陳瑞華臺灣大學：分子醫學研究所顧瑋Ku, WeiWeiKu2007-11-262018-07-092007-11-262018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/51364死亡相關蛋白激酶（Death associated protein kinase，DAPK）是一個受鈣╱攜鈣素調控的絲胺酸╱酥胺酸激酶。其生理功能已經被證實的部份主要包括會抑制細胞附著能力和促進細胞死亡。雖然有關DAPK的生理功能部份已經被解開，但其在細胞內訊息傳遞路徑有很多地方仍尚未明白。為了更進一步探討有關DAPK的生理功能，我們實驗室利用酵母菌雙雜交系統(yeast two-hybrid)尋找DAPK的結合蛋白。在這樣的策略中我們找到了DAPK的一個結合蛋白稱為LAR (leukocyte common antigen related tyrosine phosphatase)。在本篇論文的第一個部份：我們著重於討論DAPK和LAR的結合方式，發現DAPK會藉由其錨蛋白重覆序列３到６(ankyrin repeats 3-6)的區域與LAR的第一個或第二個去磷酸酶區間(phosphatase domain)進行專一性的交互作用。至於在本論文的第二部份：我們目的在找尋LAR作用於DAPK時扮演拮抗角色的蛋白，功能在拮抗LAR對DAPK所造成的影響。首先，我們發現DAPK的酪胺酸磷酸化程度會在細胞附著到細胞外基質時有上昇現象，並確定這現象的磷酸化上昇的位置是位於DAPK Y491/492。也因此我們推測出作用於DAPK上的酪胺酸激脢是Src kinase。利用正常老鼠的纖維母細胞(fibroblast)和Src/Fyn/Yes-triple knockout cells (SYF cells)做比較，我們更進一步明白在細胞內Src對DAPK的影響。不僅如此，DAPK和Src在細胞內我們也證明其間有互相結合的現象。不僅如此，因為Src表現對DAPK所造成的酪胺酸磷酸化上升結果會抑制DAPK的酵素能力，這樣的現象和LAR所造成的結果恰巧相反，顯示在對DAPK生理調控上Src的確和LAR扮演著結抗作用。概括而言，本篇論文的重點在探討DAPK訊號傳遞。我們證實DAPK上的酪胺酸磷酸化程度可以決定DAPK本身的酵素催化能力，並也知道這樣磷酸化影響的位置是位於DAPK Y491/492。其中，我們更進一步確定的這樣的調控方式是由一個酪胺酸激脢(Src)和一個酪胺酸去磷酸脢(LAR)所共同參與Death-associated protein kinase, DAPK, is a Ca2+/calmodulin-regulated Ser/Thr kinase that promotes anti-adhesion and cell death. Although the multiple physiological functions of DAPK are demonstrated, its intracellular signaling mechanism is largely unknown. To get insight into DAPK molecular signaling network, our laboratory searched for its interacting protein and identified leukocyte common antigen related tyrosine phosphatase (LAR) as one of its interacting partner. The first part of this thesis focused on studying their physical interaction. DAPK interacts with LAR in vivo through its ankyrin repeat domain, of which 3-6 repeats are both sufficient and required for interacting with LAR. On the other hand, the association of LAR and DAPK ankyrin repeats is mediated by LAR phosphatase domain, D1 or D2, as well as both of them. For the latter part of the thesis, we aimed to identify the possible antagonist of LAR, which was demonstrated in our laboratory as a DAPK activator through its dephosphorylation of DAPK at Y491/492 residues. It was discovered that there exists certain adhesion-dependent tyrosine kinase phosphorylating DAPK at Y491/492. Subsequent experiments identified this kinase as Src. By making use of Src/Fyn/Yes-triple knockout cell, we evaluated the determinant role for Src to regulate DAPK tyrosine phosphorylation at both exogenous and endogenous levels. The in vivo interaction between DAPK and Src is demonstrated. We also showed that the Y491/492 residues are the major sites for DAPK phosphorylation by Src. To investigate if this Src-mediated DAPK phosphorylation is of functional significance, we performed in vitro DAPK kinase assay and found that Src expression could inhibit DAPK catalytic activity. Therefore, it is through the direct tyrosine phosphorylation of DAPK Y491/492 that Src inhibits DAPK catalytic activity. In conclusion, Src is identified as DAPK inhibitor acting in a reverse direction in regard to LAR, revealing the reciprocal regulation of DAPK by a pair of tyrosine kinase and phosphatase, Src and LAR.Table of content 2 誌謝 5 中文摘要 7 Abstract 8 Introduction 9 DAPK in apoptosis 9 DAPK, as an actin-filament associated kinase, inactivates integrin and causes anoikis-like cell death 9 DAPK multi-domain structure 10 DAPK kinase activity is required for DAPK functions 10 Investigation of molecular context of DAPK signaling by identifying interacting proteins through protein-protein interacting modules 10 Death domain-interacting proteins positively regulate DAPK 11 The ankyrin repeat as molecular architecture for protein-protein interaction 11 Ankyrin repeats in DAPK 12 Identification of LAR as a DAPK interacting protein 12 LAR (leukocyte common antigen related tyrosine phosphatase) 13 LAR physiological function 14 LAR in cell adhesion 14 Src as a non-receptor tyrosine kinase 15 Structure and regulation of Src 15 Src physiological function 16 The interplay between Src and integrin 16 Focal adhesion kinase (FAK) as important SFK binding partner 17 Biological processes mediated by Src-integrin signaling 18 Material and Method 21 Constructs 21 Yeast two-hybrid assay 21 Generation of anti-LAR antiserum 21 Antibodies and reagents. 22 Cell culture and transfection 22 Western blotting 23 Immunoprecipitation 23 In vitro Src kinase assay 23 In vitro DAPK kinase assay 24 Result 25 DAPK interacts with LAR in vivo 25 Generation and characterization of anti-LAR antiserum 25 Endogenous LAR expression in various cell lines 26 Mapping the interacting region of LAR and DAPK 26 Identification of the adhesion-dependent tyrosine kinase phosphorylation of DAPK Y491/492 27 The adhesion-dependent tyrosine phosphorylation of DAPK can be reversed by PP2 (Src family kinase inhibitor) treatment 28 DAPK tyrosine phosphorylation is influenced by Src-FAK signaling 28 v-Src overexpression dramatically induces the tyrosine phosphorylation of DAPK but not its 491/492FF mutant 29 Constitutively active c-Src overexpression dramatically induces the tyrosine phosphorylation of DAPK but not its 491/492FF mutant 29 Src influences both DAPK Y491 and Y492 phosphorylation 29 Overexpressed DAPK exhibits higher tyrosine phosphorylation in the presence of either endogenous or ectopically introduced Src expression 30 Endogenous DAPK exhibits higher tyrosine phosphorylation in response to ectopically introduced Src overexpression 30 Endogenous DAPK exhibits higher tyrosine phosphorylation in the presence of endogenous Src family kinases 31 DAPK interacts with Src in vivo 31 DAPK Y491/492 are the major sites for in vitro phosphorylation by Src 32 Overexpression of Src inhibits DAPK catalytic activity 32 Discussions 34 Reciprocal roles of LAR and Src in regulating DAPK 34 Other possible tyrosine phosphorylation sites of DAPK under Src regulation 34 The repressed DAPK catalytic activity through Y491/492 phosphorylation 35 The signaling context at which LAR and Src antagonistically regulate DAPK 36 The spatial relationship/subcellular localization 36 Possible DAPK/Src/LAR interplay coordinated by integrin signaling 37 Possible involvement of FAK in DAPK/LAR/Src interplay 38 Possible DAPK/Src/LAR coordination in netrin signaling 38 Conclusion 39 Figure 41 Figure 1.The domain organization of DAPK 41 Figure 2.Identification of LAR as a DAPK-interacting protein in yeast two-hybrid system 42 Figure 3. DAPK interacts with LAR in vivo 43 Figure 4. Generation and characterization of anti-LAR antiserum 44 Figure 5. Endogenous LAR expression in various cell lines 45 Figure 6. Mapping the interacting region of LAR and DAPK 46 Figure 7. LAR as a novel activator of DAPK by dephosphorylating DAPK Y491/492 47 Figure 8. Identification of the adhesion-dependent tyrosine kinase phosphorylating DAPK Y491/492 48 Figure 9 . The adhesion-dependent tyrosine phosphorylation of DAPK can be reversed by PP2 (Src inhibitor) treatment 49 Figure 10. DAPK tyrosine phosphorylation is influenced by Src-FAK signaling 50 Figure 11. v-Src overexpression dramatically induces the tyrosine phosphorylation of DAPK but not its 491/492FF mutant 51 Figure 12. Constitutive active c-Src overexpression dramatically induces the tyrosine phosphorylation of DAPK but not its 491/492FF mutant 52 Figure 13. Src phosphorylates both DAPK Y491 and Y492 53 Figure 14. Overexpressed DAPK exhibits higher tyrosine phosphorylation in the presence of endogenous Src expression 54 Figure 15. Endogenous DAPK exhibits higher tyrosine phosphorylation in response to ectopically introduced Src overexpression 55 Figure 16. Endogenous DAPK exhibits higher tyrosine phosphorylation in the presence of endogenous Src expression 56 Figure 17. DAPK interacts with Src in vivo 57 Figure 18. DAPK Y491/492 as major sites for Src to phosphorylate DAPK in vitro 58 Figure 19. Overexpression of Src inhibits DAPK catalytic activity 59 Reference 601448193 bytesapplication/pdfen-US死亡相關蛋白激酶酪胺酸磷酸化酪胺酸激脢酪胺酸去磷酸脢DAPKSrcLARtyrosine phosphorylationotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51364/1/ntu-94-R92448007-1.pdf