CELL BIOLOGY--PHOSPHATIDYLCHOLINE-DERIVED DIACYLGLYCERIN

本提案的目的是阐明（在细胞培养中 模型和无细胞系统）的生物学作用 细胞信号中的磷脂酰胆碱衍生的二酰基甘油（DAG） 佛波醇酯刺激的转导和机制 这条路。 初步结果表明佛波醇酯 刺激磷脂酰胆碱（PC）特异性磷脂酶C 非转化大鼠胚胎成纤维细胞中的（PL-C）活性（参考- NT）引起DAG水平增加2至3倍。 但这 在相应的转化细胞中未观察到反应 线 在裂解物中的PC水解酶（PL-C）活性水平 两种细胞类型相似，约为2.6 nmol/mg 蛋白质/小时）。 这些结果表明，缺乏反应能力 在转化细胞系中，不是由于酶的损失，而是 可能是由于佛波酯受体/蛋白质的损伤 激酶C（PKC）途径或在生物化学调节中的作用。 转化细胞PC PL-C活性。 以确定是否 PKC调节的改变可能有助于 无反应性，我们建议比较两种细胞系， 关于PKC和佛波酯的生化特性 绑定活动。 制备三种类型的特异性抗体 兔脑PKC的类型将被用来表征潜在的 两种细胞系中PKC类型的差异。 我们还将 比较两种细胞的PC PL-C的生化特性 线 此外，我们已经确定脑细胞质是一种组织， 可以从其中纯化酶的来源。 两种细胞 裂解物和纯化的酶将用于研究中，以测试 PKC介导磷酸化对PC水解酶的影响 活动 将使用三种类型的纯化脑PKC中的每一种 在这些研究中。 PC水解在生长调节中的作用 和分化尚不清楚，尽管在 细胞生长被认为是因为天然的促有丝分裂激动剂， 加压素也刺激这些细胞中的PC水解。 此外，观察到的差异的一般性， REF-NT和REF-T细胞的反应性将通过使用 几种未转化的细胞系及其病毒或化学方法 转化的对应物 一个彻底的特征， 脂质代谢途径和相关酶， 生理效应的途径，并阐明了 佛波醇酯激活机制将有助于建立作用 这种假定的第二信使系统在正常和疾病 流程.

The objectives of this proposal are to elucidate (in cell culture models and cell-free systems) the biological role of phosphatidylcholine-derived diacylglycerol (DAG) in cell signal transduction and the mechanism by which phorbol esters stimulate this pathway. Preliminary results indicate that phorbol esters stimulate a phosphatidylcholine (PC) specific phospholipase C (PL-C) activity in nontransformed rat embryo fibroblasts (REF- NT) causing a 2- to 3-fold increase in DAG levels. However, this response was not observed in the corresponding transformed cell lines. The levels of PC hydrolase (PL-C) activity in lysates of both cell types were similar approximately 2.6 nmol/mg protein/hr). These results indicate that the lack of responsiveness in the transformed cell line is not due to loss of the enzyme, but may be due to either a lesion in the phorbol ester receptor/protein kinase C (PKC) pathway or in biochemical regulation of the transformed cell PC PL-C activity. In order to determine if alterations in regulation of PKC may contribute to the nonresponsiveness, we propose to compare the two cell lines with respect to the biochemical properties of PKC and phorbol ester binding activities. Type-specific antibodies prepared to three types of rabbit brain PKC will be used to characterize potential differences in PKC types in the two cell lines. We will also compare the biochemical properties of PC PL-C from the two cell lines. Furthermore, we have identified brain cytosol as a tissue source from which the enzyme can be purified. Both the cell lysates and the purified enzyme will be used in studies to test the effect of PKC-mediated phosphorylation on PC hydrolase activity. Each of three types of purified brain PKC will be used in these studies. The role of PC hydrolysis in regulation of growth and differentiation is not yet known, although a potential role in cell growth is suggested because a natural mitogenic agonist, vasopressin, also stimulates PC hydrolysis in these cells. Furthermore, the generality of the observed difference in responsiveness of REF-NT and REF-T cells will be tested by using several nontransformed cell lines and their virally or chemically transformed counterparts. A thorough characterization of the lipid metabolic pathways and the enzymes involved, assessment of physiological effectors of the pathway, and elucidation of the mechanism of phorbol ester activation will help establish the role of this putative second messenger system in normal and disease processes.