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

描述(申请人提供)：乔斯林糖尿病中心的Shoelson实验室已经证明，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。在介导炎症反应的生化事件方面，两条细胞内途径被平行激活，一条由c-jun氨基末端激酶(JNK)介导，另一条由ikkβ和NF-kappaB介导。有关核因子-kappaB在胰岛素抵抗中激活的研究主要集中在对促炎细胞因子和趋化因子的转录调控上，如肿瘤坏死因子-α、单核细胞趋化蛋白-1、白介素6、白介素1β和抵抗素。相比之下，对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)是一种被胰岛素结合激活的酪氨酸激酶。活化的IR与IRS-1的磷酸酪氨酸结合(PTB)结构域结合，并在分布于其初级序列的多个酪氨酸位点上依次磷酸化IRS-1。磷酸化的酪氨酸残基参与并激活含有SH2结构域的蛋白质，这些蛋白质将信号传播到细胞内。JNK在两个不同的位点，Ser307和Ser312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。Ser312的磷酸化已经被最详细地研究，并被证明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的抑制联系在一起，但它们并不提供分子机制。鉴于已知的IR和IRS-1结构，以及Ser307和Ser312位于PTB结构域(IRS-1残基156-260)的大约50个残基的事实，这些位点的磷酸化如何导致胰岛素抵抗并不明显。我曾假设IRS-1的JNK磷酸化抑制PTB结构域与IR的结合，如下所示。由于Ser307和Ser312位于蛋白质的一个无序和明显灵活的区域，这些位置可以自由地环绕PTB结构域，并与其磷酸酪氨酸结合部位相互作用。磷酸丝氨酸残基从未被证明与PTB结构域结合，但我假设这种情况发生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。PSer307或pSer312与其PTB结构域的结合将通过阻止IRS-1与胰岛素受体的pTyr960正常对接而自动抑制IRS-1。我们将通过表达蛋白连接(EPL)产生磷酸丝氨酸扩展的IRS-1 PTB结构域，并通过等温滴定热法测量结合亲和力来验证这一假设。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.