ONCOGENE PHOSPHOINOSITIDE CYCLE/KINASE C

癌基因磷酸肌醇循环/激酶 C

• 批准号: 3177313
• 负责人: IAN G MACARA
• 金额: $ 13.94万
• 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 1993-12-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3177313
• 关键词: 3T3 cells; 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.

该提案的总体目标是阐明 二酰甘油 (DAG) 代谢和蛋白激酶 C (PKC) 致癌转化活性。 DAG是内生的 PKC 的激活剂，也是肿瘤的受体 促进佛波酯。 该实验室提出了一些 癌基因可能通过解除 DAG 的调节来转化细胞 代谢，从而组成性激活 PKC。 支持 根据这一假设，DAG 水平被发现可以在 ras、src或fms转化的成纤维细胞和PKC部分 活性。 然而令人惊讶的是，这些转化细胞也 对佛波酯的作用部分致密，并且 回复系，抵抗 v-ras 的转化，显示 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型增殖信号。 确定负责构成的机制 DAG 升高，将进行代谢标记研究 使用转染温度敏感 v- 的 3T3 细胞系 src癌基因。 DAG代谢控制的研究 分子水平将重点关注DAG激酶，该激酶已被 发现存在多种同种型，并且易位迅速 膜对血清或佛波酯的反应。 单克隆 针对不同同种型的抗体正在产生 促进磷酸化、组织特异性和 加工。 其次，PKC的动力学和机制 将确定脱敏。 （是否产生脱敏 通过磷酸酶活性、PKC 抑制剂产生的变化， PKC 磷酸化，PKC 膜关联的改变 或者在 PKC 底物本身中？） PKC 在 还将研究 v-src 对转化的中介作用。 有待详细研究的表型变化包括 c- 的激活 myc 表达、DNA 合成的起始、早期变化 蛋白质磷酸化和合成以及表皮生长 因子（EGF）受体下调。 这最后的回应 似乎是独立于 PKC 的，并且涉及两个不同的、 可分离的机制，其中之一是放线菌酮敏感的。 其他蛋白激酶和自分泌因子的作用 将阐明促进下调，使用整个 细胞和膜制剂。 这些实验将提供 深入了解某些癌基因的分子机制 介导转化及其机制 可以有效地阻止转化。