation. DNA binding and hormone-induced hyperphosphorylation were not directly related, since deletion of the first zinc finger provided a hyperphosphorylated receptor. We showed that the constitutive receptor (totally deleted in the steroid binding region) exhibited only a low basal level of phosphorylation, and antagonist RU 486-receptor complexes were found to be hyperphosphorylated, leading us to conclude that the active form of the receptor was not the hyperphosphorylated one. Moreover receptor down regulation and hormone-induced receptor hyperphosphorylation were two independent phenomena. Basal phosphorylation was observed for both cytoplasmic and nuclear mutants, whereas nuclear localization was necessary but not sufficient for hyperphosphorylation. Finally, the second finger region and the hormone binding domain, which are necessary for receptor hyperphosphorylation, may be involved in the hormonally induced increased affinity of the receptor toward its kinase.
Unité de Recherches Hormones et Reproduction (Institut National de la Santé et de la Recherche Médicale U.135), Faculté de Médecine, Paris Sud, Le Kremlin Bicêtre, France.
A
A
Chauchereau
H
H
Loosfelt
E
E
Milgrom
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003488:Cyanogen Bromide; D004247:DNA / Q000378:metabolism; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000737:chemistry; D009154:Mutation; D010766:Phosphorylation; D011233:Precipitin Tests; D011817:Rabbits; D011980:Receptors, Progesterone / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D006898:Hydroxylamines; D011980:Receptors, Progesterone; D019811:Hydroxylamine; D004247:DNA; D003488:Cyanogen Bromide