Ha K S, Exton J H
Howard Hughes Medical Institute, Nashville, Tennessee.
J Biol Chem. 1993 May 15;268(14):10534-9.
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.
为了明确源自磷脂酰肌醇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，研究了蛋白激酶C（PKC）在IIC9成纤维细胞中从胞质溶胶向颗粒部分的转位。α - 凝血酶引起DAG的双相变化，分别在15 - 60秒和5 - 15分钟出现两个峰值，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PC产生单相DAG增加。α - 凝血酶还诱导肌醇1,4,5 - 三磷酸和胞质Ca2 +迅速但短暂增加，而PDGF则没有。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ζ，它们主要定位于胞质溶胶中。一部分胞质PKCα在15秒时被α - 凝血酶迅速转位，但其与膜的结合在1分钟内消失。PKCε也迅速转位；然而，其与膜的结合持续了近60分钟。PKCζ不被α - 凝血酶或佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯转位。PDGF在5分钟时转位PKCε，但在15秒时影响很小，并且不转位PKCα或ζ。用蜡样芽孢杆菌PC - 或磷脂酰肌醇特异性磷脂酶C孵育，其增加DAG但不增加磷脂酸，刺激PKCε的转位，但不刺激PKCα或ζ。添加螯合剂以抑制细胞内Ca2 +升高在很大程度上阻断了α - 凝血酶诱导的PKCα转位，但对PKCε转位没有影响。添加离子霉素使α - 凝血酶在5分钟时诱导PKCα转位。1,2 - 二辛酰甘油加离子霉素模拟了PKCα转位，但单独使用任何一种都不行。另一方面，PKCε仅被DAG转位。这些结果支持以下结论：PIP2水解在15秒时激活PKCα和ε，而PC水解在5分钟时仅激活PKCε。5分钟时的差异激活可归因于PC水解未能增加Ca2 +，而不是源自磷脂的DAG分子种类的差异。
