Leach K L, Ruff V A, Wright T M, Pessin M S, Raben D M
Department of Cell Biology, Upjohn Company, Kalamazoo, Michigan 49007.
J Biol Chem. 1991 Feb 15;266(5):3215-21.
Diacylglycerols (DAGs) derived from phosphatidylcholine (PC) hydrolysis have been shown to activate protein kinase C (PKC) in vitro, but it is not known whether this event occurs in response to DAGs generated via agonist-induced PC hydrolysis in intact cells. In this report we have addressed this question directly, using alpha-thrombin stimulation of IIC9 fibroblasts. PKC activation in intact cells was assessed in two ways, by measuring: 1) PKC membrane association as determined by kinase activity and Western blot analysis and 2) the phosphorylation of an endogenous PKC substrate, an 80-kDa protein. Treatment with 500 ng/ml alpha-thrombin has been shown to stimulate both phosphoinositide and PC hydrolysis, whereas treatment with 100 pg/ml alpha-thrombin stimulates only PC breakdown. Using these two conditions, we show that DAG produced from phosphoinositide, but not PC hydrolysis, is associated with the activation of PKC.
来源于磷脂酰胆碱（PC）水解的二酰基甘油（DAGs）已被证实在体外可激活蛋白激酶C（PKC），但尚不清楚在完整细胞中，这一事件是否会因激动剂诱导的PC水解所产生的DAGs而发生。在本报告中，我们使用α-凝血酶刺激IIC9成纤维细胞，直接解决了这个问题。通过以下两种方式评估完整细胞中的PKC激活：1）通过激酶活性和蛋白质印迹分析测定PKC膜结合；2）测定内源性PKC底物（一种80 kDa蛋白质）的磷酸化。已表明用500 ng/mlα-凝血酶处理可刺激磷酸肌醇和PC水解，而用100 pg/mlα-凝血酶处理仅刺激PC分解。利用这两种条件，我们表明由磷酸肌醇而非PC水解产生的DAG与PKC的激活相关。
