請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35688
標題: | 探討DAPK在細胞骨架及細胞移動之調控分析 Functional Characterization of DAPK in cytoskeleton regulation and cell migration |
作者: | Jean-Cheng Kuo 郭津岑 |
指導教授: | 陳瑞華 |
關鍵字: | 死亡相關蛋白激酶,細胞集中附著點,壓力纖維,細胞極性, DAPK,stress fiber,integrin,cell polarity, |
出版年 : | 2005 |
學位: | 博士 |
摘要: | 死亡相關蛋白激酶(Death-associated protein kinase,DAPK)是一個受鈣�攜鈣素所調控的絲胺酸�酪胺酸激酶。先前的研究顯示其參與許多細胞凋亡及腫瘤抑制作用,但其間機制至今仍未清楚。在這份論文的第一個部份,我們証明了DAPK可以藉由磷酸化肌凝蛋白II的調節輕鏈(regulatory light chain of myosin II,MLC)在絲胺酸19位置,進而穩定壓力纖維(stress fiber)的生成。除此之外,當細胞在沒有血清的培養條件下,表現DAPK對細胞集中附著點(focal adhesion)而言,並不能刺激其形成;當我們重新加入血清去刺激,DAPK的存在對細胞集中附著點(focal adhesion)而言,非但不能刺激其形成反倒是促使其消失但是對壓力纖維確沒有影響。我們推測這樣一種對細胞壓力纖維和集中附著點消長間不協調的作用機制,結果會抑制細胞的附著(cell adhesion)能力進而誘使細胞凋亡的進行。在論文的第二個部份,我們則著重在探討DAPK的腫瘤抑制功能。先前的證據顯示,DAPK主要是經由其誘發細胞凋亡的作用來達到腫瘤抑制。DAPK所造成的細胞凋亡是p53-dependent,但在大多數的腫瘤細胞中其p53確是沒有作用的。如此一來,顯示DAPK尚存在有另一種作用機制來造成腫瘤抑制。基於DAPK的功能在會調控細胞骨架蛋白和胞外基質受器(integrin)活性,因細胞骨架蛋白和胞外基質受器在細胞移動的能力上均扮演著重要的角色,我們推測DAPK會影響細胞的移動。我們發現表現DAPK會減弱細胞極性(polarization),藉由干擾細胞對方向性的持續力和移動力進而抑制了細胞的隨機移動(random migration)。這些DAPK對細胞移動能力的影響也被証實的確是因為DAPK抑制了胞外基質受器/cdc42的訊號傳遞。更在p53沒有作用的腫瘤細胞中證明,雖然表現DAPK並不會造成這類細胞的死亡,但當DAPK表現時還是會抑制其細胞的移動和侵入能力,更進一步證明DAPK在抑制腫瘤上的角色。最後,利用一對來源相同、卻具不同侵入能力的肺癌上皮細胞株,我們發現了DAPK的表現量和其細胞的侵入能力具有相當程度的負相關。這更進一步證實DAPK參與了p53-independent的腫瘤抑制功能。
總括來說,在探討DAPK如何執行其細胞生理功能上,我們証明DAPK會經由磷酸化肌凝蛋白II的調節輕鏈來影響細胞壓力纖維的穩定,同時並造成與細胞集中附著點形成之間作用的不協調。如此,對DAPK所參與的細胞凋亡過程上有重要性的影響。我們也找到DAPK一個p53-independent的作用機制,DAPK會藉由影響細胞的移動能力來造成腫瘤抑制功能。 Death-associated protein kinase (DAPK) is a calcium/calmodulin-dependent serine/threonine kinase. Its functions in pro-apoptosis and tumor suppression have been studied before, but detailed mechanism isn’t fully elucidated. In the first part of the study, we demonstrate that DAPK is capable of phosphorylating the regulatory light chain of myosin II (MLC) at serine 19 in vitro and in vivo, resulting in stress fibers stabilization. However, DAPK cannot stimulate the formation of focal adhesion in quiescent cells and promotes the disassembly of focal adhesions but not stress fibers in cells receiving serum factors. Thus, we proposed that DAPK functions in the uncoupling of stress fibers and focal adhesions. Such uncoupling would lead to a perturbation of the balance between contractile and adhesion forces and subsequent cell detachment, which might contribute to its pro-apoptotic activity. In the second part of the study, we focus on the mechanism through which DAPK functions as a tumor suppressor. DAPK is thought to execute tumor suppressive function by its apoptotic activity. However, the apoptotic effect of DAPK is largely p53-dependent, while many tumor cells are p53 defective. To reconcile this, we tested whether DAPK has another mechanism to suppress tumor. As DAPK has been demonstrated to the regulate cytoskeleton and integrin activity, both of which play a role in cell migration, we therefore study the function of DAPK in migration. We found that DAPK inhibits random migration by reducing directional persistence and directed migration by blocking cell polarization. These DAPK-mediated migratory defects are mainly through its suppression of integrin/Cdc42 pathway. The regulation of migration by DAPK indeed in part explains for its tumor suppressor function, exemplified by the fact that in certain p53-mutant tumor cells that are resistant to DAPK-induced apoptosis, DAPK expression can still block their migratory and invasive abilities. Furthermore, by using a paired of lung adenocarcinoma cell lines, which are of the same source but of different invasive activities, we demonstrated that DAPK expression level is a determinant factor in tumor invasiveness. There emerges the second, p53-independent, mechanism for DAPK in tumor suppression. To sum up, to get insight how DAPK exerts its physiological function, we demonstrate that DAPK uncouples stress fibers and focal adhesions, partly through its phosphorylation of MLC. Besides, we uncover a new p53-independent mechanism mediated by DAPK to affect cell motility, accounting for its tumor suppressive functions. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35688 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子醫學研究所 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-94-1.pdf 目前未授權公開取用 | 5.19 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。