Immunocytochemical evidence for translocation of protein kinase C in human megakaryoblastic leukemic cells: synergistic effects of Ca2+ and activators of protein kinase C on the plasma membrane association.
Immunological analysis using monoclonal antibodies against subspecies of protein kinase C revealed the predominant expression of the isozyme, type II, in human megakaryoblastic leukemic cells. We investigated the effects of phorbol diester 12-O-tetradecanoyl phorbol-13-acetate (TPA), the Ca2+ ionophore ionomycin and synthetic diacylglycerol 1-oleoyl-2-acetylglycerol (OAG) on the immunocytochemical localization of protein kinase C in these cells. Indirect immunofluorescence techniques revealed the enzyme to be located in a diffuse cytosolic pattern, in the intact cells. When the cells were exposed to 100 nM TPA, the immunofluorescent staining was translocated from the cytoplasm to the plasma membrane. The translocation was protracted and staining on the membrane decreased in parallel with the Ca2+, phospholipid-dependent protein kinase activity. Treatment of the cells with 500 nM ionomycin caused an apparent translocation comparable with that seen with TPA, however, this translocation was transient and most of the cytosolic staining was within 60 min. We also found that 30 micrograms/ml OAG did not have significant effects on distribution of the staining, but rather acted synergistically on the translocation with the suboptimal concentration of 100 nM ionomycin. A similar synergism was also observed with 10 nM TPA and 100 nM ionomycin. These results obtained in situ provide evidence that intracellular Ca2+ and diacylglycerol regulate membrane binding of the enzyme in vivo.
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D003593:Cytoplasm / Q000201:enzymology; D004075:Diglycerides / Q000138:chemical synthesis / Q000494:pharmacology*; D004987:Ethers / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005989:Glycerides / Q000494:pharmacology*; D006801:Humans; D007118:Immunoassay; D007150:Immunohistochemistry; D015759:Ionomycin; D007476:Ionophores / Q000494:pharmacology; D007947:Leukemia, Megakaryoblastic, Acute / Q000201:enzymology*; D011493:Protein Kinase C / Q000032:analysis* / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D000900:Anti-Bacterial Agents; D004075:Diglycerides; D004987:Ethers; D005989:Glycerides; D007476:Ionophores; D015759:Ionomycin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium