The long term goals of this proposal are to understand the role of Ca2+/CaM-dependent protein kinase II (CaM-kinase in regulating synaptic connections. Altering the amount or activity of CaM-kinase has direct effects on learning and establishing enhanced susceptibility to epileptic seizures and ischemic damage following stroke. The PI is using a molecular approach to describe CaM-kinase's biological and biochemical properties so they can accurately predict its behavior in situ and in vivo. CaM-kinase is an oligomer of alpha and beta subunits and through subunit interactions the enzyme can autophosphorylate, modifying it's activity. The properties of the alpha and beta isoforms have not been well characterized, however their unique developmental regulation, tissue distribution and subcellular localization suggests that they play distinct roles in regulating neuronal physiology. Associations between CaM-kinase and other proteins are documented, however, there has not been a systematic effort to define the nature of these interactions at a molecular level. Defining CaM-kinase association with other proteins will help explain the distribution of the enzyme within neurons. The goal of this proposal is to establish a complete understanding of the domain structure of CaM-kinase and how these domains interact with themselves and other proteins. To accomplish this foal the investigator has established expression systems for producing native and mutated forms of both the alpha and beta subunits in quantities adequate for detailed enzymatic studies. They also establish the two-hybrid system in yeast to efficiently define the mechanisms for association of individual domains of each subunit. Finally, they established methods to introduce native and mutated forms of CaM-kinase into neuronal cultures to describe the localization of these molecules within the neuronal environment. The investigator will utilize these methods to identify the structural domains essential for holoenzyme assembly and identify what structural domain(s) of the alpha and beta subunit of CaM-kinase are involved in associations with other proteins. In these studies, they will also determine how the native and structurally altered forms of the two subunits are localized within neurons. The long-term goal is to use this information to understand CaM-kinase's role in regulating synaptic physiology and neuronal pathology.
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