Hormones mediate their effects by changing biochemical events in the cell's interior. In one common scenario, a hormone elevates an intracellular second messenger called cyclic AMP (cAMP) in a particular cell compartment. Cyclic AMP then binds to an enzyme called protein kinase A (PKA). Protein kinases that are dependent on cAMP govern many biochemical events by phosphorylating target proteins. The changes in the activity of these newly phosphorylated proteins are what alters the cell's physiology. John Scott and his associates use recombinant DNA techniques, protein chemistry, and enzymology to study the actions of PKA within cells.
The biochemical effects of many peptide hormones proceed through pathways that lead to activation of PKA. However, individual hormones may promote PKA-mediated phosphorylation of distinct sets of proteins. This may be because different hormones activate different subtypes of the PKA enzyme. Alternatively, individual hormones may activate specific pools of PKA. A potential mechanism to explain this phenomenon is that individual PKA pools might be compartmentalized inside the cell at their site of action, close to the proteins that they will ultimately phosphorylate. A specific pool could be activated only when the appropriate hormone elevates cAMP in a particular microenvironment. Scott's laboratory has shown that Type II PKA is tethered at particular subcellular locations by specific A-kinase anchoring proteins (AKAPs).
Scott and his group have demonstrated that the AKAPs are a diverse family of functionally related proteins. So far, more than 75 AKAP genes have been isolated by interaction cloning techniques. Each AKAP contains a conserved amphipathic helix motif of 20 or so residues that is responsible for high affinity interaction with the regulatory subunit of PKA. Peptides corresponding to this region are antagonists of PKA/AKAP interaction and disrupt the localization of the kinase when introduced into cells. These anchoring inhibitor peptides have been used by Scott and others to uncouple certain cAMP-responsive events, such as the activation of glutamate receptors, and to suppress hormone-mediated insulin secretion from islet beta cells.
Each anchoring protein has a unique targeting site that is responsible for association with membranes or subcellular structures. These targeting domains confer specificity on each AKAP as they direct the localization of the kinase to the cytoskeleton, nuclear matrix, endoplasmic reticulum, peroxisomes, and cell membranes. One anchoring protein called AKAP79 is targeted to the nerve terminals, and experiments have shown that disruption of PKA/AKAP79 impairs the transfer of excitatory neuronal impulses from one nerve to the next. Additional studies have shown that AKAP79 also anchors two other signal transduction enzymes that participate in the transmission of neuronal impulses, the phosphatase calcineurin and protein kinase C. These observations suggest that anchoring proteins may maintain groups of enzymes involved in a particular physiological response close to their substrates. Scott and his laboratory are now working on the hypothesis that selective activation of kinases and phosphatases anchored close to their site of action may determine the specificity of intracellular signal transduction events.