Phosphoinositide-dependent Kinase: Molecular mechanisms

描述(由申请人提供)： 这项拟议研究的长期目标是了解调节磷酸肌醇依赖的激酶PDK-1的分子机制。1997年发现的PDK-1揭示了细胞信号传递的关键。该激酶单独为AGC超家族不同成员的催化功能提供“开启”开关，包括Akt(蛋白激酶B)、蛋白激酶C家族成员、p70S6激酶等。虽然PDK-1的功能自发现以来就已为人所知，但这一关键蛋白激酶的分子机制在很大程度上尚不清楚。这项研究的目的是了解PDK-1的调控、功能和亚细胞定位的分子机制。指导这一提议的中心假设是，PDK-1的亚细胞定位和大分子相互作用控制其生理功能。本部分的目的是了解PDK-1在活细胞中的时空调控。具体地说，我们深入研究以下核心问题：1)PDK-1在细胞中的哪里是活性的；2)控制这种活性的细胞输入是什么；以及3)底物磷酸化的持续时间有多长？我们将利用新的荧光方法来实时监测PDK-1的活性和亚细胞定位，以响应细胞刺激。 2.PDK-1的生化和结构分析本节的目的是了解控制PDK-1活性的生化机制。具体来说，我们提出了以下问题：1)PDK-1的膜结合机制是什么？2)PDK-1的催化活性受什么机制控制，以及3)PDK-1的结构是什么？ 3.蛋白质对PDK-1的调节：蛋白质相互作用这一目标的中心假设是：蛋白质相互作用是体内PDK-1功能的关键调节因子。我们1]阐述了PDK-1识别Akt的机制，检验了相互作用受每个激酶的PH结构域调控的假设，2]建议筛选PDK-1的PH结构域和连接区的结合伙伴，3]测试PDK-1是在与磷酸酶的复合体中的假设，4]测试PDK-1与AKAP 350信号复合体中中心体被拴住的假设。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand the molecular mechanisms that regulate the phosphoinositide-dependent kinase, PDK-1. The discovery of PDK-1 in 1997 unveiled a linchpin of cellular signaling. This kinase, alone, provides the 'on' switch for the catalytic function of diverse members of the AGC superfamily of kinases, including Akt (protein kinase B), protein kinase C family members, p70S6 kinase, among many others. Although the function of PDK-1 has been known since its discovery, the molecular mechanisms of this pivotal kinase are largely unexplored. The goal of the proposed research is to understand the molecular mechanisms governing the regulation, function, and subcellular location of PDK-1. The central hypothesis guiding this proposal is that the subcellular location and macromolecular interactions of PDK-1 control its physiological function. Three Specific Aims are: 1. Regulation of PDK-1 by Membrane Interactions The goal of this section is to understand the spatial and temporal regulation of PDK-1 in live cells. Specifically, we delve into the central questions of 1] where in the cell is PDK-1 active, 2] what are the cellular inputs that control this activity, and 3] how sustained is substrate phosphorylation? We will take advantage of novel fluorescence methodologies to monitor both the activity and subcellular location of PDK- 1 in real time in response to cellular stimuli. 2. Biochemical and Structural Analysis of PDK-1 The goal of this section is to understand the biochemical mechanisms controlling PDK-1 activity. Specifically, we ask the questions 1] what is the mechanism of membrane binding of PDK-1? 2] what mechanisms govern the catalytic activity of PDK-1, and 3] what is the structure of PDK-1? 3. Regulation of PDK-1 by Protein: Protein Interactions The central hypothesis of this aim is that protein: protein interactions are key regulators of PDK-1 function in vivo. We 1] address the mechanisms by which PDK-1 recognizes Akt, testing the hypothesis that the interaction is regulated by the PH domain of each kinase, 2] propose to screen for binding partners of the PH domain and linker region of PDK-1, 3] test the hypothesis that PDK-1 is in a complex with a phosphatase, and 4] test the hypothesis that PDK-1 is tethered at the centrosome in a signaling complex with AKAP 350.