Protein Kinase Substrates for Assays in single Neurons

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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.