Insulin Signaling in a Cell-Free System

胰岛素的作用几十年来一直是深入研究的主题。对异常胰岛素信号在几种常见代谢紊乱中的作用的认识，包括非胰岛素依赖型糖尿病和肥胖，激发了人们对这一领域的兴趣。虽然在分子水平上对胰岛素信号的理解已经取得了很大进展，但由于这种激素的作用异常复杂，许多细节仍然未知。 胰岛素信号传导不是对应于一系列简单的线性途径，而是可以更准确地表征为相互作用分子的四维网络，涉及空间和时间的坐标。由于这种复杂性，已经提出胰岛素信号传导的许多重要方面可能只能通过使用无细胞的 准确再现体内信号级联的各个片段的系统。这项建议的长期目标是阐明脂肪细胞中胰岛素主要代谢作用所涉及的分子信号级联。为了促进这一目标的实现，我们最近开发了一种新的无细胞系统，从3 T3 L1脂肪细胞， 研究胰岛素信号。该系统忠实地重建了从受体自磷酸化到Akt活化下游事件（包括GSK-3的磷酸化）的PI 3-激酶信号级联。我们建议进一步完善这种无细胞系统，并利用它来实现以下具体目标： 1)Akt是一种丝氨酸激酶，在PI 3激酶依赖性信号通路中起关键作用。它通过在苏氨酸和丝氨酸残基处的双重磷酸化来活化。苏氨酸磷酸化是由PDK 1催化的，PDK 1是一种特征良好的丝氨酸/苏氨酸激酶，然而，丝氨酸激酶尚未被鉴定。我们已经分离出一种与脂肪细胞质膜相关的新型细胞骨架亚细胞组分，相对于总细胞匀浆，其PDK 2活性富集约1000倍。我们建议从该馏分中进一步富集PDK 2活性，使用MALDI-TOF质谱鉴定激酶，然后克隆和表征酶。2)IRS-1和IRS-2是连接激活的胰岛素受体和PI 3-激酶信号通路的主要对接分子。目前的证据表明，IRS-1和IRS-2在胰岛素的代谢效应中发挥不同的作用，但对这两种IRS亚型介导不同信号传导事件的机制知之甚少。一种假说认为IRS-1和IRS-2通过不同的亚细胞定位来传播不同的信号结果。与这一假设一致，我们观察到IRS-2，而不是IRS-1，是高度富集在相同的质膜相关的细胞骨架部分，含有大量的细胞PDK 2活性。此外，使用我们的无细胞信号传导试验获得的数据表明，Akt激活依赖于质膜相关的IRS-1，IRS-2和胞质IRS-1在此过程中不起作用。我们的第二个目的是进一步表征新的PDK 2/IRS-2的亚细胞组分，并确定IRS-2靶向相应的细胞室的分子基础和功能意义。

Insulin action has been the subject of intense investigation for several decades. Interest in this field has been stimulated by an appreciation of the role of aberrant insulin signaling in several common metabolic disorders, including non-insulin-dependent diabetes mellitus and obesity. Although much progress has been made in understanding insulin signaling at the molecular level, many details remain unknown because of the unusual complexity of the effects of this hormone. Rather than corresponding to a simple series of linear pathways, insulin signaling can more accurately be characterized as a four-dimensional web of interacting molecules, involving coordinates in both space and time. Because of this complexity, it has been suggested that many important aspects of insulin signaling may only be unraveled via the use of cell-free systems that accurately reproduce various segments of the in vivo signaling cascades. The long-term goal of this proposal is to elucidate the molecular signaling cascades involved in the major metabolic effects of insulin in adipocytes. In order to facilitate the accomplishment of this goal, we have recently developed a novel cell-free system from 3T3L1 adipocytes to investigate insulin signaling. This system faithfully reconstitutes the PI 3-kinase signaling cascade from receptor autophosphorylation to events downstream of Akt activation, including the phosphorylation of GSK-3. We propose to further refine this cell-free system and to utilize it to pursue the following specific aims: 1) Akt is a serine kinase that plays a pivotal role in the PI3-kinase-dependent signaling pathway. It is activated by dual phosphorylations at a threonine and a serine residue. The threonine phoshorylation is catalyzed by PDK1, a well-characterized serine/threonine kinase, however, the serine kinase has not yet been identified. We have isolated a novel cytoskeletal subcellular fraction associated with adipocyte plasma membranes that is enriched approximately 1000-fold in PDK2 activity relative to total cellular homogenates. We propose to further enrich PDK2 activity from this fraction, identify the kinase using MALDI-TOF mass spectrometry, and then to clone and characterize the enzyme. 2) IRS-1 and IRS-2 are the major docking molecules that link the activated insulin receptor to the PI3-kinase signaling pathway. Current evidence suggests that IRS-1 and IRS-2 play distinct roles in the metabolic effects of insulin, but little is known about the mechanism by which these two IRS isoforms might mediate divergent signaling events. One hypothesis suggests that IRS-1 and IRS-2 promulgate distinct signaling outcomes by means of differential subcellular localization. Consistent with this hypothesis, we have observed that IRS-2, but not IRS-1, is highly enriched in the same plasma membrane-associated cytoskeletal fraction that contains the bulk of cellular PDK2 activity. Additionally, data obtained using our cell-free signaling assay indicate that Akt activation is dependent on plasma embrane-associated IRS-1, and that IRS-2 and cytosolic IRS-1 do not play a role in this process. Our second aim is to further characterize the novel PDK2/IRS-2-containing subcellular fraction and to determine the molecular basis and functional significance of IRS-2 targeting to the corresponding cellular compartment.