Effect of ATP on the regulation of the steroid binding activity of the oestradiol receptor.
We have observed that ATP induces a second type of oestradiol binding site with slightly lower affinity (Ka 3.3 x 10(8) M-1) and lower sedimentation coefficient (4 S) in cytosol from immature lamb uterus and MCF-7 cells. A factor isolated from immature lamb uterine nuclear extract was found to decrease the steroid binding activity of oestradiol receptor that had been purified by heparin Sepharose and oestradiol-Sepharose chromatography. Inhibition of this factor by known phosphatase inhibitors, indicated that this factor may be a phosphatase. Another factor isolated from immature lamb uterine cytosol was found to enhance the effect of ATP on receptor binding in cytosol from immature lamb uterus and MCF-7 cells. The ability of this factor to phosphorylate a partially purified cytosol receptor from immature lamb uterus when incubated with [gamma 32P]ATP, indicates that this factor is a phosphokinase. The phosphorylated products after labeling with [3H]tamoxifen aziridine were characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Three phosphorylated proteins with molecular weights 150, 97, and 67 kDa bound [3H]tamoxifen aziridine. Ammonium sulphate precipitated cytosol oestradiol receptor from immature lamb uterus was inactivated with receptor inactivating factor and then reactivated with receptor activating factor in the presence of [gamma 32P]ATP and substantially affinity labelled with [3H]tamoxifen aziridine. The affinity labelled oestradiol receptor was immunopurified with the monoclonal antibody JS 34/32. Three proteins with molecular weights 67, 50 and 43 kDa specifically bound [3H]tamoxifen aziridine and only 43 kDa receptor fragment was phosphorylated. The relevance of inactivation/reactivation of oestradiol receptor to the dephosphorylation/phosphorylation of receptor is discussed.
Lahooti
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H
Laboratory for Biochemical Endocrinology, University of Bergen, Norway.
Thorsen
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T
Aakvaag
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A
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000345:Affinity Labels; D000818:Animals; D001943:Breast Neoplasms; D002467:Cell Nucleus / Q000032:analysis; D003600:Cytosol / Q000032:analysis; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D006801:Humans; D007163:Immunosorbent Techniques; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000494:pharmacology; D011959:Receptors, Estradiol / Q000187:drug effects / Q000378:metabolism*; D012756:Sheep; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014599:Uterus / Q000648:ultrastructure
D000345:Affinity Labels; D011959:Receptors, Estradiol; D013629:Tamoxifen; D004958:Estradiol; C031859:tamoxifen aziridine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D010744:Phosphoric Monoester Hydrolases