Regulation of T-cell-receptor-stimulated bivalent-cation entry in Jurkat E6 cells: role of protein kinase C.
Stimulation of Jurkat E6 cells with anti-CD3 antibody results in a characteristic rise in [Ca2+]i which is due to both the release of Ca2+ from intracellular stores and the entry of external Ca2+. Individual components of the [Ca2+]i increase were investigated by measuring intracellular Ca2+ release in the absence of external Ca2+ and determining influx of bivalent cations by following the entry of Mn2+. The increase in [Ca2+]i induced by anti-CD3 antibody in the presence or absence of extracellular Ca2+ could be inhibited by the non-selective kinase inhibitor staurosporine, which also inhibits anti-CD3-stimulated phospholipase C activity. Staurosporine also inhibits the influx of bivalent cations induced by anti-CD3 antibody, but not that induced by depletion of intracellular Ca2+ stores using thapsigargin. The effect of staurosporine was compared with that of Ro 31-8425, a potent and selective inhibitor of protein kinase C (PKC). Ro 31-8425, at concentrations up to 10 microM, has no inhibitory effect on the anti-CD3 antibody-induced [Ca2+]i increase or phospholipase C activity. These studies are consistent with the concept that augmentation of [Ca2+]i by stimulated T-cell receptors requires activation of a kinase, probably a tyrosine kinase such as p56lck, ZAP-70 or p59fyn, and is independent of PKC. Phorbol esters inhibit the anti-CD3-stimulated [Ca2+]i increase and phospholipase C activity, showing that this can be negatively regulated by PKC. A small potentiation of the anti-CD3 antibody-induced [Ca2+]i rise in the presence of extracellular Ca2+ was detected in the presence of Ro 31-8425; this suggests that T-cell-receptor ligation can also limit the increase in [Ca2+]i via PKC activation.
Roche Research Centre, Welwyn Garden City, U.K.
L A
LA
Conroy
J E
JE
Merritt
T J
TJ
Hallam
D000470:Alkaloids / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000936:Antigen-Antibody Complex; D017252:CD3 Complex / Q000276:immunology; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D016257:Fura-2 / Q000737:chemistry; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism / Q000502:physiology*; D019311:Staurosporine; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin
D000470:Alkaloids; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D017252:CD3 Complex; D007211:Indoles; D008301:Maleimides; D011948:Receptors, Antigen, T-Cell; D013729:Terpenes; C071924:Ro 31-8425; D008345:Manganese; D019284:Thapsigargin; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2