新的人類類STE20蛋白質激酵素, MASK, 在細胞中生物功能的探討

Abstract

我們在人類的細胞中發現一個新的類STE20的蛋白質激□-MASK(MST3 and SOK1-related kinase)。STE20被發現在酵母菌對費洛蒙的感應途徑中扮演一個重要的角色，它接受上游G蛋白的刺激，接著活化下游的MAPK途徑，使酵母菌準備好進行交配反應，STE20在這樣的途徑中扮演著將外界的刺激傳遞進入細胞中的重要角色。近期在哺乳類動物細胞中也發現了許多和酵母菌的STE20具有相似性的蛋白質，其中也有幾個蛋白被認為會活化MAPK的途徑。MASK最初是從腫瘤細胞中選殖出來的一個絲胺酸/蘇胺酸蛋白質激□，分子量大小約為46.5 kDa，其激□區域位於N端，而C端則為一個調控的區域。根據之前研究結果指出截斷型的MASK比野生株的MASK更能促進蛙卵的成熟，因此我們認為MASK和細胞週期的調控有關聯。初步觀察發現MASK在細胞中所在的位置會有轉移的現象發生(細胞質到細胞核)，為了能夠即時的觀察MASK在細胞所在的位置及變化，我們將數個不同大小的MASK片段和帶有綠螢光蛋白(GFP)的表現載體(pEGFP-C2)接合在一起，使其能夠表現出帶有MASK片段的綠螢光融合蛋白(GFP-MASK)，透過螢光顯微鏡我們即時的觀察這些不同大小的融合蛋白在細胞中所在位置。我們加入不同的藥物來刺激大量表現GFP -MASK融合蛋白的細胞株，不過並沒有找到可以刺激其轉移的刺激物。接著利用我們篩選的四株穩定表現的細胞株(GFP-MASK/Wt, GFP-MASK /296t, GFP-MASK/KR, GFP-MASK/KR/t)，我們加入PMA、H2O2及UV來對細胞進行刺激，發現當我們利用高劑量的UV照射細胞時(100-200J/m2)，細胞會因受到DNA的損傷而停留在細胞週期中的G2階段，我們觀察三株細胞株和控制組對於UV所造成的傷害後發現GFP-MASK/KR死亡情形較輕微，而GFP-MASK/296t則有較嚴重的死亡現象，GFP-MASK/WT則和HEK293死亡的情形相近，因此我們推論MASK可能和細胞越過G2/M階段的作用有關聯。

We have cloned a newly identified human STE20-like protein kinase, MASK (MST3 and SOK1-related kinase), which was suspected to involve in a novel protein kinase pathway. In budding yeast, STE20 functions upstream of the MAPK pathway as a link to heterotrimeric G-protein and phosphorylates its downstream components. Recently, mammalian STE20-like protein kinases family was growing rapidly and several were characterized as potential upstream kinases of MAPK pathways. MASK was a serine/threonine protein kinase based on its amino acid composition and was characterized as one of the newly identified mammalian STE20-like protein kinases. MASK composes of 416 amino acid (46.5 kDa) and contains a N-terminal kinase domain and a C-terminal regulatory domain. It was considered as a sensor of extracellular signals, although it was previously proofed not to involved in any known MAPK pathway. MASK may consider as a cell cycle regulator because when various MASK mRNAs were injected into Xenopus oocytes, truncated form of MASK (kinase domain only) accelerated progesterone-induced oocytes maturation faster than wild type MASK. Subcellular localization analysis of MASK revealed that MASK would appear between cytoplasm and nucleus in different kinase activity stage. In order to investigate the localization of MASK immediately, we utilized the convenience of GFP-MASK fusion protein. After transfection of GFP-MASK into HEK293 and 293T cell, we stimulated cells with different reagents to see whether any stimulators would trigger the translocation of MASK from cytoplasm to nucleus. We selected 4 HEK293 cell lines which GFP-MASK/WT, GFP-MASK/296t, GFP-MASK/KR, GFP-MASK/KR/t were stable expressed in. We treated the four stable clones with PMA, H2O2 or UV, and wondered is there any difference of the viability or growth pattern between four clones. We found that GFP-MASK/296t cell possessed less survival rate and GFP-MASK/KR cell had higher survival rate than GFP-MASK/WT and HEK293 cell, under the UV damage. We proposed that MASK might involve in the G2/M phase transition.