Phosphoproteins in Insulin Signaling

胰岛素信号传导中的磷蛋白

• 批准号: 6906499
• 负责人: GUSTAV E. LIENHARD
• 金额: $ 47.08万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6906499
• 关键词: G protein; adipocytes; biological signal transduction; cell type; enzyme activity; enzyme mechanism; enzyme substrate; gene deletion mutation; gene targeting; genetically modified animals; glucose transport; glycogen synthase; hormone metabolism; hormone regulation /control mechanism; immunocytochemistry; immunoprecipitation; insulin receptor; laboratory mouse; lipolysis; messenger RNA; molecular cloning; mutant; phenotype; phosphatidylinositol 3 kinase; phosphoproteins; phosphorylation; protein kinase; protein protein interaction; protein structure function; protein tyrosine kinase; receptor expression; serine threonine protein kinase; yeast two hybrid system

DESCRIPTION (provided by applicant): The overall goal is to elucidate signaling pathways from the insulin receptor. These proceed largely through the activation of protein kinases leading to changes in protein phosphorylation. One protein kinase that plays a prominent role in insulin signaling is Akt. Recently, we have discovered five novel insulin-elicited phosphoproteins in adipocytes, four of which are likely novel Akt substrates. In addition, recently a new method for identifying many sites of phosphorylation on many proteins in one sample by mass spectrometry has been developed. This method includes a procedure for quanitating the change in phosphorylation at each site upon treatment of the cell with an agent such as insulin. Our specific aims, which build upon these advances, are: 1. To determine the cellular roles of the novel putative Akt substrates in insulin action. The initial focus will be a 250 kD protein (pp250) that contains a predicted GTPase activating protein (GAP) domain for the small G proteins Rap and Rheb. The GAP activity of recombinant pp250 toward Rap and Rheb, and the effect of phosphorylation of pp250 on the activity, will be determined. Depending upon whether pp250 is a GAP for Rap or Rheb, the effect of insulin on Rap or Rheb-regulated processes in adipocytes, and the role of pp250 in this effect, will be defined. 2. To identify other novel targets of insulin regulated phosphorylation in fat, muscle, and liver cells. Antibodies specific for phosphomotifs will be used to isolate groups of proteins that include ones phosphorylated on tyrosine by the insulin receptor and on serine/threonine by activated Akt and ERK. Novel sites of insulin-elicited phosphorylation will then be identified by the new phosphoproteomics methods. This research is directly relevant to diabetes. Detailed knowledge of insulin signaling provides a basis for understanding the changes that occur in insulin resistance and in insulin deficiency.

描述（由申请人提供）：总体目标是阐明胰岛素受体的信号通路。这些主要通过激活蛋白激酶导致蛋白磷酸化的变化来进行。在胰岛素信号传导中起重要作用的一种蛋白激酶是Akt。最近，我们在脂肪细胞中发现了五种新的胰岛素诱导的磷蛋白，其中四种可能是新的Akt底物。此外，最近已经开发了一种通过质谱法鉴定一个样品中许多蛋白质上的许多磷酸化位点的新方法。该方法包括在用诸如胰岛素的试剂处理细胞时定量每个位点的磷酸化变化的程序。我们的具体目标，建立在这些进步，是：1。确定新的假定Akt底物在胰岛素作用中的细胞作用。最初的焦点将是一个250 kD的蛋白质（pp 250），它含有一个预测的GTdR激活蛋白（GAP）结构域的小G蛋白Rap和Rheb。将测定重组pp 250对Rap和Rheb差距活性以及pp 250的磷酸化对活性的影响。根据pp 250是否是Rap或Rheb的GAP，将确定胰岛素对脂肪细胞中Rap或Rheb调节过程的影响以及pp 250在该影响中的作用。2.确定脂肪、肌肉和肝细胞中胰岛素调节磷酸化的其他新靶点。对磷酸化寡核苷酸特异的抗体将用于分离蛋白质组，所述蛋白质组包括通过胰岛素受体在酪氨酸上磷酸化的蛋白质组和通过活化的Akt和ERK在丝氨酸/苏氨酸上磷酸化的蛋白质组。新的网站胰岛素引起的磷酸化，然后将确定新的磷酸化蛋白质组学方法。这项研究与糖尿病直接相关。胰岛素信号传导的详细知识为理解胰岛素抵抗和胰岛素缺乏中发生的变化提供了基础。