A Rag GTPase dimer code defines the regulation of mTORC1 by amino acids

Abstract

Amino acid availability controls mTORC1 activity via a heterodimeric Rag GTPase complex that functions as a scaffold at the lysosomal surface, bringing together mTORC1 with its activators and effectors. Mammalian cells express four Rag proteins (RagA–D) that form dimers composed of RagA/B bound to RagC/D. Traditionally, the Rag paralogue pairs (RagA/B and RagC/D) are referred to as functionally redundant, with the four dimer combinations used interchangeably in most studies. Here, by using genetically modified cell lines that express single Rag heterodimers, we uncover a Rag dimer code that determines how amino acids regulate mTORC1. First, RagC/D differentially define the substrate specificity downstream of mTORC1, with RagD promoting phosphorylation of its lysosomal substrates TFEB/TFE3, while both Rags are involved in the phosphorylation of non-lysosomal substrates such as S6K. Mechanistically, RagD recruits mTORC1 more potently to lysosomes through increased affinity to the anchoring LAMTOR complex. Furthermore, RagA/B specify the signalling response to amino acid removal, with RagB-expressing cells maintaining lysosomal and active mTORC1 even upon starvation. Overall, our findings reveal key qualitative differences between Rag paralogues in the regulation of mTORC1, and underscore Rag gene duplication and diversification as a potentially impactful event in mammalian evolution.

Brain-enriched RagB isoforms regulate the dynamics of mTORC1 activity through GATOR1 inhibition

“…On the basis of their high sequence similarity, RagA and RagB have been assumed to be functionally equivalent. The findings presented here, together with those in an accompanying study 34 , show that this is not the case. RagA and the two RagB isoforms have distinct profiles of interaction with Raptor and GATOR1, which correspond to different levels of mTORC1 activity and result in different responses to nutrient deprivation.…”

“…1h–k ), suggesting that these two isoforms might have differential effects on a subset of mTORC1 substrates. In this manuscript we focus on the functional differences between RagA and RagB, while the accompanying manuscript by Demetriades and colleagues focuses on RagC versus RagD 34 .…”

Cell migration CRISPRi screens in human neutrophils reveal regulators of context-dependent migration and differentiation state

“…It is known that mTORC1 can regulate the activity of transcription factors, including TFEB and TFE3, via their phosphorylation that is dependent on RagC/D-FLCN and mechanistically distinct from other well-characterized mTORC1 targets 52, 53 . Notably, knockdown of TFEB and TFE3 resulted in a modest but significant decrease in sgRNA abundance in our differentiation screen, which contrasted with the positive enrichment of FLCN and LAMTOR genes (Fig.…”

“…Specifically, the phosphorylation state of the canonical mTORC1 target S6K was unchanged and instead we found substantial transcriptional changes associated with processes of lysosome biogenesis, autophagy, and cellular degranulation. This appears due to non-canonical mechanisms of mTORC1 substrate activation that have only recently begun to be elucidated 52, 53, 101 . Here, signaling via FLCN-RagC/D played a dominant role in the regulation of catabolic processes, likely altering the activity of mTORC1 toward transcription factors like TFEB and TFE3 that are master regulators of lysosomal biogenesis and autophagy.…”

The MTORC1-AHR pathway sustains translation and autophagy in tumours under tryptophan stress

“…This is surprising as PI3K is a well-known enhancer of both MTORC1 substrates (Hay and Sonenberg, 2004). Recent evidence shows that the RAG GTPases mediate MTORC1 substrate specificity towards TFEB/TFE (Alesi et al, 2021; Figlia et al, 2022; Gollwitzer et al, 2022; Li et al, 2022; Napolitano et al, 2020), but no differential effect towards RPS6KB1 versus EIF4EBP1 has been described so far. The difference in EIF4EBP1 and RPS6KB1 phosphorylation may be explained by the higher affinity of EIF4EBP1 to the MTORC1 scaffold protein RAPTOR (Bohm et al, 2021; Fumagalli and Pende, 2022), which along with increased EIF4EBP1 levels may enhance recruitment and phosphorylation by MTORC1.…”