Structural Basis for Insulin Resistance Mediated by IRS-1 Serine Phosphorylation

IRS-1 丝氨酸磷酸化介导的胰岛素抵抗的结构基础

• 批准号: 7473204
• 负责人: SangYoun Park
• 金额: $ 5.13万
• 依托单位: JOSLIN DIABETES CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7473204
• 关键词: Affinity; Binding; Binding Proteins; Binding Sites; Biochemical; C-terminal; CCL2 gene; Calorimetry; Cells; Chemicals; Condition; Crystallization; Cyclization; Cysteine; Development; Diabetes Mellitus; Disease; Docking; Escherichia coli; Event; Human; Hybrids; IKBKB; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Insulin; Insulin Receptor; Insulin Resistance; Interleukin-1 beta; Interleukin-6; Laboratories; Ligation; Link; Measures; Mediating; Methods; Modeling; Modification; Molecular; NF-kappa B; Non-Insulin-Dependent Diabetes Mellitus; Numbers; Obesity; PTB Domain; Pathway interactions; Patients; Personal Satisfaction; Phosphopeptides; Phosphoproteins; Phosphorylation; Phosphoserine; Phosphotyrosine; Process; Protein Region; Protein Tyrosine Kinase; Proteins; Resolution; Rodent Model; Serine; Signal Transduction; Site; Solubility; Structure; System; Testing; Titrations; Transcriptional Regulation; Tumor Necrosis Factor-alpha; Tyrosine; Yeasts; base; chemokine; cytokine; diabetic; experience; human TNF protein; insulin receptor substrate 1 protein; insulin signaling; intermolecular interaction; prevent; receptor binding; research study; resistin; src Homology Region 2 Domain; stress-activated protein kinase 1; thioester

DESCRIPTION (provided by applicant): The Shoelson Lab at the Joslin Diabetes Center has been instrumental in showing that a subacute inflammatory process links obesity to the development of insulin resistance and type 2 diabetes. In terms of the biochemical events that mediate the inflammatory response, two intracellular pathways are activated in parallel, one mediated by the c-Jun N-terminal kinase (JNK) and the other by IKK beta and NF-kappaB. Studies of NF-kappaB activation in insulin resistance have centered mostly on the transcriptional regulation of proinflammatory cytokines and chemokines, such as TNF-alpha, MCP-1, IL-6, IL-1 beta and resistin. In contrast, studies with JNK have focused on the direct phosphorylation of insulin receptor substrate 1 (IRS-1). Insulin receptor (IR) is a tyrosine kinase that is activated by insulin binding. Activated IR binds to the IRS-1 phosphotyrosine-binding (PTB) domain and sequentially phosphorylates IRS-1 at numerous tyrosine sites dispersed throughout its primary sequence. The phosphorylated tyrosine residues engage and activate SH2 domain containing proteins that propagate the signal into the cell. JNK phosphorylates human IRS-1 at two distinct sites, Ser307 and Ser312, and when these sites are phosphorylated; IRS-1 neither binds IR nor activates cellular signaling. Phosphorylation at Ser312 has been studied in greatest detail and shown to correlate with the development of insulin resistance in rodent models and diabetic, insulin-resistant patients. While these findings link inflammation to the inhibition of insulin signaling, they do not provide a molecular mechanism. Given what is known about IR and IRS-1 structure, and the fact that Ser307 and Ser312 are located approximately 50 residues C-terminal to the PTB domain (IRS-1 residue 156-260) in an otherwise disordered region of the protein, it is not at all obvious how phosphorylation at these sites causes insulin resistance. I have hypothesized that JNK phosphorylation of IRS-1 inhibits PTB domain binding to IR as follows. Since Ser307 and Ser312 are in a disordered and apparently flexible region of the protein, these sites are free to loop around the PTB domain and interact with its phosphotyrosine binding site. Phosphoserine residues have never been shown to bind PTB domains, but I hypothesize that this occurs because they are sufficiently close that the intermolecular interaction is energetically (entropically) favored. Engagement of its PTB domain by pSer307 or pSer312 would auto-inhibit IRS-1 by preventing it from docking as usual to pTyr960 of the insulin receptor. We will test this hypothesis by generating phosphoserine extended IRS-1 PTB domain by expressed protein ligation (EPL) and by measuring binding affinities with isothermal titration calorimetry. We will also solve the crystal structure of the self-inhibited form of the IRS-1 PTB domain.

描述（由申请人提供）：Joslin 糖尿病中心的 Shoelson 实验室在证明亚急性炎症过程将肥胖与胰岛素抵抗和 2 型糖尿病的发展联系起来方面发挥了重要作用。就介导炎症反应的生化事件而言，两条细胞内途径被并行激活，一条由 c-Jun N 末端激酶 (JNK) 介导，另一条由 IKK beta 和 NF-kappaB 介导。胰岛素抵抗中 NF-κB 激活的研究主要集中在促炎细胞因子和趋化因子（例如 TNF-α、MCP-1、IL-6、IL-1β 和抵抗素）的转录调节上。相比之下，JNK 的研究主要集中在胰岛素受体底物 1 (IRS-1) 的直接磷酸化上。胰岛素受体 (IR) 是一种由胰岛素结合激活的酪氨酸激酶。激活的 IR 与 IRS-1 磷酸酪氨酸结合 (PTB) 结构域结合，并在分散于其一级序列中的众多酪氨酸位点上依次磷酸化 IRS-1。磷酸化的酪氨酸残基接合并激活含有 SH2 结构域的蛋白质，将信号传播到细胞中。 JNK 在两个不同的位点（Ser307 和 Ser312）磷酸化人 IRS-1，并且当这些位点被磷酸化时； IRS-1 既不结合 IR 也不激活细胞信号传导。 Ser312 的磷酸化已得到最详细的研究，并显示其与啮齿动物模型和糖尿病、胰岛素抵抗患者的胰岛素抵抗的发展相关。虽然这些发现将炎症与胰岛素信号传导的抑制联系起来，但它们没有提供分子机制。鉴于对 IR 和 IRS-1 结构的了解，以及 Ser307 和 Ser312 位于 PTB 结构域 C 端约 50 个残基（IRS-1 残基 156-260）的事实，在蛋白质的其他无序区域中，这些位点的磷酸化如何导致胰岛素抵抗并不明显。我假设 IRS-1 的 JNK 磷酸化会抑制 PTB 结构域与 IR 的结合，如下所示。由于 Ser307 和 Ser312 位于蛋白质的无序且明显灵活的区域，因此这些位点可以自由地围绕 PTB 结构域并与其磷酸酪氨酸结合位点相互作用。磷酸丝氨酸残基从未被证明可以结合 PTB 结构域，但我假设这种情况发生是因为它们足够接近，分子间相互作用在能量上（熵上）是有利的。 pSer307 或 pSer312 与其 PTB 结构域的接合将通过阻止 IRS-1 像往常一样与胰岛素受体的 pTyr960 对接来自动抑制 IRS-1。我们将通过表达蛋白连接 (EPL) 生成磷酸丝氨酸延伸的 IRS-1 PTB 结构域并通过等温滴定量热法测量结合亲和力来测试这一假设。我们还将解析 IRS-1 PTB 结构域自抑制形式的晶体结构。