Kimball Scot R, Gordon Bradley S, Moyer Jenna E, Dennis Michael D, Jefferson Leonard S
The Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, United States.
The Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, PO Box 850, Hershey, PA 17033, United States.
Cell Signal. 2016 Aug;28(8):896-906. doi: 10.1016/j.cellsig.2016.03.008. Epub 2016 Mar 21.
The studies described herein were designed to explore the role of Sestrin2 in mediating the selective action of leucine to activate mTORC1. The results demonstrate that Sestrin2 is a phosphoprotein and that its phosphorylation state is responsive to the availability of leucine, but not other essential amino acids. Moreover, leucine availability-induced alterations in Sestrin2 phosphorylation correlated temporally and dose dependently with the activation state of mTORC1, there being a reciprocal relationship between the degree of phosphorylation of Sestrin2 and the extent of repression of mTORC1. With leucine deprivation, Sestrin2 became more highly phosphorylated and interacted more strongly with proteins of the GATOR2 complex. Notably, in cells lacking the protein kinase ULK1, the activation state of mTORC1 was elevated in leucine-deficient medium, such that the effect of re-addition of the amino acid was blunted. In contrast, overexpression of ULK1 led to hyperphosphorylation of Sestrin2 and enhanced its interaction with GATOR2. Neither rapamycin nor Torin2 had any effect on Sestrin2 phosphorylation, suggesting that leucine deprivation-induced repression of mTORC1 was not responsible for the action of ULK1 on Sestrin2. Mass spectrometry analysis of Sestrin2 revealed three phosphorylation sites that are conserved across mammalian species. Mutation of the three sites to phospho-mimetic amino acids in exogenously expressed Sestrin2 promoted its interaction with GATOR2 and dramatically repressed mTORC1 even in the presence of leucine. Overall, the results support a model in which leucine selectively promotes dephosphorylation of Sestrin2, causing it to dissociate from and thereby activate GATOR2, leading to activation of mTORC1.
本文所述的研究旨在探究Sestrin2在介导亮氨酸激活mTORC1的选择性作用中的角色。结果表明，Sestrin2是一种磷蛋白，其磷酸化状态对亮氨酸的可利用性有反应，但对其他必需氨基酸无反应。此外，亮氨酸可利用性诱导的Sestrin2磷酸化变化在时间和剂量上与mTORC1的激活状态相关，Sestrin2的磷酸化程度与mTORC1的抑制程度之间存在反比关系。在亮氨酸缺乏时，Sestrin2磷酸化程度更高，且与GATOR2复合物的蛋白质相互作用更强。值得注意的是，在缺乏蛋白激酶ULK1的细胞中，亮氨酸缺乏培养基中mTORC1的激活状态升高，使得重新添加氨基酸的效果减弱。相反，ULK1的过表达导致Sestrin2过度磷酸化，并增强其与GATOR2的相互作用。雷帕霉素和Torin2对Sestrin2磷酸化均无任何影响，这表明亮氨酸缺乏诱导的mTORC1抑制并非ULK1对Sestrin2作用的原因。对Sestrin2的质谱分析揭示了三个在哺乳动物物种中保守的磷酸化位点。在外源表达的Sestrin2中将这三个位点突变为模拟磷酸化的氨基酸会促进其与GATOR2的相互作用，并且即使在有亮氨酸存在的情况下也会显著抑制mTORC1。总体而言，这些结果支持了一个模型，即亮氨酸选择性地促进Sestrin2的去磷酸化，使其与GATOR2解离从而激活GATOR2，进而导致mTORC1的激活。
