Protein Kinase Substrates for Assavs in single Neurons

描述：(申请人提供) 蛋白激酶是重要的信号转导分子，几乎参与了 细胞内交流的途径。各种不同的过程如生长因子 诱导成纤维细胞增殖和神经元的长时程增强 受到蛋白激酶的显著影响。蛋白激酶的最新检测方法 活性依赖于相关激酶的免疫沉淀，并用 一种蛋白质或多肽底物加上体外的三磷酸腺苷。另一种选择是，可以使用 如果使用可回收的特定底物，则在裂解物中进行检测 细胞内蛋白质的复杂混合物(即GST-融合蛋白)。 这些方法的缺点包括制备细胞裂解物所需的时间 以及对相对大量的细胞材料的需求。化验分析 可以在单个活细胞上执行的蛋白激酶将提供一种 我们在获取有关该事件的准确信息的能力方面有了重大改进 单个细胞的行为。在这里，我们建议利用 目前可用于收获的仪器(见Meredith等人，2000)和 在单个细胞中测量底物的磷酸化。我们的目标是发展 蛋白激酶的特定底物使许多不同的蛋白激酶 可以在单个神经元中进行分析。我们将重点研究具有 与记忆、抑郁和成瘾行为有牵连，所以我们可以 从动物模型系统中测量它们在单个神经元中的活动。

DESCRIPTION: (provided by the applicant) Protein kinases are important signal transducers that participate in nearly all pathways of intracellular communication. Processes as varied as growth factor induced proliferation of fibroblasts and long term potentiation in neurons are significantly impacted by protein kinases. Current assays of protein kinase activities depend on immunoprecipitation of the relevant kinases and assay with a protein or peptide substrate plus ATP in vitro. Alternatively, kinases can be assayed in lysates, if specific substrates are used which can be recovered from the complex mixture of proteins in the cell (i.e., GST-fusion proteins). Drawbacks to these methods include the time required for preparing cell lysates and the need for relatively large amounts of cellular material. Assays of protein kinases that could be performed on single living cells would provide a major improvement in our ability to obtain accurate information about the behavior of individual cells. Here we propose to take advantage of instrumentation currently available (see Meredith et al., 2000) to harvest and measure phosphorylation of substrates in single cells. Our goal is to develop specific substrates for protein kinases so that many different protein kinases can be analyzed in single neurons. We will focus on protein kinases that have been implicated in memory, depression, and addictive behavior so that we can measure their activities in single neurons from animal model systems.