title: Covalent modification of proteins by ligands of steroid hormone receptors.
abstract: Retinoylation, acylation with retinoic acid (RA), is a covalent modification of proteins occurring in a variety of eukaryotic cell lines. In this study, we found that proteins in HL-60 cells were labeled by 17 beta-[3H]estradiol (E2), [3H]progesterone (Pg), 1 alpha,25-dihydroxy[3H]vitamin D3 [1,25(OH)2D3], [125I]triiodothyronine (T3), [125I]thyroxine (T4), and [3H]prostaglandin E2 (PGE2). All of these hormones, except PGE2, are ligands of the steroid hormone receptor family. Addition to the growth medium of 5 microM ketoconazole, an inhibitor of cytochrome P450-dependent enzymes, increased about 2-fold the labeling of proteins by T3, T4, 1,25(OH)2D3, and PGE2. In contrast, ketoconazole did not change markedly the extent of labeling by RA, E2, or Pg. Alkaline methanolysis, which cleaves ester bonds, released variable percentages of the radioactive ligands bound to protein. These values were about 80% for RA and PGE2; 50% for T3, T4, and Pg; and 20% for E2 and 1,25(OH)2D3. Treatment with thioether-cleavage reagents, iodomethane or Raney nickel catalyst, released < 2% of the covalently bound ligands. Two-dimensional polyacrylamide gel electrophoresis patterns of labeled proteins were unique for each ligand. Proteins of M(r) 47,000 and 51,000 were labeled by RA, E2, T3, and T4. These proteins had the same mobilities as RI and RII, the cAMP-binding regulatory subunits of type I and type II cAMP-dependent protein kinases. 1,25(OH)2D3 also bound to proteins of M(r) 47,000 and 51,000. However, these proteins had pI values different from those of RI or RII. These results suggest that some activities of ligands of the steroid hormone receptor family and of PGE2 may be mediated by their covalent modification of proteins.
authors.affiliation: Laboratory of Biological Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
authors.forename: N
authors.initials: N
authors.lastname: Takahashi
authors.forename: T R
authors.initials: TR
authors.lastname: Breitman
mesh_terms: D002117:Calcitriol / Q000378:metabolism*; D015232:Dinoprostone / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004958:Estradiol / Q000378:metabolism*; D006801:Humans; D007457:Iodine Radioisotopes; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008024:Ligands; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism*; D011485:Protein Binding; D011987:Receptors, Steroid / Q000378:metabolism*; D013974:Thyroxine / Q000378:metabolism*; D014284:Triiodothyronine / Q000378:metabolism*; D014316:Tritium; D014407:Tumor Cells, Cultured
chemical_list: D007457:Iodine Radioisotopes; D008024:Ligands; D009363:Neoplasm Proteins; D011987:Receptors, Steroid; D014284:Triiodothyronine; D014316:Tritium; D011374:Progesterone; D004958:Estradiol; D002117:Calcitriol; D015232:Dinoprostone; D013974:Thyroxine; D007654:Ketoconazole