Mechanism of insulin-induced ubiquitination of IRS-1

描述（由申请人提供）：本提案的总体目标是了解调节胰岛素受体信号的分子机制。胰岛素受体底物1 （Insulin receptor substrate 1, IRS-1）是胰岛素受体信号传导的重要下游效应因子，介导细胞代谢和有丝分裂发生。最近，泛素-蛋白酶体降解途径被证明参与长期暴露于胰岛素后细胞中IRS-1介导的信号转导的负调控。泛素-蛋白酶体降解途径如何被胰岛素激活并导致IRS-1转换尚不完全清楚。我们的实验将集中于确定胰岛素诱导的IRS-1泛素化的分子机制，这是蛋白酶体降解途径的关键步骤。基于我们的初步数据表明胰岛素刺激的E3连接酶活性在体内和体外IRS-1泛素化中的作用，我们提出的实验将确定IRS-1泛素化的特异性E3连接酶，并确定IRS-1泛素化的关键生化步骤。具体目的是：(1)通过亲和层析或快速高效液相色谱法纯化IRS-1的E3连接酶。具体的E3连接酶将被克隆，其在胰岛素诱导的IRS-1泛素化中的作用将进一步确定；(2)明确IRS-1中特定泛素化所必需的结构元件，包括IRS-1降解所需的泛素化位点、丝氨酸磷酸化位点和结构域；(3)明确pi3激酶在胰岛素诱导的IRS-1泛素化中的作用。我们预计我们的研究将为理解IRS-1转换在调节胰岛素受体信号转导中的作用提供新的见解，并为2型糖尿病的治疗提供新的治疗策略。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to understand molecular mechanisms regulating insulin receptor signaling. Insulin receptor substrate 1 (IRS-1) is an important downstream effector of insulin receptor signaling that mediates cell metabolism and mitogenesis. Recently, the ubiquitin-proteasome degradation pathway has been shown to be involved in negatively regulating IRS-1- mediated signal transduction in cells following prolonged exposure to insulin. How the ubiquitin-proteasome degradation pathway is activated by insulin and causes IRS-1 turnover is not completely understood. Our experiments will focus on defining the molecular mechanism of the insulin- induced ubiquitination of IRS-1, a key step in the proteasomal degradation pathway. Based on our preliminary data that indicated a role of insulin-stimulated E3 ligase activity in the ubiquitination of IRS-1 in vivo and in vitro, the proposed experiments will identify the specific E3 ligase for IRS-1 and define key biochemical steps in the ubiquitination of IRS-1. The specific aims are to: (1) Purify the E3 ligase for IRS-1 by affinity chromatography or Fast Performance Liquid Chromatography. The specific E3 ligase will be cloned and its role in the insulin-induced ubiquitination of IRS-1 will further defined; (2) Define the structural elements in IRS-1 essential for its specific ubiquitination, including ubiquitination sites, serine phosphorylation sites and domain that required for IRS-1 degradation; (3) Ascertain the role of PI3-kinase in insulin-induced ubiquitination of IRS-1. We anticipate that our studies will provide novel insights essential to understanding the role of IRS-1 turnover in regulating insulin receptor signal transduction and suggest new therapeutic strategies for treatment of Type 2 diabetes mellitus.