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.
Highlights
Choosing between life and death is one of the most important tasks of cells building up an organism
Endoplasmic reticulum (ER) stress transiently activates a pro-survival mechanism characterized by a negative balance of proteostasis, while severe stress leads to apoptotic cell death
We previously confirmed by using both theoretical and experimental techniques that ER stress induced apoptotic cell death is always preceded by autophagy-dependent survival
Summary
Choosing between life and death is one of the most important tasks of cells building up an organism. GADD34 Keeps mTOR Inactivated in ER Stress Related Autophagy environment, has key functions in synthesising and packaging secreted and membrane proteins, metabolism (such as lipid biosynthesis and carbohydrate metabolism) and several signalling processes. For these integrated roles of ER a special luminal homeostasis is required; its imbalance might result in ER stress [1,2,3]. ER stress transiently activates a pro-survival mechanism characterized by a negative balance of proteostasis (i.e. decreased translation and increased proteolysis by ERAD and autophagy), while severe stress leads to apoptotic cell death. ER stress dependent processes are regulated by a coordinated systems-level crosstalk between the complex regulatory networks of two signalling pathways [5], the unfolded protein response (UPR) [6] and the mTOR pathway [7]
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