陳瑞華臺灣大學：分子醫學研究所何宣宗Ho, Hsuan-ChungHsuan-ChungHo2007-11-262018-07-092007-11-262018-07-092004http://ntur.lib.ntu.edu.tw//handle/246246/51341死亡相關蛋白激酶(Death associated protein kinase)是一會正向促進細胞計劃性死亡並受鈣離子及攜鈣素所調控的絲胺酸/酥酪胺酸激酶。細觀結構可見其存在有多種蛋白質弁鈰炩禲A包含：激酶作用區、鈣離子結合區、ankyrin repeat、細胞骨架結合區間和死亡區塊。眾多種類的刺激，諸如干擾素、Fas、腫瘤壞死因子、細胞轉型生長因子、ceramide、c-myc和E2F等均已被證實會透過死亡相關蛋白激酶而導致細胞凋亡。在先前，有關死亡相關蛋白激酶利用其酵素催化能力所導致細胞死亡機制已被建立且證實，但其在細胞內的含量及活性如何被調控則仍未清楚。在本篇論文中，我們發現死亡相關蛋白激酶之死亡區塊的一個結合蛋白，並將其命名為DIP2。除此，我們也證實在細胞中，DIP2、死亡相關蛋白激酶及Cul3三者會形成一複合體。存在此一複合體是DIP2經由其N端的BTB區間與Cul3蛋白結合，同時也利用其C端的kelch repeats與死亡相關蛋白激酶做結合。此包含Cul3蛋白的結合模式暗喻著死亡相關蛋白激酶的蛋白質穩定程度會受到複合體活性所影響。的確，我們證實了表現DIP2會經由蛋白酶體(proteasome)來促進死亡相關蛋白激酶蛋白質的降解，進而降低細胞內死亡相關蛋白激酶的蛋白質表現量。更進一步，我們證實DIP2調控細胞內死亡相關蛋白激酶的蛋白表現是藉由泛素化(ubiquitination)作用的機制。總而言之，我們證明DIP2的弁鄏b做為Cul3連接酶複合體作用時的一個受質辨認單位，透過這樣的複合體，死亡相關蛋白激酶會被泛素化並且被蛋白酶體降解。TABLE OF CONTENT 1 中文摘要 3 ABSTRACT 4 INTRODUCTION 5 DAPK 5 Kelch-repeat superfamily 7 Ubiquitination 9 MATERIALS & METHODS 14 Cell culture and transient transfection 14 Plamids 14 Generation of DIP2 antisera 15 Other antibodies and reagents 15 Western blotting 16 Immunoprecipitation 16 Northern blotting 17 Yeast two-hybrid assay 17 In vivo degradation assay 18 In vivo ubiquitination assay 18 RESULTS 19 Identification of DIP2 as a DAPK interacting protein 19 Tissue distribution of DIP2 mRNA expression 20 DIP2 interacts with DAPK DD in yeast 20 DIP2 associates with DAPK in vivo 20 Generation and characterization of anti-DIP2 antisera 21 Endogenous DIP2 expression in various cell lines 21 Kinase activity of DAPK does not affect the interaction between DAPK and DIP2 22 DAPK interacts with DIP2 through multiple domains of DAPK 22 DAPK interacts with the kelch repeats, but not the BTB domain, of DIP2 23 DIP2 interacts with Cul3 through its BTB domain 23 DAPK complexes with DIP2 and Cul3 in vivo 24 Expression of DIP2 reduces DAPK protein level through proteasomal degradation 25 DIP2 stimulates DAPK turnover 25 DIP2 promotes the ubiquitination of DAPK in vivo 26 DISCUSSION 27 REFERENCES 32 FIGURES 39 Figure 1. The domain organization of DAPK 39 Figure 2. Schematic illustration for two mechanisms of the DAPK-mediated apoptosis 40 Figure 3. Cullin-based E3 ligases 41 Figure 4. AH3 encodes a novel BTB/kelch protein 42 Figure 5. Amino acid comparison of DIP2 with other human BTB/kelch proteins. 43 Figure 6. DIP2 mRNA expression in human fetal tissues 44 Figure 7. Interaction of DIP2 and the DD of DAPK in yeast two-hybrid system 45 Figure 8. DAPK interacts with DIP2 in vivo 46 Figure 9. Characterization of anti-DIP2 antisera 47 Figure 10. Identification of endogenous DIP2 expressed in various cell lines 48 Figure 11. Kinase activity of DAPK does not affect the interaction between DAPK and DIP2. 49 Figure 12. DAPK interacts with DIP2 through multiple domains of DAPK. 50 Figure 13. The kelch repeats of DIP2 are sufficient for association with DAPK 51 Figure 14. The BTB domain of DIP2 is required for Cul3 binding. 52 Figure 15. DAPK, DIP2 and Cul3 form a complex in vivo. 53 Figure 16. The protein level of DAPK is downregulated by DIP2 through the proteasome-dependent degradation. 54 Figure 17. DIP2 stimulates DAPK turnover. 55 Figure 18. DIP2 promotes the ubiquitination of DAPK in vivo. 561922111 bytesapplication/pdfen-US細胞凋亡死亡相關蛋白激酶泛素化DAPKapoptosisubiquitinationotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51341/1/ntu-93-R91448002-1.pdf