Sawai T, Negishi M, Nishigaki N, Ohno T, Ichikawa A
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University, Japan.
J Biol Chem. 1993 Jan 25;268(3):1995-2000.
The addition of 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly enhanced cAMP formation induced by carbacyclin, a stable prostacyclin analogue, in cultured mast cells (IC2 cells), but did not enhance basal or NaF plus AlCl3-induced cAMP formation. On the other hand, W-7, a calmodulin (CaM) inhibitor, almost completely suppressed the enhancing activity of TPA, suggesting the involvement of CaM in the enhancement by TPA of carbacyclin-induced cAMP formation. The enhancing activity of TPA disappeared in TPA-treated cells permeabilized with saponin in the presence of Ca2+, but reconstitution with CaM in the permeable cells resulted in remarkable restoration of the action of TPA. On the other hand, TPA treatment induced the phosphorylation and translocation of myristoylated alanine-rich C kinase substrate (MARCKS) from the membrane to the cytosol. Exogenously added protein kinase C (PKC) also phosphorylated MARCKS and induced its translocation in the cells permeabilized with saponin. Whereas the addition of CaM did not enhance the carbacyclin-stimulated GTPase activity and adenylate cyclase activity in the control permeable cells, in which MARCKS bound to the membrane, CaM markedly enhanced those activities in the PKC-treated permeable cells, which lost endogenous membrane-bound MARCKS. When MARCKS was added to the PKC-treated permeable cells, MARCKS bound to the membrane and inhibited the effects of CaM. These results suggest that activation of PKC enhances the prostacyclin-activated adenylate cyclase through a CaM/MARCKS system.
在培养的肥大细胞（IC2细胞）中，添加12 - O - 十四烷酰佛波醇 - 13 - 乙酸酯（TPA）可显著增强稳定的前列环素类似物卡巴前列素诱导的环磷酸腺苷（cAMP）生成，但对基础或氟化钠加氯化铝诱导的cAMP生成无增强作用。另一方面，钙调蛋白（CaM）抑制剂W - 7几乎完全抑制了TPA的增强活性，提示CaM参与了TPA对卡巴前列素诱导的cAMP生成的增强作用。在存在Ca2 + 的情况下，用皂素通透处理过的TPA处理细胞中，TPA的增强活性消失，但在通透细胞中重新加入CaM可使TPA的作用显著恢复。另一方面，TPA处理诱导了富含丙氨酸的肉豆蔻酰化C激酶底物（MARCKS）从膜向胞质溶胶的磷酸化和转位。外源添加的蛋白激酶C（PKC）也使MARCKS磷酸化并诱导其在经皂素通透处理的细胞中转位。在对照通透细胞（其中MARCKS与膜结合）中，添加CaM并未增强卡巴前列素刺激的GTP酶活性和腺苷酸环化酶活性，但在PKC处理的通透细胞（其失去了内源性膜结合的MARCKS）中，CaM显著增强了这些活性。当将MARCKS添加到PKC处理的通透细胞中时，MARCKS与膜结合并抑制了CaM的作用。这些结果表明，PKC的激活通过CaM/MARCKS系统增强了前列环素激活的腺苷酸环化酶活性。
