Intracellular Probes for Studying PKG Activity

用于研究 PKG 活性的细胞内探针

• 批准号: 6989761
• 负责人: DAVID S. LAWRENCE
• 金额: $ 33.62万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-12-15 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6989761
• 关键词: aminoacid analog; animal tissue; biological signal transduction; cGMP dependent protein kinase; enzyme activity; enzyme inhibitors; enzyme substrate; fluorescence spectrometry; fluorescent dye /probe; intracellular transport; kinase inhibitor; laboratory rat; long term potentiation; neural plasticity; nitric oxide synthase; peptide library; phosphorylation; photoactivation; technology /technique development

DESCRIPTION (provided by applicant): The cGMP-dependent protein kinase (PKG) is the key downstream participant of NO-mediated signaling pathways that regulates a range of cell-based processes, from smooth muscle relaxation to neuronal synaptic plasticity. However, its precise role during these cellular events has been difficult to evaluate due to the fact that its activity is spatially and temporally regulated. Standard techniques, such as the application of inhibitors or activators to cell populations, or ectopic overexpression of native or dominant negative mutants, are unable to probe issues related to both the when and where of protein kinase activity with respect to cellular events. The overall goal of this research program is to develop molecular tools to establish the intracellular dynamics of PKG activity during long-term potentiation (LTP) and long-term depression (LTD) of synaptic plasticity. As described in the first specific aim, we will prepare and evaluate PKG peptide substrates that display a phosphorylation-induced change in fluorescence in living cells. In the second specific aim, we describe the preparation of a PKG construct, as well as substrates and inhibitors of PKG, that are quiescent until activated by light. These species should prove useful in evaluating the temporal dynamics of intracellular PKG activity. Finally, we will employ the tools developed in specific aims 1 and 2 to sample intracellular PKG activity and assess its contribution as a biochemical mediator of LTD. A detailed understanding of the mechanisms underlying bi-directional long-term synaptic plasticity is a necessary step in unraveling its functional role(s) in neural development, processing and memory storage, as well as in treating diseases such as Alzheimer's that may involve deficits in these functions.

描述（由申请人提供）：CGMP依赖性蛋白激酶（PKG）是无介导的信号通路的关键参与者，该途径调节了一系列基于细胞的过程，从平滑肌松弛到神经元突触可塑性。但是，由于其活性在空间和时间上受到调节，因此很难评估其在这些细胞事件中的确切作用。标准技术，例如将抑制剂或激活剂应用于细胞群中，或者对天然或显性阴性突变体的异位过表达，无法探测与蛋白激酶在细胞事件相对于蛋白激酶的活性相关的问题。该研究计划的总体目标是开发分子工具，以建立长期增强（LTP）和长期抑郁（LTD）突触可塑性期间PKG活性的细胞内动力学。如第一个特定目的所述，我们将准备和评估显示磷酸化诱导的活细胞荧光变化的PKG肽底物。在第二个特定目的中，我们描述了PKG构建体的制备，以及PKG的底物和抑制剂，这些构建物是静止的，直到被光激活。这些物种应证明可用于评估细胞内PKG活性的时间动力学。最后，我们将采用特定目标1和2中开发的工具来采样细胞内PKG活性，并评估其作为LTD生化介质的贡献。对双向长期突触可塑性的基础机制的详细理解是揭示其在神经发育，加工和记忆存储中以及治疗诸如阿尔茨海默氏病等疾病的功能作用的必要步骤，该疾病可能涉及这些功能中的缺陷。