Dynamin inhibitors block activation of mTORC1 by amino acids independently of dynamin

J Cell Sci. 2018 Jan 4;131(1):jcs211755. doi: 10.1242/jcs.211755.

Abstract

mTORC1 plays a crucial role in protein synthesis and cell proliferation and growth. It is activated by growth factors and amino acids, including essential amino acids (EAAs), such as leucine; Leu enters cells via the Leu transporter LAT1-4F2hc (also known as SLC7A5-SLC3A2) 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 dynamin-1, -2 and -3. Our results show that LAT1 is the major route of Leu entry into cells and mTORC1 activation (∼70%), whereas 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 and 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 and TSC2-S939 phosphorylation; this resulted in inhibition of Rheb and mTORC1 activity. Our results suggest that these commonly used inhibitors of dynamin and endocytosis are potent suppressors of mTORC1 activation via off-target effects and not via dynamin inhibition.This article has an associated First Person interview with the first author of the paper.

Keywords: Amino acids; Dynamin; Endocytosis; Leu transporter; mTORC1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Transport
  • Cell Line, Tumor
  • Dynamins / antagonists & inhibitors
  • Dynamins / metabolism*
  • Endocytosis*
  • Fusion Regulatory Protein 1, Heavy Chain / metabolism*
  • Humans
  • Hydrazones / pharmacology
  • Large Neutral Amino Acid-Transporter 1 / metabolism*
  • Leucine / metabolism*
  • Lysosomes / metabolism
  • Mechanistic Target of Rapamycin Complex 1 / drug effects
  • Mechanistic Target of Rapamycin Complex 1 / metabolism*
  • Monomeric GTP-Binding Proteins
  • Naphthols / pharmacology

Substances

  • Fusion Regulatory Protein 1, Heavy Chain
  • Hydrazones
  • Large Neutral Amino Acid-Transporter 1
  • N'-(3,4-dihydroxybenzylidene)-3-hydroxy-2-naphthahydrazide
  • Naphthols
  • SLC3A2 protein, human
  • dyngo-4a
  • Mechanistic Target of Rapamycin Complex 1
  • RRAGA protein, human
  • Monomeric GTP-Binding Proteins
  • Dynamins
  • Leucine