(Received for publication, October 17, 1994; and in revised form, December 12, 1994)
From the
Perturbation of endoplasmic reticular (ER) function signals
increased expression of the gene encoding the ER resident chaperone
Grp78/BiP and rapid suppression of translational initiation accompanied
by phosphorylation of the -subunit of eukaryotic initiation factor
2 (eIF-2). eIF-2
phosphorylation and grp78 mRNA induction
were measured in GH
pituitary cells subjected to varied
degrees of ER stress to ascertain whether activation of an eIF-2
kinase is involved in both events. grp78 mRNA was induced at
low concentrations of ionomycin and dithiothreitol that did not provoke
eIF-2
phosphorylation or inhibition of amino acid incorporation.
Mobilization of the bulk of cell-associated Ca
and
the induction of grp78 mRNA occurred at comparable low
concentrations of ionomycin, whereas phosphorylation of eIF-2
and
inhibition of protein synthesis required higher ionophore
concentrations. Pretreatment for 1 h with cycloheximide suppressed grp78 mRNA induction and eIF-2
phosphorylation in
response to either stressor. Prolonged (17 h) cycloheximide blockade
increased eIF-2
phosphorylation without inducing grp78 mRNA. Upon release from the blockade, grp78 mRNA was
induced and eIF-2
was dephosphorylated. Translational tolerance to
ionomycin or dithiothreitol, accompanied by dephosphorylation of
eIF-2
, was observed whenever grp78 mRNA was induced.
Induction of grp78 mRNA preceded significant eIF-2
phosphorylation during treatment with brefeldin A. It is concluded that
signaling of grp78 gene transcription can occur independently
of eIF-2
phosphorylation or translational repression and that
greater degrees of ER stress are required for eIF-2
phosphorylation than for grp78 mRNA induction.
Alterations in the functional status of the endoplasmic
reticulum (ER) ()signal the increased synthesis of ER
resident protein chaperones(1, 2) . Signaling is
evoked by such conditions as depletion of ER Ca
stores, introduction of a reducing environment, suppression of
protein glycosylation, or overexpression of secretory proteins and is
regulated at the level of gene transcription. Grp78/BiP, the chaperone
induced most prominently by ER stress, has been hypothesized to
function in the correct folding and assembly of proteins during early
protein processing(3) , in the retention of improperly folded
proteins that accumulate within the ER lumen when processing is
distressed(4) , and in the translocation of proteins from the
cytosol to the ER for processing(5, 6) . The gene for
Grp78 possesses a highly conserved promoter region that confers ER
stress inducibility and binds specific transcription
factors(7, 8, 9) . Although the mechanism
whereby the stressed ER of mammalian cells signals the activation of
gene transcription is unclear, an ER transmembrane kinase appears key
to such signaling in yeast(10, 11) . Termed IRE1 or
ERN1, this kinase is structurally similar to growth factor receptor
kinases with its N terminus on the lumenal side and cytoplasmic C
terminus carrying the kinase domain. By analogy, IRE1 may phosphorylate
as yet unidentified substrate proteins on serine or threonine residues.
ER perturbants, including Ca-mobilizing and
thiol-reducing agents, also signal the rapid suppression of mRNA
translation at initiation within min(12, 13) . This
inhibition occurs in conjunction with the phosphorylation of the
-subunit of eukaryotic initiation factor 2 (eIF-2) (14, 15) which is known to constrain eIF-2B activity
and thereby reduce formation of the 43 S preinitiation
complex(16) . Several hours exposure of cells to either
Ca
-mobilizing or thiol-reducing drugs results in the
prominent induction of grp78, the dephosphorylation of
eIF-2
, and the partial (40-80%) recovery of rates of
translational initiation and amino acid
incorporation(17, 18) . All of these events are
blocked by actinomycin D. Translational recovery is associated with the
development of cross-tolerance to Ca
-mobilizing or
reducing stressors and is decreased by antisense oligonucleotides
directed against grp78 mRNA.
HCR and PKR, mammalian
eIF-2 kinases that phosphorylate the
-subunit at serine
residue 51, are strongly implicated in rapid translational repression
under conditions wherein continued protein synthesis might prove
injurious(16, 19, 20) . HCR, the
heme-regulated eIF-2
kinase of erythroid cells, is inactive when
complexed with certain cytosolic chaperones (21, 22) and may function to regulate gene
transcription(23) . PKR is interferon-inducible, activated by
double-stranded RNA produced during the replicative cycle of certain
viruses, localized to ribosomes of the rough ER, and may function
importantly in controlling growth and
differentiation(16, 19, 20) . PKR has
recently been demonstrated to activate gene transcription through
regulation of NF-
B(24) . The possibility that activation
of an eIF-2
kinase during ER stress is required for signaling of grp78 gene transcription has not been explored. The following
study was undertaken to ascertain whether phosphorylation of eIF-2
and induction of grp78 mRNA occur under identical degrees of
ER stress and whether the phosphorylation is obligatory for grp78 gene transcription.
The ionophore concentration dependencies for inhibition of
amino acid incorporation, increased eIF-2 phosphorylation,
Ca mobilization, induction of grp78 mRNA, and
development of translational tolerance were compared within the same
preparation of GH
cells ( Fig. 1and Fig. 2).
Cells suspended in medium containing phorbol ester and cholera toxin
were incubated for 15 min or 3 h with increasing concentrations of
ionomycin prior to various analyses. At 15 min of incubation, leucine
incorporation into protein was suppressed by ionomycin at
concentrations exceeding 30 nM, with 0.1 and 1 µM drug providing 48 and 88% inhibitions, respectively (Fig. 1A). eIF-2
phosphorylation increased in
parallel with inhibition of incorporation, with approximately 20% of
the factor being phosphorylated at 1 µM ionomycin (Fig. 1A and Fig. 2, upper panel).
Following 3 h of exposure, however, these parameters became resistant
to suppression by the ionophore. Some residual eIF-2
phosphorylation and inhibition of leucine incorporation was manifested
at the higher ionophore concentrations (Fig. 1B and Fig. 2, lower panel).
Figure 1:
Phosphorylation of eIF-2,
induction of grp78 mRNA, expression of translational
tolerance, and mobilization of cell-associated Ca
at
varying concentrations of ionomycin. GH
cells in medium
containing 0.6 µM PMA and 50 ng/ml cholera toxin were
treated for 15 min or 3 h with the indicated concentrations of
ionomycin. A, incubations were conducted for 15 min.
[
H]Leucine was then added to portions of each
preparation, and pulse incorporation into protein was determined after
15 min (
). Additional samples were taken to quantitate the
percentage of eIF-2
in the phosphorylated form after
immunoblotting as described under ``Experimental Procedures''
(
). B, incubations were conducted for 3 h.
[
H]Leucine incorporation (
) and the
percentage of eIF-2
in the phosphorylated form (
) were then
determined. C, incubations were conducted for 3 h. Total RNA
was extracted and subjected to dot blot analysis using radiolabeled
cDNA probes for cellular mRNAs encoding grp78 (
) or
-actin (
). The content of each mRNA in untreated cells at
time 0 was assigned a value of 1.0, and the results are expressed as
the relative concentration after 3-h incubation. Additional
preparations were subjected to a washing procedure to remove ionomycin
and were subsequently rechallenged with ionomycin (1 µM)
or solvent (Me
SO, 0.5%) for 15 min.
[
H]Leucine incorporation into protein was then
determined. The percent inhibition of incorporation provided by
ionomycin is plotted inversely on the ordinate as a measure of
translational tolerance to the ionophore (
). D, cells
preloaded with
CaCl
were challenged as above
with the indicated concentrations of ionomycin, and cell-associated
Ca was determined after 15 min (
) or 3 h (
)
of incubation.
Figure 2:
Ionomycin concentration dependence of
eIF-2 phosphorylation during brief and extended incubations.
GH
cells were treated with the indicated concentrations of
ionomycin under conditions described in the legend to Fig. 1. Upper panel, 15-min incubation; lower panel, 3-h
incubation. Preparations were denatured, subjected to slab gel
isoelectric focussing, and immunoblotted for eIF-2
as indicated
under ``Experimental Procedures.'' The position of the
phosphorylated form of eIF-2
is indicated by the arrow.
The induction of grp78 mRNA occurred at low concentrations of ionophore that did not
significantly affect eIF-2 phosphorylation or rates of amino acid
incorporation. grp78 mRNA, measured after 3 h of incubation (Fig. 1C), was induced to increasing degrees between 5
and 50 nM ionomycin. Translational tolerance, measurable in
washed preparations as an increased resistance of incorporation to
inhibition by 1 µM ionomycin, developed concomitantly with
the induction of grp78 mRNA (Fig. 1C).
Cell-associated
Ca was largely mobilized over the range of
5 and 100 nM ionomycin at either incubation time (Fig. 1D). A similar experiment was conducted to
determine the concentrations of dithiothreitol required for inhibition
of amino acid incorporation, eIF-2
phosphorylation, induction of grp78 mRNA, and development of translational tolerance (Fig. 3). Amino acid incorporation was inhibited and eIF-2
was phosphorylated within 15 min at concentrations of reducing agent in
excess of 60 µM (Fig. 3A). Effects were
half-maximal at approximately 150 µM drug. After 3 h good
recoveries of incorporation and dephosphorylation of eIF-2
were
observed for cells exposed to the previously inhibitory concentrations
of dithiothreitol (Fig. 3B). Both the induction of grp78 mRNA and the development of translational tolerance to
ionomycin occurred at low concentrations of dithiothreitol (30-50
µM) that were not inhibitory to amino acid incorporation
or productive of eIF-2
phosphorylation (Fig. 3C).
Actin mRNA concentrations did not increase during longer incubations
with either ionomycin or dithiothreitol (Fig. 1C and
3C).
Figure 3:
Phosphorylation of eIF-2, induction
of grp78 mRNA, and expression of translational tolerance at
varying concentrations of dithiothreitol. GH
cells in
medium containing 0.6 µM PMA and 50 ng/ml cholera toxin
were treated for 15 min or 3 h with the indicated concentrations of
dithiothreitol. A, incubations were conducted for 15 min.
[
H]Leucine incorporation into protein (
) and
the percentage of eIF-2
in the phosphorylated form (
) were
then determined. B, incubations were conducted for 3 h.
[
H]Leucine incorporation (
) and the
percentage of eIF-2
in the phosphorylated form (
) were then
determined. C, incubations were conducted for 3 h. Samples
were taken for extraction of total cellular RNA, and mRNAs for grp78 (
) and
-actin (
) were quantitated by
dot blot analysis. As in Fig. 1, results shown are normalized to
the basal concentration of mRNA in control cells at time 0. Additional
preparations were washed to remove dithiothreitol and were rechallenged
with ionomycin (1 µM) or solvent for 15 min.
[
H]leucine incorporation into protein was
determined, and the percent inhibition provided by ionomycin was
plotted inversely on the ordinate as a measure of
translational tolerance (
).
The ability of cycloheximide to
counteract the phosphorylation of eIF-2 and the induction of grp78 mRNA in cells responding to either ionomycin or
dithiothreitol was examined in additional incubations (Table 2).
Pretreatment for 1 h with the elongation blocker resulted in almost
complete suppression of grp78 mRNA induction in response to
either stressor. eIF-2
phosphorylation occurring in response to
the stressors was also diminished in the presence of cycloheximide.
Phosphorylation after 15-min treatment with 0.6 mM dithiothreitol was abolished, whereas phosphorylation in response
to 15 min of treatment with 1 µM ionomycin was reduced
approximately 60% by the inhibitor. The degree to which cycloheximide
suppressed eIF-2
phosphorylation depended on the concentration of
stressor (Fig. 4). Phosphorylation in response to 0.2 mM dithiothreitol or 0.2 µM ionophore was eliminated by
cycloheximide whereas that in response to 1.5 mM dithiothreitol or 2 µM ionophore was partially
suppressed. The dephosphorylation that routinely accompanies recovery
of amino acid incorporation in cells exposed for 3 h to ionophore did
not occur in incubations containing cycloheximide (Table 2).
Rather, the residual phosphorylation observed upon 15-min challenge
with ionophore in the presence of cycloheximide was preserved during
the extended incubation.
Figure 4:
Effect of cycloheximide on eIF-2
phosphorylation following treatment with two different concentrations
of dithiothreitol or ionomycin. GH
cells were pretreated in
the absence (upper panel) or presence (lower panel)
of cycloheximide (50 µM) for 1 h. Dithiothreitol (0.2 or
1.5 mM) or ionomycin (0.2 or 2 µM) was added as
indicated. After 15 min preparations were denatured, subjected to slab
gel isoelectric focussing, and immunoblotted for eIF-2
. The arrow indicates the position of the phosphorylated form of
eIF-2
.
Translational tolerance to ionomycin was expressed after 3 h, but not after 15 min, of recovery from cycloheximide pretreatment (Table 4). Development of the accommodation was prevented when cells were allowed to recover in the presence of actinomycin D.
Figure 5:
Time dependence of eIF-2
phosphorylation upon treatment with brefeldin A or ionomycin.
Conditions are as described in the legend to Table 5. GH
cells were challenged with either 5 or 10 µg/ml brefeldin A (BFA) or with 1 µM ionomycin as indicated. After
incubation for 15 min, 1 h, or 4 h, preparations were denatured,
subjected to slab gel isoelectric focussing, and immunoblotted for
eIF-2
. The arrow indicates the position of the
phosphorylated form of eIF-2
.
The event most commonly invoked in the signaling of increased grp78 gene transcription is the accumulation of unfolded proteins in the lumen of the ER. The ER perturbants used in this study to induce grp78 mRNA either cause the lumenal accumulation of unprocessed intermediates or introduce nonresident proteins into the lumen(29, 32, 33, 34, 35, 36, 37, 38) . It is less clear how release from prolonged elongation blockade, which also provoked considerable grp78 mRNA induction, could cause unfolded or alien proteins to accumulate in the ER and/or protein processing to be impaired. In this regard Dorner et al.(39) observed that expression of normal secretory proteins at higher than normal concentrations retarded processing of these proteins and stimulated grp78 gene transcription. It is conceivable, therefore, that prolonged incubation in the absence of substrates for protein processing causes down-regulation of the ER processing system and that flooding the ER with new substrates after such down-regulation causes underprocessed intermediates to accumulate.
It is clear from the findings described in this report that neither
an increase in the phosphorylation of eIF-2 nor a decrease in the
rate of mRNA translation is required for signaling of grp78 gene transcription by ER perturbants. In agreement with earlier
studies(30, 31) , grp78 gene transcription in
response to ER perturbants was actually repressed when full
translational blockade was imposed. These findings are entirely
consistent with the proposal (40) that Grp78 suppresses IRE1
activity when complexed with the kinase and that grp78 gene
transcription is signaled when unfolded processing intermediates rob
the kinase of the chaperone. Cycloheximide would be expected to block
synthesis of precursors of such unfolded intermediates that would
otherwise accumulate in the lumen of the stressed ER. An alternative
explanation for the findings described in Table 1is that
continuous synthesis of a cytosolic factor possessing a rapid turnover
rate under nonstressed conditions is obligatory for signaling of grp78 gene transcription during ER stress. Turnover would
necessarily be retarded under conditions that perturb the ER and could
conceivably involve rapid catabolism or removal from the cytosol,
either by export to the extracellular fluid or translocation into an
organelle such as the ER. An arrest of protein translocation into the
ER during stress, resulting in the accumulation of an
IRE1-phosphorylatable gene regulatory factor in the cytosol, remains
compatible with available information. Phosphorylation of eIF-2
by
Ca
ionophore or dithiothreitol was also reduced when
protein synthesis was blocked. Cycloheximide inhibitions were only
partial at higher doses of the perturbants, however, consistent with
the proposal that eIF-2
phosphorylation is signaled under
conditions wherein Grp78 is insufficient for management of stress
within the ER.
The mammalian counterpart of the protein kinase IRE1,
which is required for signaling of grp78 gene transcription in
yeast(10, 11) , has not yet been characterized. It is
improbable, however, that this enzyme signals both grp78 gene
transcription and eIF-2 phosphorylation during ER stress. To
function as an eIF-2
kinase, mammalian IRE1 would have to display
different substrate specificities at different degrees of ER stress.
Such an enzyme would be unique.
Studies are currently in progress in
our laboratory to describe the mechanism whereby the highly stressed ER
signals eIF-2 phosphorylation. We have recently identified an
eIF-2
kinase activity that is stimulated in intact cells in
response to ER perturbants and is detectible in cell lysates. (
)The properties of this kinase appear identical with those
of PKR, the interferon-inducible, double-stranded RNA-activated protein
kinase implicated in control of growth and differentiation. Further
investigations will be required to understand how the ER prompts the
activation of a protein kinase residing outside the organelle.
The
phosphorylation state of eIF-2 was found to correlate closely with
the rate of amino acid incorporation during increasing degrees of ER
stress (Fig. 1Fig. 2Fig. 3). Equally notable was
the close correlation between degree of expression of translational
tolerance to ionomycin and extent of grp78 mRNA induction. It
was also clear that dephosphorylation of eIF-2
, which always
accompanied translational recovery during extended incubations at high
(1 µM) ionophore concentrations (e.g.(14) and (15) ; Fig. 2and 5), did not
occur when grp78 mRNA induction was suppressed by
cycloheximide (Table 2). These findings, as well as those
reported previously(17, 18) , favor a role for new
Grp78 in expression of the accommodation. How the chaperone acts to
suppress eIF-2
phosphorylation and thereby maintain translational
activity remains unclear. Grp78 has been proposed to function as part
of a translocation system for protein entry into the
ER(5, 6) ; this system may conceivably bind to and
inhibit an eIF-2
kinase. The binding of cytosolic chaperones to
HCR suppresses the activity of this eIF-2
kinase in rabbit
reticulocyte lysates(21, 22) . Addition of denatured
proteins to such lysates signals activation of the kinase, presumably
as a consequence of dissociation of chaperone-enzyme
complexes(41) . It is therefore attractive to speculate that
Grp78 regulates an eIF-2
kinase in intact non-erythroid cells
during stresses that promote protein unfolding in the ER.
In many respects both eIF-2 phosphorylation and grp78 induction behave as though they are subject to inhibition by a catalytic ER pool of Grp78. Any condition involving the depletion of this pool, including the accumulation of very early ER protein folding intermediates, overproduction of high amounts of specific proteins, or interdiction of ER to Golgi vesicular traffic, would ultimately trigger the two responses. The induction of grp78 is clearly the more sensitive of the two. Under such conditions protein synthesis would become dependent on a continuing synthesis of Grp78 as is, indeed, observed.