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Partial purification and characterization of phorbol ester-regulated translational inhibitor(s) in human HL-60
Abstract:
Addition of nanomolar concentration of the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) to the human promyelocytic HL-60 leukemia cells is associated with a cessation of cellular proliferation and a subsequent differentiation into macrophage-like cells. Because the growth rate of mammalian cells is tightly coupled to the functions of the protein synthetic machinery, we have examined whether TPA induces a change in HL-60 translational functions. Addition of control HL-60 cell extracts to rabbit reticulocyte lysates results in a pronounced inhibition of protein synthesis, while TPA-treated HL-60 cell extracts are significantly less inhibitory. The reduction in TPA-induced translational inhibitory activity can be observed after a 3-6-h treatment and reaches a maximum after 24 h. Fractionation of control cell extracts on DEAE-cellulose columns reveals two inhibitory activities, eluting at 100 and 350 mM KCl, respectively. The DEAE-100 inhibitor(s) is further resolved into two activities by heparin-agarose column chromatography (HEP-100 and HEP-250). TPA treatment of HL-60 cells for 48 h completely eliminates the HEP-250 inhibitory activity and reduces the HEP-100 and the DEAE-350 inhibitory activities by 50 and 25%. Inhibition of protein synthesis in rabbit reticulocyte lysates by DEAE-100 inhibitory activities can be partially reversed by the addition of globin mRNA while translational inhibition by DEAE-350 inhibitor(s) can be reversed by addition of eukaryotic initiation factor (eIF) 2 or fructose 6-phosphate. The DEAE-100 inhibitor(s) causes extensive degradation of radioactive polynucleotides while the DEAE-350 inhibitor(s) is capable of phosphorylating both the alpha- and the beta-subunits of the highly purified rabbit reticulocyte initiation factor eIF-2. These data show that the DEAE-100 inhibitor(s) contains a nuclease while the DEAE-350 inhibitor(s) is associated with eIF-2 alpha and eIF-2 beta protein kinases.