A major goal of this research program is to discover molecules and mechanisms that govern the assembly, regulation and physiological function of distal signaling modules that medicate cAMP action and include anchored protein kinase A (PKA) isoforms. We have discovered a novel A kinase anchor protein, AKAP-KL, that (a) is co-localized with a PKA-regulated K channel protein (ROMK) at the apical surface of epithelial cells and (b) promotes the phosphorylation/activation of the channel in intact cells. The molecular and cellular mechanisms by which AKAP-KL couples cAMP generated at the basolateral surface of a polarized cell to opening channels at the distal, apical membrane will be elucidated. The structural properties of AKAP-KL that mediate the binding of regulatory subunits (RII) of PKAII and the targeting and docking of AKAP-KL RII complexes to specific intracellular sites will be elucidated by a combination of mutagenesis, transfection/expression, biochemical analysis and confocal microscopy. The physiological roles of predicated targeting and tethering domains in AKAP-KL to suppress or promote K+ channel opening in Xenopus oocytes. The precise role of AKAP- KL PKAII complexes in trans-epithelial, cAMP-medicated signaling will be established by varying the concentration and localization of anchored PKAII in intact, highly-polarized epithelial cells that are models for tubular (LLC cells) and cortical collecting duct (MDCK) regions of the nephron. Determination of the rates and levels of ROMK phosphorylation and activation at the apical membrane (as a function of hormone concentration at the basolateral plasma membrane) will provide novel insights into the mechanism by which diffuse cAMP signals are captured, amplified and focused precisely on a substrate-effector to accomplish trans-epithelial regulation. Signaling medicated by type I PKAs may also be diversified by targeting. However, nothing is known about high- affinity RI anchor proteins. We have discovered and cloned a cDNA encoding a novel, RI AKAP in C. elegans (AKAPce). We will systematically determine the biochemical properties, structure-function relationships, cell-specific and developmental patterns of expression and physiological functions for AKAPce. Knowledge of the properties and functions of AKAPce will facilitate characterization of mammalian RI AKAPs and enable the design of molecular tools to manipulate the location/function PKAI in mammalian cells.
