Inhibition of phorbol ester-induced T cell proliferation by bryostatin is associated with rapid degradation of protein kinase C.

Abstract

The bryostatins (Bryo) represent a group of immunomodulators that counteract the tumor-promoting effects of PMA. In contrast to the mitogenic effect of PMA on human peripheral blood T lymphocytes, Bryo was nonmitogenic and, furthermore, it inhibited PMA-induced T cell proliferation in a dose-dependent manner when added up to 2 days after PMA. Because both Bryo and PMA bind to, and activate, protein kinase C (PKC), we compared their effects on PKC expression and activity in human PBL or leukemic T cells (Jurkat). After treatment for 5 to 60 min, both Bryo and PMA were found to: a) activate PKC in vitro with similar dose-response curves; b) induce a nearly complete cytosol-to-membrane translocation of enzymatically active, Ca(2+)-dependent PKC and of distinct immunoreactive PKC isoforms in intact PBL; and c) stimulate similar patterns of protein phosphorylation. After a longer, 20-h treatment with PMA (20 nM), a considerable portion of PKC was still membrane-associated, and the total amount of immunoreactive PKC was not reduced considerably. In contrast, Bryo induced a marked loss of cellular immunoreactive PKC, including PKC-alpha and -beta. These results were paralleled by measurements of total cytosol- or membrane-associated PKC enzymatic activity. Thus, substantial PKC activity was associated with the particulate fraction of PMA-, but not Bryo-stimulated PBL. Furthermore, inhibition of PMA-induced T cell proliferation by Bryo also correlated with a reduction in the amount of cytosolic and membrane-bound immunoreactive PKC and enzymatic activity, demonstrating the dominance of Bryo over PMA. We propose that Bryo inhibits PMA-induced T cell proliferation by causing rapid degradation of PKC, reflecting a requirement of persistent PKC stimulation (lasting approximately 48 h) for the activation of human T cells and progression through the cell cycle.