Insulin Signaling in a Cell-Free System

几十年来，胰岛素的作用一直是密集研究的主题。对胰岛素信号异常在包括非胰岛素依赖型糖尿病和肥胖症在内的几种常见代谢紊乱中的作用的认识刺激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的进展，但由于这种激素作用的不同寻常的复杂性，许多细节仍然未知。 胰岛素信号不是对应于一系列简单的线性通路，而是可以更准确地描述为相互作用的分子的四维网，涉及空间和时间上的坐标。由于这种复杂性，有人提出，胰岛素信号的许多重要方面只能通过使用无细胞来解开 准确复制体内信号级联的不同片段的系统。这项建议的长期目标是阐明胰岛素在脂肪细胞中的主要代谢效应所涉及的分子信号级联反应。为了促进这一目标的实现，我们最近开发了一种新的从3T3L1脂肪细胞到 研究胰岛素信号。该系统忠实地重构了从受体自动磷酸化到Akt激活下游事件的PI 3-Kinase信号级联，包括GSK-3的磷酸化。我们建议进一步完善这一无牢房系统，并利用它实现以下具体目标： 1)Akt是一种丝氨酸激酶，在PI3依赖的信号通路中起着关键作用。它被苏氨酸和丝氨酸残基的双重磷酸化激活。苏氨酸磷酸化是由丝氨酸/苏氨酸激酶PDK1催化的，然而，丝氨酸激酶尚未被鉴定。我们已经分离出一种新的与脂肪细胞质膜相关的细胞骨架亚细胞组分，其PDK2活性比细胞总匀浆高约1000倍。我们建议从这一组分中进一步丰富PDK2的活性，用MALDI-TOF质谱仪鉴定该激酶，然后克隆和鉴定该酶。2)IRS-1和IRS-2是连接激活的胰岛素受体和PI3-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着不同的作用，但对这两种IRS亚型介导不同信号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发布不同的信号结果。与这一假设一致，我们观察到IRS-2，而不是IRS-1，在含有大部分细胞PDK2活性的相同的质膜相关细胞骨架部分中高度富含。此外，使用我们的无细胞信号分析获得的数据表明，Akt的激活依赖于血浆膜相关的IRS-1，而IRS-2和胞浆IRS-1在这一过程中不起作用。我们的第二个目标是进一步鉴定新的含有PDK2/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.