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Insulin Control of Gene Transcription Through Sensitin

胰岛素通过敏敏素控制基因转录

基本信息

批准号：
7437281
负责人：
BETTY C VILLAFUERTE
金额：
$ 24.59万
依托单位：
UNIVERSITY OF LOUISVILLE
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-15 至 2010-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7437281
关键词：
1-Phosphatidylinositol 3-Kinase Animals Binding Binding Proteins Biological Biological Models Cell Nucleus Cells Characteristics Cleaved cell Complementary DNA Cysteine Cytoplasmic Protein Data Defect Diabetes Mellitus Elements Endopeptidases Epidemic Event Gene Targeting Genes Genetic Transcription Genomics Goals Hepatic In Vitro Insulin Insulin Resistance Insulin-Like Growth Factor Binding Protein 3 Investigation Lead Link Liver Liver Extract Localized Mass Spectrum Analysis Mediating Metabolic Modeling Mutate Mutation Non-Insulin-Dependent Diabetes Mellitus Nuclear Translocation Obesity Pathogenesis Pathway interactions Peptide Hydrolases Peptides Phosphorylation Phosphorylation Site Phosphotransferases Physiological Play Predisposition Prevalence Process Protease Domain Protein Dephosphorylation Proteins Proteolysis Proto-Oncogene Proteins c-akt Ras/Raf Rattus Recombinants Regulation Research Personnel Resistance Role Secondary to Serine/Threonine Phosphorylation Signal Pathway Signal Transduction Site Testing Trans-Activators Transactivation Transcription Coactivator Transcriptional Activation age related base blood glucose regulation diabetic diabetic rat in vivo insight insulin signaling novel prevent programs receptor response sensitin two-dimensional

项目摘要

DESCRIPTION (provided by applicant): Insulin resistance leading to type 2 diabetes generally involve defects in insulin signaling at the postreceptor level. Activation of the PI 3'-kinase to Akt pathway plays a significant role in insulin signaling, but the events downtream of Akt are largely unknown. Recently, we identified a novel Akt substrate which binds to the insulin-responsive elements (IREs) of insulin-like growth factor binding protein-3 (IGFBP-3) and other insulin-responsive genes. The IRE-binding protein "sensitin" cDNA encodes a 120 kDa cytoplasmic protein. After adding insulin, the protein is cleaved to a 50 kDa fragment, which localizes predominantly to the nucleus and transactivates IREs. PI 3'-kinase inhibition decreases both Ser/Thr phosphorylation and proteolysis of sensitin. Based on our preliminary data, the long-term goal of our investigation is to employ the IRE of IGFBP-3 as a model system to delineate the signaling pathway by which insulin induces genomic effects through the action of a specific trans-acting factor, sensitin, that binds to the IRE. This application has the following aims: 1) To test the hypothesis that sensitin is subject to regulated phosphorylation by insulin, using 2-D phosphomapping and mass spectrometry to localize residues of sensitin phosphorylated by insulin in HepG2 cells, and the effect of phosphorylation on insulin-induced transcription assessed by mutational analysis. 2) To test the hypothesis that proteolytic cleavage of sensitin is required for transactivation of the IRE, and the corollary hypothesis that nuclear translocation of sensitin is secondary to cleavage of the protein. We will identify the cleavage sites by microsequencing, mutate the proteolytic domain to test its role in nuclear translocation and IRE transcription; and mutate the Akt phosphorylation site to determine if dephosphorylation prevents nuclear translocation or proteolysis of sensitin. 3) To determine the physiological significance of phosphorylation and proteolysis of sensitin in a rat diabetes model, we will compare the phosphorylation and proteolysis of sensitin in vitro by hepatic cytosolic extracts from diabetic, obese, and lean control rats, and determine whether sensitin mutation conferring protease- and phosphorylation-resistance increase the diabetes susceptibility of obese rats, or worsen glucose control in diabetic rats. This study may lead to better understanding of insulin-regulated gene transcription, and may provide insights into linked to the pathogenesis of type 2 diabetes mellitus.

描述（由申请人提供）：导致2型糖尿病的胰岛素抵抗通常涉及受体后水平的胰岛素信号传导缺陷。 PI 3'-激酶至 Akt 通路的激活在胰岛素信号转导中发挥着重要作用，但 Akt 下游的事件很大程度上未知。最近，我们发现了一种新型 Akt 底物，它可以与胰岛素样生长因子结合蛋白 3 (IGFBP-3) 的胰岛素反应元件 (IRE) 和其他胰岛素反应基因结合。 IRE 结合蛋白“敏感素”cDNA 编码 120 kDa 的细胞质蛋白。添加胰岛素后，该蛋白质被切割成 50 kDa 片段，该片段主要定位于细胞核并反式激活 IRE。 PI 3'-激酶抑制可降低敏敏素的 Ser/Thr 磷酸化和蛋白水解作用。根据我们的初步数据，我们研究的长期目标是利用 IGFBP-3 的 IRE 作为模型系统来描绘胰岛素通过与 IRE 结合的特定反式作用因子敏敏素的作用诱导基因组效应的信号通路。本申请具有以下目的：1) 检验敏敏素受胰岛素调节磷酸化的假设，使用二维磷酸化图谱和质谱法定位 HepG2 细胞中胰岛素磷酸化敏敏素的残基，并通过突变分析评估磷酸化对胰岛素诱导转录的影响。 2) 测试 IRE 反式激活需要敏感素蛋白水解裂解的假设，以及敏感素核易位继发于蛋白质裂解的推论假设。我们将通过微测序鉴定切割位点，突变蛋白水解结构域以测试其在核易位和IRE转录中的作用；并突变 Akt 磷酸化位点，以确定去磷酸化是否会阻止敏感素的核转位或蛋白水解。 3) 为了确定大鼠糖尿病模型中敏感素磷酸化和蛋白水解的生理意义，我们将在体外比较糖尿病、肥胖和瘦对照大鼠的肝胞质提取物对敏感素的磷酸化和蛋白水解作用，并确定敏感素突变赋予蛋白酶和磷酸化抗性是否会增加肥胖者的糖尿病易感性大鼠，或恶化糖尿病大鼠的血糖控制。这项研究可能有助于更好地了解胰岛素调节基因转录，并可能提供与 2 型糖尿病发病机制相关的见解。