Signal transduction across the plasma membrane is often mediated by second messengers. Channelling of second messengers to subcellular sites may be a mechanism for localizing hormonal and neurotransmitter effects to specific compartments of the cell. The proximate receptors for the second messenger cAMP are the cAMP-dependent protein kinases (PKA). The type II PKAs are anchored to subcellular structures through the interaction of their regulatory subunits (RII) with a number of RII- binding proteins. Our understanding of the molecular and cellular mechanisms responsible for the anchoring of RII to RII-binding proteins is incomplete. Our long term goal is to define the mechanisms responsible for targeting the type II PKAs to subcellular sites and to characterize the physiological effects of protein phosphorylation by anchored cAMP-dependent protein kinases. Oligonucleotide sit-directed mutagenesis of RII-binding site in microtubule-association protein 2 (MAP2) and the MAP2-binding site in RIIbeta will be used to characterize the structural requirements of the MAP2-RII interaction at the single amino acid level. Studies on the interaction of RII with MAP2 in vivo will be initiated. These studies will consist of transfecting plasmids containing cDNAs coding for fragments of MAP2 containing the RII-binding site but missing the tubulin binding domain into the human neuroblastoma cell line SMS-MSN. MSN cells contain MAP2, MAP2C and type II PKAs. Morphological and physiological effects will assessed in order to elucidate functional roles of RII anchoring to MAP2. These studies will provide information on the effects of displacing RII from endogenous sites on MAP2. The structure and function of a novel RII-binding protein (AKAP68) will be characterized. The cDNA coding for AKAP68 was cloned from an MSN cell library constructed in gamma gt11. A protein containing the RII-binding domain has been expressed in E. coli and a polyclonal antiserum to AKAP68 has been produced in a rabbit. Attempts to identify the physiological function of AKAP68 will be made by transfecting the gene for AKAP68 into MSN neuroblastoma cells and betaTC-3 insulinoma cells and determining the effects on protein phosphorylation patterns and hormone secretion.
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