臺灣博碩士論文加值系統

Glycogen synthase kinase-3 (GSK-3) 為一種蛋白激酶 (protein kinase) ，GSK-3 可以藉由在其許多受質上進行磷酸化作用，改變其受質的功能與活性，進而參與調控許多細胞功能。GSK-3 家族包含兩種激酶，GSK-3α與GSK-3β。細胞中的 GSK-3 常具有高度激酶活性，目前已知許多的生理刺激因子，例如像是表皮生長因子和胰島素，則會藉由引發下游特定激酶活化，進而對 GSK-3 進行磷酸化/去活化的訊號傳遞路徑，使得 GSK-3 的激酶活性下降。然而，前人的研究到目前為止，大部分的重點都是著重在 GSK-3 磷酸化機制與其受質的鑑定，對於鑑定 GSK-3 之交互作用蛋白則較少著墨。一般來說，非受質性的交互作用蛋白，也有可能會參與調控 GSK-3 的活性。本論文的目的即在找尋新的 GSK-3 交互作用蛋白。利用專一辨識 GSK-3α 抗體以及質譜儀分析技術，我們在抗體所媒介的免疫沉澱蛋白裡，找到 2-hydroxyphytanoyl-CoA lyase (2HPCL) 。2HPCL 是一個參與在3-甲基脂肪酸α-氧化代謝作用的酵素。為了證明 2HPCL 與 GSK-3α有交互作用，我們在猿猴 COS-7 細胞株中過量表現 myc-2HPCL 以及 flag-GSK-3α後，接著進行免疫共沉澱法。我們發現到，不管使用了辨識 myc 或是 GSK-3α的抗體，都可以分別在其所沉澱下來的蛋白中，發現到 myc-2HPCL 以及 flag-GSK-3α；此外，我們製作了專一性辨識 2HPCL 的抗體，並利用此抗體在人類 A431 細胞株中，藉由辨識 2HPCL 或是 GSK-3α的抗體，也一樣可以偵測到內生性的 GSK-3α以及 2HPCL 被共同免疫沉澱下來。以同樣方法，我們卻沒有偵測到 GSK-3β與 2HPCL 被共同免疫沉澱下來。此外，我們將人類 2HPCL 與 GSK-3α基因選殖出來，在大腸桿菌中表現並純化出帶有 glutathione transferase (GST) 的 2HPCL (GST-2HPCL) , 與帶有六個 histidine (His) 標記的 GSK-3α (His-GSK-3α) 。利用這些材料為工具，藉由使用 glutathione-偶合的樹脂 (GSH-bead) 與 GST-2HPCL 和 His-GSK-3α 混合物作用之後，我們也在其結合的蛋白複合物中，偵測到 His-GSK3α 的存在。藉此我們可以知道， 2HPCL 會直接與 GSK-3α結合。接著我們鑑定 2HPCL 是否為 GSK-3α的磷酸化受質。為此，我們在大腸桿菌裡表現並純化重組蛋白 GST-GSK-3α。重組蛋白 GST-GSK-3α具有與 GSK-3α一樣的激酶活性，不僅能夠進行自我磷酸化，也能對已知的受質蛋白，如腦髓鞘鹼性蛋白 (myelin basic protein) 進行磷酸化作用。然而如果將 GST-2HPCL 當作受質進行磷酸化反應，不管是重組 GST-GSK-3α，或是從組織裡所純化出來的 GSK-3α，皆無法對 GST-2HPCL 進行磷酸化的動作。總結目前所得到的結果，我們發現到， 2HPCL 會與 GSK-3α進行交互作用，然而，卻無法被其磷酸化。因此，我們猜測， 2HPCL 的確是 GSK-3α新的交互作用蛋白，但角色並非是其受質蛋白。

Glycogen synthase kinase-3 (GSK-3) is a kinase subfamily involved in regulating diverse cell functions. It can phosphorylate many substrates in vivo and thereby modulate their functions in cells. The kinase subfamily consists of two members: GSK-3α and GSK-3β. GSK-3 has a highly basal activity in resting cells, which can be down-modulated by various physiological stimuli such as epithelial growth factor (EGF) and insulin, through a kinase mediated, phosphorylation-dependent mechanism. However, little attention has been focused on its interacting proteins, which may be also involved in the regulation of GSK-3 function in cells. The aim of this study is to identify new interacting protein(s) of GSK-3. Using anti-GSK-3α antibody to immunoprecipitated GSK-3α from human A431 cell lysates, the proteins present in the immunocomplex were separated by SDS-PAGE and each candidate protein was subjected to mass spectrometric analysis. One of the proteins, 2-hydroxyphytanoyl-CoA lyase (2HPCL) was identified. 2HPCL is an enzyme participating inα-oxidation of 3-methyl group fatty acids, which allows subsequent metabolism of these fatty acids for β-oxidation. In order to confirm the interaction between 2HPCL and GSK-3α, pCMV-myc/2HPCL and pflag-CMV2/GSK-3α were constructed and transiently expressed in COS-7 cells for coimmunoprecipitation experiments. Both myc-2HPCL and flag-GSK-3α can be detected in the immunocomplex pulled down by anti-myc or anti-GSK-3α antibodies. This interaction can be also detected in A431 cells by using anti-2HPCL or anti-GSK-3α antibody to pull down endogenous 2HPCL and GSK-3α. In contract, endogenous 2HPCL could not be co-immunoprecipitated by GSK-3β. We also cloned the cDNA for human 2HPCL and GSK-3α. The GST-fusion 2HPCL and GSK-3α as well as His-tagged GSK-3α recombinant proteins were expressed and purified. We demonstrated that His-GSK-3α can be detected in the GST-2HPCL protein complex pulled down by the glutathione-conjugated bead in vitro, which indicates a direct interaction between 2HPCL and GSK-3α. Purified recombinant GST-2HPCL and GST-GSK-3α could phosphorylate itself and myelin basic protein (MBP), one of GSK-3α substrate. However, recombinant GST-GSK-3α and GSK-3α purified from pig brain failed to phosphorylate GST-2HPCL in vitro. In summary, we identified 2HPCL as a new GSK-3α-interacting protein, and 2HPCL may not be a substrate for GSK-3α.

目 錄
指導教授推薦書
口試委員會審定書
授權書 -iii-
誌謝 -iv-
中文摘要 -v-
英文摘要 -viii-
第一章 簡介 -1-
1.1 GSK-3的鑑定 -1-
1.2 GSK-3的活性調控 -1-
1.3 GSK-3為一種可調控許多受質的激酶 -2-
1.4 GSK-3參與在調控許多細胞功能 -2-
1.5 GSK-3的交互作用蛋白 -7-
1.6 GSK-3在細胞內的位置 -8-
1.7 本文研究目的 -9-
第二章 材料與方法 -11-
2.1 材料 -11-
2.2 膠體染色、膠體內水解與質譜儀分析 -11-
2.3 細胞培養 -12-
2.4 聚合酶鏈鎖反應 -13-
2.5 重組質體建構 -13-
2.6 質體短暫性轉染與共同轉染 -14-
2.7 西方墨點法 -15-
2.8 細胞蛋白萃取 -16-
2.9 免疫沉澱法 -16-
2.10 大腸桿菌重組GST融合蛋白製備 -17-
2.11 大腸桿菌重組His標定蛋白製備 -18-
2.12 激酶活性分析 -19-
2.13 2HPCL抗體製造 -20-
2.14 2HPCL-internal-15與-19胜肽偶合管柱製備 -20-
2.15 2HPCL抗體純化(兔子,多株性) -20-
第三章 實驗結果
3.1 藉由質譜分析來自A431細胞株的免疫沉澱物鑑定出2HCPL可能為潛在的GSK-3α之交互作用蛋白 -22-
3.2 製造2HPCL表現質體並在哺乳類細胞中過度表現2HPCL與GSK-3α -23-
3.3在哺乳類細胞中過度表現的2HPCL與GSK-3α彼此間能產生交互作用 -23-
3.4 內生性之2HPCL會與GSK-3α彼此間有交互作用 -24-
3.5 GSK-3α與2HPCL重組蛋白之製造 -25-
3.6 重組蛋白2HPCL無法被GSK-3α所磷酸化 -27-
3.7 GSK-3α與2HPCL會有直接的交互作用 -28-
3.8 2HPCL能些微激發GSK-3α的激酶活性 -28-
第四章 討論 -30-
第五章 引用文獻 -35-
圖一、來自A431細胞株之GSK-3α免疫複合體 -40-
圖二、藉由質譜分析鑑定出第三號蛋白位置為2HPCL -41-
圖三、在COS-7細胞株中短暫性過量表現pFLAG-CMV-2/GSK-3α與pCMV-Myc/2HPCL -43-
圖四、在短暫性過量表現pFLAG-CMV-2/GSK-3α與pCMV-Myc/2HPCL的COS-7細胞株中進行免疫共沉澱法 -44-
圖五、在人類2HPCL的胺基酸序列上分析其疏水特性 -45-
圖六、2HPCL-I-18抗體之純化結果 -46-
圖七、利用純化之2HPCL抗體在細胞萃取蛋白中進行免疫墨點法 -47-
圖八、對於內生性之2HPCL與GSK-3α進行免疫共沉澱法 -48-
圖九、預測在2HPCL上能夠被GSK-3α所磷酸化的位置 -49-
圖十、產生重組融合蛋白GST-GSK-3α -50-
圖十一、產生重組融合蛋白GST-2HPCL -51-
圖十二、對於GST-GSK-3α進行激酶活性測試 -52-
圖十三、利用GST-GSK-3α對GST-2HPCL進行激酶活性測試 -53-
圖十四、利用自豬腦純化之GSK-3α對GST-2HPCL進行激酶活性測試 -54-
圖十五、產生重組融合蛋白His-GSK-3α -55-
圖十六、His-GSK-3α與GST-2HPCL之結合測試 -56-
圖十七、對於His-GSK-3α進行激酶活性測試 -57-
圖十八、測試當加入GST-2HPCL後對His-GSK-3α的激酶活性影響 -58-

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