Thrombin-induced translocation of protein kinase C in human megakaryoblastic leukemia cells (MEG-01).
Our previous immunocytochemical study showed that Ca2+ ionophore-induced translocation of protein kinase C (PKC) in human megakaryoblastic leukemia cells (MEG-01) was potentiated by a synthetic diacylglycerol (T. Ito, T. Tanaka, T. Yoshida, K. Onoda, H. Ohta, M. Hagiwara, Y. Itoh, M. Ogura, H. Saito, and H. Hidaka, 1988, J. Cell Biol. 107, 929). In the present study, we analyzed the roles of the intracellular Ca2+ levels ([Ca2+]i) and diacylglycerol (DG) levels in thrombin-induced translocation of PKC using MEG-01 cells. When the cells were treated with thrombin (0.5 U/ml), PKC was translocated from the cytosol to the plasma membrane after 15 s, and the maximal membrane association was observed after 90 s. The [Ca2+]i of the cells rapidly increased (15 s) and reached a maximum level at 60 s which was sustained for a total of 600 s after thrombin addition. The increase in DG was biphasic with the first phase occurring in the first 15 s and the increase during the second phase lasting less than 600 s. The experiments without extracellular Ca2+ indicated that Ca2+ efflux accompanied by DG in the first phase was sufficient to initiate the membrane association of the PKC and that the large Ca2+ influx enhanced the binding. PKC returned to the cytosol within 600 s despite high levels of both [Ca2+]i and DG. We found that a relatively selective PKC inhibitor, H-7, enhanced thrombin-induced translocation of PKC without modulating [Ca2+]i or DG levels. These results indicate that certain protein phosphorylation events, potentially those mediated by PKC, may be responsible for, at least in part, inhibiting membrane association and further activation of the enzyme.
Ito
T
T
First Department of Internal Medicine, Nagoya University School of Medicine, Japan.
Kozawa
O
O
Hidaka
H
H
Saito
H
H
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology; D004075:Diglycerides / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D007947:Leukemia, Megakaryoblastic, Acute / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000187:drug effects
C011439:1,2-diacylglycerol; D004075:Diglycerides; D007546:Isoquinolines; D010879:Piperazines; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011493:Protein Kinase C; D013917:Thrombin; D002118:Calcium