(Received for publication, January 9, 1995)
From the
Perturbants of the endoplasmic reticulum (ER), including
Ca-mobilizing agents, provoke a rapid suppression of
translational initiation in conjunction with an increased
phosphorylation of the
-subunit of eukaryotic initiation factor
(eIF)-2. Depletion of ER Ca
stores was found to
signal the activation of a specific eIF-2
kinase. Analysis of
extracts derived from cultured cells that had been pretreated with
Ca
ionophore A23187 or thapsigargin revealed a
2-3-fold increase in eIF-2
kinase activity without
detectable changes in eIF-2
phosphatase activity. A peptide of
65-68 kDa, which was phosphorylated concurrently with eIF-2
in extracts of pretreated cells, was identified as the
interferon-inducible, double-stranded RNA (dsRNA)-regulated protein
kinase (PKR). Depletion of ER Ca
stores did not alter
the PKR contents of extracts. When incubated with reovirus dsRNA,
extracts derived from cells with depleted ER Ca
stores displayed greater degrees of phosphorylation of PKR and of
eIF-2
than did control extracts. The enhanced dsRNA-dependent
phosphorylation of PKR was observed regardless of prior induction of
the kinase with interferon. Lower concentrations of dsRNA were required
for maximal phosphorylation of PKR in extracts of treated as compared
to control preparations. These findings suggest that PKR mediates the
translational suppression occurring in response to perturbation of ER
Ca
homeostasis.
PKR ()(dsRNA-dependent/regulated protein kinase) is a
ubiquitously expressed enzyme induced by interferon
or
and
activated during viral infection in various cell types (reviewed in (1, 2, 3) ). The activated enzyme catalyzes
the phosphorylation of the
-subunit of eukaryotic initiation
factor (eIF)-2, resulting in the arrest of translation at initiation,
and is thought to function in both the anti-proliferative and antiviral
actions of interferon. The possibility that this enzyme possesses
multiple substrates and/or serves more broadly in cellular control
mechanisms is supported by various recent findings. For example, PKR
activates gene transcription through regulation of NF-
B (4) and is strongly implicated in the control of
differentiation and growth (5, 6, 7, 8, 9, 10) .
The endoplasmic reticulum (ER) functions critically in the early
processing of newly synthesized secretory, lysosomal and integral
membrane proteins. Metabolic perturbation of the ER with
Ca-mobilizing or thiol-reducing agents interrupts the
processing and folding of newly synthesized polypeptides within the ER
lumen(11) . These disruptions of ER function signal the
immediate suppression of translational initiation coincident with the
phosphorylation of eIF-2
(12, 13) . These
perturbants also provoke a subsequent stress response that is
characterized by the induction of certain ER resident protein
chaperones termed the glucose-regulated proteins(14) . Greater
degrees of ER stress are required for signaling of eIF-2
phosphorylation than for induction of the glucose-regulated
proteins(15) , an observation consistent with the existence of
a pathway to slow synthesis of precursors when protein processing is
seriously impaired. The mechanism whereby perturbation of the ER
signals increased phosphorylation of eIF-2
is currently undefined.
Decreased phosphatase activity has been suggested as responsible for
phosphorylation of eIF-2
in perfused liver deprived of a single
essential amino acid(16) , whereas activation of eIF-2
kinases has been proposed to regulate phosphorylation of the factor
under other conditions(1) . The present report provides
evidence that depletion of ER Ca
stores signals the
activation of an eIF-2
kinase and that the activated enzyme is
PKR.
Figure 1:
eIF-2 kinase activity of extracts
derived from GH
cells pretreated with or without
Ca
ionophore. Cells in serum-free medium were
incubated for 7.5 min with or without the addition of 1 µM A23187. Extracts (10 µg of protein) derived from each
preparation were incubated for 2 min with
[
-
P]ATP (0.5 µM) and the
indicated amounts of highly purified eIF-2. Mixtures were then
subjected to SDS-PAGE followed by autoradiography (panel A).
Incorporation of radioactive phosphate into the
-subunit of eIF-2
was quantitated for each experimental condition with an AMBIS
radioanalytic imaging system (panelB). The circles and triangles indicate pretreatments with and
without A23187, respectively.
Pretreatment of HeLa cells with interferon
modestly reduced leucine incorporation (Table 1) and
slightly increased eIF-2
phosphorylation (Table 1; Fig. 2, lanes1 and 3). Upon
challenge of the cells with either A23187 or thapsigargin, however,
leucine incorporation was inhibited approximately 80-90% and
approximately 40-50% of eIF-2
molecules became
phosphorylated regardless of interferon pretreatment (Table 1; Fig. 2, lanes2, 3, 5, and 6). Identical findings were obtained with NIH-3T3 cells (not
shown).
Figure 2:
Effects of Ca-mobilizing
drugs on eIF-2
phosphorylation in HeLa cells cultured in the
absence or presence of interferon. Cells were cultured for 18 h with or
without interferon
(IFN, 1000 units/ml). Cultures were
washed and suspended in defined medium without further additions, with
A23187 (1 µM in combination with 1 mM EGTA to
hasten depletion of HeLa Ca
stores(31) , or
with thapsigargin (Tg, 10 µM) as indicated.
Incubations without drug or with thapsigargin were conducted for 45
min. The incubation with A23187/EGTA was conducted for 20 min. After
centrifugation at 1000
g, cells were lysed and
subjected to slab gel isoelectric focusing over a pI range of
4-8. Immunoblotting for eIF-2
was performed as
described(13, 14, 16) . The arrows indicate the migration positions of the phosphorylated (eIF-2
(P)) and non-phosphorylated (eIF-2
)
subunits.
PKR when activated by dsRNA is subject to
autophosphorylation(1, 2, 3) . HeLa cells
were pretreated with interferon to increase PKR content and were
challenged with A23187 or thapsigargin to mobilize ER sequestered
Ca
. A 2-3-fold increase in eIF-2
kinase
activity was observed in extracts of challenged, as compared to
control, preparations (Fig. 3A, lanes1, 3, and 5). A 68-kDa polypeptide was
observed to phosphorylate simultaneously with eIF-2
in these
extracts. Addition of low concentrations of reovirus dsRNA to
incubations caused a marked increase in the phosphorylations of this
polypeptide and of eIF-2
(lanes1 and 2), with greater increases observed in extracts of A23187 or
thapsigargin-treated preparations (lanes2, 4, and 6). Pretreatment with interferon caused a
5-fold induction of the 68-kDa polypeptide, which was identified as PKR
by immunoblotting (Fig. 3B, lanes1 and 2). Challenge with A23187 (lane3)
or thapsigargin (lane4) did not affect the amount of
immunoreactive PKR detected in extracts of interferon-pretreated cells.
Figure 3:
dsRNA-dependent phosphorylation of
eIF-2 and PKR and the PKR contents of extracts derived from HeLa
cells treated with or without Ca
-mobilizing agents.
Cells were cultured for 18 h with interferon
(IFN, 1000
units/ml) and treated with A23187/EGTA or thapsigargin (Tg) as
described in the legend to Fig. 2. Extracts (20 µg of
protein) of each preparation were incubated for 15 min with
[
-
P]ATP (0.5 µM) and purified
eIF-2 (75 ng) with or without reovirus dsRNA (0.1 µg/ml) as
indicated. Mixtures were subjected to SDS-PAGE, and autoradiography was
performed (panelA). Migration positions of
eIF-2
and human PKR are indicated. Additional aliquots (75 µl)
of these extracts and of a lysate of cells cultured without interferon
were subjected to immunoblotting for measurement of relative PKR
concentrations (panelB). 1, no interferon (IFN); 2, with interferon; 3,
A23187-treated; 4,
thapsigargin-treated.
Extracts of HeLa cells that had not been pretreated with interferon
did not contain detectable eIF-2 kinase activity regardless of
whether dsRNA was added (not shown). Therefore, experiments were
conducted to establish whether the increased dsRNA-dependent
phosphorylations of eIF-2
and PKR in extracts of cells with
depleted ER Ca
stores were dependent on interferon
pretreatment. NIH-3T3 cells and an antibody established to efficiently
immunoprecipitate PKR from extracts of murine cells were employed for
this purpose. Cells cultured with and without interferon
were
either treated with A23187 or thapsigargin or carried as untreated
controls. Cell extracts from each preparation were incubated with or
without the addition of dsRNA, and PKR was subsequently isolated by
immunoprecipitation. Phosphorylation of PKR was determined after
SDS-PAGE and autoradiography (Fig. 4A). Phosphorylation
of the kinase in extracts of untreated controls was detectable only if
incubations were conducted with added dsRNA (lanes1 and 2). By contrast, PKR phosphorylation was
distinguishable in extracts of A23187 or thapsigargin-treated
preparations incubated without dsRNA (lanes3 and 5); addition of dsRNA to these extracts caused the kinase to
become highly phosphorylated (lanes4 and 6). When interferon-pretreated preparations were subjected to
these protocols, PKR phosphorylation was further amplified for extracts
prepared from cells treated with ER Ca
perturbants
and for incubations conducted with added dsRNA (lanes
7-12).
Figure 4:
dsRNA-dependent phosphorylation of PKR in
extracts of NIH-3T3 cells pretreated with or without interferon and
Ca-mobilizing drugs: correlation with activation of
eIF-2
kinase. Cells were cultured for 18 h with or without
interferon
(IFN, 1000 units/ml). Cells were then
incubated in serum-free medium for 7.5 min without addition, with
A23187 (1 µM), or with thapsigargin (1 µM).
Extracts (20 µg protein) derived from each preparation were
incubated for 15 min with [
-
P]ATP (0.5
µM) and purified eIF-2 (0.5 µg) with or without the
addition of reovirus dsRNA (0.1 µg/ml) as indicated. PKR was then
immunoprecipitated from each reaction mixture, immunoprecipitates were
subjected to electrophoresis, and autoradiography was performed (panelA). Supernatant fractions remaining after
immunoprecipitation of samples corresponding to lanes7, 9, and 11 in panelA were subjected to SDS-PAGE and analyzed for eIF-2
phosphorylation by autoradiography (panelB).
The increase in PKR phosphorylation attributable
to depletion of the Ca stores of interferon-treated
NIH-3T3 cells was associated with markedly increased eIF-2
kinase
activity. Supernatant fractions remaining after immunoprecipitation of
PKR from extracts corresponding to lanes7, 9, and 11 of Fig. 4A were examined
for extent of eIF-2
phosphorylation (Fig. 4B).
Extracts of cells challenged with A23187 (lane2) or
with thapsigargin (lane3) actively incorporated
phosphate into eIF-2
without addition of dsRNA, whereas extracts
of unchallenged controls did not (lane1).
Experiments were also performed to ascertain whether Ca store depletion affected the dsRNA concentration dependence of
PKR phosphorylation in vitro. Extracts derived from
A23187-treated and untreated NIH-3T3 cells were incubated with 10
µM [
-
P]ATP and increasing
concentrations of reovirus dsRNA. PKR was then isolated by
immunoprecipitation, electrophoresis and autoradiography were
performed, and PKR phosphorylation was quantitated by densitometry. The
characteristic biphasic response to changes in dsRNA concentration (2, 3) was observed (Fig. 5). Low
concentrations of dsRNA enhanced PKR phosphorylation, whereas
concentrations in excess of 0.1 µg/ml were inhibitory.
Phosphorylation of PKR in extracts of treated preparations was
significantly greater than that in extracts of control preparations
regardless of dsRNA concentration, with a 11-fold differential observed
at 0.03 µg/ml. Somewhat lower concentrations of dsRNA were required
for maximal PKR phosphorylation in extracts of A23187-treated, as
compared with control, preparations.
Figure 5:
dsRNA concentration dependence of PKR
phosphorylation in extracts of NIH-3T3 cells pretreated with or without
A23187. Cells were cultured for 18 h with interferon (1000
units/ml) and then incubated in serum-free medium for 7.5 min with or
without A23187 (1 µM). Extracts were incubated as
described in the legend to Fig. 4but with 10 µM [
-
P]ATP and the indicated
concentrations of reovirus dsRNA. PKR was immunoprecipitated from each
reaction mixture, and electrophoresis and autoradiography were
performed. Incorporation of radioactive phosphate into PKR was
quantitated by densitometry. Results are expressed as percent of the
maximum phosphorylation observed. The circles and triangles indicate pretreatments with and without A23187,
respectively.
By several criteria the phosphorylation of eIF-2 and
suppression of translational initiation occurring in eukaryotic cells
in response to ER Ca
-mobilizing agents involve the
activation of the well known, interferon-inducible, dsRNA-activated
eIF-2
kinase, PKR. Addition of dsRNA to extracts of NIH-3T3 cells
with depleted ER Ca
stores resulted in the
phosphorylation of immunoprecipitable PKR that did not occur in
extracts derived from untreated cells. Interferon pretreatment of the
cells broadened this differential. Increased phosphorylation of PKR was
also detectable in incubations without dsRNA for extracts derived from
cells treated with Ca
-mobilizing agents.
Phosphorylation of PKR in vitro invariably correlated with the
phosphorylation of eIF-2
.
While the PKR activity of the
extracts appears to be stable for some weeks when stored at -80
°C, the eIF-2 kinase activity of extracts was clearly
inefficient compared to that of intact cells. We found that the kinase
activity of crude extracts was optimally manifested during brief
incubations conducted with highly purified eIF-2
and
[
-
P]ATP of high specific activity.
Sufficient expression of eIF-2
kinase by the cells from which
extracts were derived was critical. For example, eIF-2
was not
noticeably phosphorylated in extracts of HeLa or NIH-3T3 cells unless
the cells were pretreated with interferon to induce PKR. It should be
noted that, although interferon pretreatment increased PKR
concentrations, the induced enzyme was not expressed in an activated
form. Neither the phosphorylation state of eIF-2
nor the rate of
leucine incorporation was prominently affected by interferon treatment.
Similarly, the percentage of total eIF-2
phosphorylated in
response to Ca
-mobilizing agents did not change as a
function of interferon pretreatment. Extracts derived from
interferon-pretreated cells that were challenged with
Ca
-mobilizing drugs, however, exhibited significantly
(HeLa) or markedly (NIH-3T3) increased eIF-2
kinase activity as
compared to extracts of pretreated, non-challenged controls. Treatment
with interferon, therefore, improved our detection in vitro of
the eIF-2
kinase activation occurring in vivo. While all
of the eIF-2 kinase of our extracts was apparently PKR, we could not
exclude the possibility that other eIF-2 kinase activities were
activated in vivo that were inactivated by lysis.
PKR is
reported to be modestly phosphorylated in vivo and to
phosphorylate in vitro in the absence of dsRNA(5) .
Autophosphorylation is widely viewed as essential to the mechanism
whereby dsRNA activates PKR both in vivo and in
vitro. Binding of dsRNA to the amino terminus of the latent enzyme
prompts the autophosphorylation(24) , resulting in a
conformational change that is believed necessary for expression of
eIF-2 kinase activity. Current evidence favors the hypotheses that
latent PKR exists as a non-phosphorylated, ribosome-associated monomer
and as a cytosolic dimer of partly phosphorylated subunits (25) and that cross-phosphorylation promotes
dimerization(26, 27) . Whether mobilization of
sequestered ER Ca
stores causes PKR to
autophosphorylate and/or to dimerize in vivo remains to be
established. Activation of this cytosolic protein kinase by
perturbation of the ER may represent a unique signaling mechanism.
Depletion of ER Ca
stores did not cause the enzyme to
be compartmentalized, as has been reported for eIF-2
kinase
activity after heat shock(28) . Increased cytoplasmic free
Ca
could not be implicated in the activation since
identical findings were obtained when cells were exposed to
extracellular EGTA to deplete the cytoplasmic cation pool. It is clear,
however, that activation of PKR by this pathway involves a modification
that is not destroyed during cell lysis, that favors
autophosphorylation in vitro, and that renders the enzyme more
susceptible to activation by low concentrations of dsRNA in
vitro. Further investigations will be needed to ascertain whether
an endogenous dsRNA activator, such as has been described in 3T3-F442A
cells(29) , is generated or whether an endogenous inhibitor,
such as the 58-kDa tetratricopeptide repeat protein(30) ,
dissociates from the enzyme in cells with depleted ER Ca
stores.
The data of this report indicate that PKR is subject
to activation by Ca mobilizing agents that are known
to disrupt ER protein processing and to inhibit translational
initiation in intact cells. This information, in conjunction with a
lack of other candidates for the eIF-2
kinase activity, indicates
that PKR probably functions in capacities extending beyond combating
viral infections. Inhibition of translational initiation characterized
by phosphorylation of eIF-2
occurs in eukaryotic cells
experiencing various hormonal, nutritional, or environmental
stressors(1, 2, 3) . While the exact
components of the signaling system emanating from the ER that trigger
PKR activation remain to be defined, the initiating events appear to
involve the disruption of protein processing. It is clear that
conditions or agents that either mobilize sequestered Ca
from or disrupt the oxidizing environment of the organelle
prevent the proper folding of newly synthesized proteins. Substantial
evidence suggests that ER chaperones such as GRP78 that function in
protein processing become limiting under such circumstances but are
induced within several hours. This induction is accompanied by the
dephosphorylation of eIF-2
and a limited resumption (approximately
50%) of translation(12, 13, 15) . It
therefore appears that the activation of eIF-2 kinase is inversely
proportional to the availability of GRP78.