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Abstract
Transient protein synthesis inhibition is an important protective mechanism used by cells during various stress conditions including endoplasmic reticulum (ER) stress. This response centers on the phosphorylation state of eukaryotic initiation factor (eIF)-2 alpha, which is induced by kinases like protein kinase R-like ER kinase (PERK) and GCN2 to suppress translation and is later reversed so translation resumes. GADD34 was recently identified as the factor that activates the type 1 protein serine/threonine phosphatase (PP1), which dephosphorylates eIF-2 alpha during cellular stresses. Our study delineates a negative feedback regulatory loop in which the eIF-2 alpha-controlled inhibition of protein translation leads to GADD34 induction, which promotes translational recovery. We show that activating transcription factor-4 (ATF4), which is paradoxically translated during the eIF-2 alpha-mediated translational block, is required for the transactivation of the GADD34 promoter in response to ER stress and amino acid deprivation. ATF4 directly binds to and trans-activates a conserved ATF site in the GADD34 promoter during ER stress. Examination of ATF4-/- MEFs revealed an absence of GADD34 induction, prolonged eIF-2 alpha phosphorylation, delayed protein synthesis recovery, and diminished translational up-regulation of BiP during ER stress. These studies demonstrate the essential role of GADD34 in the resumption of protein synthesis, define the pathway for its induction, and reveal that cytoprotective unfolded protein response targets like BiP are sensitive to the eIF-2 alpha-mediated block in translation.
Highlights
We show that activating transcription factor-4 (ATF4), which is paradoxically translated during the eukaryotic initiation factor (eIF)-2␣-mediated translational block, is required for the transactivation of the GADD34 promoter in response to endoplasmic reticulum (ER) stress and amino acid deprivation
Isolation and Analysis of ATF4 Null Mouse Embryonic Fibroblasts—Recently, the stress-induced GADD34 protein was shown to bind to the PP1 phosphatase and activate the dephosphorylation of eIF-2␣, thereby triggering the resumption of protein translation during cellular stresses (29 –31)
Induction of GADD34 during ER stress and amino acid deprivation stress did not occur in PERKϪ/Ϫ or GCN2Ϫ/Ϫ cells, respectively, which are defective in the initial block in protein synthesis during ER stress or amino acid deprivation [29]
Summary
ATF4 null MEFs are unable to induce GADD34 mRNA during ER or other cellular stresses, which leads to prolonged eIF-2␣ phosphorylation and diminished protein synthesis recovery. Our data revealed that BiP up-regulation at the translational level is significantly diminished in ATF4 null cells where protein synthesis inhibition persists, demonstrating that the cytoprotective UPR targets are not immune to the eIF-2␣-dependent translational inhibition and underscore the importance of translational recovery early in the response.
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