Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is required for activation of NF-kappaB in response to diverse cellular stresses - PubMed (original) (raw)

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 is required for activation of NF-kappaB in response to diverse cellular stresses

Hao-Yuan Jiang et al. Mol Cell Biol. 2003 Aug.

Abstract

Nuclear factor kappaB (NF-kappaB) serves to coordinate the transcription of genes in response to diverse environmental stresses. In this report we show that phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2) is fundamental to the process by which many stress signals activate NF-kappaB. Phosphorylation of this translation factor is carried out by a family of protein kinases that each respond to distinct stress conditions. During impaired protein folding and assembly in the endoplasmic reticulum (ER), phosphorylation of eIF2alpha by PEK (Perk or EIF2AK3) is essential for induction of NF-kappaB transcriptional activity. The mechanism by which NF-kappaB is activated during ER stress entails the release, but not the degradation, of the inhibitory protein IkappaB. During amino acid deprivation, phosphorylation of eIF2alpha by GCN2 (EIF2AK4) signals the activation of NF-kappaB. Furthermore, inhibition of general translation or transcription by cycloheximide and actinomycin D, respectively, elicits the eIF2alpha phosphorylation required for induction of NF-kappaB. Together, these studies suggest that eIF2alpha kinases monitor and are activated by a range of stress conditions that affect transcription and protein synthesis and assembly, and the resulting eIFalpha phosphorylation is central to activation of the NF-kappaB. The absence of NF-kappaB-mediated transcription and its antiapoptotic function provides an explanation for why eIF2alpha kinase deficiency in diseases such as Wolcott-Rallison syndrome leads to cellular apoptosis and disease.

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Figures

FIG. 1.

FIG. 1.

eIF2α kinase PEK is required for activation of NF-κB in response to ER stress. (A) PEK+/+ and PEK−/− MEF cells were exposed to thapsigargin for 0.25 to 3 h (as indicated) or in the absence of this ER stress agent (0 h). Phosphorylation of eIF2α was measured by immunoblot analysis by using polyclonal antibody specific to eIF2α phosphorylated at Ser-51 (eIF2α∼P). Levels of total eIF2α were assayed by using antibody that recognizes both phosphorylated and nonphosphorylated versions of the translation initiation factor. (i.e., eIF2α). Nuclear lysates were prepared from PEK+/+ (lanes 2 to 7) and PEK−/− (lanes 8 to 13) MEF cells treated with thapsigargin for the indicated times and incubated with radiolabeled DNA containing a NF-κB (B) or OCT1 (C) binding sites. Binding mixtures were separated by electrophoresis, and bound DNAs were visualized by autoradiography. Arrows indicate DNA complexed with p65/p50 or p50/p50 as defined in experiments shown in Fig. 2. OCT1 bound to DNA and an unknown protein complex are also indicated by arrows. Radiolabeled DNA at the bottom of panels B and C are unbound probe. Free probe (FP) indicates the radiolabeled NF-κB or OCT1 DNA fragments without nuclear lysate.

FIG. 2.

FIG. 2.

Phosphorylation of eIF2α at Ser-51 facilitates activation of NF-κB during ER stress. Nuclear lysates were prepared from MEF cells containing eIF2α with wild-type Ser-51 (S/S) or with alanine substituted for the eIF2α phosphorylation site (A/A) subjected to ER stress or to no stress. Equal amounts of nuclear lysate were used in each EMSA mixture containing radiolabeled DNA with an NF-κB binding site. Instances of DNA complexed with NF-κB dimers p65/p50 and p50/p50 (indicated by arrows) were visualized following autoradiography. (A) To determine the specificity for the NF-κB binding site, nonradiolabeled DNA containing the NF-κB site, URE (lanes 2 to 4), or the unrelated CREB DNA binding site (lanes 1 to 3) was added to EMSA binding mixtures containing nuclear lyates prepared from S/S MEF cells treated with thapsigargin for 6 h. Competition indicates that nonradiolabeled competitor DNA was added at a 1×, 10×, or 100× molar excess. Free probe (FP) indicates only radiolabeled NF-κB DNA fragments without nuclear lysate. Radiolabeled DNA at the bottom of panel is unbound probe. (B) Nuclear lysates were prepared from S/S (lanes 2 to 5) and A/A (lanes 6 to 9) MEF cells subjected to thapsigargin for between 0 and 6 h and assayed for NF-κB binding in the EMSA. In lanes 10 to 13, supershift indicates that polyclonal antibodies that specifically recognize p50 and/or p65 were added to the EMSA binding mixture. “None” indicates that no antibody was used in the assay (lane 10). (C) MEF cells were exposed to either thapsigargin (Tg) or tunicamycin (Tunc) as indicated by the “+” or “−” for the indicated number of hours. Nuclear lysates prepared from S/S and A/A MEF cells (as indicated) were analyzed for binding to the NF-κB probe in the EMSA.

FIG. 3.

FIG. 3.

Activation of NF-κB during amino acid conditions requires phosphorylation of eIF2α by GCN2 protein kinase. (A) GCN2+/+ and GCN2−/− MEF cells were deprived of leucine for between 1 and 6 h and subjected to thapsigargin for 1 h or to no stress (0 h) as indicated. Phosphorylation of eIF2α was assayed for by immunoblot analysis using antibody specific to eIF2α phosphorylated at Ser-51 (eIF2α∼P), and total eIF2α levels (eIF2α) were measured using antibody that recognizes both phosphorylated and nonphosphorylated versions of eIF2α. (B) Nuclear lysates were prepared from GCN2+/+ (lanes 2 to 5), GCN2−/− (lanes 6 to 9), S/S (lanes to 14), and A/A (lanes to 18) MEF cells deprived of leucine for the indicated number of hours and were assayed for binding with radiolabeled DNA containing a NF-κB binding site by the EMSA. (C) NF-κB binding was measured by the EMSA using nuclear lysates prepared from GCN2−/− and PEK−/− MEF cells that were starved for leucine or exposed to thapsigargin for the indicated number of hours. Arrows indicate DNA complexed with p65/p50 or p50/p50. Free probe (FP) indicates that only the radiolabeled NF-κB DNA was used in the assay, and the radiolabeled DNA at the bottom of panel B is unbound probe.

FIG. 4.

FIG. 4.

eIF2α kinase PEK is required for translocation of NF-κB into the nucleus during ER stress. (A and B) PEK+/+ MEF cells grown on glass slides were treated with thapsigargin for 6 h (Tg; panel A) or no stress (UT; panel B). Cells were prepared and the p65 subunit of NF-κB was visualized using rabbit polyclonal antibodies specific for this transcription factor, followed by goat anti-rabbit IgG conjugated with Rhodamine Red. NF-κB linked with Rhodamine Red was visualized by laser confocal microscopy. Cell nuclei were stained with DAPI mountain medium, and electronic images from the Rhodamine Red and DAPI were merged in the right figures. (C through F) PEK−/− cells transfected with a plasmid expressing PEK (PEK) in combination with plasmid encoding GFP were grown on glass slides. Transfected cells were exposed to thapsigargin (D and E) or were untreated (C). NF-κB was visualized by using the secondary antibody conjugated with Rhodamine Red, nuclear DNA by DAPI stain, and GFP by fluorescein isothiocyanate. All three panels were merged as indicated. PEK−/− cells transiently expressing PEK were identified by coexpression with GFP. The PEK−/− MEF cell in panel D was expressing no PEK or GFP and served as a control for eIF2α kinase dependence for NF-κB translocation to the nucleus during ER stress.

FIG. 5.

FIG. 5.

eIF2α kinase PEK is required for NF-κB transcriptional activity in response to ER stress. An NF-κB firefly luciferase reporter gene was cotransfected with a Renilla luciferase control plasmid into PEK+/+ (black bar) or PEK−/− (white bar) MEF cells. MEF cells were treated with from 0 to 2 μM thapsigargin for 6 h, and the dual luciferase assay was carried out as described in Materials and Methods. To address whether transient expression of PEK in the PEK−/− MEF cells restored NF-κB transcriptional activity, we cotransfected a PEK expression plasmid with the luciferase genes and subjected these cells to 2 μM thapsigargin (gray bar). Relative NF-κB-luciferase activity is presented in the histogram, which is normalized for PEK+/+ cells not subjected to thapsigargin treatment.

FIG. 6.

FIG. 6.

Activation of NF-κB in response to ER stress involves release but not proteolysis of IκB. S/S (lanes 1 to 7) and A/A (lanes 8 to 14) MEF cells were treated with thapsigargin for up to 6 h, with TNF-α for 30 min, or with no stress (0 h). Equal amounts of whole-cell lysates were separated by SDS-PAGE; phosphorylated eIF2α (A), total eIF2α (B), IκBα phosphorylated at Ser-32 (C), total levels of IκBα and IκBβ (D), and p65 (E) were measured by immunoblotting using specific antibodies. IκBα was immunoprecipitated and the levels of p65 (F) or IκBα (G) in the immunocomplexes were measured by immunoblot analysis. The IgG present in the immunocomplex migrated near p65 in the SDS-PAGE as indicated in panel F. Each panel is derived from one immunoblotting experiment, and the dotted line between lanes 7 and 8 is shown only for alignment purposes.

FIG. 7.

FIG. 7.

Reduced protein degradation impairs TNF-α-directed induction of NF-κB but does not reduce NF-κB activity during ER stress. PEK+/+ and PEK−/− MEF cells were treated with thapsigargin (Tg), TNF-α, and MG132 as indicated by the “+” or “−” symbols for the indicated number of hours or were subjected to no stress (0 h). Equal amounts of nuclear lysates prepared from MEF cells were analyzed for binding to the NF-κB probe by the EMSA. DNA complexed with p65/p50 or p50/p50 is indicated by arrows.

FIG. 8.

FIG. 8.

Activation of NF-κB in response to cycloheximide or actinomycin D exposure requires phosphorylation of eIF2α. (A) S/S MEF cells were exposed for the indicated number of hours to thapsigargin (Tg), actinomycin D (AD), or cycloheximide (CHX) as indicated or to no stress agent (0 h). Phosphorylation of eIF2α was measured by immunoblot analysis using polyclonal antibody specific to eIF2α phosphorylated at Ser-51 (eIF2α∼P). Levels of total eIF2α were assayed by using antibody that recognizes both phosphorylated and nonphosphorylated versions of the translation initiation factor (eIF2α). S/S and A/A MEF cells were exposed to thapsigargin, cycloheximide (B), or actinomycin D (C) as indicated by the “+” or “−” for the indicated number of hours. Nuclear lysates prepared from S/S and A/A MEF cells, as indicated, were analyzed for binding to the NF-κB probe in the EMSA. Arrows indicate DNA complexed with p65/p50 or p50/p50 in thapsigargin-stressed preparations or with p65/p65 in cycloheximide or actinomycin D-stressed cells as defined in experiments shown in panel D. In panel D, S/S MEF cells were treated with actinomycin D for 6 h and analyzed for binding with the NF-κB DNA. In lanes 1 to 4, supershift designates that antibodies that specifically recognize p50 and/or p65 were added to the EMSA binding mixture. “None” indicates that no antibody was added to the assay. In lanes 5 to 7, competition (Comp) indicates that nonradiolabeled NF-κB URE competitor DNA was added at a 10× or 100× molar excess or was not added (None). In lane 8, A/A MEF cells were similarly analyzed using actinomycin D.

FIG. 9.

FIG. 9.

p65 function is dispensable for increased expression of eIF2α kinase pathway genes in response to ER stress. p65 (RelA)+/+ (lanes 1 to 3) and p65−/− (lanes 4 to 6) MEF cells were exposed to thapsigargin (Tg) for 3 or 6 h or were subjected to no stress (0 h). Equal amounts of whole-cell lysates were separated by SDS-PAGE, and levels of p65, phosphorylated eIF2α, total eIF2α, ATF4, and Chop were measured by immunoblotting using antibodies specific to the indicated protein.

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