INTERACTIONS OF ANCHORING PROEINS WITH THEIR TARGETS

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Protein phosphorylation controls may cellular processes including hormone responsiveness, cell growth, differentiation and apoptosis. The "targeting hypothesis" processes that phosphorylation events are controlled, in part, by subcellular localization of protein kinases and phosphatases in the cell via the interaction of docking domains on the enzymes with specific anchoring proteins. The principle behind this localization hypothesis is that the protein kinase will be in close proximity to its target when the activation signal is discharged. A pressing issue then is to define the molecular basis for these targeting interactions. Thus, the long term goal of this proposal is to determine how protein:protein interactions control the localization of protein kinases in the cell. The correct intracellular targeting of protein kinases and phosphatases confers specificity, in part, by placing them in close proximity to their preferred substrates. Perhaps the best characterized of the mammalian targeting proteins is the family of A-Kinase Anchoring Proteins (AKAPs). AKAPs maintain the cyclic AMP dependent protein kinases (PKA) in specific subcellular compartments, thereby ensuring accessibility of the kinase to a limited number of substrates in a particular location. This compartmentalization occurs through the interaction of the docking domains of the regulatory subunit of PKA with a conserved "anchoring site" on the various AKAPs. New evidence suggests these AKAPs are also involved in localizing phosphatases as well as protein kinase C (PKC) at a single site, thus affording a possible mechanism for integrating signal transduction pathways. The goals of this proposal are directed towards understanding of the interactions responsible for the localization of the protein kinases PKA and PKC. We will accomplish the following in our Specific Aims: 1) We will solve the solution structure of the dimerization domain of PKA which is responsible for docking the enzyme to various AKAPs. 2) We will investigate the molecular nature of the interaction between PKA and various AKAPs by determining the solution conformation of "anchoring site" peptides with the wild-type and mutant dimerization domain of PKA. 3) We will investigate the structural motifs responsible for targeting of PKC by a combination of mutagenic and spectroscopic techniques.