| Summary: | 碩士 === 國立成功大學 === 分子醫學研究所 === 97 === AKT/protein kinase B (PKB) is involved in regulation of diverse cellular functions, including glucose metabolism, cell growth, cell proliferation, and apoptosis. After upstream phosphatidylinositide 3’-OH kinase (PI3K) is activated, Akt is phosphorylated at Thr-308 and Ser-473 by PDK1 and mTOR/rictor complex, respectively. AKT activity is negatively regulated by protein phosphatase 2A (PP2A) in various systems. PP2A is a heterotrimeric enzyme consisting of a 36-kDa catalytic C subunit, a 65-kDa structural A subunit, and a variable regulatory B subunit that is thought to control the substrate specificity and subcellular localization of the holoenzymes. We have shown that B55α, a B55 family isoform, targets the PP2A holoenzymes to selectively regulate dephosphorylation of Thr-308 of AKT and regulates Akt activity. In this report, we investigated the role of B55α in regulating the temporal and spatial dynamics of Akt activation. By using transient transfection and co-immunoprecipitation, we found that both endogenous B55α and exogenous B55α-HA interacted with exogenous Akt-FLAG. We also found that interaction between B55α and AKT occurred when HeLa cells were stimulated with epidermal growth factor. By indirect fluorescence microscopy analysis, colocalization of B55α and AKT was shown in the cytosol and plasma membrane, and serum-stimulated membrane translocation of AKT occurred at a faster rate in B55α knockdown and B55α overexpression cells, as compared to vector only cells. Since the assembly of the trimeric PP2A holoenzymes is essential for specific targeting to regulation of a specific substrate, such as Akt. We further investigated the interaction between subunits of PP2A by bimolecular fluorescence complementation (BiFC) assay. Our results demonstrate that the PP2A AB55αC holoenzyme regulates AKT activation in a temporal and spatial manner and that it is feasible to apply BiFC to sudy PP2A holoenzyme assembly in live cells.
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