Phosphoinositide-dependent Kinase: Molecular mechanisms

描述（由申请人提供）： 拟议研究的长期目标是了解调节磷酸肌醇依赖性激酶PDK-1的分子机制。1997年PDK-1的发现揭示了细胞信号传导的关键。该激酶单独为AGC超家族激酶的不同成员的催化功能提供“开启”开关，所述AGC超家族激酶包括Akt（蛋白激酶B）、蛋白激酶C家族成员、p70 S6激酶等。虽然PDK-1的功能自发现以来就已为人所知，但这一关键激酶的分子机制在很大程度上尚未探索。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的亚细胞位置和大分子相互作用控制其生理功能。三个具体目标是：1。本节的目的是了解活细胞中PDK-1的空间和时间调节。具体来说，我们深入研究了以下中心问题：1）细胞中PDK-1的活性在哪里; 2）控制这种活性的细胞输入是什么; 3）底物磷酸化的持续性如何？我们将利用新的荧光方法来监测PDK- 1的活性和亚细胞位置在真实的时间响应细胞刺激。 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。具体而言，我们提出以下问题：1] PDK-1的膜结合机制是什么？2]PDK-1的催化活性由什么机制控制，以及3] PDK-1的结构是什么？ 3.通过蛋白质：蛋白质相互作用调节PDK-1该目标的中心假设是蛋白质：蛋白质相互作用是体内PDK-1功能的关键调节剂。我们1]阐述了PDK-1识别Akt的机制，检验了相互作用受每种激酶的PH结构域调节的假设，2]提出筛选PDK-1的PH结构域和接头区的结合配偶体，3]检验了PDK-1与磷酸酶复合的假设，和4]检验PDK-1与AKAP 350在信号传导复合物中拴系在中心体的假设。

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.