Insulin Signaling in a Cell-Free System

无细胞系统中的胰岛素信号传导

• 批准号: 7368024
• 负责人: MIKE M MUECKLER
• 金额: $ 26.23万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7368024
• 关键词: 1-Phosphatidylinositol 3-Kinase; Adipocytes; Affect; Biological Assay; Cell membrane; Cell-Free System; Cells; Complex; Data; Docking; Enzymes; Event; Four-dimensional; Genes; Glycogen Synthase Kinase 3; Goals; Hormones; Insulin; Insulin Receptor; Insulin Signaling Pathway; Internet; Investigation; Link; MALDI-TOF Mass Spectrometry; Mediating; Metabolic; Metabolic Diseases; Molecular; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Outcome; PDPK1 gene; Phosphoinositide-3-Kinase, Catalytic, Gamma Polypeptide; Phosphorylation; Phosphotransferases; Plasma; Play; Process; Protein Isoforms; Protein-Serine-Threonine Kinases; Regulation; Relative (related person); Role; Series; Serine; Signal Pathway; Signal Transduction; Subcellular Fractions; System; Threonine; Time; base; forkhead protein; in vivo; insulin signaling; interest; novel; protein function; receptor; reconstitution

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.

几十年来，胰岛素作用一直是激烈调查的主题。人们对异常胰岛素信号传导在几种常见代谢疾病中的作用（包括非胰岛素依赖性糖尿病和肥胖症）的作用的作用刺激了对这一领域的兴趣。尽管在理解分子水平的胰岛素信号传导方面取得了很多进展，但由于这种激素的作用的异常复杂性，许多细节仍然未知。 胰岛素信号传导与其对应于一系列简单的线性途径，而是可以更准确地将其描述为相互作用分子的四维网络，涉及时空和时间上均匀的坐标。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只能通过无细胞的使用来揭示 准确地重现体内信号级联的各个段的系统。该提案的长期目标是阐明参与胰岛素在脂肪细胞中主要代谢作用的分子信号传导级联反应。为了促进这一目标的实现，我们最近开发了一个新型的无单元系统，从3T3L1脂肪细胞到 研究胰岛素信号传导。该系统忠实地将PI 3激酶信号传导级联反应从受体自磷酸化到AKT激活下游的事件，包括GSK-3的磷酸化。我们建议进一步完善这种无细胞的系统，并利用它来追求以下特定目标： 1）AKT是一种丝氨酸激酶，在PI3-激酶依赖性信号通路中起关键作用。它通过苏氨酸和丝氨酸残基的双重磷酸化激活。苏氨酸哲学是由一种特征良好的丝氨酸/苏氨酸激酶PDK1催化的，但是尚未鉴定出丝氨酸激酶。我们已经分离了一种与脂肪细胞质膜相关的新型细胞骨架亚细胞级分，相对于总细胞匀浆，它富集了PDK2活性约1000倍。我们建议从此馏分进一步富集PDK2活性，使用MALDI-TOF质谱法鉴定激酶，然后克隆并表征酶。 2）IRS-1和IRS-2是将活化的胰岛素受体与PI3-激酶信号通路联系起来的主要对接分子。当前的证据表明，IRS-1和IRS-2在胰岛素的代谢作用中起着不同的作用，但对这两种IRS同工型可能介导不同信号事件的机制知之甚少。一种假设表明，IRS-1和IRS-2-2颁布通过差分亚细胞定位的不同信号传导结果。与这一假设一致，我们已经观察到IRS-2而不是IRS-1在相同的质膜相关细胞骨架分数中高度富集，其中包含大部分细胞PDK2活性。此外，使用我们的无细胞信号传导分析获得的数据表明，Akt激活取决于血浆链接相关的IRS-1，而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型的含有含有IRS的PDK2/IRS-2-2的亚细胞分数，并确定IRS-2靶向相应细胞室的分子基础和功能意义。