Cabot M C, Welsh C J, Zhang Z C, Cao H T
W. Alton Jones Cell Science Center, Inc., Lake Placid, NY 12946.
FEBS Lett. 1989 Mar 13;245(1-2):85-90. doi: 10.1016/0014-5793(89)80197-8.
In this study we provide evidence for the involvement of protein kinase C (PKC) in phorbol diester-induced phosphatidylcholine (PC) hydrolysis by the phospholipase D pathway. Rat embryo fibroblasts (REF52) were prelabeled with either tritiated choline or myristic acid; these compounds are preferentially incorporated into cellular PC. Phorbol diester-induced PC degradation was determined by measuring the release of [3H]choline, and the formation of [3H]myristoyl-containing phosphatidate (PA), diacylglycerol (DG), and phosphatidylethanol (PE). Staurosporine, a PKC inhibitor, blocked from 73 to 90% of the phorbol diester-induced PC hydrolysis. The inhibition of phorbol diester-induced choline release by staurosporine was dose dependent with an approximate ED50 of 150 nM. Pretreatment of cells with phorbol diester inhibited subsequent phorbol diester-induced PC degradation by 78-92%. A close correlation between the ED50 for phorbol diester-stimulated choline release and the Kd for phorbol diester binding was demonstrated. Neither forskolin nor dibutyryl cAMP elicited cellular PC degradation. In vitro experiments using phospholipase D from Streptomyces chromofuscus showed that staurosporine did not inhibit and TPA did not stimulate enzyme activity.
在本研究中，我们提供了蛋白质激酶C（PKC）参与佛波酯通过磷脂酶D途径诱导的磷脂酰胆碱（PC）水解的证据。大鼠胚胎成纤维细胞（REF52）用氚化胆碱或肉豆蔻酸进行预标记；这些化合物优先掺入细胞PC中。通过测量[3H]胆碱的释放以及含[3H]肉豆蔻酰基的磷脂酸（PA）、二酰基甘油（DG）和磷脂酰乙醇（PE）的形成来测定佛波酯诱导的PC降解。PKC抑制剂星形孢菌素可阻断73%至90%的佛波酯诱导的PC水解。星形孢菌素对佛波酯诱导的胆碱释放的抑制作用呈剂量依赖性，其近似半数有效浓度（ED50）为150 nM。用佛波酯预处理细胞可抑制随后佛波酯诱导的PC降解78% - 92%。证明了佛波酯刺激胆碱释放的ED50与佛波酯结合的解离常数（Kd）之间存在密切相关性。福斯高林和二丁酰环磷腺苷均未引起细胞PC降解。使用来自暗褐链霉菌的磷脂酶D进行的体外实验表明，星形孢菌素不抑制且佛波醇酯不刺激酶活性。
