Hormone action heavily utilizes multifunctional serine/threonine protein kinases and phosphoprotein phosphatases. These enzymes regulate cellular events by altering the phosphorylation state of metabolic enzymes, structural proteins and ion-channels. Recently, it has been proposed that subcellular targeting of kinases and phosphatases at sites close to preferred substrates influences the specificity of these seemingly multifunctional enzymes. In support of this concept we have shown that targeting of kinases and phosphatases occurs through association with a family of """"""""anchoring proteins"""""""". During the past funding period we cloned and characterized a neuronal A-Kinase Anchoring Protein, called AKAP79, which targets protein kinase A (PKA) to the postsynaptic density (PSD), a cytoskeletal-like structure attached to the internal surface of excitatory synapses. Several lines of evidence suggest that AKAP79 is, in fact, a multifunctional anchoring protein which is capable of associating with three signaling enzymes, PKA, protein kinase C (PKC) and the phosphatase 2B, calcineurin (CaN). Each enzyme binds to distinct regions of AKAP79 and is inhibited when anchored. Therefore, we propose that AKAP79 targets these enzymes to sites just below the postsynaptic membrane where they are optimally positioned to become activated in response to the second messengers Ca2+ and cAMP and to phosphorylate neuronal substrates such as the AMPA/kainate or NMDA ion-channels. In order to test this hypothesis this proposal has two phases.
Aim 's 1-3 will conduct structure/function analysis of the AKAP79 signaling complex to define the protein-protein interactions that occur between the anchoring protein and calcineurin (aim-1), PLC (aim-2) and map the AKAP79-subcellular targeting domain (aim- 3). The functional significance of enzyme targeting via the anchoring protein will be tested in cultured hippocampal neurons in aim-4. This will be achieved by measuring changes in AMPA/kainate or NMDA evoked currents under conditions where AKAP79 derived peptides are used as reagents to inhibit CaN activity, inhibit PKC activity and disrupt targeting of the AKAP signaling complex.
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