Akt(Protein Kinase B)為一控制細胞生存、細胞凋亡、以及蛋白質轉錄之重要訊息傳遞分子，可藉由細胞表面之接受器接受訊息後促進Akt活化。當PI3K/Akt Pathway中之訊息傳遞分子因突變失去功能或活化皆可能造成此訊息傳遞路徑之過度活化。過度活化之Akt會造成蛋白質過度轉譯、細胞週期不受調控、以及細胞無法正常凋亡，最終轉型為癌化細胞。去磷酸化及部分調控蛋白之影響是抑制Akt活性的主要機制，其中，Carboxyl-Terminal Modulator Protein (CTMP)於2001年所發現。並觀察到CTMP可與Akt結合進而負向調控Akt之活性。然而，CTMP與Akt之結合之區域尚未被報導。並且，CTMP被發現可能正向或負向調控Akt之活性，因此，其對於Akt之調控尚未被確立。在本篇論文中，將進一步探討CTMP與Akt之交互作用與其活性調控。我們藉由人類乳癌細胞株確立CTMP與Akt的磷酸化以及細胞生長之相關性，並藉由免疫沉澱以及免疫螢光之分析之結果來確認CTMP與磷酸化AKT之交互作用，而 GST pull down assay 的結果證明CTMP可能包含兩個以上能與Akt之結合之區域。此外，在HeLa細胞株中穩定表現CTMP直接的增加Akt之磷酸化，並且，在細胞starvation後處理不同濃度之胰島素可發現穩定的表達CTMP顯著的增加Akt於Thr308以及Ser473位點上之磷酸化。利用cell proliferation 以及colony forming assay來觀察穩定表現CTMP之 HeLa細胞株在不同濃度之血清的處理下之生長可發現，CTMP的表現會促進細胞之生長，因此推測CTMP可能參與PI3K/Akt訊息傳遞路徑所調控之細胞生長。此外，利用soft agar assay以及在裸鼠中所建立的人類腫瘤動物模式證明，不管是在in vitro 或 in vivo 中，CTMP皆具有促進腫瘤生長之能力，並且可能促進腫瘤之轉移能力。在本篇論文中，這些結果顯示CTMP可與Akt結合進而藉由增加Akt對於胰島素之敏感性達到正向調控Akt之活性，進一步地調控細胞以及腫瘤之生長。

Akt/Protein Kinase B plays an important role in many biological responses including cell survival, proliferation, and nutrient metabolism. Activation of Akt/PKB by growth factors involves PIP3 binding, membrane translocation and the phosphorylation at Thr308 and Ser473 residues by PDK1 and PDK2, respectively. Dephosphorylation and AKT binding proteins were two inhibitory mechanisms for AKT. Carboxyl-terminal modulator protein (CTMP) has been demonstrated to bind to carboxyl-terminus of Akt and as a regulator on plasma membrane. However, the interaction region of CTMP to Akt is still unknown. On the other hand, contradict results of positive and negative effects to AKT activity were reported. In this study, we firstly demonstrated the CTMP protein level shows positive correlation with Akt phosphorylation and cell proliferation in human breast cancer cell. The interaction of CTMP and phosphorylation Akt by co-immunoprecipitation and immunofluorescence experiments shows that CTMP may be involved in regulating Akt phosphrylation. The results of GST pull down assay demonstrated that the direct binding and revealed more then one interaction region of CTMP to Akt. Interestingly, in HeLa cells, transiently or stably expressing CTMP can directly promote Akt phosphorylation even in basal state and significantly increase the phosphorylation at both Thr308 and Ser473 of Akt upon the insulin stimulation in the pre-starvation condition. When compared the cell growth of HeLa cells in 10% serum condition using cell proliferation and soft-agar colony forming assay, rapid cell proliferation and colony number increase were observed in cells stably expressing CTMP suggesting that CTMP may be involved in the regulation of Akt-mediated cell growth. The analogous results were also obtained from colony formation assay with different condition from 10% to 1% serum. The result of cell cycle distribution shows that the increasment of cell proliferation rate may due to the increasment of cell cycle progression in CTMP stable clones. Furthermore, CTMP stable clone promote tumor growth and metastasis in xenograft animal model. Together, these results indicated that the binding of CTMP positively modulate Akt and thus increase insulin sensitivity in HeLa cells.

Abstract (Chinese) …………..……………………………….……..2
Abstract ………..…………………………………………….……...4
Abbreviations………………………………………………………..6
Introduction …………………………………………………………7
Materials and Methods …………………………..……………........15
Results ……………………………………………………...…..…..28
Discussion …………………...………………………….……...…..38
References ..……….……………………..………………….……...43
Figures ………………………..…………………………….….…...50
Appendix …..……………………………………………….…...….80

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