Phosphoproteins in Insulin Signaling

胰岛素信号传导中的磷蛋白

• 批准号: 6818622
• 负责人: GUSTAV E. LIENHARD
• 金额: $ 49.22万
• 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/6818622
• 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。这主要是通过蛋白激酶的激活导致蛋白磷酸化的变化来进行的。在胰岛素信号转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酸蛋白，其中四种可能是新的Akt底物。此外，最近发展了一种新的方法，可以用质谱学鉴定一个样品中许多蛋白质上的多个磷酸化位点。这种方法包括一种程序，用于在用诸如胰岛素的试剂处理细胞时量化每个部位的磷酸化变化。在这些进展的基础上，我们的具体目标是：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250kD的蛋白质(Pp250)，它包含一个预测的小G蛋白Rap和Rheb的GTP酶激活蛋白(GAP)结构域。将测定重组pp250对Rap和Rheb的GAP活性，以及pp250的磷酸化对活性的影响。根据pp250是Rap还是Rheb的缺口，胰岛素对Rap或Rheb调节的脂肪细胞过程的影响，以及pp250在这种影响中的作用，将被确定。2.确定胰岛素调节脂肪、肌肉和肝脏细胞磷酸化的其他新靶点。针对磷酸化的抗体将被用来分离蛋白质组，其中包括被胰岛素受体磷酸化的酪氨酸和被激活的Akt和ERK激活的丝氨酸/苏氨酸的蛋白质。然后，新的磷酸蛋白质组学方法将确定胰岛素诱导的磷酸化的新位点。这项研究与糖尿病直接相关。对胰岛素信号转导的详细了解为理解胰岛素抵抗和胰岛素缺乏的变化提供了基础。