AKT/protein kinase B (PKB) 參與調節許多不同的細胞功能，包括葡萄糖代謝、細胞生長、細胞增殖和細胞凋亡等等。當細胞內上游的 phosphatidylinositide 3’ -OH kinase (PI3K)被活化之後，下游的 Akt 會被PDK1 和 mTOR/rictor complex 在 Thr-308 和 Ser-473 的位點分別磷酸化。而在許多生物系統之中， AKT的活性會被磷酸酶 protein phosphatase 2A(PP2A) 做負調控。PP2A 是一個由三個不同次單元所組成的磷酸酶，包括 36-kDa 的催化 C 次單元，65-kDa 的結構 A次單元，以及一個多變的調節 B次單元，B 次單元被認為可以控制PP2A holoenzyme 的受質特異性和PP2A holoenzyme 在細胞內的分布位置。在以前的研究中，我們已經發現 B55α (為B55 family 的 isoform 之一)，可以調節 PP2A holoenzyme 去選擇性對Akt Thr308 位點去磷酸化且調節Akt 的活性。因此在我的研究，我們想要以時間及空間的角度探討在 AKT 的活化過程中， B55α 是如何調節 AKT 活化。我們使用細胞轉染及免疫沈澱法，發現不論是內生性的 B55α 或是外生性的 B55α-HA 都會和外生性的 AKTFLAG有交互作用。我們也發現在 HeLa 細胞中，B55α 和 AKT 的交互作用會因為epidermal growth factor 的刺激而增加。經由免疫螢光染色以及螢光顯微鏡的分析，我們觀察到 B55α 和 AKT 在細胞質和細胞膜有 colocalize 的現象。同時我們也發現，經由血清刺激而引發的AKT 轉移到細胞膜的現象，和 vector only 的細胞比較，在 B55α knockdown 的細胞有增快的趨勢，然而在 B55α 過度表現的細胞也有增快的趨勢。此外，因PP2A 三個次單元的組成，對於和PP2A 下游受質的作用來說是必需的。因此我們進一步利用bimolecular fluorescence complementation (BiFC) assay 的方式來探討PP2A 次單元之間的交互作用。結論：我們結果顯示 PP2A AB55αC holoenzyme 對 AKT 活化過程具有在時間和空間上的相關性；並且我們的結果顯示 BiFC assay 可以用來研究在活細胞中 PP2A holoenzyme 的組成。

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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