Specific protein phosphorylation in human promyelocytic HL-60 leukemia cells susceptible or resistant to induction of cell differentiation by phorbol-12-myristate-13-acetate.
The pattern of protein phosphorylation induced by phorbol-12-myristate-13-acetate (PMA) was analyzed by two-dimensional gel electrophoresis in human HL-60 leukemia cells, which are susceptible to induction of cell differentiation by PMA, and in cells from an HL-60 cell variant designated R-94 that are resistant to such an induction. Protein phosphorylation was detected by observing either a rapid acid-directed charge shift of [35S]methionine-labeled protein or an increase in the amount of phosphate label in a 32P-labeled protein. The results indicated that PMA at 10(-7) M causes within 30 min after treatment the phosphorylation of at least ten different proteins in both the HL-60 and R-94 cells. Among these ten phosphorylated proteins, we identified a major cytoplasmic polypeptide (Mr approximately 64,000), a cytoskeletal protein (Mr approximately 56,000), a nonmuscle myosin light chain, and two proteins (Mr approximately 60,000 and 64,000) localized in or around the cell nucleus. Phosphoamino acid analysis of six of the ten phosphoproteins showed that they contain phosphoserine. None of these proteins contained phosphotyrosine or phosphothreonine. The R-94 cell variant was found to be capable of increased protein phosphorylation after PMA treatment; however, the level of phosphate incorporation reached only the level of the untreated HL-60 cells and thus fell far short of the level observed in the HL-60 cells after PMA treatment. It is suggested that the basis for the acquired resistance in R-94 cells towards induction of cell differentiation by PMA is a block in signal transmission involving phosphorylation of nuclear protein(s) following the binding of the inducer PMA to its receptor (protein kinase C).
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004351:Drug Resistance; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism* / Q000473:pathology; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000378:metabolism*; D010704:Phorbols / Q000494:pharmacology*; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011493:Protein Kinase C; D011955:Receptors, Drug; D011971:Receptors, Immunologic / Q000032:analysis; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D009363:Neoplasm Proteins; D010704:Phorbols; D010750:Phosphoproteins; D011955:Receptors, Drug; D011971:Receptors, Immunologic; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate