INSULIN REGULATION OF C-FOS EXPRESSION

胰岛素对 C-FOS 表达的调节

• 批准号: 3240648
• 负责人: PERRY J BLACKSHEAR
• 金额: $ 9.44万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1992-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3240648
• 关键词: DNA; DNA binding protein; affinity chromatography; autoradiography; cell free system; cell type; gel electrophoresis; gene expression; genetic transcription; hormone regulation /control mechanism; insulin; insulin receptor; laboratory mouse; laboratory rabbit; laboratory rat; liver; molecular cloning; monoclonal antibody; nucleic acid sequence; phorbols; phosphorylation; protein sequence; protooncogene; radiotracer

Insulin exerts powerful influences on the biosynthesis of many enzymes and other proteins as part of its normal role in maintaining body fuel homeostasis. In some cases, this effect has been localized to changes in the transcription rates of certain genes. In one such case, insulin rapidly stimulated the transcription of the c-fos proto-oncogene within 10 min of hormone exposure. This proto-oncogene encodes a nuclear phosphoprotein that may serve as a trans-acting regulator of the expression of other genes. In this way, the induction of c-fos by insulin might be the first or an early step in a sequential series of biosynthetic or transcriptional responses to insulin. A sequence within the 5'-flanking region of the c-fos gene which is necessary for induction of the gene by insulin and active tumor- promoting phorbol esters has recently been identified. This region was previously identified as being responsible for serum induction of c-fos in fibroblasts, and has been labelled the serum response element (SRE). Four point mutations within the SRE abolished the ability of insulin and phorbol esters to induce the transfected gene, and also abolished the specific binding of a nuclear protein which bound to the normal SRE. The overall goals of the proposed studies are to determine the molecular steps which are involved in the induction of c-fos by insulin, and perhaps other agents, such as phorbol ester, which induce the gene by a different proximal mechanism. To do this, the SRE binding protein will be purified from an insulin-responsive tissue such as rat liver, in which c-fos is induced during liver regeneration. The purified protein will be used to immunize rabbits and mice for the production of antibodies, and proteolytic peptides will be sequenced; results of these studies will be used in the molecular cloning of a cDNA for the protein. The antibodies and the specific localization of the protein on two dimensional gels will be used to determine whether insulin and other stimuli modify the protein in any way, either directly or by altering its association with other proteins. Further studies will determine whether these modifications affect c-fos transcription. Other insulin-responsive regions of the gene will also be sought, and similar techniques applied to the search for other insulin-modified proteins which might interact with these sequences. The ultimate aim of these studies is eventually to link activation of the insulin receptor with this early transcriptional response by a known series of biochemical reactions.

胰岛素对许多生物的生物合成有着强大的影响。 酶和其他蛋白质作为其正常作用的一部分， 维持体内能量平衡 在某些情况下，这种影响 被定位于某些转录速率的变化， 基因. 在一个这样的例子中，胰岛素迅速刺激了 c-fos原癌基因的转录在10分钟内 荷尔蒙暴露 这个原癌基因编码一个核 磷蛋白，可以作为一个反式作用的调节剂， 其他基因的表达。 通过这种方式， 胰岛素可能是第一个或早期步骤，在一个连续的系列， 对胰岛素的生物合成或转录反应。 在c-fos基因的5 ′-侧翼区域内的序列， 是胰岛素和活动性肿瘤诱导该基因所必需的 最近已经鉴定出促进佛波醇酯。 这一地区 先前被确定为负责血清诱导 的c-fos在成纤维细胞，并已标记的血清反应 元素（SRE）。 SRE内的四个点突变被消除 胰岛素和佛波醇酯诱导转染细胞的能力 基因，也取消了核蛋白的特异性结合， 与正常的SRE结合 拟议研究的总体目标是确定 参与c-fos诱导的分子步骤 胰岛素和可能的其它药剂，如佛波酯， 通过不同的近端机制诱导基因。 要执行此操作， SRE结合蛋白将从胰岛素应答性 组织，如大鼠肝脏，其中c-fos在肝脏过程中被诱导 再生 纯化的蛋白质将用于免疫 用于兔和小鼠的抗体生产，以及蛋白水解 将对肽进行测序;这些研究的结果将用于 蛋白质cDNA的分子克隆。 的抗体 蛋白质在二维空间的特异性定位 凝胶将用于确定胰岛素和其他刺激物是否 以任何方式修饰蛋白质，直接或通过改变其 与其他蛋白质结合。 进一步的研究将确定 这些修饰是否影响c-fos转录。 其他 还将寻找该基因的胰岛素响应区域， 类似的技术应用于寻找其他胰岛素修饰的 可能与这些序列相互作用的蛋白质。 最终 这些研究的目的是最终将胰岛素的激活 受体与这种早期的转录反应，由一个已知的系列 生化反应。