MECHANISM OF PHORBOL ESTER-INDUCED LIPID METABOLISM

蛋白激酶C途径的信号转导紊乱 与肿瘤过程的发展有关。目的 这项提议是为了定义一条潜在的蛋白质抑制途径。 激酶C，游离的狮身人面像碱基已被证明具有拮抗作用 蛋白激酶C在体外和在完整细胞中的激活。然而， 目前还没有证据表明，一条涉及 鞘磷脂代谢到狮身人面像碱基起到灭活蛋白质的作用 生理条件下的蛋白激酶C。最近的这方面的研究 实验室证明，1，2-二酰甘油，内源性 蛋白激酶C的激活剂，被刺激降解 GH3垂体细胞中的鞘磷脂鞘磷脂。此事件已发生 通过鞘磷脂酶(EC 3.1.4.12)。佛波酯，肿瘤- 促进蛋白激酶C的激活剂，但未能刺激 事件。此外，鞘磷脂酶的作用足以灭活。 蛋白激酶C。这些研究表明，1，2-二酰甘油可能 激活蛋白激酶C未被利用的负效应通路 佛波尔酯。这项提议的目的是探索 对这一概念进行了详细阐述。 三种不同的细胞系将用于这些研究；GH3大鼠 垂体细胞、3T3小鼠成纤维细胞和HL-60人白血病细胞。 1，2-二酰基甘油和佛波酯对合成的影响 和含胆碱的磷脂的降解， 将对磷脂酰胆碱和鞘磷脂进行评估。的作用 这些过程中的cAMP和蛋白激酶C系统将是 下定决心。通过鞘磷脂酶降解鞘磷脂将是 与蛋白激酶C的激活/失活有关。受体介导 鞘磷脂酶激活与游离狮身人面像碱基的产生 将对利用肌醇磷脂途径的配体进行评估 用于信号转导。将进行研究以确定是否 1，2-二氯苯酚引起的生物反应模式的差异 甘油二酯和佛波酯可归因于鞘磷脂酶 行动。希望这些研究能揭示出一种与生理相关的 蛋白激酶C的抑制途径，并解释了一些 1，2-二酰基甘油对细胞激活的差异 和佛波酯。

Disordered signal transduction via the protein kinase C pathway has been implicated in the development of the neoplastic process. The purpose of this proposal is to define a potential inhibitory pathway for protein kinase C. The free sphingoid bases have been shown to antagonize activation of protein kinase C in vitro and in intact cells. However, evidence has not been presented to suggest that a pathway involving sphingolipid metabolism to sphingoid bases serves to inactivate protein kinase C under physiologic conditions. Recent studies from this laboratory demonstrated that 1,2-diacylglycerols, the endogenous activators of protein kinase C, stimulated degradation of the sphingolipid sphingomyelin in GH3 pituitary cells. This event occurred via a sphingomyelinase (EC 3.1.4.12). Phorbol esters, the tumor- promoting activators of protein kinase C, failed to stimulate this event. Further, sphingomyelinase action was sufficient to inactivate protein kinase C. These studies suggest that 1,2-diacylglycerols may activate a negative effector pathway for protein kinase C not utilized by the phorbol esters. The intention of this proposal is to explore this concept in detail. Three distinct cell lines will be utilized for these studies; GH3 rat pituitary cells, 3T3 mouse fibroblasts and HL-60, human leukemia cells. The effect of 1,2-diacylglycerols and phorbol esters on the synthesis and degradation of the choline-containing phospholipids, phosphatidylcholine and sphingomyelin, will be evaluated. The role of the cAMP and protein kinase C systems in these processes will be determined. Sphingomyelin degradation via a spingomyelinase will be related to protein kinase C activation/inactivation. Receptor-mediated sphingomyelinase activation and the generation of free sphingoid bases will be assessed for ligands that utilize the phosphoinositide pathway for signal transduction. Studies will be performed to determine if differences in the pattern of biologic responses after 1,2- diacylglycerols and phorbol esters can be ascribed to sphingomyelinase action. Hopefully, these studies will reveal a physiologically relevant inhibitory pathway for protein kinase C and explain some of the differences observed between cellular activation by 1,2-diacylglycerols and phorbol esters.