人類似STE20蛋白質激酵素，MST3，其在細胞中之功能探討

Abstract

MST3是一哺乳動物細胞中的激酵素，相似另一於酵母菌中發現的激酵素，STE20。STE20自1992年在酵母菌中所發現後，目前已在許多物種中發現類似的酵素，酵母菌中的STE20是細胞中對費落蒙的感應器，而哺乳動物細胞中STE20激酵素很可能扮演著對環境改變或外界刺激的初期感應器，MST3的分子量約52kDa,屬於絲胺酸/蘇胺酸蛋白質激酵素，其N端具激酵素功能區域，C端為調節功能區域。MST3已發現普遍存在於老鼠、猴子及人體的各組織中，但目前對其在細胞中的生理功能尚未知；初步研究中發現在細胞凋亡的過程中，MST3是半光胺酸水解酵素 (caspase) 3，7，及8的受質，會被切斷成40kDa（主要為激酵素功能區域）及13kDa（主要為調節功能區域）兩片段；為了更進一步瞭解MST3在細胞中所扮演的角色，我們建構MST3的突變株，即主要含激酵素功能區域的片段。我們將突變株與野生株MST3分別送入細胞中大量表現，進而發現此突變株比野生株易造成細胞大量的凋亡。在螢光顯微鏡下觀察的結果發現此MST3的突變株會由細胞質轉移到細胞核中，經氨基酸序列比對，發現在半光胺酸切位點之前有一段細胞核轉移序列（nuclear localization signal），即278KKTSYLTELIDRYKRWK294 ，此序列在功能上扮演著重大的影響。根據這些結果，我們推測MST3在細胞中受到半光胺酸水解酵素的切割後，而活化其激酵素功能區域，並且暴露出細胞核轉移序列，進而轉移到細胞核中，傳遞細胞凋亡的訊息，造成細胞的凋亡。

MST3, a 52KDa protein, is one of mammalian STE20-like serine/threonine protein kinases with unknown physiological functions. It contains a kinase domain at its N-terminus, while a regulatory domain at its C-terminus. Previous studies have shown that MST3 is a cytoplasmic protein and ubiquitously expressed in many species, including human, mouse, and monkey. Endogenous MST3 was shown to be specifically cleaved by caspase3, caspase7, and 8 during staurosporine or tumor necrosis factor α (TNF-α) - induced apoptosis. Caspase-mediated cleavage of MST3 removes regulatory domain and correlates with a decreased viability of cells expressed with wild-type MST3 and truncated MST3. Overexpression of either wild-type MST3 or a truncated mutant induces a characteristic related to apoptosis. Upon alignment, MST3 contains a potential nuclear localization signal (NLS) sequence located prior to the caspase cleavage site (D313) of MST3. In this study, we demonstrated that amino acids sequence, 278KKTSYLTELIDRYKRWK294, of MST3 play an important role in the nuclear localization of the protein. Wild type of full length MST3 was present predominantly in the cytoplasm, but the truncated form of MST3 was mainly localized in the nucleus. In conclusion, our findings suggest that MST3 could be activated by caspase through cleavage site at D313. Subsequently, the NLS domain of MST3 is exposed and facilitates the nuclear translocation of the protein. The significance of the nuclear translocation of truncated MST3, however, is not clear so far.