利用HeLa細胞探討Mst3誘導細胞凋亡之分子機制

Abstract

Mst3為52KDa蛋白質，隸屬人類STE20相似蛋白激酵素 (human Ste20 like protein kinase)家族中的一員，在細胞凋亡過程中扮演重要角色。它具有N端的激酵素功能區域及C端的調控功能區域，之前研究指出Mst3一般存在於細胞質且會被caspase活化，且大量表現Mst3野生株及截斷株，皆可誘發細胞凋亡之產生。 實驗室便利用廣泛性caspase抑制劑 (Z-VAD-fmk) 處理細胞，結果我們發現Mst3所導致的細胞凋亡並不會被抑制，而且Mst3與caspase-3共同表現也不會促進細胞凋現象產生亡，因而推測Mst3引起的細胞凋亡或許並非透過caspase所誘發或者兩者機制是並行存在的。 另一造成細胞凋亡之訊息傳遞途徑則是透過粒腺體途徑 (mitochondrial pathway)，之前研究指出，當細胞遭遇細胞凋亡時，粒腺體內的膜電位 (membrane potential) 會改變、滲透壓及鈣離子濃度亦會提高，進而導致部份粒腺體凋亡因子，如cytochrome C、AIF游離出來。而這些凋亡因子則會受到一些粒腺體蛋白調控，我們研究以免疫染色發現Cytochrome C是會與Mst3重疊，但AIF及Endo G則無此現象發生。而Bcl-2蛋白質會於Mst3存在時，細胞質中的量也會相對減少。我們更意外發現：Mst3不僅存在於細胞質中，也可在粒腺體中發現。故便需進一步以釐清Mst3誘發的凋亡現象中，粒腺體所扮演的角色。

Mst3, a novel human Ste20-like serine/threonine protein kinase, plays an important role in the process of cell apoptosis. It contains a kinase domain at its N-terminus and a regulatory domain at the C-terminus. Pervious studies have shown that overexpression of Mst3 or its truncated mutant induced apoptosis of HeLa and HEK293 cells. Further studies showed that the Mst3-indued apoptosis did not involve in any known MAPK cascades. Caspases are the central executioner of most classical apoptosis pathway, but its role in Mst3-induced apoptosis of HeLa cells is controversial. The result from this work showed that the universal caspase inhibitor, Z-VAD-fmk, could not supposed Mst3-induced HeLa cell apoptosis suggesting a caspase-independent pathway might exist. Mitochondria play an essential role in trigging caspase-dependent or independent apoptotic process. Many apoptotic factors are present in the mitochondrial intermembrane space. Once activated by apoptosis stimulus, these apoptotic factors may translocate from mitochondria to the cytoplasm or nucleus. The translocation of these apoptotic factors can be regulated by the Bcl-2 family proteins. Our results demonstrated that Mst3 is co-localized with cytochrome C in the mitochondria. This observation indicates that Mst3 may induce apoptosis by altering the cellular localization of cytochrome C. Interestingly the protein level of Bcl-2 was also reduced with the overexpression of Mst3. The subcellular redistribution of other apoptotic factors in response to the overexpression of Mst3 is unknown. Subsequent studies, we found that staurosporine may act differently to induce nuclear translocation of Mst3 as well as AIF proteins. Bcl-2 in nucleus was also increased with the stimulation of staurosporine. The role of Mst3 in mitochondria is unknown so far. The existence of Mst3 in mitochondria may provide a new aspect about the physiological roles of Mst3.