INSULIN REGULATION OF C-FOS EXPRESSION

胰岛素对许多物质的生物合成有很强的影响 酶和其他蛋白质作为其正常作用的一部分 维持身体的燃料平衡。在某些情况下，这种影响具有 被定位于某些转录速率的变化 基因。在一个这样的案例中，胰岛素迅速刺激了 C-fos原癌基因在10分钟内转录 荷尔蒙暴露。这种原癌基因编码一种核 磷酸蛋白可能作为一种反式作用调节蛋白。 其他基因的表达。通过这种方式，c-fos的诱导通过 胰岛素可能是一系列胰岛素的第一步或早期步骤 对胰岛素的生物合成或转录反应。 C-fos基因5‘侧翼区内的序列 是胰岛素和活动性肿瘤诱导基因所必需的- 最近发现了促进佛波醇酯的方法。这一地区 先前被确定为对血清诱导负责 C-fos在成纤维细胞中的表达，并被标记为血清反应 元素(SRE)。SRE中的四个点突变被废除 胰岛素和佛波酯诱导转染体的能力 基因，还取消了核蛋白的特异性结合 它与正常的SRE结合。 拟议研究的总体目标是确定 C-fos诱导过程中的分子步骤 胰岛素，也许还有其他药物，如佛波酯，它 通过不同的近端机制诱导基因。要做到这点， SRE结合蛋白将从一种胰岛素反应的 C-fos在肝脏中被诱导的组织，如大鼠肝脏 再生。纯化的蛋白将用于免疫 兔和小鼠用于产生抗体和蛋白水解物 将对多肽进行测序；这些研究的结果将用于 该蛋白的一个cdna的分子克隆。抗体 以及蛋白质在二维上的特异性定位 凝胶将被用来确定胰岛素和其他刺激是否 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 这些修饰是否会影响c-fos转录。其他 还将寻找该基因的胰岛素响应区，以及 类似的技术适用于寻找其他胰岛素修饰的药物 可能与这些序列相互作用的蛋白质。终极的 这些研究的目的是最终将胰岛素的激活联系起来 通过已知的一系列具有这种早期转录反应的受体 生化反应。

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.