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实验室在证明亚急性炎症过程与胰岛素抵抗和2型糖尿病的发展有关方面发挥了重要作用。在介导炎症反应的生化事件中，两种细胞内通路平行激活，一种由c-Jun n -末端激酶（JNK）介导，另一种由IKK β和NF-kappaB介导。NF-kappaB活化在胰岛素抵抗中的研究主要集中在促炎细胞因子和趋化因子的转录调控上，如tnf - α、MCP-1、IL-6、IL-1 β和抵抗素。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（Insulin receptor， IR）是一种酪氨酸激酶，由胰岛素结合激活。激活的IR结合到IRS-1磷酸酪氨酸结合（PTB）结构域，并依次磷酸化分散在其初级序列中的IRS-1的许多酪氨酸位点。磷酸化的酪氨酸残基参与并激活含有SH2结构域的蛋白质，将信号传播到细胞中。JNK磷酸化人类IRS-1的两个不同位点，Ser307和Ser312，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。Ser312位点的磷酸化已被详细研究，并在啮齿类动物模型和糖尿病、胰岛素抵抗患者中被证明与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。考虑到已知的IR和IRS-1结构，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，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磷酸化抑制PTB结构域与IR的结合如下。由于Ser307和Ser312位于蛋白的一个无序且明显灵活的区域，这些位点可以自由地环绕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与PTB结构域结合，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上（熵上）更有利。pSer307或pSer312参与其PTB结构域将通过阻止IRS-1像往常一样与胰岛素受体的pTyr960对接而自动抑制IRS-1。我们将通过表达蛋白连接（EPL）产生磷酸化丝氨酸延伸的IRS-1 PTB结构域，并通过等温滴定量热法测量结合亲和力来验证这一假设。我们还将求解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.