MOLECULAR AND CELLULAR BIOLOGY, Dec. 2004, p. 10161–10168
0270-7306/04/$08.00?0 DOI: 10.1128/MCB.24.23.10161–10168.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 23
Translational Repression Mediates Activation of Nuclear Factor
Kappa B by Phosphorylated Translation Initiation Factor 2
Jing Deng,1Phoebe D. Lu,1Yuhong Zhang,1Donalyn Scheuner,2Randal J. Kaufman,2,3
Nahum Sonenberg,4Heather P. Harding,5and David Ron1*
Skirball Institute of Biomolecular Medicine1and Departments of Cell Biology, Medicine, and Pharmacology,5
New York University School of Medicine, New York, New York; Department of Biochemistry2and Howard
Hughes Medical Institute,3University of Michigan School of Medicine, Ann Arbor, Michigan;
and Department of Biochemistry, McGill University, Montreal, Quebec, Canada4
Received 11 May 2004/Returned for modification 6 July 2004/Accepted 31 August 2004
Numerous stressful conditions activate kinases that phosphorylate the alpha subunit of translation initia-
tion factor 2 (eIF2?), thus attenuating mRNA translation and activating a gene expression program known as
the integrated stress response. It has been noted that conditions associated with eIF2? 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-?B) and that eIF2? phosphorylation is required for NF-?B
activation by ER stress. We have used a pharmacologically activable version of pancreatic ER kinase (PERK,
an ER stress-responsive eIF2? kinase) to uncouple eIF2? phosphorylation from stress and found that
phosphorylation of eIF2? is both necessary and sufficient to activate both NF-?B DNA binding and an NF-?B
reporter gene. eIF2? phosphorylation-dependent NF-?B activation correlated with decreased levels of the
inhibitor I?B? protein. Unlike canonical signaling pathways that promote I?B? phosphorylation and degra-
dation, eIF2? phosphorylation did not increase phosphorylated I?B? levels or affect the stability of the protein.
Pulse-chase labeling experiments indicate instead that repression of I?B? translation plays an important role
in NF-?B activation in cells experiencing high levels of eIF2? phosphorylation. These studies suggest a direct
role for eIF2? phosphorylation-dependent translational control in activating NF-?B during ER stress.
Diverse stressful conditions lead to the phosphorylation of
translation initiation factor 2 on its alpha subunit (eIF2?).
Phosphorylated eIF2 inhibits its guanine nucleotide exchange
factor, eIF2B, and thereby inhibits the exchange reaction re-
quired to generate active GTP-bound eIF2. As a consequence,
regulated phosphorylation of eIF2? serves to modulate mRNA
translation rates (18, 20). In addition to its negative impact on
global protein synthesis, eIF2 phosphorylation also promotes
gene-specific upregulation of the translation of certain
mRNAs. The two known examples of this involve the yeast
transcription factor GCN4 (19) and the mammalian transcrip-
tion factor ATF4 (12). Regulated gene expression appears to
be an important consequence of eIF2? phosphorylation, as
mutations that interfere with eIF2? phosphorylation lead to an
important defect in stress-induced gene expression (16, 28, 39).
Four known eIF2? kinases couple seemingly unrelated
stressful conditions to the aforementioned common transla-
tional regulatory event. PKR responds to double-stranded
RNA in virally infected cells (23), GCN2 is activated by un-
charged tRNAs in amino acid-starved cells (20), HRI is acti-
vated by heme depletion in erythroid precursor cells (3), and
PERK is activated by unfolded proteins in the endoplasmic
reticulum (ER), or ER stress (37). Mutations in each of these
four kinases have been produced, and their phenotypes reveal
the importance of eIF2? phosphorylation in stressed cells (6).
Nuclear factor kappa B (NF-?B) encompasses a family of
stress-induced transcription factors. Like the more ancient
eIF2? phosphorylation-dependent signaling, NF-?B signaling
is also triggered by diverse stressful conditions, and activated
NF-?B has broad effects on gene expression (38). Several stud-
ies have suggested cross talk between the eIF2? phosphor-
ylation pathway and NF-?B activation. The double-stranded-
RNA-activated eIF2? kinase PKR was noted to phosphorylate
the NF-?B inhibitor, I?B (26), and genetic and pharmacolog-
ical interventions that interfere with PKR activity attenuated
NF-?B activation by cytokines (4, 27, 47) or viruses (9, 43).
There is some uncertainty regarding the role of eIF2? phos-
phorylation in NF-?B activation by PKR, as the latter contrib-
utes to NF-?B activation by both kinase-dependent (9) and
kinase-independent (8) mechanisms.
Conditions that promote accumulation of unfolded proteins
in the endoplasmic reticulum lead to high levels of eIF2?
phosphorylation (34, 35), which is mediated by the ER-local-
ized kinase PERK (14, 15). These same conditions activate
NF-?B (32). A recent study has found that ER stress-mediated
NF-?B activation was attenuated both in PERK?/?cells and,
importantly, in cells bearing two mutant alleles of EIF2A in
which serine 51 (the substrate of the stress-inducible kinases)
had been mutated to an alanine. These mutant eIF2?A/Acells
were also defective in NF-?B activation by amino acid starva-
tion, as were cells lacking GCN2 (21), the kinase that phos-
phorylates eIF2? in amino acid-starved cells.
Together these observations point to a nonredundant role
for eIF2? phosphorylation in NF-?B activation under various
stress conditions. But they provide little insight into the mech-
anisms involved. One of the best-characterized aspects of
* Corresponding author. Mailing address: New York University
Medical Center, SI 3-10, 540 First Ave., New York, NY 10016. Phone:
(212) 263-7786. Fax: (212) 263-8951. E-mail: firstname.lastname@example.org
NF-?B regulation is the phosphorylation-dependent, protea-
some-mediated degradation of its inhibitor, I?B. However, it is
not clear if and how eIF2? phosphorylation ties in to I?B
levels. Because the stressful conditions used to promote eIF2?
phosphorylation have multiple other effects (reviewed in ref-
erence 17), it is not even known whether eIF2? phosphoryla-
tion plays a permissive role or an instructive role in NF-?B
activation, nor is it known whether the phosphorylated form of
eIF2? is affecting NF-?B activation as a modified translation
initiation factor or by some other means. In an effort to answer
some of these questions, we have probed NF-?B activation in
an experimental system that uncouples eIF2? phosphorylation
from stress signaling and discovered that translational repres-
sion of I?B can account for activation of NF-?B under condi-
tions of eIF2? phosphorylation.
MATERIALS AND METHODS
Cell culture, cell transfection, and treatment. The wild-type cells and
EIF2AA/Amutant cells in which the serine at position 51, the regulatory phos-
phorylation site, had been replaced by an alanine have been previously described
(39). They were cultured in Dulbecco’s modified Eagle’s medium supplemented
with glutamine, nonessential amino acids, 55 ?M ?-mercaptoethanol, and 10%
fetal calf serum. The establishment of stable clones of mouse fibroblasts express-
ing Fv2E-PERK with defined EIF2A genotypes has been previously described
The Fv2E-PERK?wild-type mouse embryonic fibroblasts described above
were transiently transfected using Fugene lipid-based gene transfer reagent (cat-
alog no. 1814443; Roche, Indianapolis, Ind.) with luciferase reporter plasmids
containing a minimal rat angiotensinogen promoter driven by four wild-type or
mutant NF-?B binding sites from the rat angiotensinogen gene, as previously
described (36). One day later the cells were treated for 1 h with the indicated
concentration of AP20187 (gift of ARIAD Pharmaceuticals, Cambridge, Mass.),
washed free of the activator (to allow translation to recover), and harvested for
use in a luciferase assay 24 h later.
Cells were treated with thapsigargin (catalog no. T9033; Sigma, St. Louis, Mo.)
at a final concentration of 400 nM or cycloheximide (catalog no. C7698; Sigma)
at 20 ?g/ml. Unless otherwise indicated, AP20187 was used at a concentration of
10 nM. Cells were treated with 20 ng of tumor necrosis factor alpha (TNF-?;
catalog no. T7539; Sigma)/ml with or without the proteasome inhibitor MG132
(catalog no. 474790; Calbiochem-Novobiochem, San Diego, Calif.) at 10 ?M.
Immunoblotting and immunoprecipitation. Total I?B? was detected with a
purified rabbit immune serum (catalog no. 9242; Cell Signaling, Beverly, Mass.),
and I?B? phosphorylated on serine 32 and 36 was detected with an epitope-
specific antiserum (catalog no. 9246; Cell Signaling). GADD34 was detected with
an antiserum directed to the N terminus of the mouse protein raised in our lab
(30). PERK was detected with a 1:1 mixture of two rabbit antisera (NY97, which
detects the unphosphorylated form of the protein, and NY201, which detects
predominantly the hyperphosphorylated forms of the protein) as described pre-
viously (2). Total eIF2? was detected with a monoclonal antibody to human
eIF2?, a gift of the late Edward Henshaw (40), and phosphorylated eIF2? was
detected with an epitope-specific antiserum (catalog no. RG0001; Research
Genetics, Huntsville, Ala.).
Pulse-chase labeling experiments were carried out in the Fv2E-PERK?wild-
type mouse embryonic fibroblasts described above. Cells were switched to Dul-
becco’s modified Eagle’s medium containing 10% of the normal content of
methionine and cysteine (these levels of methionine and cysteine are sufficient to
suppress activation of the eIF2? kinase GCN2 yet are compatible with high-level
incorporation of labeled amino acids into newly synthesized proteins) 15 min
before addition of TRANSlabel (MP Biomedical, Irvine, Calif.)
methionine-cysteine mixture at 200 ?Ci/ml for 10 min. The labeling pulse was
terminated by washing the unincorporated label and flooding the cells with
complete medium. Following the indicated chase period, during which cells were
exposed to AP20187 and/or MG132, the cells were lysed in RIPA buffer (20 mM
Tris [pH 8.5], 100 mM NaCl, 0.2% sodium deoxycholate, 0.2% NP-40, 0.2%
Triton X-100, 0.1% sodium dodecyl sulfate, 1 mM EDTA, 1 mM dithiothreitol,
1 mM phenylmethylsulfonyl fluoride, 4 ?g of aprotinin/ml, 2 ?g of pepstatin/ml),
and the lysate was clarified by centrifugation at 14,000 ? g for 15 min, precleared
on protein A-Sepharose beads (catalog no. 10-1042; Zymed, South San Fran-
cisco, Calif.), and subjected to immunoprecipitation with prebound anti-I?B?
rabbit immunoglobulin G (catalog no. SC-371 AC; Santa Cruz Biotech, Santa
Cruz, Calif.). Radiolabeled proteins found in the immunoprecipitate were re-
solved by reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
and the dried gel was exposed to autoradiography using a phosphoimaging
cassette (Molecular Dynamics, Sunnyvale, Calif.).
EMSA. NF-?B DNA binding activity in nuclear extracts was detected by an
electrophoretic mobility shift assay (EMSA) performed as previously described
(21, 36). The indicated molar excess of unlabeled competitor probe or 1 ?l of
purified anti-p65 (catalog no. SC-7151; Santa Cruz Biotech) or anti-CHOP
antiserum (45) was added to the binding reaction together with the radiolabeled
To confirm the previously reported role of eIF2? phosphor-
ylation in NF-?B activation (21), we performed EMSA on
nuclear extracts prepared from unstressed cells and cells that
had been treated with thapsigargin (Fig. 1A). Thapsigargin-
mediated ER calcium depletion leads to rapid onset of ER
FIG. 1. NF-?B activation during ER stress depends on eIF2? phos-
phorylation and is associated with declining levels of the NF-?B inhib-
itor I?B?. (A) Autoradiogram of an NF-?B EMSA performed with
nuclear extracts of thapsigargin-treated mouse fibroblasts (Tg) with
wild-type (EIF2AS/S) or mutant (EIF2AA/A) EIF2A genotypes. The free
radiolabeled probe and the labeled NF-?B/DNA complex are indi-
cated. (B) Immunoblots of I?B?, GADD34, phosphorylated eIF2?,
and total eIF2? from extracts of the cells shown in panel A, detected
with specific antibodies.
10162 DENG ET AL.MOL. CELL. BIOL.
stress, eIF2? phosphorylation (detected here by immunoblot-
ting with an antiserum specifically reactive with the phosphor-
ylated form), and subsequent ATF4-mediated activation of
downstream gene expression, measured here by accumula-
tion of the GADD34 target gene. A protein complex rapidly
formed on the NF-?B binding site in nuclear extracts of treated
wild-type cells but not in extracts from cells homozygous for
the EIF2AA/Amutation that substitutes the serine at position
51 of eIF2? with an alanine and thereby prevents regulatory
phosphorylation. Reduced levels of the NF-?B inhibitory pro-
tein I?B?, detected by immunoblotting, preceded the induc-
tion of NF-?B EMSA activity in thapsigargin-treated cells. The
recovery of I?B? levels at longer treatment points correlated
phorylation of eIF2? (Fig. 1B).
To more closely examine the role of eIF2? phosphorylation
in NF-?B activation, we made use of an experimental system
that uncouples eIF2? phosphorylation from stress signaling.
PERK, the ER stress-inducible eIF2? kinase, is normally ac-
tivated by oligomerization in the plane of the ER membrane
(2). We fused PERK’s eIF2? kinase domain to a protein mod-
ule 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 eIF2? phos-
phorylation in Fv2E-PERK?cells but had no effect on the
parental cells lacking the artificial kinase (Fig. 2A). Fv2E-
PERK was readily activated in mutant EIF2AA/Acells, but this
predictably failed to induce eIF2? phosphorylation. EMSA of
nuclear extracts showed that AP20187 induced NF-?B activity
in Fv2E-PERK?wild-type (EIF2AS/S) cells but not in the mu-
tant EIF2AA/Acells (Fig. 2B). Homologous competition bind-
ing assays and antibody supershift experiments confirmed the
identity of the NF-?B protein-DNA complex detected in the
assay (Fig. 2C).
To gauge the functional significance of Fv2E-PERK-medi-
ated eIF2? phosphorylation and activation of NF-?B DNA
binding activity, we measured the activity of a transfected re-
porter gene driven by four copies of a wild-type NF-?B binding
site. A brief (60-min) pulse of AP20187 induced marked acti-
vation of the wild-type reporter gene (measured 24 h later [Fig.
3]). No activation of a reporter gene driven by mutant NF-?B
sites was observed. In addition, endogenous NF-?B target
genes, such as those encoding the major histocompatibility
complex heavy chains (H2-Q8, H2-2KF, H2-K2, and H2-D1)
and ?2 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).
Fv2E-PERK-mediated eIF2? phosphorylation and NF-?B
activation correlated with a time-dependent decrease in I?B?
levels that was not observed in the mutant EIF2AA/Acells (Fig.
4A). Interestingly, Fv2E-PERK activation had no measurable
effect on levels of the p65 NF-?B subunit, which is consistent
with the known stability of that protein (24) and with the
induction of NF-?B binding activity that we observe. eIF2?
levels were similarly stable, attesting to the effect’s specificity to
I?B? (Fig. 4B). Canonical activators of NF-?B access signal
FIG. 2. Phosphorylation of eIF2? on serine 51 is sufficient to acti-
vate NF-?B DNA binding activity in vivo. (A) Immunoblots of ligand-
activable Fv2E-PERK (upper panel), phosphorylated eIF2? (P-eIF2?;
middle panel), and total eIF2? (lower panel) in extracts of mouse fi-
broblasts of wild-type (S/S) and EIF2AA/Amutant (A/A) genotypes that
do and do not stably express the chimeric eIF2? 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-?B EMSA performed with
nuclear extracts of cells treated as described for panel A. (C) Autora-
diogram of an NF-?B EMSA with nuclear extract obtained from
AP20187-treated Fv2E-PERK?cells performed in the presence of the
indicated excess of an unlabeled homologous competitor oligonucleo-
tide (left panel) or in the presence of antisera to CHOP (a negative
control) or p65 (a component of the NF-?B DNA binding complex)
(right panel). The positions of the free radiolabeled probe, the NF-
?B/DNA complex, and antiserum supershifted complex are indicated.
VOL. 24, 2004eIF2? PHOSPHORYLATION AND NF-?B ACTIVATION 10163
transduction pathways that promote phosphorylation of the
inhibitor I?B? on serines 32 and 36 (38). A ubiquitin ligase
complex recognizes the phosphorylated form of I?B?, and
polyubiquitinated I?B? is degraded by the proteasome. Fv2E-
PERK activation by AP20187 did not promote a measurable
increase in levels of phosphorylated I?B?, which remained
undetectable. However, phosphorylated I?B? was readily de-
tectable in lysates of cells treated with the proteasome inhibi-
tor, MG132, which stabilizes the phosphorylated form of the
protein (Fig. 4B).
Because it is rapidly degraded, signal-dependent accumula-
tion of phosphorylated I?B? is difficult to detect, rendering
an Fv2E-PERK-mediated increase in I?B? phosphorylation
potentially easy to miss. Therefore, to determine if the eIF2?
phosphorylation-dependent decline in I?B? 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 in-
creased the levels of phosphorylated I?B? in tumor necrosis
factor alpha-treated cells (Fig. 5A). Interestingly, proteasome
inhibition led to only modest stabilization of total I?B?, an
observation that is consistent with the existence of proteasome-
independent mechanisms for I?B? degradation (5, 11).
MG132 treatment led to a progressive increase in phosphor-
ylated I?B? levels in cells that were otherwise unperturbed
(Fig. 5A, 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
I?B? in these cells. The decline in I?B? levels effected by
Fv2E-PERK was only slightly attenuated by proteasome inhi-
bition (compare Fig. 4, lanes 4 to 6, with 5B, lanes 7, 9, and 11).
Furthermore, proteasome inhibition promoted some eIF2?
phosphorylation (Fig. 5, lanes 8 and 10), presumably mediated
by proteotoxic stress. Remarkably, however, Fv2E-PERK ac-
tivation and eIF2? phosphorylation not only failed to increase
I?B? phosphorylation but also significantly attenuated the ac-
cumulation of phosphorylated I?B? in proteasome-inhibited
cells (Fig. 5B, compare odd- and even-numbered lanes). These
observations indicate that eIF2? phosphorylation does not
activate NF-?B by accessing one of the canonical I?B? phos-
phorylation-promoting pathways and must use a different
The original descriptions of I?B emphasized the lability of
the factor, as translational inhibitors were noted to promote
NF-?B DNA binding activity (1, 42). Given that eIF2? phos-
phorylation also inhibits protein synthesis, we decided to ex-
plore this facet of NF-?B activation in more detail. NF-?B
DNA binding activity was increased by cycloheximide treat-
ment of wild-type cells, as previously reported (42), and this
correlated with reduced levels of the inhibitor, I?B? (Fig. 6A).
Cycloheximide treatment led to no measurable decrease in p65
or eIF2? protein levels, attesting to the stability of these pro-
teins. The effects of cycloheximide on levels of phosphorylated
I?B? also resembled those of Fv2E-PERK activation (Fig. 4B)
in that no increase in the phosphorylated protein was observed
FIG. 3. eIF2? phosphorylation is sufficient to activate an NF-?B
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-?B binding sites in mouse fibroblasts stably express-
ing Fv2E-PERK is shown following treatment with the indicated con-
centration 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 re-
FIG. 4. eIF2? phosphorylation reduces cellular levels of I?B?. (A)
Immunoblots of total I?B? (upper panel), phosphorylated eIF2? (P-
eIF2?; middle panel), and total eIF2? (lower panel) in extracts of
wild-type (EIF2AS/S) and mutant (EIF2AA/A) Fv2E-PERK?mouse
fibroblasts following treatment with the activating ligand AP20187 for
the indicated periods of time. (B) Immunoblots of total I?B?, phos-
phorylated I?B? (P-I?B?), p65 NF-?B subunit, and total eIF2? in
extracts of wild-type (EIF2AS/S) Fv2E-PERK?mouse fibroblasts fol-
lowing treatment with the activating ligand AP20187 or the protea-
some inhibitor (MG132) for the indicated periods of time.
10164 DENG ET AL.MOL. CELL. BIOL.
in cells treated with cycloheximide alone. Proteasome inhibi-
tor, by itself, led to a progressive increase in levels of phos-
phorylated I?B?, whereas the addition of cycloheximide
strongly attenuated this increase (Fig. 6B).
As previously noted, cycloheximide treatment induced eIF2?
phosphorylation (21, 22) (Fig. 6A), an effect that might be
attributed to loss of the labile eIF2? phosphatase CReP (22).
To study the role of eIF2? phosphorylation in cycloheximide-
mediated activation of NF-?B, we treated mutant EIF2AA/A
cells with the protein synthesis inhibitor and studied NF-?B
activation by EMSA and I?B? levels by immunoblotting. The
EIF2AA/Agenotype, which inhibits regulatory phosphorylation
of eIF2?, had no measurable effect on NF-?B activation, I?B?
phosphorylation, or total I?B? levels in cycloheximide-treated
cells (Fig. 6C). These observations suggest that inhibition of
new protein synthesis can adequately explain the effects of
cycloheximide on NF-?B activity without evoking an additional
role for eIF2? phosphorylation.
Induced degradation of I?B? plays an important role in
FIG. 5. Reduction in levels of I?B? in cells with elevated eIF2?
phosphorylation occurs independently of I?B? phosphorylation. (A)
Immunoblots of I?B? phosphorylated on serines 32 and 36 (P-I?B?;
upper panel) and total I?B? (lower panel) in extracts of mouse fibro-
blasts treated with TNF-? and/or the proteasome inhibitor MG132.
(B) Immunoblots of phosphorylated I?B? (P-I?B?), total I?B?, phos-
phorylated eIF2? (P-eIF2?), and total eIF2? in extracts of Fv2E-
PERK?mouse fibroblasts treated with the activating ligand AP20187
and/or the proteasome inhibitor MG132 for the indicated periods of
FIG. 6. Reduction in levels of I?B? in cells treated with the protein
synthesis inhibitor cycloheximide occurs independently of I?B? phos-
phorylation or eIF2? phosphorylation. (A) The top panel is an auto-
radiogram of an NF-?B EMSA from nuclear extracts of untreated and
cycloheximide (CHX)-treated mouse fibroblasts. The lower panels are
immunoblots (IB) of total I?B?, phosphorylated I?B? (P-I?B?), phos-
phorylated eIF2? (P-eIF2?), total eIF2?, and the p65 NF-?B subunit
from the same cells. (B) Immunoblots of phosphorylated I?B? (P-
I?B?), total I?B?, and total eIF2? in extracts of wild-type (EIF2AS/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 con-
ducted with mutant (EIF2AA/A) Fv2E-PERK?cells.
VOL. 24, 2004eIF2? PHOSPHORYLATION AND NF-?B ACTIVATION10165
canonical activation of NF-?B. To address the possibility that
eIF2? phosphorylation might affect this aspect of I?B? metab-
olism (independently of I?B? phosphorylation), we performed
pulse-chase labeling experiments, tracking the fate of newly
synthesized I?B?. The basal turnover of I?B? in murine fibro-
blasts 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 dur-
ing the chase had no measurable effect on the decay of the
I?B? signal (Fig. 7A). Addition of proteasome inhibitor during
the chase stabilized I?B? somewhat; however, in that context,
too, activation of Fv2E-PERK during the chase did not accel-
erate I?B? degradation and may have even contributed mod-
estly to its stability (Fig. 7B). We conclude that I?B? turns over
rapidly in murine fibroblasts and that eIF2? phosphorylation
does not exert its effects on the levels of the inhibitor by further
enhancing its degradation.
Next we compared the rates of synthesis of I?B? in un-
treated cells with those in cells treated with AP20187, cyclo-
heximide, the ER stress-promoting agent thapsigargin, and
the canonical NF-?B activator TNF-?. The amount of radio-
labeled I?B? immunoprecipitated with a specific antibody
following a short labeling pulse was markedly diminished by
activation of the eIF2? kinase Fv2E-PERK by AP20187, by
treatment with cycloheximide, or by exposure to conditions
that cause ER stress (thapsigargin) (Fig. 7C). The effect of
thapsigargin on I?B? synthesis depended on eIF2? phosphor-
ylation, since it was abolished in the EIF2AA/Amutant cells
(Fig. 7C), and the decline in I?B? synthesis paralleled the
global inhibition in protein synthesis in the cells exposed to
conditions promoting eIF2? phosphorylation (Fig. 7D). By
contrast, exposure to the canonical NF-?B activator, TNF-?,
increased I?B? synthesis, suggesting a completely different
mechanism of action. These observations are consistent with a
role for inhibited synthesis of I?B? in mediating the effects of
eIF2? phosphorylation on NF-?B activation both in ER-
stressed cells and following activation of Fv2E-PERK.
Signaling through stress-induced phosphorylation of eIF2?
is conserved among the eukaryotes and represents one of the
oldest pathways for stress-induced gene expression. Further-
more, eIF2? phosphorylation is concerned mostly with auton-
omous cell adaptations to stress. NF-?B signaling, on the other
hand, is found in metazoans, and canonical activators of NF-?B
signaling, such as cytokines, are intercellular signaling mole-
cules. However, over the years evidence that autonomous cell
phenomena, such as ER stress, are also associated with NF-?B
activation has accrued, with the suggestion that ancient, au-
tonomous cell signaling pathways might be linked to NF-?B
This study confirms the established role of eIF2? phosphor-
ylation in NF-?B activation by ER stress (21). Using an induc-
ible system that uncouples eIF2? phosphorylation from other
stress signals, we find that eIF2? phosphorylation can have an
instructive role in NF-?B activation. In other words, activation
of an eIF2? kinase provides a signal sufficient for NF-?B ac-
tivation in cultured mouse fibroblasts. Our study also reveals
significant differences between the mechanism used by canon-
FIG. 7. eIF2? phosphorylation inhibits synthesis of I?B? but does
not destabilize the preexisting protein. (A) Autoradiogram of I?B?
immunoprecipitated from wild-type (EIF2AS/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 I?B? 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) Autora-
diogram of the radiolabeled I?B? present at the end of the 10-min
labeling pulse in wild-type (EIF2AS/S) or mutant (EIF2AA/A) Fv2E-
PERK?mouse fibroblasts treated with the indicated concentration of
AP20187 ligand (in nM), cycloheximide (in ?g/ml), thapsigargin (in ?M),
or TNF-? (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.
10166 DENG ET AL.MOL. CELL. BIOL.
ical inducers of NF-?B and the consequences of eIF2? phos-
phorylation. Unlike canonical inducers of NF-?B, eIF2? phos-
phorylation promoted neither phosphorylation nor degradation
of I?B?. Instead, our data argue that the major impact of eIF2?
phosphorylation on NF-?B activation is inhibition of the syn-
thesis of the labile inhibitor I?B?.
The mechanism uncovered in this study suggests that the
link between eIF2? phosphorylation and NF-?B activation de-
pends on the lability of the inhibitor, which, in turn, likely
depends on basal levels of signaling through the canonical
pathway(s) that activates NF-?B. Indeed, the rapid accumula-
tion of phosphorylated I?B? in mouse fibroblasts treated with
proteasome inhibitor is consistent with high basal levels of
I?B? kinase activity in these cells. It is worth noting that both
eIF2? phosphorylation and cycloheximide treatment dispro-
portionately reduced the levels of phosphorylated I?B?, com-
pared with their effect on the levels of total I?B?. Inhibited
protein synthesis may attenuate basal activity of an I?B? ki-
nase and account for some of this effect. Alternatively, newly
synthesized I?B? might constitute a preferred substrate for its
kinases. The plausibility of the latter explanation is supported
by evidence for the existence of multiple pools of I?B? in cells
(25, 33, 41). The existence of more than one pool of I?B?
might also explain the discrepancy between the short half-life
of newly synthesized I?B? (measured by the pulse-chase meth-
od [Fig. 7A and B]) and the much longer half-life inferred from
the gradual decline in total I?B? protein levels in the cyclo-
heximide-treated and Fv2E-PERK-activated cells (Fig. 4, 5B,
and 6A and B). However, these potential complexities of I?B?
metabolism do not weaken our conclusion that attenuated
synthesis of the inhibitor plays a major role in mediating acti-
vation of NF-?B by eIF2? phosphorylation in mouse fibro-
Our findings are at odds with those reported by Jiang and
colleagues, who found no decrease in steady-state I?B? levels
in thapsigargin-treated cells and instead uncovered evidence
for dissociation of the I?B?–NF-?B complex under those con-
ditions (see Fig. 6 in reference 21). We have no explanation for
these differences; however, we do note that since the submis-
sion of the present study Wu and colleagues have reported that
induction of NF-?B DNA binding activity in cells exposed to
UV light is also associated with eIF2? phosphorylation-depen-
dent repression of I?B? synthesis (46).
Our study does not address the physiological significance of
the link between eIF2? phosphorylation and NF-?B activation.
It is worth noting that we have but an incomplete understand-
ing of the relative significance of regulated protein synthesis
versus activation of gene expression programs as readouts of
eIF2? phosphorylation. In yeast it is fairly clear that mutations
in the transcription factor GCN4 phenocopy mutations in the
upstream kinase GCN2 or in the gene encoding its substrate
SUI2 (yeast eIF2?) (6, 7). In mammalian cells too, some of the
phenotypes of loss of PERK gene function or the EIF2AA/A
genotype are mimicked by mutations in the gene encoding the
downstream transcription factor ATF4 (16, 29, 39). Further-
more, in both yeast and mammalian cells, translation activation
of the transcription factors GCN4 and ATF4 occurs at levels of
eIF2? phosphorylation that have only a modest impact on
global protein synthesis (7, 44; Lu et al., unpublished observa-
tion). By contrast, our proposed mechanism of cross talk be-
tween eIF2? phosphorylation and NF-?B signaling is propor-
tional to the repression of I?B? translation. Such levels of
repression are easily attained in thapsigargin-treated cells (14)
or in Fv2E-PERK?cells activated by AP20187 (28) and are
clearly sufficient to activate NF-?B in cultured mouse fibro-
blasts (Fig. 1A and 2B) (21).
The extent to which translational repression contributes to
NF-?B activation in more physiological contexts in which
eIF2? kinases are activated is not known. However, we note
that endogenous proinflammatory NF-?B target genes, such
as those encoding the major histocompatibility complex heavy
and light chains, the interleukin 17 receptor, and a comple-
ment receptor-related protein, were all induced in the Fv2E-
PERK?cells by AP20187 treatment and in wild-type mouse
fibroblasts by exposure to tunicamycin (National Center for
Biotechnology Information GEO data set GDS405). The PERK-
dependent induction of NF-?B target genes by tunicamycin is
potentially significant, as global repression of mRNA transla-
tion is relatively modest under those conditions (14), mimick-
ing physiological stress situations. Furthermore, loss-of-func-
tion mutations in the eIF2? kinase PERK or HRI or the
EIF2AA/Agenotype all predispose cells to programmed cell
death under physiologically stressful conditions (10, 13, 14, 39,
48); however, the role of defective activation of NF-?B in this
phenotype, if any, remains to be explored.
Translational repression in response to activation of eIF2?
kinases tends to be transient (34, 35). Translational recovery is
mediated in part by activation of GADD34, an eIF2?-specific
regulatory subunit of a holophosphatase complex (30, 31),
which is itself a target of the eIF2? phosphorylation-dependent
gene expression program, the integrated stress response (16,
29, 30). GADD34-mediated translational recovery is therefore
likely to reestablish I?B? translation and reverse the effects of
eIF2? phosphorylation on NF-?B activity, since the stress re-
sponse is attenuated (Fig. 1B). Furthermore, while activation
of NF-?B proceeds through utilization of preformed compo-
nents, the response in terms of target gene expression depends
on new protein synthesis. Thus, the biphasic nature of the
inhibition of protein synthesis, which is inherent to stressful
conditions that promote eIF2? phosphorylation, is also pre-
dicted to contribute to the expression of NF-?B target genes.
In conclusion, our study indicates that the pathways promot-
ing eIF2? phosphorylation and those that activate NF-?B in-
teract through translational repression of the inhibitor I?B?.
Our study also suggests that the importance of this link is likely
to be influenced by signaling through canonical NF-?B activa-
tion pathways that define the turnover rate of I?B?. As such,
eIF2? phosphorylation and the consequent inhibition of eIF2B
might modulate NF-?B signaling by parallel pathways active in
We thank Yinon Ben Neriah and Haoyuan Jiang for scientific advice
and the ARIAD Corporation for the gift of the inducible dimerization
This work was supported by NIH grants ES08681 and DK47119
(to D.R.) and DK42394 (to R.J.K.). D.R. is a Scholar of the Ellison
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