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

描述（由申请人提供）：乔斯林糖尿病中心的Shoelson实验室有助于表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。在介导炎症反应的生化事件方面，两种细胞内途径平行激活，一种由c-Jun N-末端激酶（JNK）介导，另一种由IKK β和NF-κ B介导。胰岛素抵抗中NF-κ B活化的研究主要集中在促炎细胞因子和趋化因子如TNF-α、MCP-1、IL-6、IL-1 β和β-内酰胺酶的转录调节上。相反，JNK的研究集中在胰岛素受体底物1（IRS-1）的直接磷酸化。胰岛素受体（IR）是一种酪氨酸激酶，通过与胰岛素结合而被激活。活化的IR结合IRS-1磷酸酪氨酸结合（PTB）结构域，并在分散在其一级序列中的许多酪氨酸位点连续磷酸化IRS-1。磷酸化的酪氨酸残基接合并激活含有SH 2结构域的蛋白质，该蛋白质将信号传播到细胞中。JNK在两个不同的位点（Ser 307和Ser 312）磷酸化人IRS-1，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。Ser 312的磷酸化已被最详细地研究，并显示与啮齿动物模型和糖尿病胰岛素抵抗患者中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的抑制联系起来，但它们并没有提供分子机制。已知IR和IRS-1结构，以及Ser 307和Ser 312位于PTB结构域（IRS-1残基156-260）C-末端约50个残基的事实，在蛋白质的其他无序区域中，这些位点的磷酸化如何引起胰岛素抗性根本不明显。我假设IRS-1的JNK磷酸化抑制PTB结构域与IR的结合，如下所述。由于Ser 307和Ser 312位于蛋白质的无序和明显的柔性区域，这些位点可以自由地围绕PTB结构域形成环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，但我假设这是因为它们足够接近，分子间的相互作用在能量（熵）上是有利的。其PTB结构域与pSer 307或pSer 312的结合将通过阻止IRS-1与胰岛素受体的pTyr 960正常对接而自动抑制IRS-1。我们将通过表达蛋白连接（EPL）产生磷酸丝氨酸延伸的IRS-1 PTB结构域和用等温滴定量热法测量结合亲和力来测试这一假设。我们还将解决IRS-1 PTB结构域的自抑制形式的晶体结构。

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.