ONCOGENE PHOSPHOINOSITIDE CYCLE/KINASE C

癌基因磷酸肌醇循环/激酶 C

• 批准号: 3177314
• 负责人: IAN G MACARA
• 金额: $ 12.32万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-06-01 至 1993-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3177314
• 关键词: DNA replication; alpharetrovirus; diacylglycerols; enzyme mechanism; epidermal growth factor; gene expression; high performance liquid chromatography; laboratory mouse; laboratory rabbit; laboratory rat; lipid metabolism; molecular biology; neoplasm /cancer genetics; neoplastic transformation; oncogenes; phosphatidylinositols; phosphorylation; phosphotransferases; protein kinase C; radionuclides; temperature; thin layer chromatography; tissue /cell culture; viral carcinogenesis

The overall goal of this proposal is to elucidate the role of diacylglycerol (DAG) metabolism and of protein kinase C (PKC) activity in oncogenic transformation. DAG is the endogenous activator of PKC, which is also the receptor for the tumor- promoting phorbol esters. This laboratory has proposed that some oncogenes might transform cells by de-regulating DAG metabolism, so as to constitutively activate PKC. In support of this hypothesis, DAG levels were found to be evaluated in fibroblasts transformed by ras, src or fms and PKC was partially activated. Surprisingly, however, these transformed cells are also partially densensitized to the effects of phorbol esters, and revertant lines, resistant to transformation by v-ras, display a more exaggerated desensitization. These results suggest that desensitization may represent an attempt by the cell to overcome the constitutive proliferative signals generated by the oncogenes. To determine the mechanism responsible for constitutive elevation of DAG, metabolic labeling studies will be performed using a 3T3 cell-line transfected with a temperature-sensitive v- src oncogene. Investigation of the control of DAG metabolism at the molecular level will focus on DAG kinase, which has been found to exist in multiple isotypes, and which translocates rapidly to membranes in response to serum or phorbol esters. Monoclonal antibodies are being generated to the different isotypes to facilitate studies of phosphorylation, tissue specificity and processing. Secondly, the kinetics and mechanism of PKC desensitization will be determined. (Is desensitization produced by changes in phosphatase activity, PKC inhibitor production, PKC phosphorylation, alterations in membrane association of PKC or in the PKC substrates themselves?) The role of PKC in the mediation of transformation by v-src will also be investigated. Phenotypic changes to be studied in detail include activation of c- myc expression, initiation of DNA synthesis early changes in protein phosphorylation and synthesis, and epidermal growth factor (EGF) receptor down-modulation. This last response appears to be PKC-independent and involves two distinct, separable mechanisms, one of which is cycloheximide-sensitive. The role of other protein kinases, and of autocrine factors, in promoting down-modulation will be elucidated, using both whole cell and membrane preparations. These experiments will provide insight into the molecular mechanism by which certain oncogenes mediate transformation, and the mechanism by which transformation can be effectively blocked.

本提案的总体目标是阐明 甘油二酯（DAG）代谢和蛋白激酶C（PKC） 致癌转化的活性。 DAG是内源性的 PKC的激活剂，也是肿瘤的受体， 促进佛波醇酯。 该实验室提出， 癌基因可能通过去调节DAG来转化细胞 代谢，从而组成性激活PKC。 支持 在这一假设中，发现DAG水平在 ras、src或fms和PKC部分转化成纤维细胞， 激活 然而，令人惊讶的是，这些转化细胞也 对佛波醇酯的作用部分脱敏，和 对v-ras转化具有抗性的回复突变株系显示出 更夸张的脱敏 这些结果表明 脱敏可能代表细胞试图克服 由癌基因产生的组成性增殖信号。 确定负责构成性的机制 DAG升高，将进行代谢标记研究 使用转染有温度敏感性v- src癌基因 研究了对DAG代谢的控制， 分子水平将集中在DAG激酶， 发现存在于多个同种型中，并且迅速易位 对血清或佛波醇酯的反应。 单克隆 针对不同的同种型产生抗体， 促进磷酸化、组织特异性和 处理. 第二，PKC的动力学和作用机制 将确定脱敏。 (Is减敏产生 通过改变磷酸酶活性，PKC抑制剂的产生， PKC磷酸化，PKC膜结合的改变 还是PKC底物本身？） 蛋白激酶C在肿瘤细胞凋亡中的作用 还将研究V-SRC对转化的介导作用。 待详细研究的表型变化包括c- myc的表达，DNA合成的起始， 蛋白质磷酸化和合成以及表皮生长 EGF受体下调。 最后一个回答 似乎是PKC独立的，涉及两个不同的， 可分离的机制，其中之一是环己酰亚胺敏感。 其他蛋白激酶和自分泌因子在 将使用两个整体来阐明促进下调制。 细胞和膜制备。 这些实验将提供 深入了解某些致癌基因 介导的转化，以及 可以有效地阻止转化。