Isolation of Hyperactive Mutants of Mammalian Target of Rapamycin
Isolation of Hyperactive Mutants of Mammalian Target of Rapamycin
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DOI：
10.1074/jbc.m801546200
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发表时间：
2008-11-14
影响因子：
4.8
通讯作者：
Maeda, Tatsuya
中科院分区：
文献类型：
--
作者：
Ohne, Yoichiro;Takahara, Terunao;Maeda, Tatsuya
哺乳动物的雷帕霉素靶标(MTOR)是一种丝氨酸/苏氨酸(Ser/Thr)激酶，在蛋白质合成、细胞大小和自噬等一系列与生长相关的过程中发挥重要作用。MTOR形成两个功能不同的复合体，称为mTOR复合体1(MTORC1)和2(MTORC2)；只有前者被雷帕霉素抑制。基于雷帕霉素处理和营养饥饿引起的细胞反应之间的相似性，人们普遍认为，对营养状态的反应调节mTORC1活性直接影响这些细胞反应，尽管直接证据很少。在此，我们报道了mTOR高活性突变体的分离。分离的mTOR突变体在体外表现出增强的激酶活性，并使细胞对氨基酸饥饿时mTORC1底物的去磷酸化不起作用。表达高活性mTOR突变体的细胞在正常生长条件下表现出较大的细胞尺寸，并且在氨基酸饥饿的条件下抵抗细胞尺寸缩小和自噬诱导。这些结果表明，mTORC1的活性实际上指导了这些细胞过程对营养状况的反应，并证实了mTORC1的生物学功能，这是完全通过使用雷帕霉素和(分子)基因技术进行功能丧失分析而提出的。此外，高活性的mTOR突变体没有诱导NIH/3T3细胞的细胞转化，这表明肿瘤发生需要伴随着额外途径的激活。这种高度活跃的mTOR突变体将是在细胞和生物体中建立mTOR激活的生理后果的有价值的工具。
The mammalian target of rapamycin(mTOR) is a Ser/Thr kinase that plays essential roles in the regulation of a wide array of growth-related processes such as protein synthesis, cell sizing, and autophagy. mTOR forms two functionally distinct complexes, termed the mTOR complex 1(mTORC1) and 2 (mTORC2); only the former of which is inhibited by rapamycin. Based on the similarity between the cellular responses caused by rapamycin treatment and by nutrient starvation, it has been widely accepted that modulation in the mTORC1 activity in response to nutrient status directs these cellular responses, although direct evidence has been scarce. Here we report isolation of hyperactive mutants of mTOR. The isolated mTOR mutants exhibited enhanced kinase activity in vitro and rendered cells refractory to the dephosphorylation of the mTORC1 substrates upon amino acid starvation. Cells expressing the hyperactive mTOR mutant displayed larger cell size in a normal growing condition and were resistant to cell size reduction and autophagy induction in an amino acid-starved condition. These results indicate that the activity of mTORC1 actually directs these cellular processes in response to nutrient status and confirm the biological functions of mTORC1, which had been proposed solely from loss-of-function analyses using rapamycin and(molecular) genetic techniques. Additionally, the hyperactive mTOR mutant did not induce cellular transformation of NIH/3T3 cells, suggesting that concomitant activation of additional pathways is required for tumorigenesis. This hyperactive mTOR mutant will be a valuable tool for establishing physiological consequences of mTOR activation in cells as well as in organisms.