臺灣博碩士論文加值系統

死亡相關蛋白激酶(Death Associated Protein Kinase, DAPK)，屬於絲氨酸/蘇氨酸蛋白激酶家族，受鈣離子及攜鈣素共同調控，具有鈣離子/攜鈣素調控區塊。DAPK會抑制致癌因子(如c-myc或E2F)所引起的細胞癌化，也影響腫瘤的轉移，被認為是一個重要的腫瘤抑制因子。目前已知DAPK在多個氨基酸上的磷酸化修飾會造成其結構與功能上的改變，在已知的磷酸化位點中，尤以Ser-308位點最為特別，DAPK會對自身S308位點進行磷酸化。研究發現當S308位點被磷酸化後，會導致DAPK的活性降低。我們實驗室在先前細胞(in vitro)實驗中觀察到S308的磷酸化會造成粒線體型態的改變，但目前對於其在生物體內(in vivo)的作用機制仍了解甚少。同時我們也有興趣去探討是否還有其他激酶會影響S308的磷酸化。

為瞭解DAPK S308位點磷酸化程度在生物體內的作用機制，我們進一步以動物實驗來研究觀察。本研究選用果蠅肌肉組織進行粒線體型態實驗。為探討DAPKS308磷酸化程度對肌肉組織粒線體的影響，我們建立了不同DAPKS308點突變(野生型；S308A: 磷酸化抑制型；S308D: 模擬磷酸化型)的轉殖果蠅。當大量表現在肌肉時會導致果蠅個體不同程度的死亡率(S308A>WT>S308D)，此現象與先前細胞實驗結果相吻合。同時我們發現DAPK的活性與果蠅肌肉組織中粒線體的型態變化有相關性。

有趣的是，當我們表現點突變的 DAPKK42A(失去自我磷酸化能力)在肌肉與眼睛組織中，可以偵測到S308位點仍呈現高度磷酸化，顯示果蠅體內還有其他的激酶可以磷酸化此位點。因此我們利用果蠅遺傳學方式進行篩選，試圖找出也能造成S308位點磷酸化的激酶，目前初步篩選已找到會影響DAPK功能的候選激酶，未來還會回到哺乳動物細胞中再做探討確認。

DAPK (Death Associated Protein Kinase) is a Ca2+/calmodulin-regulated serine/threonine kinase. DAPK can suppress carcinogenic factors (such as c-myc or E2F) and also affects tumor metastasis. It is also considered to be an important tumor suppressor. Phosphorylation on different amino acid residues in DAPK causes its structural and functional changes. Previous studies showed that autophosphorylation on Serine 308 results in decreasing kinase activity of DAPK. Our lab has discovered that phosphorylation of S308 site causes mitochondrial morphological changes in culture cells. However, the detail molecular mechanism is still unclear. In addition, it is not known whether there are other kinases capable of phosphorylating DAPK S308 site.

Drosophila muscle tissue has been widely used in studying mitochondrial dynamics. To investigate the effect of DAPKS308 phosphorylation on mitochondria, we established transgenic flies bearing different DAPKS308 point mutations (Wild Type; S308A: blocks phosphorylation; S308D: mimics phosphorylation). When overexpressing different forms of DAPK in muscle tissue, we found that the transgenic animals exhibit different extent of mortality (S308A>WT>S308D). These results are consistent with previous experiments conducted in culture cells. Furthermore, we found that the kinase activity of DAPK nicely correlates with mitochondrial elongation in fly muscle tissue. These results suggest that DAPK kinase activity might regulate mitochondrial fusion/fission in vivo.

Intriguingly, we still observed the S308 being phosphorylated in muscles and eyes when overexpressing kinase dead mutant DAPKK42A (loss of autophosphorylation). This indicates that other unknown kinases are able to phosphorylate S308 in Drosophila. Therefore, we conducted a kinome screening to look for kinases that might phosphorylate DAPK S308 residue in vivo. Currently, our preliminary screening has discovered several candidate S308 kinases which are needed for further validation in the future.

正文目錄
正文目錄 I
圖表目錄 V
附錄目錄 VI
中文摘要 VII
英文摘要 IX
第一章 緒論 1
第一節、死亡相關蛋白激酶(Death-Associated Protein Kinase, DAPK) 之介紹 1
壹、死亡相關蛋白激酶背景及分子結構 1
貳、死亡相關蛋白激酶之磷酸化位點與影響之簡介 2
第二節、淺談死亡相關蛋白激酶與粒線體的關係 4
第三節、粒線體(Mitochondrion)之介紹 5
壹、粒線體之研究背景及結構簡介 5
貳、粒線體之生理功能與重要性 6
參、粒線體分裂與融合特性之簡介 8
第四節、果蠅(Drosophila)實驗模型之簡介 9
第五節、研究背景與目的 10

第二章　材料與方法 12
第一節、果蠅品系(Drosophila strains) 12
壹、DAPK過度表達之果蠅 12
貳、驅動特定組織基因表現之果蠅 13
參、Canton S品系果蠅 13
第二節、果蠅培養 13
第三節、抗體(Antibodies) 14
壹、免疫螢光染色使用 14
貳、西方點墨法使用 14
第四節、基因轉殖果蠅(Transgenic flies) 14
第五節、UAS-GAL4基因表現系統 15
第六節、免疫螢光染色(Immunostaining) 16
第七節、果蠅眼睛表徵觀察(Drosophila eye phenotype) 16
第八節、激酶篩選(Kinome Screening) 17
第九節、西方點墨法(Western Blot) 18
壹、果蠅樣本準備(Drosophila sample preparation) 18
貳、裂解液置備(Lysate preparation) 18
參、西方點墨法(Western Blot) 19
第十節、果蠅成蟲存活率之計算 20
第三章　結果 23
第一節、 DAPK與粒線體之間的交互作用 23
第二節、 DAPK活性對於果蠅成蟲的影響 25
壹、DAPK活性影響果蠅存活率 25
貳、DAPK活性對果蠅發育的影響 26
第三節、 發現果蠅體內另有其他激酶可對DAPK的S308位點進行磷酸化作用 27
壹、建立果蠅品系以篩選可使pDAPKS308磷酸化的激酶 27
貳、DAPKK42A對果蠅所產生的影響 28
參、以遺傳學方式進行候選激酶之篩選 30
肆、七個初步候選激酶之個別介紹（果蠅基因名/人類同源基因名） 33
伍、重覆驗證最具潛力之兩個激酶－Gprk2與Madm 40
第四章　討論 42
第一節、DAPK活性對於果蠅成蟲的影響 42
第二節、實驗過程所遇之問題 44
壹、DAPK蛋白可能有聚集(aggregate)之特性 44
貳、DAPK可能因降解造成蛋白表現量不穩 45
參、激酶缺陷實驗中某些組別無存活之果蠅成蟲 46
第三節、本實驗之未來方向 47
第四節、DAPK與粒線體關係之研究潛力 47
第五節、實驗討論 49
第五章　結論 50
第六章　參考文獻 52
第七章　結果圖表 56
附錄 88

圖表目錄

圖 1、建立穩定的DAPK果蠅品系 56
圖 2、觀察已建立好的果蠅品系幼蟲之粒線體型態 58
圖 3、在肌肉組織中DAPKS308磷酸化表現程度會影響果蠅成蟲的存活率 61
圖 4、DAPK過度表達促使果蠅眼睛的退化表現 63
圖 5、S308位點的磷酸化不只有透過自我磷酸化作用 65
圖 6、利用UAS-Gal4 system將DAPK表現在果蠅不同部位中並觀察其蛋白量的表現 66
圖 7、DAPK失去磷酸化能力會增加果蠅的存活率 69
圖 8、在三齡蟲中DAPKWT與DAPKK42A對粒線體形態的影響 71
圖 9、進行kinome screening前置作業 72
圖 10、本實驗已完成之kinome screening詳細資訊列表 74
圖 11、計算子代之存活率篩選出候選激酶 80
圖 12、目前篩選出之候選激酶 84
圖 13、由數個候選激酶中再篩出最具潛力的候選激酶 85


附錄目錄

附錄一、死亡相關蛋白激酶家族蛋白質結構示意圖 88
附錄二、死亡相關蛋白激酶上的四個磷酸化位點 90
附錄三、廠商進行微量注射之果蠅詳細資訊 92
附錄四、常用之balancer資訊列表 93
附錄五、S308A、S308D組別無法順利進行電泳實驗 94
附錄六、蛋白表現量無法穩定 96

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