Protein Kinase Substrates for Assays in single Neurons

描述：（申请人提供） 蛋白激酶是重要的信号转导子，参与几乎所有的 细胞内通讯的途径。生长因子变化的过程 诱导的成纤维细胞增殖和神经元中的长时程增强是 受到蛋白激酶的显著影响。蛋白激酶的最新检测方法 活性取决于相关激酶的免疫沉淀和用 蛋白质或肽底物加体外ATP。或者，激酶可以是 在裂解物中测定，如果使用可从 细胞中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 以及需要相对大量的细胞材料。的测定 可以在单个活细胞上进行的蛋白激酶将提供 在我们的能力，以获得准确的信息， 单个细胞的行为。在这里，我们建议利用 目前可用的仪器（参见Meredith等人，（2000年）收获， 测量单个细胞中底物的磷酸化。我们的目标是开发 蛋白激酶的特异性底物， 可以在单个神经元中进行分析。我们将重点关注蛋白激酶， 与记忆、抑郁和成瘾行为有关， 在动物模型系统的单个神经元中测量它们的活性。

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.