Intracellular Probes for Studying PKG Activity

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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.