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. 2010 Apr;30(8):2006-16.
doi: 10.1128/MCB.01350-09. Epub 2010 Feb 12.

OGFOD1, a novel modulator of eukaryotic translation initiation factor 2alpha phosphorylation and the cellular response to stress

Karen A Wehner  1 , Sylvia Schütz, Peter Sarnow
Affiliations

OGFOD1, a novel modulator of eukaryotic translation initiation factor 2alpha phosphorylation and the cellular response to stress

Karen A Wehner et al. Mol Cell Biol. 2010 Apr.

Abstract

Cells possess mechanisms that permit survival and recovery from stress, several of which regulate the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). We identified the human OGFOD1 protein as a novel stress granule component that regulates the phosphorylation of eIF2alpha and the resumption of translation in cells recovering from arsenite-induced stress. Coimmunoprecipitation studies revealed that OGFOD1 associates with a small subset of stress granule proteins (G3BP1, USP10, Caprin1, and YB-1) and the ribosome in both unstressed and stressed cells. Overexpression of OGFOD1 led to increased abundance of phosphorylated eIF2alpha, both in unstressed cells and in cells exposed to arsenite-induced stress, and to accelerated apoptosis during stress. Conversely, knockdown of OGFOD1 resulted in smaller amounts of phosphorylated eIF2alpha and a faster accumulation of polyribosomes in cells recovering from stress. Finally, OGFOD1 interacted with both eIF2alpha and the eIF2alpha kinase heme-regulated inhibitor (HRI), which was identified as a novel stress granule resident. These findings argue that OGFOD1 plays important proapoptotic roles in the regulation of translation and HRI-mediated phosphorylation of eIF2alpha in cells subjected to arsenite-induced stress.

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Figures

FIG. 1.
FIG. 1.
OGFOD1 colocalizes with G3BP1 in arsenite-induced and thapsigargin-induced stress granules. Endogenous OGFOD1 and G3BP1 were detected by indirect immunofluorescence in untreated HeLa cells, cells treated with 1 mM arsenite for 30 min at 37°C, or cells treated with 10 μM thapsigargin for 1 h at 37°C. An overlap of OGFOD1 and G3BP1 appears yellow in the merged image. White arrows point to stress granules.
FIG. 2.
FIG. 2.
OGFOD1 interacts with specific stress granule proteins in HeLa cells. (A) OGFOD1 coimmunoprecipitates specific stress granule proteins. Extracts from untreated or arsenite-treated HeLa cells were subjected to immunoprecipitation (IP) with an antibody against OGFOD1. Proteins were separated by SDS-PAGE and detected by Western blot analysis using the indicated antibodies. Immunoprecipitation of Hif1α served as a control for nonspecific interactions. (B) OGFOD1 coimmunoprecipitation of G3BP1, USP10, and Caprin1 is not RNase A sensitive. Extracts from untreated HeLa cells were subjected to immunoprecipitation with an antibody against OGFOD1 and then incubated with RNase A (RNase A treated). Proteins were separated by SDS-PAGE and detected by Western blot analysis using the indicated antibodies. Beads without added antibody served as a control for nonspecific interactions.
FIG. 3.
FIG. 3.
OGFOD1 band 1 interacts with stress granule proteins and the ribosome. (A) Detection of multiple forms of OGFOD1 in HeLa lysates by Western blot analysis. (B) OGFOD1 band 1 coimmunoprecipitates with stress granule proteins and the ribosome. Immunoprecipitations were performed on HeLa cell lysates, using antibodies against YB-1, USP10, G3BP1, and the large ribosomal subunit 5.8S rRNA (Y10B). Beads without added antibody and a lamin A/C immunoprecipitation were used as controls for nonspecific interactions.
FIG. 4.
FIG. 4.
G3BP1 regulates OGFOD1 band 1 levels. (A) Depletion of G3BP1 by RNAi results in loss of OGFOD1 band 1. HeLa cells were treated with a negative-control siRNA (neg. cntrl.) and siRNAs against OGFOD1, G3BP1, or USP10 for 72 h prior to preparation of extracts. Endogenous proteins were detected by Western blot analysis. (B) Overexpression of G3BP1 leads to a larger amount of OGFOD1 band 1. HeLa cells were transiently transfected with a plasmid expressing a G3BP1 cDNA. Whole-cell protein extracts were prepared at 72 h posttransfection, separated by SDS-PAGE, and analyzed by Western blotting. (C) OGFOD1 band 1 is not degraded by serine and cysteine proteases during G3BP1 knockdown. HeLa cells were treated with a control siRNA (neg. cntrl.) and siRNAs against OGFOD1 and G3BP1 for 72 h. During the last 6 h of siRNA-mediated knockdown, cells were treated with normal medium (untreated) or medium containing a cocktail of serine and cysteine protease inhibitors (inhibitor). Endogenous proteins present in lysates were analyzed by Western blotting. Migration of novel OGFOD1 band 1.5 in the presence of protease inhibitors is indicated.
FIG. 5.
FIG. 5.
OGFOD1 regulates the phosphorylation of eIF2α during recovery from arsenite-induced stress. (A) Abundance of phosphorylated eIF2α decreases upon knockdown of OGFOD1 or G3BP1. Cells were treated with a negative-control siRNA (neg. cntrl.) and siRNAs against G3BP1 and OGFOD1 72 h prior to harvest. Extracts were prepared from unstressed cells (untreated), arsenite-treated cells, and cells recovering from arsenite-induced stress. Proteins were analyzed by Western blotting. (B) Abundance of phosphorylated eIF2α increases after overexpression of OGFOD1 or G3BP1. Extracts were prepared as described above from HeLa cells transiently transfected with an empty DNA vector (neg. cntrl.) or with plasmids expressing G3BP1 and OGFOD1 cDNAs and subjected to Western blot analysis. (C) Abundance of phosphorylated eIF2α in cells recovering from arsenite-induced stress changes upon knockdown or overexpression of OGFOD1 and G3BP1. Levels of phosphorylated eIF2α and total eIF2α were quantitated for three independent experiments, using Image J software. Representative Western blots are shown in panels A and B. Percent changes in phosphorylated eIF2α were calculated relative to total eIF2α levels and normalized to the negative control. Error bars represent the standard errors of the means.
FIG. 6.
FIG. 6.
OGFOD1 regulates formation of polysomes during recovery from arsenite-induced stress. Examination of polysome formation during stress recovery was performed by sucrose gradient sedimentation analysis. HeLa cells were treated with a negative-control siRNA (black line) or with an siRNA against OGFOD1 (red line) for a total of 72 h. Cells were treated with 1 mM arsenite for 30 min at 37°C and allowed to recover for the indicated times. Sucrose gradient analyses were performed as described in Materials and Methods. Absorbance peaks that correspond to the ribosomal 40S and 60S subunits as well as to 80S monosomes and polysomes are indicated. The Isco UA-6 detector was set to a sensitivity of 0.5 for all gradients except the arsenite-treated sample, which was collected at a sensitivity of 1.0. The asterisk indicates a user-initiated change in the UA-6 baseline setting so that all absorbance data could be retained.
FIG. 7.
FIG. 7.
OGFOD1 regulates survival of arsenite-induced stress. (A) Knockdown of OGFOD1 reduces accumulation of the 89-kDa PARP cleavage product in cells recovering from arsenite-induced stress. Cells were treated with a negative-control siRNA (neg. cntrl.) and siRNAs against G3BP1 or OGFOD1 72 h prior to harvest. Extracts were prepared from unstressed cells (untreated), arsenite-treated cells, and cells recovering from arsenite-induced stress. Proteins were analyzed by Western blotting. (B) Overexpression of OGFOD1 increases accumulation of the 35-kDa caspase-9 and 89-kDa PARP cleavage products in cells exposed to arsenite. Extracts were prepared as described above from HeLa cells transiently transfected with an empty DNA vector (neg. cntrl.) or with plasmids expressing G3BP1 and OGFOD1 cDNAs and subjected to Western blot analysis.
FIG. 8.
FIG. 8.
OGFOD1 interacts with eIF2α kinase HRI. Extracts from untreated or arsenite-treated HeLa cells were subjected to immunoprecipitation with an antibody against OGFOD1. Proteins were separated by SDS-PAGE and detected by Western blot analysis using the indicated antibodies. Beads without added antibody and Hif1α immunoprecipitation served as controls for nonspecific interactions.
FIG. 9.
FIG. 9.
OGFOD1 and HRI interact with eIF2α. (A) HRI colocalizes with G3BP1 in arsenite-induced stress granules. Endogenous HRI and G3BP1 were detected by indirect immunofluorescence in untreated HeLa cells or in cells treated with 1 mM arsenite for 30 min at 37°C. An overlap of HRI and G3BP1 appears white in the merged image. White arrows point to stress granules. (B) OGFOD1 band 2 coimmunoprecipitates with HRI. Extracts from untreated or arsenite-treated HeLa cells were subjected to immunoprecipitation with an antibody against HRI. Beads without added antibody and Hif1α immunoprecipitation were used as controls for nonspecific interactions. Proteins were separated by SDS-PAGE and detected by Western blot analysis using the indicated antibodies. (C) OGFOD1 and HRI coimmunoprecipitate eIF2α and phosphorylated eIF2α. Extracts from untreated or arsenite-treated HeLa cells were subjected to immunoprecipitation with antibodies against OGFOD1 and HRI. Beads without added antibody and Hif1α immunoprecipitation were used as controls for nonspecific interactions. Proteins were separated by SDS-PAGE and detected by Western blot analysis using the indicated antibodies.
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