PI 3-Kinase Isoforms and Insulin Action

描述(由申请人提供)：这是一项竞争性更新，重点是PI 3-激酶在胰岛素作用中的作用。在过去的4年里，我们已经确定了不同亚型的PI 3-激酶调节亚基在胰岛素作用中的异同和互补作用。这是通过建立和鉴定特定PI3-激酶亚型缺失或过度表达的小鼠和细胞系来实现的。这导致了新的假说，关于调节亚基和催化亚基之间的化学计量在胰岛素作用中的重要作用，调节PIP3半衰期和应激激酶通路的PI 3-激酶调节亚基发出的其他信号的可能性，以及Akt作为PI 3-激酶下游效应因子的作用。在接下来的五年期间，我们建议扩大我们以前的观察，并在一系列新的具体目标中侧重于这些假设： 1.明确p85α/β的N-末端结构域及其短亚型p50α和p55α/AS53在胰岛素信号转导中的作用，包括这些结构域在不依赖PI 3-激酶活性的调节亚基介导的信号转导中的潜在作用，特别是JNK和p38 MAPK通路，以及P85与脂磷酸酶PTEN之间的联系，以及它们产生和稳定PIP3的不同能力。 2.通过酵母和细菌双杂交筛选，确定与p85、p55和p50调节亚基N-末端区域相互作用的特异性分子，确定它们在pI-3-K酶信号转导和亚细胞定位中的作用，以及它们与rac1和cdc42信号的关系。 3.通过分析p85a、p55a和p50a基因启动子在体内和体外的表达，明确不同亚型调控亚基在正常和病理状态下表达的调控机制，探讨化学计量学在胰岛素敏感性调节中的作用。 4.确定PI 3-激酶催化亚基p110pha和p110beta(相当于线虫中的AGE-1)的化学计量比在胰岛素信号、胰岛素敏感性和寿命中的作用，并通过单独创建和表征组织特异性基因敲除小鼠以及与Akt2基因敲除相结合的方法，确定主要下游效应因子Akt1在啮齿动物和细胞系中的作用。

DESCRIPTION (provided by applicant): This is a competitive renewal which has focused on the role of PI 3-kinase in insulin action. Over the past 4 years, we have defined the similarities, differences and complementary roles of various isoforms of PI 3-kinase regulatory subunits in insulin action. This has been accomplished by creation and characterization of mice and cell lines in which specific isoforms of PI 3-kinase have been deleted or overexpressed. This has led to new hypotheses about the important role of stoichiometry between regulatory and catalytic subunits in insulin action, the potential for other signals emanating from the regulatory subunit of PI 3-kinase involved in regulation of PIP3 half-life and the stress kinase pathways, and the role of Akt as a downstream effector of PI 3-kinase. In the next five-year period, we propose to extend our previous observations and focus on these hypotheses in a series of new specific aims: 1. Define the role of the N-terminal domains of p85alpha/beta and the short isoforms p50alpha and p55alpha/AS53 in insulin signaling, including the potential roles of these domains in signaling mediated by the regulatory subunits independent of PI 3-kinase activity, especially the JNK and p38 MAPK pathways, and the link between p85 and lipid phosphatase PTEN, and their differential ability to generate and stabilize PIP3. 2. Define the specific molecules interacting with the N-terminal region of p85, p55 and p50 regulatory subunits through yeast and bacterial two-hybrid screening, determine their role in signaling and subcellular localization of the PI 3-kinase enzyme, and their relationship to signaling through Racl and cdc42. 3. Explore the role of stoichiometry in regulation of in insulin sensitivity by defining the mechanisms regulating expression of various isoforms of regulatory subunit in normal and pathological states and analyzing the promoters of the p85a, p55a and p50a gene in vivo and in vitro. 4. Determine the role of stoichiometry of PI 3-kinase catalytic subunits p110alpha and p110beta (equivalent to age-1 in C. elegans) in insulin signaling, insulin sensitivity and longevity, and define the role of the major downstream effector Aktl in rodents and cell lines by creating and characterizing tissue specific knockout mice alone and in combination with Akt2 knockout.