From the Department of Molecular Biology and Diabetes Unit, Medical Services, Massachusetts General Hospital, Boston, Massachusetts 02114 and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 2014 Jan 31;289(5):2658-74. doi: 10.1074/jbc.M113.528505. Epub 2013 Dec 11.
Activation of mammalian target of rapamycin complex 1 (mTORC1) by amino acids is mediated in part by the Rag GTPases, which bind the raptor subunit of mTORC1 in an amino acid-stimulated manner and promote mTORC1 interaction with Rheb-GTP, the immediate activator. Here we examine whether the ability of amino acids to regulate mTORC1 binding to Rag and mTORC1 activation is due to the regulation of Rag guanyl nucleotide charging. Rag heterodimers in vitro exhibit a very rapid, spontaneous exchange of guanyl nucleotides and an inability to hydrolyze GTP. Mutation of the Rag P-loop corresponding to Ras(Ser-17) abolishes guanyl nucleotide binding. Such a mutation in RagA or RagB inhibits, whereas in RagC or RagD it enhances, Rag heterodimer binding to mTORC1. The binding of wild-type and mutant Rag heterodimers to mTORC1 in vitro parallels that seen with transient expression, but binding to mTORC1 in vitro is entirely independent of Rag guanyl nucleotide charging. HeLa cells stably overexpressing wild-type or P-loop mutant RagC exhibit unaltered amino acid regulation of mTORC1. Despite amino acid-independent raptor binding to Rag, mTORC1 is inhibited by amino acid withdrawal as in parental cells. Rag heterodimers extracted from (32)P-labeled whole cells, or just from the pool associated with the lysosomal membrane, exhibit constitutive [(32)P]GTP charging that is unaltered by amino acid withdrawal. Thus, amino acids promote mTORC1 activation without altering Rag GTP charging. Raptor binding to Rag, although necessary, is not sufficient for mTORC1 activation. Additional amino acid-dependent steps couple Rag-mTORC1 to Rheb-GTP.
哺乳动物雷帕霉素靶蛋白复合物 1（mTORC1）的激活部分由 Rag GTPases 介导，这些 GTPases 以受氨基酸刺激的方式与 mTORC1 的 raptor 亚基结合，并促进 mTORC1 与 Rheb-GTP（直接激活剂）的相互作用。在这里，我们研究了氨基酸调节 mTORC1 与 Rag 结合和 mTORC1 激活的能力是否归因于 Rag 鸟苷酸充电的调节。 Rag 异二聚体在体外表现出非常快速、自发的鸟苷酸交换，并且不能水解 GTP。 Rag 中的 P 环突变对应于 Ras(Ser-17)，从而消除了鸟苷酸结合。 RagA 或 RagB 中的这种突变抑制，而 RagC 或 RagD 中的这种突变增强 Rag 异二聚体与 mTORC1 的结合。 Rag 野生型和突变异二聚体在体外与 mTORC1 的结合与瞬时表达所见的结合相似，但在体外与 mTORC1 的结合完全独立于 Rag 鸟苷酸充电。稳定过表达野生型或 P 环突变 RagC 的 HeLa 细胞表现出 mTORC1 的氨基酸调节不变。尽管存在与 Rag 的氨基酸独立的 raptor 结合，但 mTORC1 如在亲本细胞中一样被氨基酸撤出所抑制。从（32）P 标记的整个细胞中提取的或仅从与溶酶体膜相关的池提取的 Rag 异二聚体表现出组成型 [(32)P]GTP 充电，这种充电不受氨基酸撤出的影响。因此，氨基酸促进 mTORC1 激活而不改变 Rag GTP 充电。 Raptor 与 Rag 的结合虽然是必需的，但不足以激活 mTORC1。其他氨基酸依赖性步骤将 Rag-mTORC1 与 Rheb-GTP 偶联。
