鈉鉀ATP酶和蛋白激酶A調控線蟲計劃性細胞死亡

Abstract

計畫性細胞死亡的主要功能是在維持體內細胞數的平衡以及移除掉不想要的細胞，如果計畫性細胞死亡的調控過程中發生問題可能會造成癌症以及某些疾病。在過去30年中，已知有數十個基因，包括主要進行細胞死亡的基因: EGL-1(a BH3-containing protein)，CED-9(Bcl-2)，CED-4(Apaf-1)以及CED-3(Caspase)已經被知道參與在線蟲計畫性細胞死亡中執行死亡的階段，然而這4個主要的基因是如何被調控的以及還有什麼樣的基因參與在其中還不是非常清楚。在這篇論文中，我發現鈉鉀ATP酶α單元EAT-6可以促進線蟲的計畫性細胞死亡。哺乳動物的鈉鉀ATP酶由2個含有ATP酶活性的α單元和2個β單元以及1個在特定組織中才有的γ單元所組成，它主要的功能是在維持細胞膜電位的平衡。而線蟲有2個α單元eat-6和catp-4和3個β單元nkb-1，nkb-2和nkb-3，但是沒有γ單元。在這裡我發現失去任何的β單元不會造成胚胎發育過程中計畫性細胞死亡的問題，但是一旦失去α單元eat-6就會造成胚胎發育過程中應該要死的細胞沒死掉，這個結果顯示α單元與β單元參與在計畫性細胞死亡的角色並不相同。除此之外，當大量表現eat-6時會促進細胞死亡。很有趣的是，當EAT-6的ATP酶活性位置如果發生突變的話，仍然會促進細胞死亡，因此代表EAT-6除了在運送鈉鉀離子的功能之外還有另一個新的角色，也就是促進細胞死亡。另外在特定細胞中，沒有結合β單元NKB-1或 NKB-2的α單元EAT-6也會促進細胞死亡，代表在這些細胞中單獨α單元EAT-6就有促進死亡的功能了。除了eat-6之外，我還有發現蛋白激酶A (cAMP-dependent protein kinase)的調控單元kin-2也會促進線蟲的計畫性細胞死亡。在哺乳動物中，蛋白激酶A是由2個調控單元以及2個活化單元所組成，但是線蟲只有1個調控單元kin-2和 1個活化單元kin-1。在這裡我發現，一旦失去調控單元kin-2就會造成胚胎發育過程中應該要死的細胞沒死掉，但是當失去活化單元kin-1反而會更增加細胞的死亡。除此之外，當大量表現kin-2時也會促進細胞死亡。然而我還發現在dying cell中表現eat-6 或kin-2會挽救eat-6 或kin-2突變蟲減少細胞死亡的問題，因此顯示eat-6 和kin-2是會作用在dying cell的。另外eat-6和kin-2是會透過egl-1幫助去促進計畫性細胞死亡的。

Programmed cell death (apoptosis) functions in maintaining homeostasis and eliminating unwanted cells. Defects in programmed cell death has been implicated in cancerous growth and diseases. Over the past 30 years, about a dozen of genes, including the core cell death genes EGL-1 (a BH3-containing protein), CED-9 (Bcl-2), CED-4 (Apaf-1) and CED-3 (Caspase), have been identified to function in the execution of programmed cell death in C. elegans. However, how the cell death process is regulated is still unclear. Here, I report that EAT-6, a sodium-potassium ATPase alpha subunit, can promote programmed cell death in C. elegans. A typical mammalian sodium-potassium ATPase is composed of two alpha subunits containing an ATPase activity and two beta subunits with/without one gamma subunit, and functions to maintain membrane potential across plasma membrane. C. elegans has two alpha subunits, eat-6 and catp-4, and three beta subunits, nkb-1, nkb-2 and nkb-3, but has no gamma subunit. I found that loss of any beta subunit does not result in a detectable cell death defect during embryogenesis and, in contrast, loss of the alpha subunit eat-6, significantly reduced cell death during embryogenesis. This result shows differential involvement of alpha and beta subunits in promoting cell death. In addition, overexpression of eat-6 promotes cell death. Interestingly, the “pump dead” EAT-6 with a mutation disrupting the ATPase activity, can still promote cell death, suggesting that EAT-6 has a function in promoting cell death independent of its pumping activity. In addition, EAT-6 can promote cell death in the presence or absence of beta subunit NKB-1/NKB-2 in a cell-specific manner. And I also found KIN-2, a protein kinase A (cAMP-dependent protein kinase) regulatory subunit can promote programmed cell death in C. elegans. A typical mammalian protein kinase A is composed of two regulatory subunits and two catalytic subunits. C. elegans has one regulatory subunit, kin-2, and one catalytic subunit, kin-1. I found that loss of the regulatory subunit, kin-2 reduced cell death during embryogenesis and, in contrast, loss of the catalytic subunit kin-1 increased cell death during embryogenesis. In addition, overexpression of kin-2 promotes cell death. And I found that expression of eat-6 or kin-2 in dying cells rescued the cell death defeat in eat-6 or kin-2 mutants, indicating that eat-6 and kin-2 function in dying cells. And I found that eat-6 and kin-2 promote cell death through egl-1.