- 學位論文
GSK-3利用差異性的N端與C端執行特異化功能
Diverse N-terminal and C-terminal domains of GSK-3 play on its specificity
摘要
肝醣合成激酶3(GSK-3)為一個多功能蛋白質激酶，在細胞中不僅參與肝醣合成，也調控許多訊息傳遞、基因表現、細胞週期調控及細胞增生等。在哺乳動物中GSK-3有兩種同型異構物(isoform)分別為GSK-3α和GSK-3β，除了在激酶區域有很高的相似性外，GSK-3α有一段較長且富含甘氨酸(Glycine-rich)的N 端，且兩者在C端的76個氨基酸也呈現差異。這兩種異構物雖然結構很相似但功能卻不盡相同，而近年來GSK-3β的受質與功能被陸續報導，但關於GSK-3α的研究卻相對較少。因此本研究利用酵母菌雜交系統(Yeast Two-Hybrid system)找尋新的GSK-3α結合蛋白，發現了一個新的GSK-3α特異性結合蛋白，並以此為基礎合併已知蛋白將GSK-3結合蛋質分為三大類：GSK-3α特異結合蛋白、GSK-3β特異結合蛋白以及GSK-3α/β結合蛋白，結果顯示GSK-3是以C端區域與它們的結合蛋白作用。In vitro Kinase Assay的結果顯示並非所有GSK-3的結合蛋白都是它們的受質，而與受質是否結合也跟GSK-3的催化能力不成正相關，顯示蛋白質結合與受質催化可能是獨立事件。利用Confocal Microscopy觀察發現GSK-3β在間期時會均勻散佈在細胞內，而GSK-3α則主要分佈在核外並與中心體共同座落，但去除掉N端的GSK-3α分佈情形則與GSK-3β類似，暗示GSK-3α N端的功能可能與細胞內的分佈有關。綜合上述結果，本研究發現GSK-3利用N端與C端執行不同的功能，而我們可藉由進一步研究新發現的GSK-3α結合蛋白以了解GSK-3α更多的重要功能。
關鍵字
肝醣合成激酶酵母菌雜交實驗並列摘要
Glycogen Synthase Kinase 3 (GSK-3) is a multifunctional serine/theronine kinase involved in a wide range of cellular process, including cellular response to Wnt signaling, cell cycle regulation and proliferation. There are two mammalian GSK-3 isoforms: GSK-3α and GSK-3β, although structurally similar, they are not functionally identical. These two isoforms are highly homologous within their kinase domains but differing in the Glycine-rich(47 over 94 aa) N-terminal extension of GSK-3α and the last 76 C-terminal residues. Recently, many binding proteins and putative substrates of GSK-3β have been reported whereas less studies are on GSK-3α.In this thesis, the aim is to investigate whether the diverse N-terminal and C-terminal domains play a role in regulating their functions. Using yeast-two hybrid system, a new GSK-3α-specific binding proteins has been found. We classify 7 GSK-3 binding proteins into three categories : (1) binds to GSK-3α , (2) binds to GSK-3β, (3) binds to both GSK-3α and GSK-3β.The data showed that the C-terminal domain of GSK-3 may be involved in the interaction with their binding proteins while the N-terminal extension of GSK-3α has no function for binding. In vitro kinase assay data showed that not all binding proteins of GSK-3 undergo phosphorylation and act as a substrate. In addition, binding and phosphorylation are independent. Using confocal microscopy we observed that GSK-3β dispersed in the cytoplasm and nucleus, whereas GSK-3α located in the cytoplasm and colocalized with centrosome. However, the N-terminal deleted form of GSK-3α also located smoothly through the cell just like GSK-3β did, suggesting that the N-terminal of GSK-3α may be involved in regulating its localization. Taken together, these data indicated that the N-terminal and C-terminal domains of GSK-3 may be involved in different functions. The ongoing studies on GSK-3α-specific binding protein may provide more information about the physiological function of GSK-3α.