Chen Xianwei, Zhu Yinghui, Wang Zhaohui, Zhu Huishan, Pan Qingfei, Su Siyuan, Dong Yusheng, Li Li, Zhang Hongbing, Wu Lin, Lou Xiaomin, Liu Siqi
CAS Key Laboratory of Genome Sciences and Information, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
CAS Key Laboratory of Genome Sciences and Information, China Gastrointestinal Cancer Research Center, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China.
J Proteomics. 2016 Mar 16;136:13-24. doi: 10.1016/j.jprot.2016.01.021. Epub 2016 Feb 2.
Mammalian target of rapamycin complex 1 (mTORC1) plays important roles in regulating cell growth and proliferation, and the aberrant activation of mTORC1 has been observed in many human diseases. However, the proteins regulated by mTORC1 activation and their roles in mTORC1 downstream functions are still poorly understood. Using proteomic analysis, we found that proteins regulated by mTORC1 in MEFs could be categorized into eight functional groups including protein nuclear import and glycolysis. The positive regulation of Karyopherin subunit alpha-2 (KPNA2), an importer protein involved in protein nuclear import, by mTORC1 was verified in several other mouse and human cell lines. The regulation occurred at the transcriptional level, rather than at the level of S6K1- and 4E-BP1-dependent protein synthesis. KPNA2 knockdown partially blocked upregulation of glycolytic genes by mTORC1 activation, indicating that mTORC1 activation enhanced expression of glycolytic genes by increasing KPNA2 abundances. Furthermore, KPNA2 knockdown had no effects on the expression and subcellular localization of HIF1α, a transcription factor involved in regulating glycolytic genes downstream of mTORC1. In conclusion, our results proved that KPNA2 regulated the expression of glycolytic genes downstream of mTORC1 in a HIF1α-independent manner.
Identifying mTORC1-regulated proteins through proteomic method is a feasible way to study the downstream functions of mTORC1. In this study, we identified many mTORC1-regulated proteins using proteomic analysis by overlapping two different high vs low/no mTORC1 activity comparisons, TSC2(-/-) vs WT MEFs and TSC2(-/-) with/without rapamycin treatment. We found the abundances of many enzymes in glycolysis pathway and several proteins involved in protein nuclear import were positively regulated by mTORC1. More importantly, we first discovered that mTORC1 positively regulated the importer protein KPNA2, which participated in glycolysis regulation downstream of mTORC1 in a HIF1α-independent manner, indicating that mTORC1 regulates glycolysis through multiple ways.
雷帕霉素复合物1的哺乳动物靶点（mTORC1）在调节细胞生长和增殖中起重要作用，并且在许多人类疾病中都观察到mTORC1的异常激活。然而，受mTORC1激活调节的蛋白质及其在mTORC1下游功能中的作用仍知之甚少。通过蛋白质组学分析，我们发现mTORC1在小鼠胚胎成纤维细胞（MEFs）中调节的蛋白质可分为八个功能组，包括蛋白质核输入和糖酵解。mTORC1对核转运蛋白α-2（KPNA2）的正向调节在其他几种小鼠和人类细胞系中得到了验证，KPNA2是一种参与蛋白质核输入的输入蛋白。这种调节发生在转录水平，而不是在依赖S6K1和4E-BP1的蛋白质合成水平。敲低KPNA2部分阻断了mTORC1激活对糖酵解基因的上调作用，表明mTORC1激活通过增加KPNA2丰度来增强糖酵解基因的表达。此外，敲低KPNA2对缺氧诱导因子1α（HIF1α）的表达和亚细胞定位没有影响，HIF1α是一种参与mTORC1下游糖酵解基因调节的转录因子。总之，我们的结果证明，KPNA2以不依赖HIF1α的方式调节mTORC1下游糖酵解基因的表达。
通过蛋白质组学方法鉴定mTORC1调节的蛋白质是研究mTORC1下游功能的一种可行方法。在本研究中，我们通过重叠两种不同的高与低/无mTORC1活性比较（TSC2（-/-）与野生型MEFs以及TSC2（-/-）有/无雷帕霉素处理），利用蛋白质组学分析鉴定了许多受mTORC1调节的蛋白质。我们发现糖酵解途径中的许多酶以及几种参与蛋白质核输入的蛋白质的丰度受到mTORC1的正向调节。更重要的是，我们首次发现mTORC1正向调节输入蛋白KPNA2，KPNA2以不依赖HIF1α的方式参与mTORC1下游的糖酵解调节，表明mTORC1通过多种方式调节糖酵解。
