The cAMP-dependent protein kinase (PKA) is a member of the signal transduction pathways that regulate a wide variety of cell-based processes, including glycogenolysis, apoptosis, and the cell division cycle. Recently, PKA has come under scrutiny as a component of the pathways that drive cell motility. However, its precise role during cellular locomotion has been difficult to evaluate due to the fact that its' activity is apparently 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. For example, EGF-stimulated motility is blocked in the presence of agents that inhibit PKA activity as well as in the presence of agents that activate PKA activity. The work described in this proposal seeks to develop new methodologies that can be used to explore the intracellular dynamics of PKA activity in appropriately stimulated cells. The proposed experiments are grouped according to three specific aims. In the first aim, we will synthesize and evaluate PKA substrates that can fluorescently report intracellular PKA action. These substrates will be prepared and identified using an array of different library-based synthesis and screening strategies previously developed in our laboratory. The corresponding membrane permeable analogs as well as derivatives that can spatially report PKA activity will also be prepared.
In specific aim 2, we will synthesize light-activatable (""""""""caged"""""""") derivatives of the agents developed in specific aim 1 as well as caged derivatives of PKA inhibitors. These caged analogs are designed to allow us to probe PKA activity with temporal and spatial control in living cells. Finally, in specific aim 3 we will evaluate the intracellular action of PKA with respect to known molecular and sub-cellular markers of motility and determine which of these molecular and sub-cellular events is dependent upon the spatial and/or temporal action of PKA. ? ?
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