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Title: | Caveolin-1與Integrin Complex在S-ras (Q61K ) Balb/3T3轉型細胞中的互動 The Interaction between Caveolin-1 and Integrin Complex in S-ras (Q61K) Balb/3T3 cells |
Authors: | Fan Jun 一 Huei 範潤薈 |
Publication Year : | 2002 |
Degree: | 碩士 |
Abstract: | 本論文藉由S-ras (Q61K) Balb / 3T3細胞,去探討當細胞內膽固醇的生合成路徑遭到阻礙時,細胞內與訊息傳導相關的蛋白integrin βl , caveolin-l (Cav-l)以及FAK之間的交互作用。 本論文首先利用不同濃度的SB-14分次萃取pcDNA 3.1 Balb/3T3細胞以及shrimp-ras (Q61K) pcDNA3.1 Balb/3T3轉型細胞之膜蛋白,發現無論在pcDNA 3.1Balb/3T3細胞中或者 shrimp-ras (Q61K) pcDNA3.1 Balb/3T3轉型細胞中,絕大多數(~99%)的integrinβ1都出現在以0.1% SB-14處理後的細胞膜之蛋白液中。當細胞分別處理10μM mevastatin以阻斷 mevalonate pathway,在0.1% SB-14所抽取的pcDNA 3.1 Balb/3T3細胞之細胞膜蛋白萃取液中,Cav-l的量上升。在免疫沈澱法實驗中,未處理過mevastatin的shrimp-ras(Q61K) pcDNA 3.1 Balb/3T3轉型細胞,其在integrin βl上有Cav-l量的增加而形成 integrin βl-Cav-l complex,以及integrin βl上降解的FAK;經由mevastatin的處理後,在轉殖pcDNA 3.1質體的Balb/3T3細胞中的integrin β1上也有Cav-1量的增加,形成integin βl-Cav-1 complex,同樣integrin βl上也出現降解的FAK。推論經過mevastatin處理的pcDNA 3.1 Balb/3T3細胞,在0.1% SB-14所抽取的細胞膜之蛋白液中增加的Cav-l,可能用以協助細胞形成integrin βl-Cav-1 complex。 PKD為golgi 中參與蛋白質傳遞的分子,而細胞中的Cav-l必須經由golgi傳送。為了推測integrin complex上增量的Cav-1來自於何處,所以分別將經過或未經過mevastatin處理的細胞染測PKD以及phospho-PKD。結果顯示pc DNA 3.1 Balb/3T3細胞和S-ras (Q61K) Balb/3T3轉型細胞中,存在未磷酸化的PKD,此種PKD不具有運輸蛋白質的能力以推論integrin complex上增量的Cav-l是由Caveolae而來。再進一步以GGTI-286以及FTI-277處理細胞。實驗結果顯示,經過GGTI-286或FTI-277抑制prenylation作用後的轉殖或未轉殖S-ras (Q61K)的細胞中,染測不到活化的integrin complex。所以推論當細胞中prenylation 作用被抑制後,Cav-1無法在膜上被傳送。 BALB/3T3 cells transfected with DNA encoding shrimp Penaeus japonicus S-ras (Q61K) were transformed successfully. The interaction among integrin βl, caveolin-1 (Cav-1), and focal adhesion kinase (FAK) was studied while the biosynthesis of cholesterol was blocked. The amount of caveolin-1 in the membrane fraction of the transformed BALB/3T3 cells that was extractable with 0.1% Sulfobetaine-14 was significant larger than in pcDNA 3.l-transfected BALA/3T3 cells. The possible interaction of caveolin-1 with β1 integrin was investigated. Most of the caveolin-1 in the 0.1% Sulfobetaine-14 lysates of S-ras(Q61)-transformed BALB/3T3 cells was associated with β1 integrin, but the signal transmission of βl integrin-FAK was inhibited by the proteolysis of FAK. On the other hand, caveolin-1 in the 0.1% Sulfobetaine-14 lysates of mock-transfected BALB/3T3 cells was found to be free from binding with β1 integrin and the β1 integrin-FAK was intact for signal transmission. Following treatment by mevastatin ( a HMG -CoA reductase inhibitor) to prevent the mevalonate pathwav, mock-transfected BALB/3T3 cells exhibited additional amounts of caveolin-1 in the 0.1% Sulfobetaine-14 extractable membrane lysates, but transformed BALB/3T3 cells did not. Most of the 0.1% Sulfobetaine-1-extractable caveolin-1 in the mock-transfected and mevastatin treated BALB/3T3 was bound with β1 integrin, but the signal transmission of β1 integrin-FAK was inhibited by the proteolysis of FAK, mimicking the results in cells transformed with S-ras (Q61K). However, electron microscopy revealed no morphological identity between mevastatin-treated and S-ras (Q61K)-transformed BALB/3T3 cells. In addition, the recruitment of caveoli-1 with β1 integrin was not detectable after further incubation of S-ras (Q61K)-transformed BALB/3T3 cells with 10μM mevastatin for 3.5 hrs. However, the signal transmission of βl integrin-FAK was sustained, as arrested by the proteolysis of FAK. In other words, the recruitment of caveolin-1 with β1 integrin functions as a molecular switch that responds to cholesterol deprivation, but the proteolytic degradation of FAK does not. Furthermore, pcDNA3.l-transfected BALA/3T3 cells and S-ras(Q61K)- transformed Balb/3T3 cells were treated with 10μM GGTI-286 or 10μM GGTI-277 for 1 hr. The results show that there is no active integrin complex in pcDNA3.l-transfected BALB/3T3 cells and S-ras (Q6lK)-transformed BALB/3T3 cells. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75348 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 動物學研究所 |
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