Translational Repression Mediates Activation of Nuclear Factor Kappa B by Phosphorylated Translation Initiation Factor 2

Article (PDF Available)inMolecular and Cellular Biology 24(23):10161-8 · January 2005with38 Reads
DOI: 10.1128/MCB.24.23.10161-10168.2004 · Source: PubMed
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.
MOLECULAR AND CELLULAR BIOLOGY, Dec. 2004, p. 10161–10168 Vol. 24, No. 23
0270-7306/04/$08.000 DOI: 10.1128/MCB.24.23.10161–10168.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Translational Repression Mediates Activation of Nuclear Factor
Kappa B by Phosphorylated Translation Initiation Factor 2
Jing Deng,
1
Phoebe D. Lu,
1
Yuhong Zhang,
1
Donalyn Scheuner,
2
Randal J. Kaufman,
2,3
Nahum Sonenberg,
4
Heather P. Harding,
5
and David Ron
1
*
Skirball Institute of Biomolecular Medicine
1
and Departments of Cell Biology, Medicine, and Pharmacology,
5
New York University School of Medicine, New York, New York; Department of Biochemistry
2
and Howard
Hughes Medical Institute,
3
University of Michigan School of Medicine, Ann Arbor, Michigan;
and Department of Biochemistry, McGill University, Montreal, Quebec, Canada
4
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 IB protein. Unlike canonical signaling pathways that promote IB phosphorylation and degra-
dation, eIF2 phosphorylation did not increase phosphorylated IB levels or affect the stability of the protein.
Pulse-chase labeling experiments indicate instead that repression of IB 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, IB (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/A
cells
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: ron@saturn.med.nyu
.edu.
10161
NF-B regulation is the phosphorylation-dependent, protea-
some-mediated degradation of its inhibitor, IB. However, it is
not clear if and how eIF2 phosphorylation ties in to IB
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 IB 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
EIF2A
A/A
mutant 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
(28).
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 IB was detected with a
purified rabbit immune serum (catalog no. 9242; Cell Signaling, Beverly, Mass.),
and IB 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.)
35
S-labeled
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-IB
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 lof
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
probe.
RESULTS
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 IB. (A) Autoradiogram of an NF-B 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-B/DNA complex are indi-
cated. (B) Immunoblots of IB, GADD34, phosphorylated eIF2,
and total eIF2 from extracts of the cells shown in panel A, detected
with specific antibodies.
10162 DENG ET AL. M
OL.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 EIF2A
A/A
mutation 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 IB, detected by immunoblotting, preceded the induc-
tion of NF-B EMSA activity in thapsigargin-treated cells. The
recovery of IB levels at longer treatment points correlated
with the induction of the GADD34 phosphatase and the dephos-
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 EIF2A
A/A
cells, but this
predictably failed to induce eIF2 phosphorylation. EMSA of
nuclear extracts showed that AP20187 induced NF-B activity
in Fv2E-PERK
wild-type (EIF2A
S/S
) cells but not in the mu
-
tant EIF2A
A/A
cells (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 IB
levels that was not observed in the mutant EIF2A
A/A
cells (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
IB (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 EIF2A
A/A
mutant (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.
V
OL. 24, 2004 eIF2 PHOSPHORYLATION AND NF-B ACTIVATION 10163
transduction pathways that promote phosphorylation of the
inhibitor IB on serines 32 and 36 (38). A ubiquitin ligase
complex recognizes the phosphorylated form of IB, and
polyubiquitinated IB is degraded by the proteasome. Fv2E-
PERK activation by AP20187 did not promote a measurable
increase in levels of phosphorylated IB, which remained
undetectable. However, phosphorylated IB 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 IB is difficult to detect, rendering
an Fv2E-PERK-mediated increase in IB phosphorylation
potentially easy to miss. Therefore, to determine if the eIF2
phosphorylation-dependent decline in IB 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 IB in tumor necrosis
factor alpha-treated cells (Fig. 5A). Interestingly, proteasome
inhibition led to only modest stabilization of total IB,an
observation that is consistent with the existence of proteasome-
independent mechanisms for IB degradation (5, 11).
MG132 treatment led to a progressive increase in phosphor-
ylated IB 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
IB in these cells. The decline in IB 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
IB phosphorylation but also significantly attenuated the ac-
cumulation of phosphorylated IB 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 IB phos-
phorylation-promoting pathways and must use a different
mechanism.
The original descriptions of IB 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, IB (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
IB 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-
produced twice.
FIG. 4. eIF2 phosphorylation reduces cellular levels of IB. (A)
Immunoblots of total IB (upper panel), phosphorylated eIF2 (P-
eIF2; middle panel), and total eIF2 (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 IB, phos-
phorylated IB (P-IB), p65 NF-B subunit, and total eIF2 in
extracts of wild-type (EIF2A
S/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. M
OL.CELL.BIOL.
in cells treated with cycloheximide alone. Proteasome inhibi-
tor, by itself, led to a progressive increase in levels of phos-
phorylated IB, 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 EIF2A
A/A
cells with the protein synthesis inhibitor and studied NF-B
activation by EMSA and IB levels by immunoblotting. The
EIF2A
A/A
genotype, which inhibits regulatory phosphorylation
of eIF2, had no measurable effect on NF-B activation, IB
phosphorylation, or total IB 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 IB plays an important role in
FIG. 5. Reduction in levels of IB in cells with elevated eIF2
phosphorylation occurs independently of IB phosphorylation. (A)
Immunoblots of IB phosphorylated on serines 32 and 36 (P-IB;
upper panel) and total IB (lower panel) in extracts of mouse fibro-
blasts treated with TNF- and/or the proteasome inhibitor MG132.
(B) Immunoblots of phosphorylated IB (P-IB), total IB, 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
time.
FIG. 6. Reduction in levels of IB in cells treated with the protein
synthesis inhibitor cycloheximide occurs independently of IB 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 IB, phosphorylated IB (P-IB), phos-
phorylated eIF2 (P-eIF2), total eIF2, and the p65 NF-B subunit
from the same cells. (B) Immunoblots of phosphorylated IB (P-
IB), total IB, and total eIF2 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 con-
ducted with mutant (EIF2A
A/A
) Fv2E-PERK
cells.
V
OL. 24, 2004 eIF2 PHOSPHORYLATION AND NF-B ACTIVATION 10165
canonical activation of NF-B. To address the possibility that
eIF2 phosphorylation might affect this aspect of IB metab-
olism (independently of IB phosphorylation), we performed
pulse-chase labeling experiments, tracking the fate of newly
synthesized IB. The basal turnover of IB 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
IB signal (Fig. 7A). Addition of proteasome inhibitor during
the chase stabilized IB somewhat; however, in that context,
too, activation of Fv2E-PERK during the chase did not accel-
erate IB degradation and may have even contributed mod-
estly to its stability (Fig. 7B). We conclude that IB 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 IB 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 IB 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 IB synthesis depended on eIF2 phosphor-
ylation, since it was abolished in the EIF2A
A/A
mutant cells
(Fig. 7C), and the decline in IB 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 IB synthesis, suggesting a completely different
mechanism of action. These observations are consistent with a
role for inhibited synthesis of IB in mediating the effects of
eIF2 phosphorylation on NF-B activation both in ER-
stressed cells and following activation of Fv2E-PERK.
DISCUSSION
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
activation.
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 IB but does
not destabilize the preexisting protein. (A) Autoradiogram of IB
immunoprecipitated from wild-type (EIF2A
S/S
) Fv2E-PERK
mouse
fibroblasts following a brief, 10-min [
35
S]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 IB 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 IB 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 g/ml), thapsigargin (in M),
or TNF- (in ng/ml) starting 30 min before and continuing throughout
the pulse. (D) Autoradiogram ([
35
S]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. M
OL.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 IB. 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 IB.
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 IB in mouse fibroblasts treated with
proteasome inhibitor is consistent with high basal levels of
IB kinase activity in these cells. It is worth noting that both
eIF2 phosphorylation and cycloheximide treatment dispro-
portionately reduced the levels of phosphorylated IB, com-
pared with their effect on the levels of total IB. Inhibited
protein synthesis may attenuate basal activity of an IB ki-
nase and account for some of this effect. Alternatively, newly
synthesized IB 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 IB in cells
(25, 33, 41). The existence of more than one pool of IB
might also explain the discrepancy between the short half-life
of newly synthesized IB (measured by the pulse-chase meth-
od [Fig. 7A and B]) and the much longer half-life inferred from
the gradual decline in total IB protein levels in the cyclo-
heximide-treated and Fv2E-PERK-activated cells (Fig. 4, 5B,
and 6A and B). However, these potential complexities of IB
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-
blasts.
Our findings are at odds with those reported by Jiang and
colleagues, who found no decrease in steady-state IB levels
in thapsigargin-treated cells and instead uncovered evidence
for dissociation of the IB–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 IB 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 EIF2A
A/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 IB 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
EIF2A
A/A
genotype 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 IB 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 IB.
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 IB. As such,
eIF2 phosphorylation and the consequent inhibition of eIF2B
might modulate NF-B signaling by parallel pathways active in
stressed cells.
ACKNOWLEDGMENTS
We thank Yinon Ben Neriah and Haoyuan Jiang for scientific advice
and the ARIAD Corporation for the gift of the inducible dimerization
system.
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
Medical Foundation.
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10168 DENG ET AL. MOL.CELL.BIOL.
    • "The detailed mechanism underlying reduced NF-jB signaling, but not IFN-c signaling, in Atf6a À/À microglia remains to be resolved, but it may include enhancement of NF-jB p65 degradation through the proteasome (Fig. 6). It is noteworthy that other UPR branches, such as PERK and Ire1a, were also reported to activate NF-jB signaling through the translational suppression of IjBa (Deng et al. 2004; Lin et al. 2013) and the recruitment of IKK (Hu et al. 2006), respectively. We propose a scenario wherein each branch of the UPR regulates NF-jB signaling by different mechanisms . "
    [Show abstract] [Hide abstract] ABSTRACT: Accumulating evidence suggests a critical role for the unfolded protein response (UPR) in multiple sclerosis (MS) and in its animal model, experimental autoimmune encephalomyelitis (EAE). In this study, we investigated the relevance of activating transcription factor 6α (ATF6α), an upstream regulator of part of the UPR, in EAE. The expressions of ATF6α-target molecular chaperones such as glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94) were enhanced in the acute inflammatory phase after induction of EAE. Deletion of Atf6α suppressed the accumulation of T cells and microglia/macrophages in the spinal cord, and ameliorated the clinical course and demyelination after EAE induction. In contrast to the phenotypes in the spinal cord, activation status of T cells in the peripheral tissues or in the culture system was not different between two genotypes. Bone marrow transfer (BMT) experiments and adoptive transfer of autoimmune CD4(+) T cells to recipient mice (passive EAE) also revealed that CNS-resident cells are responsible for the phenotypes observed in Atf6α(-/-) mice. Further experiments with cultured cells indicated that inflammatory response was reduced in Atf6α(-/-) microglia, but not in Atf6α(-/-) astrocytes, and was associated with proteasome-dependent degradation of NF-κB p65. Thus, our results demonstrate a novel role for ATF6α in microglia-mediated CNS inflammation. This article is protected by copyright. All rights reserved.
    Full-text · Article · Jun 2016
    • "Of note, salubrinal acts at a local level and does not modify the inflammation markers in blood, as reveals the lack of response of C-reactive protein in the blood. UPR inhibits NF-jB activation by inflammatory stimuli (canonical pathway) but ER stress is able to activate NF-jB through the non-canonical pathway, as described in cell culture (Jiang et al. 2003; Deng et al. 2004). Thus, the similarity in the NF-jB levels in Cx and CA1, despite their striking differences in the TNF-a inflammatory stimulus, suggests an almost exclusively non-canonical expression of NF-jB in Cx, in a similar way as reported in cell culture (Nakajima et al. 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: This study describes the neuroprotective effect of treatment with salubrinal 1 and 24 h following 15 min of ischemia in a two-vessel occlusion model of global cerebral ischemia. The purpose of this study was to determine if salubrinal, an enhancer of the unfolded protein response (UPR), reduces the neural damage modulating the inflammatory response. The study was performed in CA1 and CA3 hippocampal areas as well as in the cerebral cortex whose different vulnerability to ischemic damage is widely described. Characterization of proteins was made by Western blot, immunofluorescence and ELISA while mRNA levels were measured by qPCR. The salubrinal treatment decreased the cell demise in CA1 at 7 days as well as the levels of matrix metalloprotease 9 (MMP-9) in CA1 and cerebral cortex at 48 h and ICAM-1 and VCAM-1 cell adhesion molecules. However, increases in tumour necrosis factor α (TNF-α) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inflammatory markers were observed at 24 h. Glial fibrillary acidic protein (GFAP) levels were not modified by salubrinal treatment in CA1 and cerebral cortex. We describe a neuroprotective effect of the post-ischemic treatment with salubrinal, measured as a decrease both in CA1 cell demise and in the blood brain barrier impairment. We hypothesize that the ability of salubrinal to counteract the CA1 cell demise is due to a reduced ability of this structure to elicit UPR which would account for its greater ischemic vulnerability. Data of both treated and non treated animals suggest that the neurovascular unit present a structure-dependent response to ischemia and a different course time for CA1/cerebral cortex compared with CA3. Finally, our study reveals a high responsiveness of endothelial cells to salubrinal in contrast to the limited responsiveness of astrocytes. This article is protected by copyright. All rights reserved.
    Article · Apr 2016
    • "Activation of the first mediator, PERK, results in the phosphorylation and inhibition of elf2α, leading to inhibition of protein translation (Fig. 4b). This inhibition of protein translation via elf2α can result in further NF-κB activation [32]. The second, ATF6, which is responsible for XBP1 and BiP transcription, has also been shown to activate Akt and NF-κB [33] (Fig. 4b). "
    [Show abstract] [Hide abstract] ABSTRACT: Inherently defective immunity typically results in either ineffective host defense, immune regulation, or both. As a category of primary immunodeficiency diseases, those that impair immune regulation can lead to autoimmunity and/or autoinflammation. In this review we focus on one of the most recently discovered primary immunodeficiencies that leads to immune dysregulation: "Copa syndrome". Copa syndrome is named for the gene mutated in the disease, which encodes the alpha subunit of the coatomer complex-I that, in aggregate, is devoted to transiting molecular cargo from the Golgi complex to the endoplasmic reticulum (ER). Copa syndrome is autosomal dominant with variable expressivity and results from mutations affecting a narrow amino acid stretch in the COPA gene-encoding COPα protein. Patients with these mutations typically develop arthritis and interstitial lung disease with pulmonary hemorrhage representing a striking feature. Immunologically Copa syndrome is associated with autoantibody development, increased Th17 cells and pro-inflammatory cytokine expression including IL-1β and IL-6. Insights have also been gained into the underlying mechanism of Copa syndrome, which include excessive ER stress owing to the impaired return of proteins from the Golgi, and presumably resulting aberrant cellular autophagy. As such it represents a novel cellular disorder of intracellular trafficking associated with a specific clinical presentation and phenotype.
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