Rapid protein phosphorylation induced by phorbol ester in HL-60 cells. Unique alkali-stable phosphorylation of a 17,000-dalton protein detected by two-dimensional gel electrophoresis.
Treatment of the leukemic promyelocytic cell line, HL-60, with phorbol-12-myristate-13-acetate (PMA), has been shown to induce terminal differentiation of the cells into monocytes. We found that this effect involves a rapid increase in phosphorylation of a 17-kDa protein (pp17, pI approximately 5.5) and a 27-kDa protein (pp27, pI approximately 5.5) as detected by two-dimensional gel electrophoresis. Within 15 min there was 5- to 7-fold and 3- to 4-fold increase in the phosphorylation of pp17 and pp27, respectively. The rate of phosphorylation remained accelerated over a period of 60-min incubation with PMA. In contrast to these specific changes, there was no effect of PMA on total 32Pi incorporation into HL-60 cells during the same period. The phosphoester bond of the pp17, but not of pp27, revealed a remarkable stability to alkali treatment of the electrophoretic gels. However, phosphoamino acid analysis showed that both pp17 and pp27 proteins are phosphorylated only at serine residues. These two phosphoproteins were located exclusively in the cytosol and were absent from the crude membrane fraction. We further demonstrated that several inactive derivatives of phorbol ester failed to cause an increase in phosphorylation of pp17 and pp27, suggesting that these specific phosphorylation events are intimately related to the induction of differentiation in HL-60 cells by PMA.
D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000032:analysis*; D008970:Molecular Weight; D010704:Phorbols / Q000494:pharmacology*; D010766:Phosphorylation; D011506:Proteins / Q000032:analysis*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D010704:Phorbols; D011506:Proteins; D013755:Tetradecanoylphorbol Acetate