Immunocytochemical Evidence for Translocation of Protein Kinase C in Human Megakaryoblastic Leukemic Cells: Synergistic Effects of Ca 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 Ca 2÷ ionophore ionomycin and synthetic diacylglycerol 1-oleoyl-2acetylglycerol (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 Ca 2÷, 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 ~tg/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 syngergism was also observed with 10 nM TPA and 100 nM ionomycin. These results obtained in situ provide evidence that intracellular Ca 2+ and diacylglycerol regulate membrane binding of the enzyme in vivo. I t is generally accepted that Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) plays an important role in transmembrane signaling (27). The enzyme is active in the presence of both Ca 2+ and phospholipids. Diacylglycerol, which is transiently generated as a consequence of the receptor-mediated hydrolysis of inositol phospholipids, and tumor-promoting phorbol diester, which have a chemical structure similar to that of diacylglycerol, increase the affinity of the enzyme for Ca 2÷ and phospholipid (20). Several agonists including phorbol diesters (23) and those noted to promote inositol phospholipids hydrolysis, such as gonadotropin-releasing hormone (17), thyrotropin-releasing hormone (11), interleukin 2 (8), interleukin 3 (9), and certain antibodies (4) cause a translocation of protein kinase C activity from the cytosol to the particulate (membrane) fraction in various cells and cell lines, which seems to be involved in the activation of the enzyme. Inositol phosphates, the other products of hydrolysis of inositol phospholipids, increase intracellular free Ca 2+ ([Ca2+]i) I by releasing Ca 2+ from intracellular Ca 2÷ stores (38). Ca 2+ ionophore such as ionomycin affect the permeability of the cell membrane to Ca 2+ and are pertinent for use in studies on the role of [Ca2+]i in the cell regulation. Moreover, the synthetic diacylglycerol or phorbol diester has been noted in various cellular responses (27, 35), including platelet secretion (13, 20), lymphocyte activation (39), smooth muscle contraction (34), and cell proliferation and differentiation (40). Although in vitro studies suggested that protein kinase C is activated in a component of a quarternary complex consisting of the kinase, Ca 2+, diacylglycerol, and phospholipid associated with cellular membrane structure (12), direct demonstration of the distribution or redistribution of the enzyme has been technically difficult, and the Ca 2÷ ionophore or diacylglycerol-induced translocation of 1. Abbreviations used in this paper: [Ca2+]i, cytosolic free calcium concentration; OAG, l-oleoyl-2-acetylglycerol. © The Rockefeller University Press, 0021-9525/88/09/929/9 $2.00 The Journal of Cell Biology, Volume 107, September 1988 929-937 929 on A ril 2, 2017 D ow nladed fom Published September 1, 1988