Intracellular Probes for Studying PKG Activity

描述（由申请人提供）：cGMP依赖性蛋白激酶（PKG）是NO介导的信号通路的关键下游参与者，其调节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。然而，由于其活性在空间和时间上受到调节的事实，其在这些细胞事件中的确切作用一直难以评估。标准技术，如应用抑制剂或激活剂的细胞群体，或异位过表达的天然或显性负突变体，是无法探测的问题，无论是在何时何地蛋白激酶活性相对于细胞事件。本研究计划的总体目标是开发分子工具，以建立突触可塑性的长时程增强（LTP）和长时程抑制（LTD）过程中PKG活性的细胞内动力学。如第一个具体目标中所述，我们将制备和评价在活细胞中显示磷酸化诱导的荧光变化的PKG肽底物。在第二个具体目标，我们描述了PKG构建体的制备，以及PKG的底物和抑制剂，其是静止的，直到被光激活。这些物种应该证明是有用的，在评估细胞内PKG活性的时间动态。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的生化介质的贡献。详细了解双向长时程突触可塑性的机制是揭示其在神经发育，加工和记忆储存中的功能作用的必要步骤，以及治疗可能涉及这些功能缺陷的疾病，如阿尔茨海默氏症。

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.