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研究生:柯程耀
研究生(外文):Cheng-Yao Ko
論文名稱:探討GCIP蛋白在調控細胞週期所扮演的可能角色
論文名稱(外文):Assessment of GCIP, a human grap2 and cyclin D interacting protein, its potential role in regulation of cell cycle progression
指導教授:張敏政張敏政引用關係
指導教授(外文):Ming-Chung Chang
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物科技研究所碩博士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:86
中文關鍵詞:細胞週期調控
外文關鍵詞:cyclin DGCIPribosomal protein P0Grap2cell cycle control
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  哺乳類細胞生長週期的調控是經由細胞外受器接受刺激以後,經由細胞內訊息傳遞的網絡將調控細胞生長的訊息傳到與調控細胞週期相關的分子上。在細胞週期從一個時期進入到下一個時期的過程當中,需要許多調控分子的參與。當細胞週期要由first gap phase (G1 phase)進行到DNA合成的 (S phase)過程時,retinoblastoma (Rb)蛋白質會被Cyclin D-dependent protein kinases 給磷酸化,Rb蛋白質的磷酸化會抑制Rb蛋白質的功能,並且促使細胞週期由 G1時期進行到S時期。
  P0蛋白質是構成真核細胞核糖體大次單元 (large subunit) 的重要分子。在2001年時,實驗室先前的成員利用P0蛋白質作為探針,在yeast two-hybrid 的實驗中找到與之接合的GCIP蛋白質。GCIP蛋白質的全名為human Grap2 and Cyclin D Interacting Protein。根據之前的研究,GCIP蛋白質在人體的每個組織中都會表現;然而在一些分化到末期的組織中,例如心臟、肌肉、周邊血液系統中的白血球以及大腦,GCIP蛋白質的表現會達到最高。除此之外,先前的研究發現,GCIP蛋白質會減少Rb蛋白質的磷酸化,並且抑制E2F1轉錄因子的轉錄能力,而這兩個部分是廣泛被接受在調控細胞週期由 G1時期進行到S時期中,最重要的調控步驟。
  因此,根據上述的原因,為了進一步地去探討cyclin D-dependent protein kinases的調控、Rb蛋白質的磷酸化調控、E2F轉錄能力的調控以及細胞週期的調控機制,本論文將專注於GCIP以及與之相關的蛋白質的研究。我們的實驗結果,發現在in vitro及in vivo的共同免疫沈澱實驗中,GCIP分別會與cyclin D1和cyclin D3進行交互作用。除此之外,在GST-pull down與in vivo共同免疫沈澱的實驗當中,我們發現GCIP蛋白質的N端與C端的區域是主要與P0進行交互作用的區域;同時,我們也確認P0蛋白質其N端第39-114的氨基酸片段,是與GCIP進行交互作用的區域。將此片段做進一步的電腦分析,預測這39-114氨基酸的區域含有兩個潛在的磷酸化區域,分別是Protein Kinase C(PKC)與Casein Kinase II (CK II)辨識的序列。在我們所建立可持續表現GCIP蛋白質的細胞株中,我們發現大量地表現GCIP蛋白,會促使細胞生長的速率下降。以流氏細胞儀做進一步細胞週期的分析發現,GCIP蛋白質促使細胞生長緩慢的原因是導致細胞週期停止在G1時期。因此根據上面的實驗結果,我們認為GCIP蛋白在調控細胞週期上,是扮演負調控者的角色,會使得細胞週期停在G1時期。因此,根據之前的文獻與本論文的實驗結果,我們推測GCIP與P0蛋白的交互作用可能在細胞的DNA修復過程,扮演重要的角色。
  Regulation of mammalian cell growth and proliferation is administered through receptor-mediated signaling networks that ultimately converge on the cell cycle machinery. Multiple components are implicated in the transition from one cell cycle phase to another. As cells progress through the first gap phase (G1 phase) to the initiation of DNA synthesis (S phase), the protein retinoblastoma (Rb) is phosphorylated in a cyclin D-dependent protein kinases manner and the phosphorylation of Rb results in the functional inactivation of Rb and the progression of the cell cycle through the late G1 restriction point into the S phase.
  Previously, our lab demonstrated that P0, which is the critical component of ribosomal stalk of eukaryotic ribosomal large subunit, interacts with GCIP, using P0 as a bait protein in the yeast two-hybrid assay. GCIP is a novel human Grap2 and Cyclin D Interacting Protein, which is reported first in 2000. The expression of GCIP was found in all human tissues with the highest level of expression in heart, muscle, peripheral blood leukocytes and brain. Furthermore, overexpression of GCIP was found to reduce the phosphorylation of retinoblastoma protein and inhibit E2F1-mediated transcription activity, which is generally considered the critical step for cell cycle progression from the G1 phase into the S phase.
  In this study, we further assessed the potential role of GCIP in regulation of cell cycle progress and its association with ribosomal protein P0. Our results showed GCIP interacting with cyclin D1 and D3 both in vitro and in vivo. We found that P0 bound to GCIP in both GST-pull down assay and in mammalian cells through simultaneous binding to the N- and C-terminal regions of GCIP. GCIP bound to P0 through binding to the N-terminal domain (39-114 amino acids) of protein P0. Through the computational prediction, there are two potential phosphorylation sites located at this N-terminal domain; each potential site is recognized by Protein Kinase C (PKC) and Casein Kinase II (CK II), respectively. By establishment of stable cell line overexoressing GCIP, we observed that overexpression of GCIP arrests the growth of the transfected human kidney cells (HEK-293) in contrast with that of un-transfected control cells. In addition, through FACS cell cycle analysis, we found that the growth arrest is due to the accumulation of G0/G1 phase arrest, suggesting GCIP influences on cell cycle progression by acting on G0/G1 phase. Taken together, our results suggested that GCIP acts as a negative regulator in controlling cell proliferation on G0/G1 phase through vague pathways. In addition, we proposed a hypothesis that GCIP/P0 interaction is important to cellular DNA repair mechanism.
摘要......................................................................................1
目錄......................................................................................5
圖目錄....................................................................................6
縮寫檢索表................................................................................9
序論.....................................................................................11
材料與方法...............................................................................20
結果.....................................................................................40
一、 GCIP與D-type cyclin在試管內(in vitro)與活體內(in vivo)交互作用.................40
二、 GCIP與核糖體蛋白P0交互作用的區域...............................................42
三、 核糖體P0蛋白與GCIP交互作用的區域...............................................44
四、 GCIP蛋白對於細胞生長的影響.....................................................46
討論.....................................................................................49
參考文獻.................................................................................55
圖.......................................................................................61
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