Wang Xuemin, Fonseca Bruno D, Tang Hua, Liu Rui, Elia Androulla, Clemens Michael J, Bommer Ulrich-Axel, Proud Christopher G
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver V6T 1Z3, Canada.
J Biol Chem. 2008 Nov 7;283(45):30482-92. doi: 10.1074/jbc.M803348200. Epub 2008 Aug 1.
Signaling through mammalian target of rapamycin complex 1 (mTORC1) is stimulated by amino acids and insulin. Insulin inactivates TSC1/2, the GTPase-activator complex for Rheb, and Rheb.GTP activates mTORC1. It is not clear how amino acids regulate mTORC1. FKBP38 (immunophilin FK506-binding protein, 38 kDa), was recently reported to exert a negative effect on mTORC1 function that is relieved by its binding to Rheb.GTP. We confirm that Rheb binds wild type FKBP38, but inactive Rheb mutants showed contrasting abilities to bind FKBP38. We were unable to observe any regulation of FKBP38/mTOR binding by amino acids or insulin. Furthermore, FKBP38 did not inhibit mTORC1 signaling. The translationally controlled tumor protein (TCTP) in Drosophila was recently reported to act as the guanine nucleotide-exchange factor for Rheb. We have studied the role of TCTP in mammalian TORC1 signaling and its control by amino acids. Reducing TCTP levels did not reproducibly affect mTORC1 signaling in amino acid-replete/insulin-stimulated cells. Moreover, overexpressing TCTP did not rescue mTORC1 signaling in amino acid-starved cells. In addition, we were unable to see any stable interaction between TCTP and Rheb or mTORC1. Accumulation of uncharged tRNA has been previously proposed to be involved in the inhibition of mTORC1 signaling during amino acid starvation. To test this hypothesis, we used a Chinese hamster ovary cell line containing a temperature-sensitive mutation in leucyl-tRNA synthetase. Leucine deprivation markedly inhibited mTORC1 signaling in these cells, but shifting the cells to the nonpermissive temperature for the synthetase did not. These data indicate that uncharged tRNA(Leu) does not switch off mTORC1 signaling and suggest that mTORC1 is controlled by a distinct pathway that senses the availability of amino acids. Our data also indicate that, in the mammalian cell lines tested here, neither TCTP nor FKBP38 regulates mTORC1 signaling.
通过雷帕霉素哺乳动物靶点复合物1（mTORC1）的信号传导受氨基酸和胰岛素刺激。胰岛素使TSC1/2失活，TSC1/2是Rheb的GTP酶激活复合物，而Rheb.GTP激活mTORC1。目前尚不清楚氨基酸如何调节mTORC1。FKBP38（亲免蛋白FK506结合蛋白，38 kDa）最近被报道对mTORC1功能有负面影响，而其与Rheb.GTP的结合可解除这种影响。我们证实Rheb与野生型FKBP38结合，但无活性的Rheb突变体显示出与FKBP38结合的能力不同。我们未能观察到氨基酸或胰岛素对FKBP38/mTOR结合的任何调节作用。此外，FKBP38并未抑制mTORC1信号传导。果蝇中的翻译控制肿瘤蛋白（TCTP）最近被报道可作为Rheb的鸟嘌呤核苷酸交换因子。我们研究了TCTP在哺乳动物TORC1信号传导中的作用及其受氨基酸的调控。降低TCTP水平在氨基酸充足/胰岛素刺激的细胞中并未可重复性地影响mTORC1信号传导。此外，过表达TCTP并不能挽救氨基酸饥饿细胞中的mTORC1信号传导。另外，我们未能观察到TCTP与Rheb或mTORC1之间有任何稳定的相互作用。先前有人提出未负载的tRNA积累参与氨基酸饥饿期间mTORC1信号传导的抑制。为了验证这一假设，我们使用了一种在中国仓鼠卵巢细胞系中，其亮氨酰 - tRNA合成酶存在温度敏感突变。亮氨酸剥夺显著抑制了这些细胞中的mTORC1信号传导，但将细胞转移至合成酶的非允许温度时则不然。这些数据表明未负载的tRNA（Leu）不会关闭mTORC1信号传导，并表明mTORC1受一条感知氨基酸可用性的独特途径控制。我们的数据还表明，在此处测试的哺乳动物细胞系中，TCTP和FKBP38均不调节mTORC1信号传导。
