Translational repression mediates activation of nuclear factor kappa B by phosphorylated translation initiation factor 2

Abstract

Numerous stressful conditions activate kinases that phosphorylate the alpha subunit of translation initiation factor 2 (eIF2alpha), thus attenuating mRNA translation and activating a gene expression program known as the integrated stress response. It has been noted that conditions associated with eIF2alpha phosphorylation, notably accumulation of unfolded proteins in the endoplasmic reticulum (ER), or ER stress, are also associated with activation of nuclear factor kappa B (NF-kappaB) and that eIF2alpha phosphorylation is required for NF-kappaB activation by ER stress. We have used a pharmacologically activable version of pancreatic ER kinase (PERK, an ER stress-responsive eIF2alpha kinase) to uncouple eIF2alpha phosphorylation from stress and found that phosphorylation of eIF2alpha is both necessary and sufficient to activate both NF-kappaB DNA binding and an NF-kappaB reporter gene. eIF2alpha phosphorylation-dependent NF-kappaB activation correlated with decreased levels of the inhibitor IkappaBalpha protein. Unlike canonical signaling pathways that promote IkappaBalpha phosphorylation and degradation, eIF2alpha phosphorylation did not increase phosphorylated IkappaBalpha levels or affect the stability of the protein. Pulse-chase labeling experiments indicate instead that repression of IkappaBalpha translation plays an important role in NF-kappaB activation in cells experiencing high levels of eIF2alpha phosphorylation. These studies suggest a direct role for eIF2alpha phosphorylation-dependent translational control in activating NF-kappaB during ER stress.

FIG. 1.

NF-κB activation during ER stress depends on eIF2α phosphorylation and is associated with declining levels of the NF-κB inhibitor IκBα. (A) Autoradiogram of an NF-κB EMSA performed with nuclear extracts of thapsigargin-treated mouse fibroblasts (Tg) with wild-type (EIF2A^S/S) or mutant (EIF2A^A/A) EIF2A genotypes. The free radiolabeled probe and the labeled NF-κB/DNA complex are indicated. (B) Immunoblots of IκBα, GADD34, phosphorylated eIF2α, and total eIF2α from extracts of the cells shown in panel A, detected with specific antibodies.

FIG. 2.

Phosphorylation of eIF2α on serine 51 is sufficient to activate NF-κB DNA binding activity in vivo. (A) Immunoblots of ligand-activable Fv2E-PERK (upper panel), phosphorylated eIF2α (P-eIF2α; middle panel), and total eIF2α (lower panel) in extracts of mouse fibroblasts of wild-type (S/S) and EIF2A^A/A mutant (A/A) genotypes that do and do not stably express the chimeric eIF2α kinase, Fv2E-PERK. Where indicated, the cells had been treated with the Fv2E ligand, AP20187. Endogenous PERK is not detected at this exposure. The asterisk marks the position of a nonspecific band reactive with the anti-PERK sera. (B) Autoradiogram of an NF-κB EMSA performed with nuclear extracts of cells treated as described for panel A. (C) Autoradiogram of an NF-κB EMSA with nuclear extract obtained from AP20187-treated Fv2E-PERK⁺ cells performed in the presence of the indicated excess of an unlabeled homologous competitor oligonucleotide (left panel) or in the presence of antisera to CHOP (a negative control) or p65 (a component of the NF-κB DNA binding complex) (right panel). The positions of the free radiolabeled probe, the NF-κB/DNA complex, and antiserum supershifted complex are indicated.

FIG. 3.

eIF2α phosphorylation is sufficient to activate an NF-κB reporter gene. The activity of a transiently transfected reporter gene consisting of a minimal promoter driven by four wild-type (wt) or mutant (mut) NF-κB binding sites in mouse fibroblasts stably expressing Fv2E-PERK is shown following treatment with the indicated concentration of the activating ligand AP20187. The results are expressed as relative light units, and the activity of the reporter in untreated cells is arbitrarily set at 1. Shown are means and standard errors of the means of results from an experiment performed in triplicate and reproduced twice.

FIG. 4.

eIF2α phosphorylation reduces cellular levels of IκBα. (A) Immunoblots of total IκBα (upper panel), phosphorylated eIF2α (P-eIF2α; middle panel), and total eIF2α (lower panel) in extracts of wild-type (EIF2A^S/S) and mutant (EIF2A^A/A) Fv2E-PERK⁺ mouse fibroblasts following treatment with the activating ligand AP20187 for the indicated periods of time. (B) Immunoblots of total IκBα, phosphorylated IκBα (P-IκBα), p65 NF-κB subunit, and total eIF2α in extracts of wild-type (EIF2A^S/S) Fv2E-PERK⁺ mouse fibroblasts following treatment with the activating ligand AP20187 or the proteasome inhibitor (MG132) for the indicated periods of time.

FIG. 5.

Reduction in levels of IκBα in cells with elevated eIF2α phosphorylation occurs independently of IκBα phosphorylation. (A) Immunoblots of IκBα phosphorylated on serines 32 and 36 (P-IκBα; upper panel) and total IκBα (lower panel) in extracts of mouse fibroblasts treated with TNF-α and/or the proteasome inhibitor MG132. (B) Immunoblots of phosphorylated IκBα (P-IκBα), total IκBα, phosphorylated eIF2α (P-eIF2α), and total eIF2α in extracts of Fv2E-PERK⁺ mouse fibroblasts treated with the activating ligand AP20187 and/or the proteasome inhibitor MG132 for the indicated periods of time.

FIG. 6.

Reduction in levels of IκBα in cells treated with the protein synthesis inhibitor cycloheximide occurs independently of IκBα phosphorylation or eIF2α phosphorylation. (A) The top panel is an autoradiogram of an NF-κB EMSA from nuclear extracts of untreated and cycloheximide (CHX)-treated mouse fibroblasts. The lower panels are immunoblots (IB) of total IκBα, phosphorylated IκBα (P-IκBα), phosphorylated eIF2α (P-eIF2α), total eIF2α, and the p65 NF-κB subunit from the same cells. (B) Immunoblots of phosphorylated IκBα (P-IκBα), total IκBα, and total eIF2α in extracts of wild-type (EIF2A^S/S) Fv2E-PERK⁺ mouse fibroblasts treated with cycloheximide and/or the proteasome inhibitor MG132 for the indicated periods of time are shown. (C) The same assays as shown in panels A and B were conducted with mutant (EIF2A^A/A) Fv2E-PERK⁺ cells.

FIG. 7.

eIF2α phosphorylation inhibits synthesis of IκBα but does not destabilize the preexisting protein. (A) Autoradiogram of IκBα immunoprecipitated from wild-type (EIF2A^S/S) Fv2E-PERK⁺ mouse fibroblasts following a brief, 10-min [³⁵S]methionine- and cysteine-labeling pulse and cold chase of the indicated duration. The chase was conducted in the presence or absence of the activating ligand AP20187. The IκBα signal intensity is expressed as a fraction of that present at the end of the labeling pulse and is depicted beneath each lane. (B) Same assay as shown in panel A except that the proteasome inhibitor, MG132, was included during the chase where indicated. (C) Autoradiogram of the radiolabeled IκBα present at the end of the 10-min labeling pulse in wild-type (EIF2A^S/S) or mutant (EIF2A^A/A) Fv2E-PERK⁺ mouse fibroblasts treated with the indicated concentration of AP20187 ligand (in nM), cycloheximide (in μg/ml), thapsigargin (in μM), or TNF-α (in ng/ml) starting 30 min before and continuing throughout the pulse. (D) Autoradiogram ([³⁵S]methionine) of equal fractions of the cell lysates used in panel C. The right panel is of a gel that was run longer than the left panel, accounting for differences in appearance of the two. (E) Coomassie stain of the gels shown in panel D.