Differential translocation of protein kinase C isozymes by thrombin and platelet-derived growth factor. A possible function for phosphatidylcholine-derived diacylglycerol.
Differential translocation of protein kinase C isozymes by thrombin and platelet-derived growth factor. A possible function for phosphatidylcholine-derived diacylglycerol.
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DOI：
10.1016/s0021-9258(18)82231-1
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发表时间：
1993-05
期刊：
影响因子：
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通讯作者：
K. Ha;J. Exton
中科院分区：
文献类型：
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作者：
K. Ha;J. Exton
研究了 IIC9 成纤维细胞中蛋白激酶 C (PKC) 从胞质部分到颗粒部分的易位，以确定源自磷脂酰肌醇 4,5-二磷酸 (PIP2) 和磷脂酰胆碱 (PC) 水解的 1,2-二酰基甘油 (DAG) 的功能。 α-凝血酶引起 DAG 的双相变化，在 15-60 秒和 5-15 分钟有两个峰值，分别源自 PIP2 和 PC，而血小板衍生生长因子 (PDGF) 在 5-15 分钟诱导来自 PC 的单相 DAG 增加。 α-凝血酶还诱导肌醇 1,4,5-三磷酸和胞质 Ca2+ 快速但短暂的增加，而 PDGF 则不然。通过蛋白质印迹法在 IIC9 细胞中鉴定出三种 PKC 同工酶：α、ε 和 zeta，它们主要位于细胞质中。 15 秒时，一小部分胞浆 PKC α 被 α-凝血酶快速移位，但其膜结合在 1 分钟内消失。 PKC epsilon 也迅速易位；然而，其膜结合持续了近 60 分钟。 PKC zeta 不会被 α-凝血酶或佛波醇 12-肉豆蔻酸酯 13-乙酸酯移位。 PDGF 在 5 分钟时使 PKC epsilon 易位，但在 15 秒时几乎没有作用，并且不使 PKC α 或 zeta 易位。与蜡样芽胞杆菌 PC 或磷脂酰肌醇特异性磷脂酶 C 一起孵育，会增加 DAG 而不会增加磷脂酸，刺激 PKC epsilon 易位，但不会刺激 PKC α 或 zeta 易位。添加螯合剂来抑制细胞内 Ca2+ 的升高很大程度上阻断了 α-凝血酶诱导的 PKC α 易位，但对 PKC ε 易位没有影响。添加离子霉素使得 α-凝血酶在 5 分钟时诱导 PKC α 易位。 1,2-二辛酰甘油加离子霉素可以模拟 PKC α 易位，但单独使用任何一种都不能模拟。另一方面，PKC epsilon 仅由 DAG 易位。这些结果支持以下结论：PIP2 水解在 15 秒时激活 PKC α 和 epsilon，而 PC 水解在 5 分钟时仅激活 PKC epsilon。 5 分钟时的活化差异可归因于 PC 水解未能增加 Ca2+，而不是源自磷脂的 DAG 分子种类的差异。
The translocation of protein kinase C (PKC) from the cytosolic to the particulate fraction in IIC9 fibroblasts has been studied to define the functions of 1,2-diacylglycerol (DAG) derived from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylcholine (PC). alpha-Thrombin caused a biphasic change in DAG, with two peaks at 15-60 s and 5-15 min, derived from PIP2 and PC, respectively, while platelet-derived growth factor (PDGF) induced a monophasic DAG increase from PC at 5-15 min. alpha-Thrombin also induced a rapid, but transient, increase of inositol 1,4,5-trisphosphate and cytosolic Ca2+, whereas PDGF did not. Three PKC isozymes, alpha, epsilon, and zeta, were identified by Western blotting in IIC9 cells and were mainly localized in the cytosol. A fraction of cytosolic PKC alpha was rapidly translocated by alpha-thrombin at 15 s, but its membrane association was lost within 1 min. PKC epsilon was also rapidly translocated; however, its membrane association was sustained for almost 60 min. PKC zeta was not translocated by alpha-thrombin or phorbol 12-myristate 13-acetate. PDGF translocated PKC epsilon at 5 min but had little effect at 15 s and did not translocate PKC alpha or zeta. Incubation with Bacillus cereus PC- or phosphatidylinositol-specific phospholipase C, which increased DAG but not phosphatidic acid, stimulated translocation of PKC epsilon, but not PKC alpha or zeta. Addition of chelators to inhibit the rise in intracellular Ca2+ largely blocked PKC alpha translocation induced by alpha-thrombin but had no effect on PKC epsilon translocation. Addition of ionomycin allowed alpha-thrombin to induce PKC alpha translocation at 5 min. PKC alpha translocation was mimicked by 1,2-dioctanoylglycerol plus ionomycin, but not by either alone. On the other hand, PKC epsilon was translocated by the DAG alone. These results support the conclusion that PIP2 hydrolysis activates both PKC alpha and epsilon at 15 s, whereas PC hydrolysis activates only PKC epsilon at 5 min. The differential activation at 5 min can be attributed to the failure of PC hydrolysis to increase Ca2+ and not to a difference in the molecular species of DAG derived from the phospholipids.