DNA-dependent protein kinase在細胞有絲分裂的功能(1/3)

許多的實驗顯示DNA-PKcs 在細胞內具有多重且廣泛的功能，包括在DNA 傷害修復、 V(D)J 重組、DNA 複製、細胞週期檢查點 (checkpoint) 的調控和細 胞凋亡等。但由於DNA-PKcs 不易操作且缺乏適當的in vivo 研究工具，因此在 這些作用機制內，DNA-PKcs 扮演的角色和作用的模式仍然是不清楚的。本計劃 實行中，我們成功的製備了數種能辨識DNA-PKcs 的專一性抗體，其中包括能 辨識於T2609 胺基酸呈磷酸化的DNA-PKcs 之單株抗體，而得以在in vivo 的情 況下，研究活化態的DNA-PKcs 於細胞週期運轉和外在環境壓力下所扮演的生 理角色。首先我們在DNA 複製時，發現磷酸化DNA-PKcs 會位於複製後染色體 的中心體(centrosome)上，且對於微小管的形成(microtubule nucleation)扮演正面 調控的角色。另外，我們亦發現磷酸化DNA-PKcs 在細胞分裂前期會標第至中 心粒(centromere) ，與AuroraB 發生交互作用，並影響其磷酸激酶活性。這些發 現賦予DNA-PKcs 前所未見的功能，並可望對細胞核質協調及細胞週期調控提 供新的解釋。Since its first description in 1990, DNA-PK catalytic subunit has been one of the most attractive and intriguing molecules in the research field. The bulk of studies has implicated its participation in a wide array of cellular functions, including DNA damage repair, V(D)J recombination, DNA replication, cell-cycle checkpoint and apoptosis. Nevertheless, the role and mode of action of DNA-PKcs in these pathways still remain obscure, mainly because of its difficulty in manipulation, as well as the lack of reagents for tracking the active molecule in vivo. In the course of our study we raised several antibodies against different portions of DNA-PKcs, and most importantly, antibodies that can recognize specifically phosphorylated DNA-PKcs, which represents the active kinase in in vivo. These antibodies enable us to describe for the first time the behavior of activated DNA-PKcs in physiological conditions, as well as during cell cycle progression. We detected the presence of phosphorylated DNA-PKcs on duplicated centrosomes, and described its positive role in microtubule nucleation. Furthermore, we found that phosphorylated DNA-PKcs localize to the prometaphase kinetochores to interact with the AuroraB complex, modulating its kinase activity. These findings reveal unforeseen functions for DNA-PKcs, and may offer the key to unravel new insights into cell nuclear/cytoplasmic coordination and cell cycle progression checkpoints