Insulin Control of Gene Transcription Through Sensitin

胰岛素通过敏敏素控制基因转录

• 批准号: 6973085
• 负责人: BETTY C VILLAFUERTE
• 金额: $ 26.46万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-15 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6973085
• 关键词: binding proteins; body weight; cell line; diabetes mellitus genetics; gene mutation; genetic regulatory element; genetic strain; genetic susceptibility; genetic transcription; hormone regulation /control mechanism; insulin; insulin sensitivity /resistance; insulinlike growth factor; laboratory rat; liver metabolism; mass spectrometry; noninsulin dependent diabetes mellitus; nuclear transport; phosphorylation; protein localization; protein sequence; proteolysis; serine threonine protein kinase

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胰岛素信号的缺陷。pi3’-激酶对Akt通路的激活在胰岛素信号传导中起着重要作用，但Akt下游的事件在很大程度上是未知的。最近，我们发现了一种新的Akt底物，它与胰岛素样生长因子结合蛋白-3 （IGFBP-3）的胰岛素反应元件（IREs）和其他胰岛素反应基因结合。ire结合蛋白“sensitin”cDNA编码一个120 kDa的细胞质蛋白。在加入胰岛素后，该蛋白被切割成一个50 kDa的片段，该片段主要定位于细胞核并激活IREs。pi3 '-激酶抑制降低了敏感蛋白的丝氨酸/苏氨酸磷酸化和蛋白水解。基于我们的初步数据，我们研究的长期目标是利用IGFBP-3的IRE作为模型系统来描述胰岛素通过与IRE结合的特定交易作用因子（敏化蛋白）的作用诱导基因组效应的信号通路。本应用的目的如下：1)为了验证敏化蛋白受胰岛素调控磷酸化的假设，利用二维磷酸化和质谱技术定位HepG2细胞中敏化蛋白被胰岛素磷酸化的残基，并通过突变分析评估磷酸化对胰岛素诱导转录的影响。2)验证IRE的转激活需要敏化蛋白的蛋白水解裂解的假设，以及敏化蛋白的核易位继发于蛋白裂解的推论。我们将通过微测序确定裂解位点，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；并使Akt磷酸化位点突变，以确定去磷酸化是否能阻止敏感蛋白的核易位或蛋白水解。3)为了确定致敏蛋白磷酸化和蛋白水解在大鼠糖尿病模型中的生理意义，我们将比较糖尿病、肥胖和瘦肉对照大鼠肝细胞质提取物对致敏蛋白的体外磷酸化和蛋白水解，并确定致敏蛋白突变是否会增加肥胖大鼠的糖尿病易感性，或使糖尿病大鼠的血糖控制恶化。该研究可能有助于更好地了解胰岛素调控基因的转录，并可能为2型糖尿病的发病机制提供新的见解。