Complementary Roles of GADD34- and CReP-Containing Eukaryotic Initiation Factor 2α Phosphatases during the Unfolded Protein Response

Abstract

Phosphorylation of eukaryotic initiation factor 2α (eIF2α) controls transcriptome-wide changes in mRNA translation in stressed cells. While phosphorylated eIF2α (P-eIF2α) attenuates global protein synthesis, mRNAs encoding stress proteins are more efficiently translated. Two eIF2α phosphatases, containing GADD34 and CReP, catalyze P-eIF2α dephosphorylation. The current view of GADD34, whose transcription is stress induced, is that it functions in a feedback loop to resolve cell stress. In contrast, CReP, which is constitutively expressed, controls basal P-eIF2α levels in unstressed cells. Our studies show that GADD34 drives substantial changes in mRNA translation in unstressed cells, particularly targeting the secretome. Following activation of the unfolded protein response (UPR), rapid translation of GADD34 mRNA occurs and GADD34 is essential for UPR progression. In the absence of GADD34, eIF2α phosphorylation is persistently enhanced and the UPR translational program is significantly attenuated. This "stalled" UPR is relieved by the subsequent activation of compensatory mechanisms that include AKT-mediated suppression of PKR-like kinase (PERK) and increased expression of CReP mRNA, partially restoring protein synthesis. Our studies highlight the coordinate regulation of UPR by the GADD34- and CReP-containing eIF2α phosphatases to control cell viability.

FIG 1

Basal expression of GADD34 mRNA and protein. (A) mRNA levels, translational efficiency, and total translation of GADD34 mRNA in WT MEFs were assessed by ribosome profiling and RNA-seq. (B) GADD34 protein levels in unstressed WT MEFs and increased levels in MEFs subjected to ER stress (1 μM Tg for 6 h) were detected by immunoblotting with anti-GADD34 antibody. Lysates from unstressed and stressed GADD34^−/− MEFs lacked the immunoreactive band. (C) Levels of eIF2α phosphorylation in WT and GADD34^−/− MEFs were detected by immunoblotting with anti-P-eIF2α antibody. Total levels of eIF2α used as a loading control are also shown. (D) Box plot indicating the percentage of change in basal eIF2α phosphorylation in GADD34^−/− MEFs relative to WT MEFs assessed by immunoblotting and quantification by ImageJ (n = 5).

FIG 2

Regulation of protein synthesis by GADD34 in unstressed MEFs. (A) Comparison of total levels of mRNA translation, analyzed by ribosome profiling, in unstressed WT and unstressed GADD34^−/− MEFs (n = 2). The shaded area represents 5 standard deviations from the mean of results from WT biological replicates, with genes with significantly different results (at least 2-fold change and P < 0.05 by Student's t test [52]) highlighted in magenta (enhanced translation in the absence of GADD34) or green (suppressed translation in the absence of GADD34). (B) Percentages of changes in total translation in unstressed GADD34^−/− cells relative to WT cells attributable to changes in mRNA levels and translation efficiency. Percentages were calculated as described in Materials and Methods. (C) Relationship between changes in translation in unstressed GADD34^−/− cells relative to WT cells and changes in translation in WT cells after 30 min of induction of ER stress by the use of 1 μM Tg as reported previously (22). (D) Gene ontologies with changes in total translation in the absence of GADD34, with P values (p-val) determined by bootstrapping. (E) Histogram of differences in total translation between GADD34^−/− and WT MEFs for mRNAs encoding ER-targeted proteins (containing signal sequence or transmembrane domain) (red) and cytosolic proteins (green). (F) Moving averages were calculated for the proportions of mRNAs encoding ER-targeted proteins as a function of GADD34-mediated changes in translation. Enrichment among the highly suppressed and highly enhanced genes highlights mRNAs encoding ER-targeted proteins as particularly sensitive to loss of GADD34. (G) Cumulative density plot representing the fraction of each mRNA encoding ER-targeted proteins associated with ER in WT and GADD34^−/− cells.

FIG 3

Functions of GADD34 and CReP in early stages of UPR. (A) Translational activity in WT, GADD34^−/−, and CReP^−/− cells during Tg-induced ER stress, as measured by pulse-labeling with [³⁵S]Met-Cys (n = 3). Data for WT cells are reproduced from reference . max, maximum. (B) Time-dependent changes in GADD34 mRNA and translation in WT MEFs following exposure to 1 μM Tg, assessed by RNA-seq and ribosome profiling (n = 2).

FIG 4

UPR gene expression is suppressed in the absence of GADD34. (A) Levels of UPR-induced proteins analyzed by immunoblotting following Tg treatment of WT and GADD34^−/− MEFs. Tubulin is shown as a loading control. KO, knockout. (B and C) mRNA levels of CHOP (B) and ATF4 (C) were analyzed by quantitative PCR (qPCR) in MEFs following Tg exposure (n = 3). (D) Time-dependent changes in the expression of XBP1 protein in WT and GADD34^−/− MEFs following Tg treatment. (E) XBP1 splicing following Tg treatment of WT and GADD34^−/− MEFs was analyzed by exon-spanning PCR as described in Materials and Methods.

FIG 5

GADD34 is required for timely progression of the UPR program. (A) A heat map of changes in total translation following Tg treatment of WT and GADD34^−/− MEFs is shown. The mRNAs were sorted based on mean changes in translation levels across all time points and cell types. (B) Median changes in translation of 5% of the genes most highly translationally enhanced after 30 min of Tg treatment in WT cells (n = 2) are shown. (C) Translation responses for 5% of the genes most highly translationally enhanced in WT cells following 4 h of Tg treatment relative to the mean for untreated cells and cells treated with Tg for 30 min (n = 2). (D) Temporal changes in ER enrichment for translation of mRNAs encoding ER-targeted proteins are shown for Tg-treated WT, GADD34^−/− (n = 2), and CReP^−/− (n = 1) cells.

FIG 6

Loss of GADD34 function protects mice against tunicamycin-induced renal toxicity. WT and GADD34^−/− mice were injected intraperitoneally with Tm (1 mg/kg body weight) or DMSO as described in Materials and Methods. (A) Representative images (magnification, ×200) of kidney slices from WT and GADD34^−/− mice 4 days after Tm administration and after staining with H&E are shown. Scale bar, 200 μm; n = 4 to 12. (B) Paraffin-embedded sections of fixed mouse kidneys were stained with anti-cleaved caspase-3 antibody. Representative images (×200) show cells positive for cleaved caspase-3 (dark brown) at sites of renal lesions observed 4 days after Tm injection. Scale bar, 200 μm; n = 2 to 4. (C) Quantification of cleaved caspase-3 in mouse kidneys as a percentage of the image stained for cleaved caspase-3 using ImageJ. Error bars represent standard deviations (SD) (n = 4). Veh, vehicle. (D) An immunoblot of apoptotic markers in WT and GADD34^−/− MEFs 30 h after Tm (2 μg/ml) treatment is shown. (E) RNA was extracted from kidney samples and assayed by qPCR, normalizing against β-actin mRNA. ISR, integrated stress response.

FIG 7

Late recovery of UPR suppression in GADD34-null MEFs. (A) eIF2α phosphorylation and selected UPR proteins were analyzed by immunoblotting following Tg treatment in WT and GADD34^−/− MEFs. (B) Protein synthesis in MEFs treated with Tg was measured by puromycin labeling. MEFs were treated with Tg, and after 30 min with 10 μg/ml puromycin, lysates were analyzed by immunoblotting with an antipuromycin antibody. (C) Quantification of puromycin labeling used ImageJ (n = 3; error bars represent SD). (D) AKT and PERK phosphorylation in WT and GADD34^−/− MEFs is shown. (E and F) AKT and PERK phosphorylation was quantified by ImageJ in WT (E) and GADD34^−/− (F) MEFs (n = 3; error bars represent SD). (G) CReP mRNA levels were quantified by qRT-PCR following Tg treatment of WT and GADD34^−/− MEFs (n = 3; error bars represent SD). (H) Expression levels of GADD34 and CReP mRNA in human tissues from the Human Protein Atlas (38) are shown. Each point represents a different human tissue.