ONCOGENE PHOSPHOINOSITIDE CYCLE/KINASE C

这项提案的总体目标是阐明 二酰甘油(DAG)代谢和蛋白激酶C(PKC)代谢 致癌转化活性。DAG是内源性的 PKC的激活物，它也是肿瘤的受体- 推广佛波醇酯。这个实验室提出了一些 癌基因可能通过去调节DAG而转化细胞 代谢，从而结构性地激活PKC。为了支持…… 这一假设，DAG水平被发现评估在 Ras、src或FMS和PKC联合转化的成纤维细胞部分 激活了。然而，令人惊讶的是，这些转化的细胞也是 对佛波醇酯的影响部分致密，以及 抗v-ras转化的回复系表现为 更夸张的脱敏。这些结果表明， 脱敏可能代表细胞试图克服 癌基因产生的结构性增殖信号。 确定本构关系的责任机制 DAG升高，将进行代谢标记研究 用温度敏感型v-1基因转染的3T3细胞系 SRC癌基因。糖尿病大鼠DAG代谢调控的研究 分子水平将聚焦于DAG激酶，它已经被 发现存在多种同工型，并且移位迅速 对血清或佛波酯有反应的膜。单克隆 针对不同同型的抗体正在产生，以 促进磷酸化、组织特异性和 正在处理。第二，PKC的动力学和机理 脱敏作用将被确定。(是否产生脱敏 通过磷酸酶活性的变化，蛋白激酶C抑制剂的产生， PKC的磷酸化、膜结合的改变 还是在PKC底物本身？)PKC在全球经济一体化中的作用 还将调查v-src对转化的调解。 有待详细研究的表型变化包括c-受体的激活 MYC的表达，启动DNA合成的早期变化 蛋白质的磷酸化和合成与表皮生长 因子(EGF)受体下调。这是最后一个回答 似乎是独立于PKC的，并且涉及两个不同的、 可分离的机制，其中之一是环己亚胺敏感。 其他蛋白激酶和自分泌因子在 促进下调制将被阐明，使用两个整体 细胞和膜制剂。这些实验将提供 洞察某些癌基因的分子机制 中介转化，以及通过什么机制 转变可以被有效地阻止。

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.