探討DAPK引發細胞凋亡的機制及其受LAR之調控

The Apoptotic Mechanism of Death-associated Protein Kinase and its Regulation by Protein Tyrosine Phosphatase LAR

王琬菁(Won-Jing Wang)

指導教授 ： 陳瑞華

國立臺灣大學/醫學院/分子醫學研究所/博士(2005年)

https://doi.org/10.6342/NTU.2005.00931

摘要

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。

關鍵字

死亡相關蛋白激酶細胞凋亡

並列摘要

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.

並列關鍵字

integrin ； DAPK ； LAR ； apoptosis

參考文獻

Abraham,M.C. and Shaham,S. (2004). Death without caspases, caspases without death. Trends Cell Biol. 14, 184-193.

Almeida,E.A., Ilic,D., Han,Q., Hauck,C.R., Jin,F., Kawakatsu,H., Schlaepfer,D.D., and Damsky,C.H. (2000). Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH(2)-terminal kinase. J. Cell Biol. 149, 741-754.

Andersen,J.N., Mortensen,O.H., Peters,G.H., Drake,P.G., Iversen,L.F., Olsen,O.H., Jansen,P.G., Andersen,H.S., Tonks,N.K., and Moller,N.P. (2001). Structural and evolutionary relationships among protein tyrosine phosphatase domains. Mol. Cell Biol. 21, 7117-7136.

Arnoult,D., Gaume,B., Karbowski,M., Sharpe,J.C., Cecconi,F., and Youle,R.J. (2003). Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization. EMBO J. 22, 4385-4399.

Aznar,S., Fernandez-Valeron,P., Espina,C., and Lacal,J.C. (2004). Rho GTPases: potential candidates for anticancer therapy. Cancer Lett. 206, 181-191.