To confirm the previously reported role of eIF2α phosphorylation in NF-κB activation (21
), we performed EMSA on nuclear extracts prepared from unstressed cells and cells that had been treated with thapsigargin (Fig. ). Thapsigargin-mediated ER calcium depletion leads to rapid onset of ER stress, eIF2α phosphorylation (detected here by immunoblotting with an antiserum specifically reactive with the phosphorylated form), and subsequent ATF4-mediated activation of downstream gene expression, measured here by accumulation of the GADD34 target gene. A protein complex rapidly formed on the NF-κB binding site in nuclear extracts of treated wild-type cells but not in extracts from cells homozygous for the EIF2AA/A
mutation that substitutes the serine at position 51 of eIF2α with an alanine and thereby prevents regulatory phosphorylation. Reduced levels of the NF-κB inhibitory protein IκBα, detected by immunoblotting, preceded the induction of NF-κB EMSA activity in thapsigargin-treated cells. The recovery of IκBα levels at longer treatment points correlated with the induction of the GADD34 phosphatase and the dephosphorylation of eIF2α (Fig. ).
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 (more ...)
To more closely examine the role of eIF2α phosphorylation in NF-κB activation, we made use of an experimental system that uncouples eIF2α phosphorylation from stress signaling. PERK, the ER stress-inducible eIF2α kinase, is normally activated by oligomerization in the plane of the ER membrane (2
). We fused PERK's eIF2α kinase domain to a protein module with two high-affinity binding sites for the otherwise inert bivalent compound AP20187. When expressed in cells, this artificial kinase, Fv2E-PERK, is subordinate to AP20187 treatment (28
) and is activated independently of any stress signaling. AP20187 treatment led to high-level eIF2α phosphorylation in Fv2E-PERK+
cells but had no effect on the parental cells lacking the artificial kinase (Fig. ). Fv2E-PERK was readily activated in mutant EIF2AA/A
cells, but this predictably failed to induce eIF2α phosphorylation. EMSA of nuclear extracts showed that AP20187 induced NF-κB activity in Fv2E-PERK+
) cells but not in the mutant EIF2AA/A
cells (Fig. ). Homologous competition binding assays and antibody supershift experiments confirmed the identity of the NF-κB protein-DNA complex detected in the assay (Fig. ).
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 (more ...)
To gauge the functional significance of Fv2E-PERK-mediated eIF2α phosphorylation and activation of NF-κB DNA binding activity, we measured the activity of a transfected reporter gene driven by four copies of a wild-type NF-κB binding site. A brief (60-min) pulse of AP20187 induced marked activation of the wild-type reporter gene (measured 24 h later [Fig. ]). No activation of a reporter gene driven by mutant NF-κB sites was observed. In addition, endogenous NF-κB target genes, such as those encoding the major histocompatibility complex heavy chains (H2-Q8, H2-2KF, H2-K2, and H2-D1) and β2 microglobulin (Qb-1), were induced in the Fv2E-PERK+ cells by AP20187 treatments and in wild-type mouse fibroblasts by exposure to tunicamycin (National Center for Biotechnology Information Gene Expression Omnibue [GEO] data set GDS405).
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 (more ...)
Fv2E-PERK-mediated eIF2α phosphorylation and NF-κB activation correlated with a time-dependent decrease in IκBα levels that was not observed in the mutant EIF2AA/A
cells (Fig. ). Interestingly, Fv2E-PERK activation had no measurable effect on levels of the p65 NF-κB subunit, which is consistent with the known stability of that protein (24
) and with the induction of NF-κB binding activity that we observe. eIF2α levels were similarly stable, attesting to the effect's specificity to IκBα (Fig. ). Canonical activators of NF-κB access signal transduction pathways that promote phosphorylation of the inhibitor IκBα on serines 32 and 36 (38
). A ubiquitin ligase complex recognizes the phosphorylated form of IκBα, and polyubiquitinated IκBα is degraded by the proteasome. Fv2E-PERK activation by AP20187 did not promote a measurable increase in levels of phosphorylated IκBα, which remained undetectable. However, phosphorylated IκBα was readily detectable in lysates of cells treated with the proteasome inhibitor, MG132, which stabilizes the phosphorylated form of the protein (Fig. ).
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 (more ...)
Because it is rapidly degraded, signal-dependent accumulation of phosphorylated IκBα is difficult to detect, rendering an Fv2E-PERK-mediated increase in IκBα phosphorylation potentially easy to miss. Therefore, to determine if the eIF2α phosphorylation-dependent decline in IκBα levels correlated with any increased phosphorylation on serines 32 and 36, we exposed the AP20187-treated cells to the proteasome inhibitor MG132. As expected, proteasome inhibition markedly increased the levels of phosphorylated IκBα in tumor necrosis factor alpha-treated cells (Fig. ). Interestingly, proteasome inhibition led to only modest stabilization of total IκBα, an observation that is consistent with the existence of proteasome-independent mechanisms for IκBα degradation (5
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; (more ...)
MG132 treatment led to a progressive increase in phosphorylated IκBα levels in cells that were otherwise unperturbed (Fig. , compare lanes 1 and 3, and B, compare lane 1 with lanes 2, 4, 6, 8, and 10). This observation is consistent with a relatively high basal phosphorylation-dependent turnover of IκBα in these cells. The decline in IκBα levels effected by Fv2E-PERK was only slightly attenuated by proteasome inhibition (compare Fig. , lanes 4 to 6, with 5B, lanes 7, 9, and 11). Furthermore, proteasome inhibition promoted some eIF2α phosphorylation (Fig. , lanes 8 and 10), presumably mediated by proteotoxic stress. Remarkably, however, Fv2E-PERK activation and eIF2α phosphorylation not only failed to increase IκBα phosphorylation but also significantly attenuated the accumulation of phosphorylated IκBα in proteasome-inhibited cells (Fig. , compare odd- and even-numbered lanes). These observations indicate that eIF2α phosphorylation does not activate NF-κB by accessing one of the canonical IκBα phosphorylation-promoting pathways and must use a different mechanism.
The original descriptions of IκB emphasized the lability of the factor, as translational inhibitors were noted to promote NF-κB DNA binding activity (1
). Given that eIF2α phosphorylation also inhibits protein synthesis, we decided to explore this facet of NF-κB activation in more detail. NF-κB DNA binding activity was increased by cycloheximide treatment of wild-type cells, as previously reported (42
), and this correlated with reduced levels of the inhibitor, IκBα (Fig. ). Cycloheximide treatment led to no measurable decrease in p65 or eIF2α protein levels, attesting to the stability of these proteins. The effects of cycloheximide on levels of phosphorylated IκBα also resembled those of Fv2E-PERK activation (Fig. ) in that no increase in the phosphorylated protein was observed in cells treated with cycloheximide alone. Proteasome inhibitor, by itself, led to a progressive increase in levels of phosphorylated IκBα, whereas the addition of cycloheximide strongly attenuated this increase (Fig. ).
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 (more ...)
As previously noted, cycloheximide treatment induced eIF2α phosphorylation (21
) (Fig. ), an effect that might be attributed to loss of the labile eIF2α phosphatase CReP (22
). To study the role of eIF2α phosphorylation in cycloheximide-mediated activation of NF-κB, we treated mutant EIF2AA/A
cells with the protein synthesis inhibitor and studied NF-κB activation by EMSA and IκBα levels by immunoblotting. The EIF2AA/A
genotype, which inhibits regulatory phosphorylation of eIF2α, had no measurable effect on NF-κB activation, IκBα phosphorylation, or total IκBα levels in cycloheximide-treated cells (Fig. ). These observations suggest that inhibition of new protein synthesis can adequately explain the effects of cycloheximide on NF-κB activity without evoking an additional role for eIF2α phosphorylation.
Induced degradation of IκBα plays an important role in canonical activation of NF-κB. To address the possibility that eIF2α phosphorylation might affect this aspect of IκBα metabolism (independently of IκBα phosphorylation), we performed pulse-chase labeling experiments, tracking the fate of newly synthesized IκBα. The basal turnover of IκBα in murine fibroblasts proved very high. Less than 30% of the signal measured at the end of the 10-min labeling pulse was present after a 20-min chase. Furthermore, activation of Fv2E-PERK during the chase had no measurable effect on the decay of the IκBα signal (Fig. ). Addition of proteasome inhibitor during the chase stabilized IκBα somewhat; however, in that context, too, activation of Fv2E-PERK during the chase did not accelerate IκBα degradation and may have even contributed modestly to its stability (Fig. ). We conclude that IκBα turns over rapidly in murine fibroblasts and that eIF2α phosphorylation does not exert its effects on the levels of the inhibitor by further enhancing its degradation.
FIG. 7. eIF2α phosphorylation inhibits synthesis of IκBα but does not destabilize the preexisting protein. (A) Autoradiogram of IκBα immunoprecipitated from wild-type (EIF2AS/S) Fv2E-PERK+ mouse fibroblasts following (more ...)
Next we compared the rates of synthesis of IκBα in untreated cells with those in cells treated with AP20187, cycloheximide, the ER stress-promoting agent thapsigargin, and the canonical NF-κB activator TNF-α. The amount of radiolabeled IκBα immunoprecipitated with a specific antibody following a short labeling pulse was markedly diminished by activation of the eIF2α kinase Fv2E-PERK by AP20187, by treatment with cycloheximide, or by exposure to conditions that cause ER stress (thapsigargin) (Fig. ). The effect of thapsigargin on IκBα synthesis depended on eIF2α phosphorylation, since it was abolished in the EIF2AA/A mutant cells (Fig. ), and the decline in IκBα synthesis paralleled the global inhibition in protein synthesis in the cells exposed to conditions promoting eIF2α phosphorylation (Fig. ). By contrast, exposure to the canonical NF-κB activator, TNF-α, increased IκBα synthesis, suggesting a completely different mechanism of action. These observations are consistent with a role for inhibited synthesis of IκBα in mediating the effects of eIF2α phosphorylation on NF-κB activation both in ER-stressed cells and following activation of Fv2E-PERK.