Dynamin inhibitors block mTORC1 activation by amino acids independently of dynamin

mTORC1 plays a critical role in protein synthesis and cell proliferation and growth. It is activated by growth factors and amino acids, including essential amino acid (EAA), such as Leu; Leu enters cells via the Leu transporter LAT1-4F2hc and potentially via endocytosis. Here we investigated the contribution of the different routes of Leu entry into cells to mTORC1 activation using pharmacological inhibitors and cells that lack LAT1 or dynamin1/2/3. Our results show that LAT1 is the major route of Leu entry into cells and mTORC1 activation (∼70%), while dynamin-dependent endocytosis and macropinocytosis contribute minimally to both (5-15%). However, macropinocytosis contributes significantly (∼40%) to activation of mTORC1 by other EAAs. Surprisingly, the dynamin inhibitors dynasore or Dyngo 4A, which minimally inhibited Leu uptake, abolished mTORC1 activation independently of dynamin. Instead, dynasore inhibited RagA binding to Raptor, reduced mTORC1 recruitment to the lysosome, and inhibited Akt activation & TSC2-S939 phosphorylation; this resulted in inhibition of Rheb and mTORC1 activity. Our results suggest that these commonly used dynamin/endocytosis inhibitors are potent suppressors of mTORC1 activation via off-target effects and not via dynamin inhibition.