Retinoic acid acylation (retinoylation) of a nuclear protein in the human acute myeloid leukemia cell line HL60.
all-trans-Retinoic acid is a potent inducer in vitro of the differentiation of the human acute myeloid leukemia cell line HL60 and of fresh cells from patients with acute promyelocytic leukemia. The recent discovery of nuclear retinoic acid receptors provides a basis for understanding how retinoic acid acts at the genetic level. We have now found that retinoic acid is incorporated into HL60 cells in a form that is not removed by extraction with CHCl3:CH3OH. About 90% of this labeled retinoic acid is trichloroacetic acid-soluble after digestion with proteinase K or after hydrolysis with either NH2OH or CH3OH:KOH under mild conditions. Methyl retinoate is the major product of hydrolysis with CH3OH:KOH. These results are consistent with retinoylation of protein with the formation of an ester, probably thioester, bond. The extent of the retinoylation of HL60 protein is dependent on both time and retinoic acid concentration. A major fraction of the retinoylation is of protein that has a molecular mass of 55 kDa after reduction with dithiothreitol. On two-dimensional gels, the retinoylated protein has a pI of about 4.9 and a molecular mass of 55-60 kDa. These characteristics and its localization in the cell nucleus are consistent with retinoylation of the HL60 nuclear retinoic acid receptor or a closely related protein.
Takahashi
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Laboratory of Biological Chemistry, National Cancer Institute, Bethesda, Maryland 20892.
Breitman
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TR
D000215:Acylation; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism*; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013347:Subcellular Fractions; D014212:Tretinoin / Q000378:metabolism* / Q000494:pharmacology
D009687:Nuclear Proteins; D014212:Tretinoin