GADD34 Keeps the mTOR Pathway Inactivated in Endoplasmic Reticulum Stress Related Autophagy

PLoS One. 2016 Dec 16;11(12):e0168359. doi: 10.1371/journal.pone.0168359. eCollection 2016.

Abstract

The balance of protein synthesis and proteolysis (i.e. proteostasis) is maintained by a complex regulatory network in which mTOR (mechanistic target of rapamycin serine/threonine kinase) pathway and unfolded protein response are prominent positive and negative actors. The interplay between the two systems has been revealed; however the mechanistic details of this crosstalk are largely unknown. The aim of the present study was to investigate the elements of crosstalk during endoplasmic reticulum stress and to verify the key role of GADD34 in the connection with the mTOR pathway. Here, we demonstrate that a transient activation of autophagy is present in endoplasmic reticulum stress provoked by thapsigargin or tunicamycin, which is turned into apoptotic cell death. The transient phase can be characterized by the elevation of the autophagic marker LC3II/I, by mTOR inactivation, AMP-activated protein kinase activation and increased GADD34 level. The switch from autophagy to apoptosis is accompanied with the appearance of apoptotic markers, mTOR reactivation, AMP-activated protein kinase inactivation and a decrease in GADD34. Inhibition of autophagy by 3-methyladenine shortens the transient phase, while inhibition of mTOR by rapamycin or resveratrol prolongs it. Inhibition of GADD34 by guanabenz or transfection of the cells with siGADD34 results in down-regulation of autophagy-dependent survival and a quick activation of mTOR, followed by apoptotic cell death. The negative effect of GADD34 inhibition is diminished when guanabenz or siGADD34 treatment is combined with rapamycin or resveratrol addition. These data confirm that GADD34 constitutes a mechanistic link between endoplasmic reticulum stress and mTOR inactivation, therefore promotes cell survival during endoplasmic reticulum stress.

MeSH terms

  • Autophagy* / drug effects
  • Autophagy* / genetics
  • Cell Survival / drug effects
  • Cell Survival / genetics
  • Cells, Cultured
  • Down-Regulation / drug effects
  • Down-Regulation / physiology
  • Endoplasmic Reticulum Stress / drug effects
  • Endoplasmic Reticulum Stress / physiology*
  • HEK293 Cells
  • Hep G2 Cells
  • Humans
  • Protein Phosphatase 1 / antagonists & inhibitors
  • Protein Phosphatase 1 / genetics
  • Protein Phosphatase 1 / physiology*
  • RNA, Small Interfering / pharmacology
  • Signal Transduction / drug effects
  • Signal Transduction / physiology
  • TOR Serine-Threonine Kinases / metabolism*

Substances

  • RNA, Small Interfering
  • MTOR protein, human
  • TOR Serine-Threonine Kinases
  • PPP1R15A protein, human
  • Protein Phosphatase 1

Grant support

Support was provided by János Bolyai Research Scholarship of the Hungarian Academy of Sciences (to OK); Hungarian Scientific Research Fund (OTKA-PD 104110 and OTKA 112696); and MedInProt grant of the Hungarian Academy of Sciences.