Insulin Control of Gene Transcription Through Sensitin

描述(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缺陷。PI3‘-激酶对Akt通路的激活在胰岛素信号转导中起着重要作用，但Akt信号转导途径的作用机制目前尚不清楚。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“敏感素”编码120 kDa的细胞质蛋白。加入胰岛素后，蛋白质被切割成50 kDa的片段，主要定位于细胞核并反式激活IRES。PI 3‘-激酶抑制会降低Ser/Thr的磷酸化和敏感素的蛋白分解。基于我们的初步数据，我们研究的长期目标是使用IGFBP-3的IRE作为一个模型系统来描述胰岛素通过与IRE结合的特定反式作用因子敏感素的作用来诱导基因组效应的信号通路。本研究的目的有以下几个方面：1)验证胰岛素调节敏感素磷酸化的假说，利用二维磷酸化图谱和质谱仪定位胰岛素磷酸化敏感素在HepG2细胞中的残基，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的反式激活需要敏感素的蛋白水解性切割的假说，以及敏感素的核转位次于蛋白质的裂解的推论。我们将通过显微测序确定裂解位点，突变蛋白水解区以测试其在核转位和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可以阻止敏感素的核转位或蛋白分解。3)通过比较糖尿病大鼠、肥胖大鼠和瘦身对照组大鼠肝细胞胞液提取液体外对敏感素的磷酸化和蛋白分解的影响，确定敏感蛋白的磷酸化和蛋白分解在糖尿病大鼠模型中的生理学意义，并确定导致蛋白水解酶和磷酸化抗性的敏感素突变是增加肥胖大鼠的糖尿病易感性，还是恶化糖尿病大鼠的血糖控制。这项研究可能有助于更好地理解胰岛素调节的基因转录，并可能为深入了解与2型糖尿病的发病机制有关的基因提供依据。

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.