Coordinated Regulation of the Neutral Amino Acid Transporter SNAT2 and the Protein Phosphatase Subunit GADD34 Promotes Adaptation to Increased Extracellular Osmolarity

Dawid Krokowski,Raul Jobava,Bo-Jhih Guan,Kenneth Farabaugh,Jing Wu,Mithu Majumder,Massimiliano G Bianchi,Martin D Snider,Ovidio Bussolati,Maria Hatzoglou

doi:10.1074/jbc.m114.636217

Abstract

Cells respond to shrinkage induced by increased extracellular osmolarity via programmed changes in gene transcription and mRNA translation. The immediate response to this stress includes the induction of expression of the neutral amino acid transporter SNAT2. Increased SNAT2-mediated uptake of neutral amino acids is an essential adaptive mechanism for restoring cell volume. In contrast, stress-induced phosphorylation of the α subunit of the translation initiation factor eIF2 (eIF2α) can promote apoptosis. Here we show that the response to mild hyperosmotic stress involves regulation of the phosphorylation of eIF2α by increased levels of GADD34, a regulatory subunit of protein phosphatase 1 (PP1). The induction of GADD34 was dependent on transcriptional control by the c-Jun-binding cAMP response element in the GADD34 gene promoter and posttranscriptional stabilization of its mRNA. This mechanism differs from the regulation of GADD34 expression by other stresses that involve activating transcription factor 4 (ATF4). ATF4 was not translated during hyperosmotic stress despite an increase in eIF2α phosphorylation. The SNAT2-mediated increase in amino acid uptake was enhanced by increased GADD34 levels in a manner involving decreased eIF2α phosphorylation. It is proposed that the induction of the SNAT2/GADD34 axis enhances cell survival by promoting the immediate adaptive response to stress.

Highlights

Increased phosphorylation of the translation initiation factor eIF2␣ promotes apoptosis during hyperosmotic stress
GADD34 Is Induced during Hyperosmotic Stress in an ATF4independent Manner—Exposure of Mouse embryonic fibroblasts (MEFs) to mild hyperosmotic stress (500 mosmol) led to an initial (1-h) decrease in the rates of protein synthesis followed by gradual recovery (Fig. 1A)
Signaling pathways known to regulate mRNA translation were modified by stress. eIF2␣ was phosphorylated rapidly, followed by dephosphorylation after 1.5 h of treatment (Fig. 1B). p70S6K, the protein kinase activated by mTOR, and the downstream target, the ribosomal protein S6, were transiently dephosphorylated (Fig. 1B)

Summary

Introduction

Increased phosphorylation of the translation initiation factor eIF2␣ (eIF2␣-P) promotes apoptosis during hyperosmotic stress. Increased SNAT2-mediated uptake of neutral amino acids is an essential adaptive mechanism for restoring cell volume. We show that the response to mild hyperosmotic stress involves regulation of the phosphorylation of eIF2␣ by increased levels of GADD34, a regulatory subunit of protein phosphatase 1 (PP1). The induction of GADD34 was dependent on transcriptional control by the c-Jun-binding cAMP response element in the GADD34 gene promoter and posttranscriptional stabilization of its mRNA. This mechanism differs from the regulation of GADD34 expression by other stresses that involve activating transcription factor 4 (ATF4). The SNAT2-mediated increase in amino acid uptake was enhanced by increased GADD34 levels in a manner involving decreased eIF2␣ phosphorylation. It is proposed that the induction of the SNAT2/ GADD34 axis enhances cell survival by promoting the immediate adaptive response to stress

Methods

Results

Conclusion