陳瑞華臺灣大學：分子醫學研究所王琬菁Wang, Won-JingWon-JingWang2007-11-262018-07-092007-11-262018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/51329死亡相關蛋白激酶（Death associated protein kinase，DAPK）是一個由鈣╱攜鈣素調控的絲胺酸╱酪胺酸激酶，同時參與各式的細胞凋亡系統。在這份論文中，主要研究死亡相關蛋白激酶引起的細胞凋亡機制及其訊息傳遞，並且探討其功能上與某種磷酸化酪胺酸去磷酸酶（phosphotyrosine phosphatase）間的交互作用。在研究的第一部份，本論文顯示死亡相關蛋白激酶能藉著一個由內向外影響的機制（inside-out mechanism）來調降胞外基質受器（integrin）的活性，進一步抑制胞外基質受器所造成的細胞附著（cell adhesion）。死亡相關蛋白激酶經抑制細胞附著作用阻斷胞外基質受器所傳遞的存活訊息（survival signals）同時增加p53的活性，進而誘使細胞凋亡。為了証實這個觀點，本論文証明：無論是藉由胞外基質受器本身或是其下游的作用者FAK來增加胞外基質受器所傳邊的存活訊息路徑，均能中止死亡相關蛋白激酶誘發的細胞凋亡反應，同時死亡相關蛋白激酶也不能在無法進行失巢凋亡（anoikis resistant）的細胞中促使細胞凋亡現象的產生。因此，本論文探討死亡相關蛋白激酶的細胞凋亡機制，同時確認死亡相關蛋白激酶是失巢凋亡的誘發者。在研究的第二部份，試著找尋在死亡相關蛋白激酶的訊息傳導途徑中，能與死亡相關蛋白激酶相互作用分子。以死亡相關蛋白激酶的錨蛋白重覆區塊（ankyrin-repeat domain）為餌進行酵母菌雙雜交（yeast two-hybrid）系統分析，發現白血球共通抗原相關酪胺酸去磷酸酶（leukocyte common antigen related tyrosine phosphatase，LAR）能與死亡相關蛋白作用。本論文証明：死亡相關蛋白激酶能藉由錨蛋白重覆序列３到６的區域與LAR的去磷酸酶區間進行專一性的交互作用。更進一步發現死亡相關蛋白激酶對白血球共通抗原相關酪胺酸去磷酸酶的受質受限突變株（substrate-trapping mutant）具有較高的接合親合力，這意味著死亡相關蛋白激酶是白血球共通抗原相關酪胺酸去磷酸酶的受質。事實上，無論在試管內或活體中分析，死亡相關蛋白激酶均能有效地被白血球共通抗原相關酪胺酸去磷酸酶移除其酪胺酸491及492（Y491/492）上的磷酸根。除此之外，亦證明白血球共通抗原相關酪胺酸去磷酸酶能藉由這兩個胺基酸來增加死亡相關蛋白激酶的催化活性。因此，死亡相關蛋白激酶的各種生物活性，包括抑制細胞附著與誘發細胞凋亡，能在LAR過量表現下明顯增加。相形之下，利用RNA干擾技術（RNA interference）降低細胞內既有的白血球共通抗原相關酪胺酸去磷酸酶表現，會造成酪胺酸491╱492磷酸化的增加及抑制死亡相關蛋白激酶的生物活性。這些結果顯示：經由酪胺酸491╱492磷酸化，白血球共通抗原相關酪胺酸去磷酸酶是一個新發現的DAPK活化分子。因此，揭櫫於本論文中的死亡相關蛋白激酶－白血球共通抗原相關酪胺酸去磷酸酶相互作用及此激酶誘發細胞凋亡機制，均為死亡相關蛋白激酶的訊息傳導與其生物功能有了更進一步的認知與瞭解。Death associated protein kinase (DAPK) is a calcium/calmodulin-dependent serine/threonine kinase, and participates in various apoptotic systems. In this thesis, we studied the apoptotic mechanism of DAPK and its signaling and functional crosstalk with a phosphotyrosine phosphatase. In the first part of study, we demonstrated that DAPK suppresses integrin-mediated cell adhesion by down-regulating integrin activity through an inside-out mechanism. This adhesion-inhibitory effect of DAPK blocks integrin survival signals and up-regulates p53 protein, thereby inducing apoptosis. In support of this notion, we demonstrated that enforced activation of integrin survival pathways from either integrin itself or its downstream effector FAK abolishes the apoptotic effect of DAPK, and that DAPK can no longer induce apoptosis in the anoikis resistant cells. Thus, our study unravels that apoptotic mechanism of DAPK and identifies DAPK as an inducer of anoikis. In the second part of this thesis, we searched for DAPK interaction partners as part of our work to dissect DAPK signaling network. A yeast two-hybrid screen using the anykrin-repeat domain of DAPK as bait identified the leukocyte common antigen related tyrosine phosphatase (LAR) as a DAPK interacting protein. We showed that DAPK interacts specifically with LAR, through the ankyrin repeats 3-6 of DAPK and the phosphatase domain of LAR. The higher binding affinity of DAPK towards a substrate-trapping mutant of LAR suggests that DAPK functions as a substrate of LAR. Indeed, DAPK can be efficiently tyrosine-dephosphorylated by LAR both in vitro and in vivo, and this dephopshorylation event occurs at Y491/492 residues of DAPK. Furthermore, we demonstrated that LAR up-regulates the catalytic activity of DAPK through an Y491/492-dependent manner. As a consequence, the various biological activities of DAPK, such as anti-adhesion and apoptosis induction, are significantly promoted by overexpression of LAR. In contrast, knockdown of endogenous LAR by RNA interference technique results in an elevation of DAPK tyrosine phosphorylation at Y491/492 and inhibition of DAPK biological functions. These data indicate that LAR functions as a novel activator of DAPK through dephosphorylating DAPK at Y491/492. The uncovering of DAPK-LAR interplay and DAPK apoptotic mechanism in this thesis would shed light on the molecular mechanisms of DAPK signaling and biological functions.TABLE CONTENTS..................................1 ABSTRACT........................................7 中文摘要..........................................8 ABBREVIATIONS.....................................9 1. APOPTOSIS........................................10 1.1 OVERVIEW.........................................10 1.2 CONNECTION OF CASPASE TO APOPTOSIS...............11 1.3 THE COMMON APOPTOTIC SIGNALING PATHWAYS............11 1.3.1 The mitochondrion-dependent cascade............11 1.3.2 The mitochondrion- independent cascade..........12 1.3.3 The crosstalk between these two cascades.........12 1.4 THE DEATH STAR: CONNECTION OF P53 TO APOPTOSIS.....13 1.4.1 Transcription-dependent pathways.................13 1.4.2 Transcription-independent pathways...............14 2.AUTOPHAGY............................................15 2.1 AUTOPHAGY IN CELL DEATH............................15 2.2 AUTOPHAGIC MECHANISM...............................16 3. DEATH ASSOCIATED PROTEIN KINASE(DAPK)...............17 3.1 DAPK FAMILY AND THE BASIC PROPERTIES...............17 3.2 DAPK IN CELL DEATH.................................18 3.3 THE TUMOR SUPPRESSION AND POSSIBLE NEURONAL FUNCTIONS OF DAPK.........20 4. INTEGRIN.............................................21 4.1 CELL ADHESION AND DE-ADHESION.......................21 4.2 INTEGRIN AND THE EXTRACELLULAR MATRIX...............22 4.3 FROM INTEGRIN STRUCTURE VIEW TO INTEGRIN ACTIVITY…....24 4.4 INTEGRIN SIGNALING NETWORK..........................24 4.4.1 Inside-out signaling..............................25 4.4.2 Outside-in signaling..............................26 4.4.2.1 Integrin signaling to actin cytoskeleton........26 4.4.2.2 Tyrosine phosphorylation-mediated signal transduction.........................................26 4.4.2.3 Activation of small G proteins.............27 4.4.2.4 Integrin and growth factor receptor cross-talk...28 5. ANOIKIS..............................................28 5.1 PHYSIOLOGICAL SIGNIFICANCE.......................28 5.2 ANOIKIS MECHANISM...................................29 5.2.1 Role of integrin in anoikis.....................29 5.2.2 Loss of FAK and PI3K/Akt signaling plays a key role in anoikis induction....................................30 5.2.3 Other molecules regulating anoikis work in coordination with integrins..........30 6. PROTEIN TYROSINE PHOSPHATASE (PTP)...................31 6.1 THE IMPORTANCE OF TYROSINE PHOSPHORYLATION........................................31 6.2 REGULATION BY KINASES AND PTPS.......................32 6.3 PTP STRUCTURE AND FUNCTION......................33 6.3.1 PTP classification............................33 6.3.2 Features of PTP active site....................34 CHAPTER I.......................35 DAP-KINASE INDUCES APOPTOSIS BY SUPPRESSING INTEGRIN ACTIVITY AND DISRUPTING MATRIX SURIVIAL SIGNALS.....................35 ABSTRACT.............................................35 INTRODUCTION.........................................36 RESULTS..............................................39 DAPK INDUCES APOPTOSIS-INDEPENDENT MORPHOLOGICAL CHANGES IN 293T CELLS...........................................39 DAPK SUPPRESSES INTEGRIN-MEDIATED CELL ADHESION AND SIGNAL TRANSDUCTION............................................40 DAPK SUPPRESSES INTEGRIN FUNCTION THROUGH AN INSIDE-OUT MECHANISM...............................................41 THE ANTI-ADHESION FUNCTION OF DAPK IS NOT DEPENDENT ON CELL TYPES..............................................42 DAPK PROMOTES APOPTOSIS BY BLOCKING ECM SURVIVAL SIGNALS...............................................42 DAPK INDUCES AN ANOIKIS-LIKE APOPTOSIS IN EPITHELIAL CELLS, WHICH IS REVERSED BY INTEGRIN ACTIVATION.........44 ACTIVATION OF INTEGRIN OR FAK BLOCKS DAPK-INDUCED UPREGULATION OF P53....................................46 DISCUSSION.............................................47 MATERIALS AND METHODS...............................................50 PLASMIDS..............................................50 CELL CULTURE, TRANSFORMATION AND RETROVIRAL INFECTION............................50 ANTIBODIES AND REAGENTS...............................50 ADHESION ASSAYS.................................................51 FLOW CYTOMETRY ANALYSIS..............................................51 IMMUNOPRECIPITATIONS..................................51 APOPTOSIS AND CASPASE ACTIVITY ASSAYS.................52 REPORTER ASSAYS.......................................52 SUPPLEMENTAL MATERIALS AVAILABLE......................52 CHAPTER II:...........................................53 ACTIVATION OF DAPK CATALYTIC ACTIVITY AND BIOLOGICAL FUNCTIONS BY LAR.......................................53 ABSTRACT...............................................53 INTRODUCTION...........................................54 DEATH-ASSOCIATED PROTEIN KINASE (DAPK) FAMILY..........54 IDENTIFICATION OF DAPK AND ITS DOMAIN PROPERTIES.............................................54 DAPK’S TARGETED SUBSTRATE AND CELLULAR FUNCTION..............................................55 PTP FAMILY............................................57 FEATURES OF THE PTP ACTIVATION........................58 THE RPTP-LAR..........................................58 LAR’S CELLULAR FUNCTIONS.............................59 SIGNIFICANCE..........................................61 MATERIALS AND METHODS.................................62 YEAST TWO-HYBRID SCREEN...............................62 CONSTRUCTS............................................62 CELL CULTURE AND TRANSFECTIONS........................62 ANTIBODIES............................................63 ADHESION ASSAYS:......................................63 IMMUNOPRECIPITATION:..................................63 IN VITRO DEPHOSPHORYLATION OF RECOMBINAT LAR-D1:......63 DAPK KINASE ASSAY:....................................64 APOPTOSIS ASSAYS.............................64 REDUCTION OF ENDOGENOUS LAR EXPRESSION USING SIRNA.................................64 RESULTS:............................................66 IDENTIFICATION OF LAR AS A DAPK-INTERACTING PROTEIN..................................66 MAPPING THE INTERACTING REGION OF LAR AND DAPK.........66 DAPK AND LAR INTERACT IN VIVO.........................67 LAR PROMOTES TYROSINE DEPHOSPHORYLATION OF DAPK....................................67 DAPK IS A DIRECT SUBSTRATE OF LAR..................68 MAPPING THE LAR-MEDIATED TYROSINE-DEPHOSPHORYLATION SITES ON DAPK............................................69 LAR PROMOTES DAPK CATALYTIC ACTIVITY THROUGH AN Y491/492-DEPENDENT MANNER....................................70 LAR PROMOTES THE ANTI-ADHESION EFFECT OF DAPK..........71 LAR PROMOTES DAPK-INDUCED ANOIKIS-TYPE CELL DEATH.......71 KNOCKDOWN OF ENDOGENOUS LAR ENHANCES DAPK TYROSINE PHOSPHORYLATION AND SUPPRESSES DAPK INDUCED ANTI-ADHESION FUNCTION.............................................72 DISCUSSION:..........................................73 TABLES..........................................77 TABLE 1. EXPRESSION OF HUST-21 EPITOPE ON 293T CELLS TRANSFECTED WITH VARIOUS CONSTRUCTS AND INCUBATED WITH OR WITHOUT MN2+.................77 TABLE 2: DAPK DELETION MUTANTS AND THEIR TYROSINE DEPHOSPHORYLATION STATE INFLUENCED BY LAR OVEREXPRESSION..................78 TABLE 3: SUMMARY OF THE EXTENT OF LAR-MEDIATED TYROSINE DEPHOSPHORYLATION OF DIFFERENT DAPK Y/F MUTANTS.......................................78 FIGURES:.......................................79 FIG. 1. DAPK INDUCES MORPHOLOGICAL CHANGES IN 293T CELLS.......................79 FIG. 2. DAPK INDUCES APOPTOSIS-INDEPENDENT MORPHOLOGICAL CHANGES IN 293T CELLS...................................80 FIG. 3. DAPK INHIBITS INTEGRIN-MEDIATED CELL ADHESION..................................81 FIG. 4. DAPK INHIBITS INTEGRIN-MEDIATED ECM SIGNAL TRANSDUCTION........................................82 FIG. 5. DAPK DOES NOT AFFECT CELL SURFACE EXPRESSION OF THE FIBRONECTIN AND LAMININ RECEPTORS..................83 FIG. 6. DAPK SUPPRESSES CELL ADHESION BY MODULATING INTEGRIN Β1 ACTIVITY.............................84 FIG. 7. DAPK SUPPRESSES INTEGRIN ACTIVITY............85 FIG. 8. DAPK INHIBITS ADHESION OF NIH3T3 CELLS BY INSIDE-OUT MODULATION OF INTEGRIN ACTIVITY...........86 FIG. 9. DAPK INDUCES APOPTOSIS IN CELLS PLATED ON MATRIX.............................87 FIG. 10. DAPK PROMOTES APOPTOSIS BY BLOCKING ECM SURVIVAL SIGNALS.88 FIG. 11. ACTIVATION OF INTEGRIN RESTORES FAK TYROSINE PHOSPHORYLATION.......................................89 FIG. 12. ACTIVATION OF INTEGRIN RESTORES FAK TYROSINE PHOSPHORYLATION AND PROTECTS CELLS FROM DAPK-INDUCED APOPTOSIS........................90 FIG. 13. DAPK INDUCES AN ANOIKIS-LIKE APOPTOSIS IN EPITHELIAL CELLS..................91 FIG. 14. DAPK CAN NO LONGER PROMOTE APOPTOSIS IN CELLS RESISTANT TO ANOIKIS....................92 FIG. 15. INTEGRIN OR FAK ACTIVATION BLOCKS THE INDUCTION OF P53 BY DAPK......................93 FIG. 16. A MODEL ILLUSTRATING THE MECHANISM BY WHICH DAPK INDUCES APOPTOSIS......................95 FIG. 17: IDENTIFICATION OF LAR AS DAPK-BINDING PROTEIN IN YEAST TWO-HYBRID SYSTEM..............................96 FIG. 18: GST-DAPK ANKYRIN REPEATS COULD PULL DOWN IN VITRO-TRANSLATED LAR PROTEIN.................................97 FIG. 19: ANKYRIN REPEATS 3-6 IS SUFFICIENT TO INTERACT WITH LAR (1680-1898).........................98 FIG. 20: NOT ONLY LAR D2 DOMAIN BUT ALSO LAR D1 DOMAIN CAN INTERACT WITH DAPK ANKYRIN REPEAT.......................99 FIG. 21: DAPK INTERACTS WITH LAR IN VIVO............100 FIG. 22: LAR INTERACTS WITH ENDOGENOUS DAPK.........101 FIG. 23: ENHANCEMENT OF DAPK BINDING AFFINITY BY LAR D/A MUTANT...102 FIG. 24: REDUCTION OF DAPK TYROSINE PHOSPHORYLATION IN RESPONSE TO OVER- EXPRESSION OF LAR.............103 FIG. 25: LAR OVEREXPRESSION DOES NOT AFFECT FAK TYROSINE PHOSPHORYLATION...................104 FIG. 26: OVEREXPRESSION OF LAR DOES NOT GENERALLY AFFECT TYROSINE PHOSPHORYLATION OF CELLULAR PROTEINS.......105 FIG. 27: SCHEMATIC REPRESENTATION OF THE DOMAIN STRUCTURE OF DIFFERENT DAPK DELETION MUTANTS.................106 FIG. 28: DELETION OF THE DEATH DOMAIN (DD) OF DAPK DOES NOT AFFECT LAR-INDUCED TYROSINE DEPHOSPHORYLATION OF DAPK.....................................107 FIG. 29: DAPK K-AR MUTANT CAN STILL BE DEPHOSPHORYLATED BY LAR OVEREXPRESSION................108 FIG. 30: DAPK K-CAM MUTANT IS INSENSITIVE TO LAR........................................109 FIG. 31: COMPARISON OF THE TYROSINE DEPHOSPHORYLATION STATES AMONG DIFFERENT DAPK Y/F MUTANTS IN RESPONSE TO LAR OVEREXPRESSION........110 FIG. 32: LAR DIRECTLY DEPHOSPHORYLATES DAPK IN VITRO.............................111 FIG. 33: ECTOPIC EXPRESSION OF LAR PROMOTES DAPK CATALYTIC ACTIVITY IN VIVO...................112 FIG. 34: LAR PROMOTES THE CATALYTIC ACTIVITY OF DAPK IN VIVO..............113 FIG. 35: OVEREXPRESSED LAR MODESTLY DECREASES THE ADHESION ABILITY OF 293T CELLS.................................114 FIG. 36: LAR PROMOTES DAPK-INDUCED CELL DETACHMENT............................115 FIG. 37: LAR PROMOTES DAPK-INDUCED APOPTOSIS..............................................116 FIG. 38: ESTABLISHMENT OF LAR SIRNA STABLE CLONES IN 293T CELLS…........117 FIG. 39: KNOCKDOWN OF LAR EXPRESSION ENHANCES THE TYROSINE PHOSPHORYLATION OF DAPK..................118 FIG. 40: KNOCKDOWN OF ENDOGENOUS LAR SUPPRESSES DAPK-MEDIATED CELL DETACHMENT.....................119 APPENDIX.............................................120 TABLE S1. PROTEIN IDENTIFIED TO INTERACT WITH DAPK ANKYRIN REPEAT IN THE YEAST TWO-HYBRID SCREEN..................120 FIG. S1: REDUCTION OF DAPK TYROSINE PHOSPHORYLATION IN RESPONSE TO OVER-EXPRESSION OF BOTH LARWT AND ITS PHOSPHATASE DOMAIN 2 DELETED MUTANT.................................................121 REFERENCES.............................................1223188463 bytesapplication/pdfen-US死亡相關蛋白激酶細胞凋亡integrinDAPKLARapoptosisotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51329/1/ntu-94-D89448002-1.pdf