磷酸肌醇激酶(PI-3 K)/Akt激酶訊息傳遞路徑在細胞中扮演著十分重要的角色，許多細胞的生理作用包含葡萄糖代謝、細胞增生、血管新生、轉錄作用、以及細胞凋亡都與其訊息傳遞路徑息息相關。Akt激酶又稱之為蛋白質基酶B型(Protein kinase B/PKB)，以下簡稱Akt，為一個蘇氨酸以及絲氨酸的激酶，主要是作用在磷酸肌醇激酶(PI-3 K)的下游。過去的研究指出，Akt的過度活化與許多人類的癌症有相關性。Akt能夠在生長因子的刺激之下而活化，而且Akt的活化與本身磷酸化的狀態有關，已知Akt可藉由兩個位點:第308蘇氨酸以及第473絲氨酸的磷酸化來達到其活化。近年來有相當多的證據顯示，在不同的細胞中磷酸酶PP2A具有去磷酸化Akt來達到負調節Akt活性的能力。磷酸酶PP2A，以下簡稱PP2A，為異型三次單元體所組成的三元複合體的磷酸酶。主要是由次單元體A、次單元體B以及次單元體C所組成的。次單元體A的功能為骨架蛋白，次單元體C則是具有著催化反應能力的蛋白，而為數眾多的次單元體B則是被認為扮演著決定PP2A的受質專一性以及PP2A在細胞內分佈位置的調節蛋白的角色。雖然，研究顯示PP2A能夠負調節Akt的活性，但是對於PP2A是否可以藉由負調節Akt而影響到Akt所調節細胞存活訊息傳遞路徑，目前仍是尚未明朗。因此，我們的研究目標即是要了解PP2A是否能夠調節Akt的抗細胞凋亡功能，而我們主要是以倚靠IL-3而存活的類淋巴細胞的FL5.12細胞作為我們研究的模型。我們的研究結果發現，在移除IL-3後Akt產生去磷酸化的現象也同時伴隨著FL5.12細胞的死亡。這樣的去磷酸化現象則可藉由PP2A的選擇性抑制劑，okadaic acid，而抑制住，這意味著在FL5.12細胞中PP2A具有調節Akt的功能。我們的研究更進一步的利用免疫沉澱法來確定在細胞中PP2A與Akt之間的交互作用，我們發現在NIH 3T3、COS-7以及FL5.12細胞中PP2A與Akt是存在同一個免疫沉澱下來的複合體內。接著，我們也發現Akt是存在於含有B55α的PP2A複合體裡面。我們也利用Microcystin pull-down的方法，將PP2A的三元複合體從FL5.12細胞中沉澱下來，同樣地也觀察到Akt存在於PP2A的三元複合體內。綜合來說，我們的研究結果顯示PP2A可能是藉由其次單元體B55α來調節Akt的活性。

　The phosphatidylinositide 3’-OH kinase (PI3K)/Akt signaling pathway plays a major role in regulation of multiple cellular processes such as glucose metabolism, cell proliferation, angiogenesis, transcription, and apoptosis. Akt, also termed protein kinase B, is a serine/threonine kinase that acts downstream of PI3K. Elevated Akt activity has been linked to several human cancers. Phosphorylation of Akt at two regulatory residues, Thr308 and Ser473, is required for its full activation in response to stimulation by survival factors and mitogens in a PI3K-dependent manner. A significant amount of evidence shows that Akt activity is negatively regulated by protein phosphates 2A in various cell types. PP2A is a heterotrimeric enzyme which consists of a scaffold subunit A, a catalytic subunit C, and a variable regulatory subunit B. However, the regulatory role of PP2A in Akt-mediated survival pathway is unclear. The aim of this project is to characterize the role of PP2A in regulating the anti-apoptosis activity of Akt in IL-3 dependent FL5.12 lymphoid cells. Our finding demonstrates that IL-3 withdrawal, a death stimulus to FL5.12 cells, triggers Akt dephosphorylation. Okadaic acid, a selective inhibitor of PP2A, prevented Akt dephosphorylation during IL-3 withdrawal, suggesting a role of PP2A in regulating Akt in FL5.12 cells. To further characterize the role of PP2A in regulating Akt, we investigated whether physical interaction of PP2A and Akt exists, we found that PP2A and Akt were co-immunoprecipitated in COS-7, NIH 3T3 cells. Moreover, Akt is present in the same complex with trimeric PP2A holoenzyme pulled down by microcystin sepharose in FL5.12 cells. Microcystin is known to pull down PP2A holoenzyme complex through the binding to C subunit of PP2A. Furthermore, Akt is found in association with the B55α-containing PP2A trimeric holoenzyme comlex in FL5.12 cells by co-immunoprecipitation. Taken together, our data demonstrate that PP2A may regulate Akt activity through the regulatory subunit B55α.

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