An mTORC1-mediated negative feedback loop constrains amino acid-induced FLCN-Rag activation downstream of TSC2 loss

The mechanistic target of rapamycin complex 1 (mTORC1) integrates inputs from growth factors and nutrients, but how mTORC1 autoregulates its activity remains unclear. The MiT/TFE transcription factors are phosphorylated and inactivated by mTORC1 following lysosomal recruitment by RagC/D GTPases in response to amino acid stimulation. We find that starvation-induced lysosomal localization of the RagC/D GAP complex, FLCN:FNIP2, is markedly impaired in a mTORC1-sensitive manner in cells with TSC2 loss, resulting in unexpected TFEB hypophosphorylation and activation upon feeding. TFEB phosphorylation in TSC2-null cells is partially restored by destabilization of the lysosomal folliculin complex (LFC) induced by FLCN mutants and is fully rescued by forced lysosomal localization of the FLCN:FNIP2 dimer. Our data indicate that a negative feedback loop constrains amino acid-induced, FLCN:FNIP2-mediated RagC activity in cells with constitutive mTORC1 signaling, and the resulting MiT/TFE hyperactivation may drive oncogenesis with loss of the TSC2 tumor suppressor. Overall design: RNAseq profiling of human xenografts with TSC2 KO, TSC2/TFE3 KO, TSC2/TFEB KO, TSC2/TFE3/TFEB KO, and HEK293T WT genotypes. RNAseq of CRISPR-Cas9 TSC1/2 KO and WT HEK293T cells.