絲氨酸/蘇氨酸磷酸酶2A (Ser/Thr Protein phosphatase 2A;簡稱PP2A)在某些細胞中佔有所有細胞蛋白的百分之一，而且在酵母菌及老鼠的模式中，如果將PP2A的催化次單元體除去會造成生物體的胚胎發育的死亡，證明了PP2A在一般生物的功能上是一個不可或缺的基因。PP2A在細胞內扮演了很多角色例如:細胞凋亡、細胞週期的調控以及細胞骨架的重組。PP2A的組成包含了一個core enzyme:扮演骨架功能的A次單元體與催化的C次單元體，此外，還有一個調節功能的B次單元體。不同的B次單元體結合到中心二元複合體時會辨識不同的受質。Akt又稱為蛋白質激酶B簡稱PKB，它扮演了細胞內的許多調節例如:糖類的代謝、細胞生長、細胞分化以及細胞凋亡。Akt在試管中可以被PP2A去活化，而且當細胞處理PP2A的選擇型抑制劑Okadaic acid時Akt會活化。但是目前並不了解Akt被PP2A調節的機制。所以我們的目標在於去了解PP2A的次單元體與Akt之間的交互作用。首先，我們利用免疫沉澱法用有大量表達B55α-HA的NIH 3T3細胞去證明PP2A-B55α的holoenzyme與Akt之間的交互作用。從Microcystin pull-down 實驗中我們也可以看到細胞內的PP2A-B55α與PP2A-B55-HA都可以與細胞內的PP2A core enzyme形成複合體。在Microcystin pull-down 實驗中又如我們所預期的Akt會結合在PP2A的複合體中。更進一步地，我們將一系列的GST融合蛋白在大腸桿菌中表達純化出來包括: GST-PP2A-A、GST-PP2A- B55 α、GST-PP2A-C以及GST-Akt。此外，我們還表達純化了帶有His-Tag的Akt與B55α。藉由這些基因重組的蛋白質的純化來研究Akt與PP2A之間的交互作用。在GST pull-down的實驗中我們可以發現不論在短時間或長時間的反應下His-Akt都可以與GST-PP2A- B55α結合，但是，只有在長時間的反應下His-Akt才可以與PP2A-C結合。有趣的是，在試管中的試驗，不論是Akt的N端、催化區域或是C端都可以結合到His-B55α。我們的結果說明了PP2A跟Akt的交互作用至少會藉著PP2A-B55α這個次單元體。

　Serine/Threonine phosphatase PP2A accounts for 1% of total cellular proteins in some cell types. Deletion of the PP2A catalytic subunit is lethal in yeast and mouse, demonstrating that PP2A is essential for normal cell function. PP2A is involved in multiple cellular events such as regulation of apoptosis, cell cycle progression and cytoskeleton reorganization. PP2A consists of a heterodimer core enzyme, which includes a catalytic subunit C, a scaffold subunit A, and a variable regulatory subunit B. Different B subunits bind to the core dimer targeting to different substrates. Akt, also named protein kinase B, is involved in regulation of diverse cellular processes including glucose metabolism, cell growth, cell proliferation, and apoptosis. Akt is inactivated by PP2A in vitro and is stimulated in cells upon treatment with okadaic acid, which is a selective inhibitor of PP2A. Yet the mechanism of regulation of Akt by PP2A is unclear. We aim to characterize the interactions between individual subunits of PP2A and Akt. First, we demonstrated physical interaction of PP2A AB55αC holoenzyme and Akt in NIH 3T3 cells overexpressing HA-tagged PP2A-B55α by co-immunoprecipitation assay. Microcystin pull-down assays showed that both endogenous PP2A-B55α and exogenous PP2A-B55α-HA formed complexes with the PP2A core enzyme. As expected, Akt was present in the micirocystin pull-downed phosphatase complexes. Furthermore, we have expressed and purified a series of GST fusion proteins including GST-PP2A-A, GST-PP2A-B55α, GST-PP2A-C, GST-Akt from E. coli. In addition, we also expressed and purified the His-tagged Akt and His-tagged PP2A-B55α. These recombinant proteins were used to investigate interactions between Akt and PP2A in vitro. In GST pull-down assays, the His-Akt protein are pull downed by GST-PP2A-B55α in both short incubation and longer incubation time. However, the His-Akt protein are also pull downed with GST-PP2A-C in a longer incubation time. Interestingly, the N-terminal domain, the catalytic domain and C-terminal domain of Akt are all involved in binding to PP2A-B55 in vitro. Our results indicated that the interaction between PP2A and Akt was mediated at least through the PP2A B55α subunit.

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