cAMP-dependent protein kinase (PKA) is a component of an intracellular signalling pathway that is implicated in many examples of neural plasticity including presynaptic facilitation of the sensorimotor synapse in Aplysia, a cellular correlate of a simple form of learning and memory. The enzyme transduces signals carried by neuromodulatory transmitters, which are released as the result of an animal's experience, to sites within target neurons altering their properties and their interactions with other neurons. PKA is involved in several aspects of facilitation that are spatially and temporally separated. For example, during short- term facilitation, PKA mediates the closure of potassium channels, prolonging the action potential, which results in increased transmitter release. In long-term facilitation, synaptic growth is activated and requires the phosphorylation of transcription factors by PKA. How can a single enzyme control such diverse events, without causing chaos through indiscriminate phosphorylation? The answer many lie in the complexity of Aplysia PKA, which is composed of at least five regulatory (R) and four catalytic (C) subunits that generate multiple holoenzymes (R2C2). Recent work from this laboratory shows that these forms of PKA differ in substrate specificity, regulation, and subcellular location. Our hypothesis is that this accounts for the physiological versatility of PKA, by providing enzymes with multiple physiological roles, both overlapping and distinct. To establish this idea, the properties of the various R and C subunits of Aplysia neuronal PKA will be examined both in vitro and in intact sensory neurons and cell extracts, with emphasis on differences in behavior between the various forms of the subunits. In the cellular studies, the effects of treatments that induce short- and long-term facilitation will be determined. The following questions will be addressed: 1, What is the nature of the diversity of the R and C subunits of PKA in Aplysia sensory neurons? cDNAs encoding presently uncharacterized subunits will be cloned and sequenced. 2. What are the substrates of the C subunits? Substrate specificity will be examined in vitro, in sensory neurons and homogenates, and by electrophysiological recording. 3. How is PKA regulated in Aplysia neurons? Particular attention will be given to regulation by R subunits and by """"""""autophosphorylation"""""""" as well as phosphorylation by other kinases. 4. Where are PKA subunits in Aplysia neurons and do they change location? The subcellular locations of individual forms of R and C subunits will be determined, before and after treatments that induce facilitation, by using subcellular fractionation, immunofluorescence microscopy and fluorescence imaging. It is likely that general principles revealed in these studies will be applicable and fluorescence imaging. It is likely that general principles revealed in these studies will be applicable to vertebrates. Thus, this work lead to a better understanding of plasticity in normal and disease brain.
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