Oncoprotein18是一種主要分布在細胞質內，大小約為19 kDa的蛋白。在前人的研究中指出Op18主要透過磷酸化的方式調控微管聚合和崩解。在進入細胞週期時，其表現量的上升與磷酸化將使得Op18失去使tubulin維持單體狀態的能力。目前已知Op18蛋白上有四個磷酸化的位點: ser16、ser25、ser38和ser63，分別受到不同蛋白激酶的磷酸化以調控其功能。在本實驗中，我們想知道除了磷酸化修飾之外，尚有其他會影響其功\\能的修飾類型。首先將His-tagged泛素表現質體與GFP-Op18表現載體共轉染後，利用免疫沉澱法證實Op18蛋白具有泛素化修飾的現象。為了研究Op18泛素化對Op18參與微管蛋白聚合調節功能的影響，我們將Op18蛋白的五個可能被修飾的lysine位點序列K42、K53、K75、K104及K119同時突變成Arginine，結果發現其泛素化程度有顯著比野生型Op18呈現下降的趨勢。我們將細胞進行同期化，使停留在有絲分裂期，再觀察過度表達野生型Op18及突變型Op18時，是否影響細胞內聚合型與非聚合型的微管蛋白含量。結果顯示，表現突變型Op18-M5K的有絲分裂期細胞內，其聚合型微管蛋白含量比在表現野生型Op18細胞中的含量有顯著提升的現象。同時也發現突變型Op18-M5主要為分子量較大的的磷酸化型態，有別於野生型Op18。這些結果可能暗示Op18的泛素化修飾可能影響Op18蛋白被磷酸化的狀態，而影響其與微管蛋白結合能力，進而調節微管蛋白形成聚合型的步驟，未來將利用活體外微管蛋白聚合能力分析加以確認上述發現。

Oncoprotein18 (Op18) is a 19 kDa cytosolic phosphoprotein critical for cell growth and differentiation. Unphosphorylated Op18 associates with αβ-tubulin heterodimer to form 2 tubulin-1 stathmin T2S complex and promotes microtubule catastrophe in interphase. Numerous cellular signals activate downstream protein kinases result in the phosphorylation of Ser16, Ser25, Ser38 and Ser 63 residues in Op18 that lowers its affinity for tubulin thereby increases the stability of microtubule and facilitates the formation of spindle during mitosis. Here, we found in addition to phosphorylation, Op18 could also be ubiquitin modified in vivo. An expression plasmid encodes for mutant EGFP-Op18-M5K protein whose potential lysine residues K42, K53, K75, K104, and K119 were mutated to arginines was generated to investigate the effect of ubiquitin modification of Op18 on the tubulin polymerization. Our results revealed a decrease of ubiquitin modification of mutant EGFP-Op18-M5K in comparison with that of wildtype EGFP-Op18. The expression of mutant but not the wildtype Op18 resulted in a significant increase of polymerized tubulin in mitotic cell implying that they might exhibit differential tubulin binding affinity. Moreover, the result of western blotting showed that the mutant Op18 detected in the mitotic cell corresponds to the phosphorylated version of Op18. In summary, these results imply the ubiquitination of Op18 might interfere with its phosphorylation and decrease its tubulin binding potential, thereby facilitates the polymerization of tubulin in mitotic cells. The in vitro tubulin polymerization assay will be performed to further confirm the above finding.

中文摘要 3
Abstract 5
前言 7
研究動機 14
材料與方法 15
結果 28
討論與未來研究方向 32
參考文獻 40
圖表 45
附錄 54

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