In-depth quantification and characterisation of PI 3kinase signalling networks

我们研究一组称为磷酸肌醇3-激酶（简称PI 3 Ks）的信号转导蛋白，这些蛋白在健康生物体的许多生物功能中发挥重要作用，并且还与糖尿病，过敏和癌症等疾病有关。有8种不同的PI 3 K基因，并且越来越清楚它们具有不同的功能，并且它们参与不同的疾病。PI 3 K基因的作用的描述不仅在提供对基本生物过程的洞察方面具有纯粹的学术重要性，而且对于正在进行的抑制特定形式的PI 3 K以治疗不同疾病的药物的开发也具有巨大的兴趣。尽管最近在该领域取得了许多科学进展，但关于不同形式的PI 3 K如何发挥其独特的生物学功能的信息很少。我们想要检验的假设是，不同的PI 3 K影响一组称为蛋白激酶的蛋白质及其底物的方式不同。为了验证这一假设，我们将使用我们在实验室开发的一种新方法，基于一种称为质谱的技术，来量化细胞中蛋白激酶通路的活性。质谱法的优点是它可以全面地分析蛋白激酶的活性，可以同时定量数千种活性。这种深度的分析使我们能够量化蛋白激酶活性，而无需预先考虑细胞中的许多激酶中哪些可能会或可能不会受到PI 3 K的影响。因此，它还允许发现PI 3 K信号转导的未知机制。在本申请中提出的工作中，我们将通过药理学和遗传学手段检测细胞中不同的PI 3 K，并与正常或未处理的细胞进行比较。这项工作的重点将是3种不同的PI 3 K，即p110 alpha，p110 beta和p110 delta。这些结果可能导致发现不同PI 3 K基因特异性影响的途径。这些结果将在该领域具有很高的影响力，并且还允许发现选择性抑制这些PI 3 K的药物的标记物。该项目的第二阶段涉及研究这些新发现的蛋白质作为PI 3 K功能的潜在参与者的作用，这将通过基于细胞的研究中的标准遗传和生化方法来实现。

We work on a group of signal transduction proteins called phosphoinositide 3-kinases (or PI3Ks in short) that play important roles in many biological functions in the healthy organism, and are also implicated in diseases such as diabetes, allergy, and cancer. There are 8 different PI3K genes and it is becoming clear that they have different functions and that they are involved in different diseases. The delineation of the roles of PI3K genes not only has purely academic importance in providing insight into fundamental biological processes but is also of huge interest for the ongoing development of drugs that inhibit specific forms of PI3Ks to treat different diseases. Despite many recent scientific advances in the field, there is little information on how the different forms of PI3K perform their distinct biological functions. The hypothesis we would like to test is that the different PI3Ks differ in the way by which they affect a group of proteins called protein kinases and their substrates. To test this hypothesis we will use a novel method that we have developed in the laboratory, based on a technique known as mass spectrometry, to quantify how active protein kinase pathways are in cells. The advantage of mass spectrometry is that it analyses the activities of protein kinases in a comprehensive fashion, allowing to quantify thousands of activities simultaneously. This depth of analysis is allowing us to quantify protein kinase activity without preconceptions of which of the many kinases in cells may or may not be affected by PI3K. It therefore also allows to discover unknown mechanisms of PI3K signal transduction. In the work proposed in this application we will inactivate the different PI3Ks in cells by pharmacological and genetic means, and compare to normal or untreated cells. The focus of this work will be on 3 different PI3Ks known as p110alpha, p110beta and p110delta. These results are likely to lead to the discovery of pathways specifically affected by the different PI3K genes. These results would be of high impact in the field and also allow to discover markers for drugs that inhibit these PI3Ks selectively. The second stage of the project involves investigating the role of these newly identified proteins as potential players in the function of PI3K, which will be achieved by standard genetic and biochemical approaches in cell-based studies.