Effect of tumour-promoting phorbol ester, thrombin and vasopressin on translocation of three distinct protein kinase C isoforms in human platelets and regulation by calcium.
Protein kinase C (PKC) acts in synergy with Ca2+ mobilization for the activation of platelets. Three different PKC subtypes that specifically react with antibodies to alpha- beta- and zeta-PKC have been detected in human platelets. We have compared the subcellular redistribution of these isoforms in platelets after exposure to the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) and to two physiological agonists, thrombin and vasopressin. In the presence of PMA, beta-PKC is most rapidly translocated to membranes, followed by zeta-PKC and alpha-PKC [membrane contents of 39 +/- 6, 31 +/- 4 and 24 +/- 4% (means +/- S.E.M.) respectively after 2 min incubation]. In contrast, both thrombin and vasopressin induced a biphasic translocation of PKC isoforms. For both agonists, the first phase of translocation occurred within 1 min and was identical for the three isoforms. However, during the second phase, the translocation of zeta-PKC by thrombin and vasopressin differed [membrane contents (mean +/- S.E.M.) of 24 +/- 3 and 46 +/- 4% respectively after 10 min]. These results suggest a differential activation of zeta-PKC by vasopressin and thrombin. PMA-induced translocation of alpha-PKC was decreased from 278 +/- 27 to 198 +/- 24 (mean +/- S.E.M., P = 0.02; percentage increase over control value) in the presence of 1 mM-EDTA, whereas chelation of intracellular Ca2+ by Quin2-AM does not influence this response. These results suggest that the PMA-induced translocation of alpha-PKC depends on the presence of 1 mM concentration of extracellular Ca2+. In addition, the chelation of either extracellular or intracellular Ca2+ inhibited both vasopressin- and thrombin-induced translocation of all three isoforms, suggesting that Ca2+ is an important requirement for the translocation of alpha-, beta- and zeta-PKC by physiological agonists. In conclusion, the translocation of PKC varies between different isoforms and between different agonists.
Crabos
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Department of Research, University Hospital, Basel, Switzerland.
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D000595:Amino Acid Sequence; D001127:Arginine Vasopressin / Q000494:pharmacology*; D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology / Q000502:physiology; D002118:Calcium / Q000097:blood* / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007527:Isoenzymes / Q000097:blood* / Q000235:genetics; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000097:blood* / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology*; D014178:Translocation, Genetic / Q000187:drug effects*
D007527:Isoenzymes; D001127:Arginine Vasopressin; D011493:Protein Kinase C; D013917:Thrombin; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium