Polarized epithelial cells segregate the function and activities of their apical and basolateral membranes. This necessitates the translation of hormonal and neurotransmitter signals at the basolateral membrane into signals which can be utilized at the apical membrane. Transduction of the cyclic AMP second message relies on multifunctional serine-threonine protein kinases and phosphoprotein phosphatases. These proteins regulate events such cellular events as ion channel activation and enzyme activity by altering the phosphorylation status of these enzymes and ion channels. Recently it has been proposed that targeting and sequestration of kinases and phosphatases at sites close to specific substrates influences the specificity of these seemingly multifunctional enzymes. Recently, it has been recognized that CFTR chloride ion channels bind EBP50 through PDZ domain interactions. In other systems, it is known that EBP50 also binds ezrin, an AKAP initially identified in gastric parietal cells. We have isolated clathrin coated vesicles as a membrane fraction enriched in CFTR. These vesicles also contain EBP50, ezrin and type II PKA, leading us to speculate that such a multimeric protein complex may be involved in targeting PKA to CFTR. Although ezrin has been identified as an AKAP in polarized cells, it is by no means the only AKAP known. Thus, we have identified other AKAPs present in clathrin coated vesicles using an RII overlay assay. Recent studies have also indicated that certain AKAPs may be multifunctional proteins, serving as scaffolds to target both kinases and phosphatase activities. Therefore we propose that targeting of kinase and phosphatase activities to CFTR allows for the selective and specific regulation of CFTR via phosphorylation and dephosphorylation. Thus the overall aim of the proposal is to identify the biochemical and molecular basis for the anchoring of kinase and phosphatase isoforms at the apical membrane of polarized epithelial cells necessary for the regulation of CFTR.
Aims 1 and 2 are directed at elucidating the scaffold molecules that target kinase activity to CFTR.
In aim 1 we test the hypothesis that ezrin serves this function by establishing the association of ezrin, RII, EBP50 and CFTR by performing co-immunoprecipitation and immunocytochemical co-localization.
Aim 2 is directed at evaluating the role of other AKAPs in targeting kinase the role of other AKAPs in targeting kinase activity to CFTR.
In aim 1 we test the hypothesis that ezrin serves this function by establishing the association of ezrin, RII, EBP50 and CFTR by performing co-immunoprecipitation and immunocytochemical co-localization.
Aim 2 is directed at evaluation the role of other AKAPs in targeting kinase activity to CFTR. RII overlay and cAMP-affinity chromatography will be performed to delineate AKAPs present in clathrin coted vesicles.
Aim 3 is directed at elucidating the scaffold proteins that target phosphatase activity to CFTR. Immunogold electron microscopy and immunoblot on subcellular fractionations (including isolation of clathrin coated vesicles) will be performed using antibodies against the B56alpha subunit isoform which is expressed in lung and pancreas. These investigations will provide important insights into the general mechanisms by which epithelial cells segregate their second messenger-dependent signaling pathways, and specifically into the mechanisms by which CFTR is regulated.
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