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INTRODUCTION |
Chaperones resident to the endoplasmic reticulum
(ER)1 catalyze the folding of
nascent polypeptides to tertiary structures that are competent for ER
to Golgi transport. Protein folding and processing within the ER are
dependent upon Ca2+ sequestration and the maintenance of a
redox potential that permits the formation of specifically placed
disulfide bonds (1-3). Protein processing is inhibited within several
minutes following exposure to Ca2+-mobilizing or
sulfhydryl-reducing agents at concentrations that do not lower ATP
(4-6). Accumulation of unfolded protein in response to these agents is
thought to signal a slowing of translational initiation through
activation of the double-stranded RNA-stimulated eIF-2
kinase, in
conjunction with eIF-2
phosphorylation and the inhibition of eIF-2
cycling (6).
Continued inhibition of processing (ER stress) results in the
subsequent induction of various ER chaperones (7-9). GRP78/BiP, the
chaperone induced most prominently and rapidly by ER stress, has been
hypothesized to function in the correct folding and assembly of
proteins during early protein processing (10), in the retention of
improperly folded proteins that accumulate within the ER lumen when
processing is distressed (11), and in the co-translational translocation of nascent polypeptides from the cytosol to the ER for
processing (12, 13). The gene encoding GRP78 possesses a highly
conserved promoter region that confers ER stress inducibility (8, 9).
Induction or overexpression of the chaperone confers tolerance to
translational inhibition and eIF-2 phosphorylation in response to ER
stress (6, 14). In cell types overproducing proteins that translocate
to the ER but that are incapable of ER to Golgi transport, GRP78 is
chronically elevated, and mRNA translation is sustained upon
challenge with ER stressors (6).
GRP78 is reported to undergo post-translational modification by
mono-ADP-ribosylation and by phosphorylation (15, 16). Both
modifications involve oligomerization of the chaperone to an inactive
form. When complexed with other proteins, GRP78 is not subject to
covalent modification or oligomerization (17). Post-translational
modifications of GRP78 are generally observed during conditions that
deplete the ER of processible protein. For example, the fractional
ADP-ribosylation of GRP78 is increased by lowered temperature, amino
acid starvation, and treatments with cycloheximide or amino acid
analogs (18-20). In contrast, the modification of GRP78 is suppressed
by conditions that inhibit glycoprotein processing within the ER such
as glucose depletion or treatments with tunicamycin, glucose analogs,
or Ca2+ ionophores (15, 19, 21, 22). It is also suppressed
by hormones that stimulate growth (22). The unmodified, unbound form of
GRP78 is thought, therefore, to function as the active form of the
chaperone that is available to interact with processing intermediates
derived from co-translational translocation. It is this form of the
chaperone that must be subject to inactivation through covalent
modification. Signaling of grp78 gene expression correlates
inversely with the extent to which the chaperone is covalently modified
(7-9, 18, 22).
The present report describes an investigation of the relationship of
GRP78 modification to protein processing and mRNA translation in
GH3 pituitary cells utilizing isoelectric focusing
methodology. ADP-ribosylation of the chaperone was increased by
treatments that slow the rate of mRNA translation relative to that
of protein processing. By contrast, ADP-ribosylation was decreased or
reversed by treatments that slow the rate of protein processing
relative to that of mRNA translation. A dynamic relationship was
found to prevail between the fractional ADP-ribosylation of GRP78 and the degree of eIF-2 phosphorylation that was explicable in terms of
changing contents of the active monomer.
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EXPERIMENTAL PROCEDURES |
Materials--
Monoclonal antibody to eIF-2
was the gift of
Dr. Lynn O'Brien, University of Rochester. Polyclonal rabbit
anti-GRP78 and recombinant GRP78 were purchased from StressGen
Biotechnologies. Ampholines were purchased from Gallard Schlesinger (pH
4-8) and from ESA Inc. (pH 3.5-10). Urea was purchased from
Boehringer Mannheim. [2-3H]Adenosine and
[
-32P]ATP were obtained from Amersham Pharmacia
Biotech, and L-[4,5-3H]-leucine was from ICN
Radiochemicals. Thapsigargin and okadaic acid were obtained from
Calbiochem. Pactamycin was donated by The Upjohn Co. Ionomycin,
brefeldin A, puromycin, dithiothreitol, cycloheximide, emetine,
novobiocin, nicotinamide, and calf intestinal alkaline phosphatase were
purchased from Sigma.
General Methodology--
GH3 pituitary cells were
maintained in suspension and utilized as described (23). Cultures were
normally provided with fresh growth medium for approximately 16 h
before harvest. Prior to treatments, cells were equilibrated for 15 min
with serum-free Ham's F-10 medium modified to contain 0.2 mM Ca2+. Leucine pulse incorporations were
measured as described previously (24) for 10- or 15-min incubations of
2 × 106 cells per experimental condition.
One-dimensional 7.5% polyacrylamide gel electrophoresis (SDS-PAGE) of
detergent-solubilized extracts of variously treated cell preparations
was conducted as described previously (25).
Determinations of the Covalent Modifications of GRP78 and of
eIF-2
Phosphorylation--
Cells (106) were harvested
by centrifugation and lysed with 300 µl of urea buffer containing 2%
ampholines (pH 3.5-10, two-dimensional), 4% Triton X-100, 100 mM dithiothreitol, and 9.9 M urea. Lysates were
applied to a 6% acrylamide slab gel (30% acrylamide, 1.5% bisacrylamide) containing ampholines (1:1, pH 3.5-10 to pH 4-8) and
subjected to slab gel isoelectric focusing in the presence of 9.5 M urea to separate the modified and unmodified forms of eIF-2
and of GRP78. Gels were treated with 1 M Tris, pH
8.8, and blotted onto polyvinylidene difluoride membranes under basic conditions. Phosphorylated and non-phosphorylated eIF-2
were immunodetected with monoclonal antibody to eIF-2 and chemiluminescence (26). The membranes were then treated with 1% sodium dodecyl sulfate
and 0.2%
-mercaptoethanol in phosphate-buffered saline at room
temperature for 15-30 min to remove the secondary antibody. GRP78 was
immunodetected with primary antibody (1:5000), with goat anti-rabbit
antiserum (1:5000) serving as the secondary antibody.
Phosphorylation of Recombinant GRP78--
The incubation
conditions for in vitro phosphorylation of GRP78 were as
described previously (27), except that the reaction was conducted at
37 °C for 2 h in 250 µl of 50 mM Hepes, pH 7.0, containing 20 mM [
-32P]ATP, 100 mM CaCl2, and 30 µg of recombinant GRP78.
Labeling of GH3 Cells with
[2-3H]Adenosine--
Cells were suspended to 4 × 106/ml of Ham's F-10 supplemented with 0.4 µM phorbol 12-myristate 13-acetate to maintain
translational rates in the absence of serum and were incubated for
2 h in the presence of [2-3H]adenosine (100 µC/ml). Cells were then diluted 2-fold with fresh medium containing
[2-3H]adenosine (100 µC/ml) and incubated in the
absence or presence of 50 µM cycloheximide for 1 h.
Samples were then prepared for isoelectric focusing and analysis of
GRP78 modification.
Treatment of Lysates with Alkaline
Phosphatase--
GH3 cells (2 × 106/ml)
were incubated in Ham's F-10 medium with or without 50 µM cycloheximide for 2 h. Cells were collected by
centrifugation and placed on ice, and lysis buffer (50 mM
Tris·HCl, 150 mM NaCl, 0.5% deoxycholate, 0.5% Triton
X-100, 0.5 mM phenylmethylsulfonyl fluoride) was added at a
ratio of 0.5 ml per 106 cells. The preparations were then
subjected to homogenization and sonication. Lysates were centrifuged at
15,000 × g for 10 min at 4 °C, and supernatant
fractions were stored at
70 °C until use. Lysates (200 µl)
and/or recombinant [32P]GRP78 (1 µg) were incubated for
2 h with 10 units of calf intestinal alkaline phosphatase. Samples
were treated for 18 h with 5 volumes of ice-cold acetone, and the
precipitates were collected, dried, and resuspended in 100 µl urea buffer.
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RESULTS |
Modification of GRP78 in Response to the Inhibition of mRNA
Translation by Various Agents--
Good evidence supports the
hypothesis that GRP78 functions in part to coordinate the respective
rates of ER protein processing and mRNA translation (6). It was
therefore of interest to examine GRP78 for modifications occurring in
response to different classes of agents that inhibit translation.
GH3 pituitary cells were chosen as a model system for the
experiments in view of their extensive characterization in terms of
Ca2+ homeostasis and translational control. Analyses of
GRP78 modification were conducted in conjunction with determinations of
amino acid incorporation into protein and measurements of the
phosphorylation of eIF-2
as an index of the suppression of
initiation. Relatively short incubations (30-90 min) were utilized to
minimize induction of chaperone by chemical stressors that inhibit
mRNA translation. Isoelectric focusing procedures permitted the
separation of eIF-2
from its phosphorylated form concurrently with
the separation of the unmodified and modified forms of GRP78. Both
types of analyses were conducted by sequential Western blotting of the
same transfer membrane.
GH3 cells were initially exposed for 30 min to various
direct-acting inhibitors of translational elongation (puromycin,
cycloheximide, and emetine) or to pactamycin, an acknowledged
inhibitor of translational initiation (Fig.
1). All of these agents were strongly
inhibitory to amino acid incorporation. None of them promoted eIF-2
phosphorylation (B, lanes 2-5) with respect to an untreated
control (B, lane 1), but all generated a separable modified
form of GRP78 (A, lanes 2-5) as compared with an untreated
control (A, lane 1). The ER Ca2+-mobilizing
agents, ionomycin and thapsigargin, and the reducing agent,
dithiothreitol, strongly inhibit both ER protein processing and
translational initiation. These agents increased eIF-2 phosphorylation (B, lanes 6-8) without generating modified GRP78
(A, lanes 6-8). During longer term exposures, these agents
act as ER stressors that induce the synthesis of GRP78 but not heat
shock proteins. Sodium arsenite, in contrast, modifies the sulfhydryl
groups of cytoplasmic proteins. This event produces cytoplasmic stress
associated with the complexing of cytoplasmic chaperones, the
activation of double-stranded RNA-activated eIF-2
kinase, and the
inhibition of translational initiation, and the subsequent induction of
the heat shock proteins but not GRP78 (28). Arsenite treatment produced phosphorylation of eIF-2 (B, lane 10) in conjunction with
GRP78 modification (A, lane 10). The modification of GRP78
therefore differed according to the class of agent producing
translational inhibition.

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Fig. 1.
Phosphorylation of eIF-2 and the
modification of GRP78 in response to the inhibition of mRNA
translation by various agents. Samples 1-8,
GH3 cells were incubated under standard conditions (see
"Experimental Procedures") for 30 min without addition (lane
1) or with 100 µM puromycin (lane 2), 20 µM cycloheximide (lane 3), 0.1 µM emetine (lane 4), 0.1 µM
pactamycin (lane 5), 1 µM ionomycin
(lane 6), 0.2 µM thapsigargin (lane
7), or 600 µM dithiothreitol (lane 8).
Aliquots were removed for protein separation by isoelectric focusing
and for measurements of [3H]leucine pulse incorporation.
Inhibitions of leucine incorporation by various agents ranged from 90 to 98%. Samples 2-5 were then adjusted to 1 µM ionomycin, and the incubation was continued for an
additional 30 min. Aliquots were removed at 60 min for protein
separation by isoelectric focusing. Western blotting: A,
GRP78 at 30 min; B, eIF-2 at 30 min; C, GRP78
at 60 min; D, eIF-2 at 60 min. Samples 9 and
10, a different preparation of GH3 cells was
incubated for 45 min either without addition (lane 9) or
with 50 µM (lane 10) sodium arsenite. Aliquots
were removed for protein separation by isoelectric focusing and for
measurements of [3H]leucine pulse incorporation.
Inhibitions of protein synthesis were 88% at 50 µM
arsenite, respectively. Samples 9 and 10 were then adjusted to 1 µM ionomycin, and the incubation was continued for 30 min, followed by sampling for isoelectric focusing. Western blotting:
A, GRP78 at 45 min; B, eIF-2 at 45 min;
C, GRP78 at 75 min; D, eIF-2 at 75 min. The
positions of modified (GRP78(m)) and unmodified GRP78 and of the
phosphorylated (eIF2 (P)) and non-phosphorylated forms of eIF-2
are indicated by the short arrows.
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During the subsequent 30 min of incubation the fractional modification
of GRP78 increased in the nontreated controls (C, lane 1), a
typical finding associated with longer term incubations without serum.
Addition of ionomycin to mobilize sequestered Ca2+ reversed
this modification (C, lane 9). Replicate samples undergoing treatments that inhibited protein synthesis (C and D,
lanes 2-8, and 10) were also adjusted with ionomycin
to ascertain whether the cells remained responsive to an ER stressor.
Ionomycin treatment decreased pre-existing modification of GRP78
resulting from conventional inhibitors of translation at either
elongation (C, lanes 2-4) or at initiation (C, lane
5). The modification of GRP78 in response to arsenite was also
abolished by ionomycin (C, lane 10). Cells that had been
pretreated with ER stressors (C and D, lanes
6-8) were not further affected by the addition of
ionomycin. Phosphorylation of eIF-2
developed following the addition
of ionomycin to a previously untreated sample (D, lane 9)
but not in an untreated control (D, lane 1) as well as in
samples containing conventional elongation inhibitors (D, lanes
2-4). Pactamycin treatment invariably precluded eIF-2
phosphorylation in response to ER stressors (D, lane 5). Cells treated with arsenite displayed intensified eIF-2 phosphorylation in response to ionomycin (D, lane 10).
Both the modified and unmodified forms of GRP78 focused as discrete
protein bands without evidence of multiple banding, as would have been
anticipated if multiple sites were covalently modified or if more than
one type of modification was occurring. Migration of the modified GRP78
to a more acidic isoelectric position was compatible with modification
by either phosphorylation or by ADP-ribosylation, each of which have
been reported to occur for this chaperone. Several lines of evidence
indicate that the modification of GRP78 occurring in GH3
cells was compatible with ADP-ribosylation, but not with
phosphorylation, of the protein. Cells that were equilibrated with
[2-3H]adenosine were found to possess radioactivity in
the modified but not in the unmodified form (Fig.
2A, right lanes). To generate significant radiolabeling, high cell density in conjunction with a
protracted incubation without serum was utilized. This condition resulted in a substantial degree of GRP78 modification (A, left lanes). Both the fractional degree of modification and the extent of radiolabeling were further increased by cycloheximide. While these
results were supportive of ADP-ribosylation, they did not preclude a
potential contribution of phosphorylation to the modification of
GRP78.

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Fig. 2.
Evidence that GRP78 of GH3 cells
is modified by ADP-ribosylation rather than by phosphorylation.
A, incorporation of adenosine into the modified form of
GRP78. Cells (4 × 106/ml) were incubated as described
under "Experimental Procedures" with [2-3H]adenosine
(100 µCi/ml) for 2 h. Cells were then diluted 2-fold with fresh
[2-3H]adenosine-containing medium with or without 50 µM cycloheximide (CHX). After an additional
hour of incubation, samples were collected for separation of proteins
by isoelectric focusing. The same blot was used for analysis of GRP78
by Western blotting (left) and for autoradiography (1 month,
right). B, stability of the modified form of
GRP78 to alkaline phosphatase. Cells were incubated with 50 µM cycloheximide for 2 h. Lysates (200 µl) and/or
recombinant [32P]GRP78 (1 µg) were incubated for 2 h either without further addition or with calf intestinal alkaline
phosphatase (Alk. P'tase, 10 units), okadaic acid (1 µM), or trypsin (0.001%). Samples were treated for
18 h with 5 volumes of ice-cold acetone, and the precipitates were
collected, dried, and resuspended in 100 µl urea buffer, 30 µl of
which was subjected to isoelectric focusing. Lower, Western
blots of GRP78; upper, autoradiography of the same blots.
C, inhibition of GRP78 modification by inhibitors of
ADP-ribosylation. Cells were incubated for 30 min without addition,
with 0.5 or 1 mM novobiocin (Novo), or with 20, 40, or 80 mM nicotinamide (Nico). Samples were
then divided into two portions, one of which received cycloheximide (20 µM). All samples were incubated for an additional 90 min
and analyzed for GRP78 by isoelectric focusing followed by Western
blotting. Upper blots, without cycloheximide; lower
blots, with cycloheximide. Short arrows in
A-C indicate the positions of modified (GRP78(m)) and
unmodified GRP78.
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The role of phosphorylation in the generation of modified GRP78 was
examined in more detail. Purified recombinant GRP78 was autophosphorylated with [
-32P]ATP. The stability of
the phosphate moiety was then tested by the addition of a cell lysate
derived from GH3 cells that had been pretreated with
cycloheximide to promote modification of GRP78. Isoelectric focusing
and Western blotting procedures revealed one major band of recombinant
GRP78 migrating to the same position as the unmodified GRP78 derived
from the cell lysates (Fig. 2B, lower lanes). The
radiolabeled recombinant protein (Fig. 2B, upper lanes), as
determined by radioautography, displayed the same isoelectric behavior
as the modified form of GRP78 detected in cell lysates by Western
blotting. Treatment of lysates with alkaline phosphatase did not affect
the proportion of GRP78 in the modified form, whereas the
32P-labeled recombinant protein was stripped of
radioactivity. Dephosphorylation of recombinant GRP78 also occurred in
incubations with lysate but without okadaic acid or alkaline
phosphatase (not shown). Both the modified and unmodified forms of
GRP78 were fully accessible and susceptible to proteolysis by trypsin.
ADP-ribosylation of various proteins has been reported to be inhibited
by novobiocin and nicotinamide (29). The small amount of GRP78 that was
modified in control cells was found to be abolished by either agent
(Fig. 2C, upper lanes). The much larger fractional
modification of GRP78 in response to cycloheximide was also completely
suppressed by either novobiocin or nicotinamide (Fig. 2C, lower
lanes). Novobiocin at 50 µM was sufficient for full
suppression of ADP-ribosylation (not shown). Collectively, these
findings emphasize that the covalent modification of GRP78 occurring in
intact GH3 cells is consistent with ADP-ribosylation of the
protein as described by others (15-22).
ADP-ribosylation of GRP78 and the Phosphorylation of eIF-2
in
Response to ER Stressors--
The time dependence of the
ADP-ribosylation of GRP78 in response to cycloheximide was examined
over 90 min (Fig. 3). ADP-ribosylation did not change in untreated controls throughout this period
(lanes A, left). Cells exposed to cycloheximide developed
increasing degrees of ADP-ribosylation within 15 min that approached
maximal values at 60 min (lanes B, left). Neither of these
conditions affected eIF-2 phosphorylation (lanes A and
B, right). Cycloheximide-treated cells were then washed to
remove the drug such that amino acid incorporation was no longer
inhibited, and the cells were analyzed for the reversal of the
ADP-ribosylation of GRP78. Reversal occurred with the same time
dependence as seen for the development of ADP-ribosylation of GRP78
(lanes C, left) without eIF-2 phosphorylation (lanes C, right). The reversal of ADP-ribosylation was
accelerated approximately 2-fold by either ionomycin (lanes D,
left) or by dithiothreitol (lanes E, left) at
concentrations established to suppress protein processing and amino
acid incorporation. Phosphorylation of eIF-2 in response to either
agent became maximal within 15 min (lanes D and
E, right). It was therefore apparent that eIF-2
phosphorylation occurred more rapidly than either ADP-ribosylation or
deribosylation of GRP78.

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Fig. 3.
Reversible modification of GRP78 as a
function of time following cycloheximide addition and removal.
GH3 cells were incubated under standard conditions for 90 min with (B) or without (A) 5 µM
cycloheximide and sampled for isoelectric focusing at the indicated
times. The remaining cells were collected, washed, and resuspended in
fresh medium without cycloheximide, all of which required 15 min.
Sample B was divided into 3 portions (C-E). C
was incubated without further addition; D was adjusted to 1 µM ionomycin; and E was adjusted to 600 µM dithiothreitol. C-E were incubated an
additional 90 min and sampled at the indicated times. Following
isoelectric focusing of proteins, GRP78 and eIF-2 were detected by
Western blotting for A-E. As in Fig. 1, the modified form
of GRP78 and the phosphorylated form of eIF-2 migrated to slightly more
acidic positions. [3H]Leucine incorporations revealed
that cycloheximide (B) was 93% inhibitory with respect to
A, that all of the cycloheximide was removed from
C with respect to the washed A control, and that
inhibitions by ionomycin (D) and dithiothreitol
(E) were 94 and 93%, respectively.
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Shorter term incubations were designed to minimize deactivation of
GRP78 from ADP-ribosylation in response to cycloheximide while
providing sufficient time (10 min) for eIF-2 phosphorylation in
response to ER stressors. It was anticipated that slowed rates of
translational elongation should result in the depletion of ER-processible protein as proteins completed processing in the absence
of continuing translocation. Some accumulation of the active
(unmodified) form GRP78 dissociating from proteins completing processing would be anticipated in view of the relatively slow rate of
inactivation of GRP78 by ADP-ribosylation. The relationship of protein
synthetic rates to the ADP-ribosylation of GRP78 and the
phosphorylation of eIF-2 was examined at a series of cycloheximide concentrations (0.2, 0.75, and 10 µM) (Fig.
4). These concentrations produced graded
inhibitions of amino acid incorporation ranging from 50 to 93%. Over
30 min ADP-ribosylation was increased by each cycloheximide
concentration (lanes 1, B-D, left) as compared with that of
the untreated control (lane 1A, left).
Phosphorylation of eIF-2 was not altered at any cycloheximide
concentration in incubations without ER stressor (lanes 1, right). Phosphorylation of eIF-2 in response to a 15-min exposure
to either ionomycin (1 µM, lanes 2, right) or
dithiothreitol (600 µM, lanes 3, right) was
also examined in cells that had been pretreated with cycloheximide for
15 min. Under these conditions eIF-2 phosphorylation in response to
stressors was sharply reduced by increasing concentrations of
cycloheximide (lanes 2 and 3, right).
Phosphorylation of eIF-2 in response to ER stressors was completely
suppressed at cycloheximide concentrations that largely inhibited
translation (lanes D, right). Modest reductions of
ADP-ribosylation of GRP78 occurred in response to ionomycin
(lanes 2, left) or dithiothreitol (lanes 3, left) as compared with incubations with cycloheximide alone
(lanes 1, B-D, left).

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Fig. 4.
Sensitivity of eIF-2 phosphorylation and
GRP78 modification to ER stressors as a function of increasing degrees
of inhibition of mRNA translation by cycloheximide.
GH3 cells were incubated under standard conditions for 15 min with 0 (A), 0.2 µM (B), 0.75 µM (C), or 10 µM cycloheximide
(D). Replicate cell samplings at each cycloheximide
concentration were either not treated further (lane 1) or
were adjusted with 1 µM ionomycin (lane 2) or
600 µM dithiothreitol (lane 3). The incubation
was continued for 15 min and sampling conducted for isoelectric
focusing and Western blotting of GRP78 and eIF-2 and for
[3H]leucine incorporation. As in Fig. 1, the modified
form of GRP78 and the phosphorylated form of eIF-2 migrated to slightly
more acidic positions. Amino acid incorporations were inhibited 50, 71, and 93% by 0.2, 0.75, and 10 µM cycloheximide,
respectively.
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The effect of cycloheximide on the sensitivity of eIF-2 phosphorylation
to ER stressors was examined at various times of incubation (0-90 min)
at a high concentration of the elongation inhibitor (20 µM) (Fig. 5). Samples for
each incubation time were adjusted with either ionomycin or
dithiothreitol 15 min before collection. Concentrations of each agent
were tested that were known to produce either submaximal or maximal
degrees of eIF-2
phosphorylation. Cells incubated as controls
without cycloheximide displayed high degrees of eIF-2 phosphorylation
with either concentration of either stressor (lanes A, 2-5,
right) as compared with the unstressed control (lane
A1, right) which did not change throughout the course of the experiment (lanes 1, right). Samples adjusted
simultaneously with cycloheximide and ER stressors for 15 min displayed
much less eIF-2 phosphorylation (lanes B, 2-5, right).
Samples pretreated with cycloheximide for 15 min before the addition of
stressors (lanes C, 2-5, right) displayed minimal degrees
of phosphorylation of eIF-2. Phosphorylation in samples adjusted at 30 min of cycloheximide treatment (lanes D, 2-5, right) was
depressed to a slightly lesser extent. By 45 min of cycloheximide
treatment before adjustment with stressor, eIF-2 phosphorylation had
nearly recovered (lanes E, 2-5, right) to the original
values observed without cycloheximide (lanes A, right). This
pattern was maintained at 90 min (lanes F, 2-5,
right). The simultaneous analysis of ADP-ribosylation of GRP78 was
also conducted. GRP78 was not significantly modified in controls
lacking cycloheximide (lanes A, 1-5, left).
ADP-ribosylation of GRP78 increased with time of incubation in cells
exposed to cycloheximide without ER stressors (lanes 1, B-F,
left) to approximately 50% at the longest treatment period with
cycloheximide (lane 1, F, left). At each time the addition
of either ionomycin or dithiothreitol lowered the fraction of GRP78
that was ADP-ribosylated. Marked reductions of ADP-ribosylation
occurred during shorter pretreatment periods with cycloheximide (0-30
min) (lanes B-D, 2-5, left). Cells treated for 45 or 90 min with cycloheximide before the addition of stressors exhibited more
modest reversals of ADP-ribosylation of GRP78 (lanes E-F, 2-5
left). Cycloheximide-treated samples with high degrees of
ADP-ribosylation of GRP78 were invariably more subject to eIF-2
phosphorylation in response to ER stressors.

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Fig. 5.
Sensitivity of eIF-2 phosphorylation to ER
stressors as a function of time following inhibition of mRNA
translation by cycloheximide. GH3 cells were suspended
for incubation under standard conditions. Initial samplings
(A) were conducted at the beginning of the incubation, and
the remaining cells were adjusted to 20 µM cycloheximide
for samplings immediately (B), and at 15, 30, 45, and 90 min
of incubation (lanes C-F, respectively). The samplings were
either left unadjusted (lane 1) or immediately adjusted with
0.2 µM ionomycin (lane 2), 1 µM
ionomycin (lane 3), 200 µM dithiothreitol
(lane 4), or 600 µM dithiothreitol (lane
5). Cells were then incubated for 15 min with these adjustments
and prepared for isoelectric focusing and Western blotting of GRP78 and
eIF-2 . As in Fig. 1, the modified form of GRP78 and the
phosphorylated form of eIF-2 migrated to slightly more acidic
positions. Prior to addition of cycloheximide (as in A),
samples were also removed for measurements of [3H]leucine
pulse incorporation after 15 min with or without stressor. Inhibitions
of incorporation by 0.2 and 1 µM ionomycin or by 200 or
600 µM dithiothreitol were 77, 92, 89, and 95%,
respectively.
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The ADP-ribosylation of GRP78 and the phosphorylation of eIF-2 in
GH3 cells were examined for coordination during a
restricted time frame (Fig. 6). Additions
of cycloheximide were staged at various times with respect to
adjustments with ionomycin over a total incubation period of 30 min.
The 30-min control for untreated cells possessed a marginal degree of
ADP-ribosylation (A, lane 1) and undetectable eIF-2
phosphorylation (B, lane 1). Samples exposed to
cycloheximide alone for 30 min developed moderate ADP-ribosylation (A, lane 2) without eIF-2 phosphorylation (B, lane
2). In contrast, samples treated with ionophore for 30 min
displayed minimal ADP-ribosylation of GRP78 (A, lane 3) in
conjunction with increased eIF-2
phosphorylation (B, lane
3). Some samples that were incubated with ionomycin for a total of
30 min were challenged at 10 min (lane 4) or 5 min (lane 5) of incubation with cycloheximide, providing 20 and
25 min of drug overlap, respectively. These incubations provided an
index of how readily cycloheximide interdicted the actions of
ionomycin. Cycloheximide, when added 10 min after ionomycin, produced
little, if any, alteration in GRP78 ADP-ribosylation (A, lane
4) or eIF-2 phosphorylation (B, lane 4) from that
observed in the ionomycin control (lanes 3). When added 5 min after ionomycin, however, cycloheximide suppressed eIF-2
phosphorylation about 50% (B, lane 5) without
much increase in ADP-ribosylation of GRP78 (A, lane 5). It
was therefore apparent that the effects of ionophore on eIF-2
phosphorylation became refractory to modification by cycloheximide
within 10 min. This time frame corresponds closely with that required
for ionomycin to generate eIF-2 phosphorylation in GH3
cells. Phosphorylation is half-maximal at 4 min and approaches maximal
values by 8 min (24). The ability of ionomycin to reverse cycloheximide-generated changes was assessed in additional incubations. Samples were treated with cycloheximide for 30 min with ionophore being
added at 2, 5, and 10 min of incubation corresponding to overlap
periods of 28, 25, and 20 min, respectively. With these increasingly
later times of ionomycin addition, graded increases in ADP-ribosylation
(A, lanes 6-8) and graded decreases in eIF-2 phosphorylation (B, lanes 6-8) were observed. The 2-min
delay in ionophore addition reduced eIF-2 phosphorylation about 50% (B, lane 6) as compared with the ionomycin control (B,
lane 3), while a 10-min delay in the addition almost abolished
phosphorylation (B, lane 8). Only a brief exposure to
cycloheximide, therefore, was needed to arrest eIF-2 phosphorylation in
response to the subsequent addition of ionomycin. These data are
consistent with the time required for maximal inhibition of amino acid
incorporation in response to cycloheximide (1-2 min).

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Fig. 6.
Alteration of the sensitivity of eIF-2
phosphorylation to ionomycin as a function of the time of addition of
cycloheximide relative to that of the ionophore. GH3
cells were suspended in replicate for incubation under standard
conditions. Ionomycin (IM, 0.2 µM) and/or
cycloheximide (CHX, 20 µM) were added at
various times, as indicated, during the subsequent 30-min incubation.
At 30 min, all samples were collected for analysis of GRP78
(A) and eIF-2 (B) by isoelectric focusing and
Western blotting. As in Fig. 1, the modified form of GRP78 and the
phosphorylated form of eIF-2 migrated to slightly more acidic
positions. The indicated times (min) represent the periods of exposure
to each drug (i.e. 30 min indicates that the drug was added
at the start of the incubation, whereas 20 min indicates that the drug
was added 10 min into the incubation). [3H]Leucine
incorporation was inhibited 97% by cycloheximide and 90% by ionomycin
when these substances were added separately.
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Phosphorylation of eIF-2 and the ADP-ribosylation of GRP78 in
Longer Term Incubations--
The preceding results collectively
emphasize that a continuing flow of processible protein to the ER is
required to generate high degrees of phosphorylation of eIF-2 in
response to stressors that retard protein folding. Folding
intermediates that accumulate in response to ER stressors provide high
affinity sites that sequester GRP78 and retard recycling of the
chaperone. In contrast inhibitors of mRNA translation, such as
cycloheximide, abolish the provision of new processible protein to the
ER while permitting completion of protein processing already in
progress within the organelle. As proteins complete processing, GRP78
accumulates in the catalytically active form that is subject to
ADP-ribosylation and inactivation during the following 90 min. The
fractional ADP-ribosylation of GRP78 in response to cycloheximide
therefore represents a measure of the chaperone that had formerly been
catalytically active in protein processing.
Brefeldin A inhibits ER to Golgi traffic while permitting the
retrograde coalescence of cis-, medial-, and
trans-Golgi components into the ER. Protein synthesis
continues unabated for several hours but thereafter declines modestly
in conjunction with increasing phosphorylation of eIF-2 (30, 31). It
was of interest to determine whether ER protein retention in response
to brefeldin A affected the ADP-ribosylation of GRP78 observed during
cycloheximide treatment. Replicate samples of GH3 cells
were adjusted with brefeldin A for different periods ranging from 0 to
4.5 h. Cycloheximide was added to half of the replicates 90 min
before collection (Fig. 7B),
while the remaining samples were carried as brefeldin-treated controls
(Fig. 7A). Slowly increasing degrees of eIF-2
phosphorylation were observed in response to brefeldin, which were not
affected by cycloheximide (right panels). ADP-ribosylation
of GRP78, by contrast, decreased with increasing exposure times to
brefeldin (left panels). Cycloheximide generated a high
fractional ADP-ribosylation of GRP78 (about 50%) in samples adjusted
with brefeldin at the time of collection (B, left, 0 h). Longer term incubations with brefeldin resulted in declining
fractional ADP-ribosylation of GRP78. At 4.5 h of brefeldin
treatment, the fractional ADP-ribosylation of the protein was
comparable for samples treated with or without cycloheximide. The
accumulation of Golgi proteins in the ER presumably retarded recycling
of GRP78 sufficiently that less chaperone was available for
ADP-ribosylation. The reduction in ADP-ribosylation paralleled the
increased degree of eIF-2 phosphorylation. Depletion of nutrients
present in growth medium during extended incubations slowly depresses
protein synthesis relative to protein processing capability.
ADP-ribosylation provides a device whereby GRP78 in excess of needs can
be sidelined to an inactive pool as processing capability is balanced
with protein synthesis (20). Over a period of 26 h,
GH3 cells that were initially adjusted with fresh growth medium exhibited gradually increasing degrees of ADP-ribosylation of
GRP78 without alteration of eIF-2 phosphorylation (Fig.
8). By 26 h approximately a third of
the GRP78 was ADP-ribosylated, but the total chaperone content was
unchanged as determined by Western blotting following either
isoelectric focusing (Fig. 8A) or SDS-PAGE (Fig.
8B). Treatment of the same cell preparation with
cycloheximide resulted in increasing degrees of ADP-ribosylation of
GRP78 that maximized to roughly 60% at 2-4 h (Fig. 8A).
After 10 h, the ADP-ribosylated form began to disappear without
apparent conversion to the deribosylated form. Total chaperone content, as determined by SDS-PAGE and Western blotting, declined dramatically after 16 h of incubation with cycloheximide (Fig. 8B).
Phosphorylation of eIF-2 was modestly increased at later times of
incubation with cycloheximide (Fig. 8A). The sensitivity of
eIF-2 to phosphorylation in response to challenge with ionomycin for 20 min was also examined after a long term (24 h) incubation conducted in
the absence or presence of cycloheximide (Fig.
9). Cycloheximide-treated cells, as
opposed to untreated controls, displayed eIF-2 phosphorylation in the
absence of stressor and greater eIF-2 phosphorylations in response to
low ionomycin concentrations (lower panels). Approximately half of the GRP78 in cells incubated without cycloheximide became ADP-ribosylated, whereas cells incubated with cycloheximide had selectively lost all detectable ADP-ribosylated chaperone (upper panels).

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Fig. 7.
Effect of brefeldin A on eIF-2
phosphorylation and modification of GRP78. GH3 cells
in replicate samples were incubated for 3 h in Ham's F-10 medium
with 10% fetal calf serum. Brefeldin A (5 µg/ml) was added at
various times (0, 1.5, and 2.5 h of incubation). At the end of the
3-h incubation, the cells were collected and resuspended under standard
conditions either with (B) or without (A)
adjustment to 20 µM cycloheximide. Samples previously
adjusted with brefeldin A received additional drug. The incubation was
then continued and additional replicates were adjusted with brefeldin
at 3.5 h and at 4.5 h total incubation time. All samples were
then collected for analysis of GRP78 and eIF-2 by isoelectric
focusing and Western blotting. Time (h) with brefeldin A is indicated.
As in Fig. 1, the modified form of GRP78 and the phosphorylated form of
eIF-2 migrated to slightly more acidic positions.
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Fig. 8.
Selective loss of the modified form of GRP78
during prolonged incubation of GH3 cells with
cycloheximide. A, disappearance of modified GRP78.
Cultures at 7 days growth were divided into two portions, one of which
was adjusted to 20 µM cycloheximide (CHX).
Both portions were adjusted with 10% fresh growth medium and incubated
for 2 h to lower the fractional phosphorylation eIF-2 and
fractional modification of GRP78. Culturing of the cells was then
continued for 26 h with periodic sampling for isoelectric focusing
and analysis of GRP78 and eIF-2 by Western blotting. B,
decrease in total GRP78 content. Urea sample preparations from
A above were diluted with two parts of 5× Laemmli's sample
buffer. Samples were subjected to SDS-PAGE (7.5%) followed by Western
blotting onto polyvinylidene difluoride membranes and immunostaining
with anti-GRP78 antibodies as described under "Experimental
Procedures."
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Fig. 9.
Phosphorylation of eIF-2 after prolonged
incubation of GH3 cells with cycloheximide. Cells
cultured as described in the legend to Fig. 8 were adjusted with 10%
fresh growth medium and incubated for 23 h with or without 20 µM cycloheximide (CHX). The cells were then
resuspended under standard conditions; replicates were immediately
challenged for 20 min with the indicated concentrations of ionomycin
(IM); and samples were taken for isoelectric focusing and
analysis of GRP78 and eIF-2 by Western blotting. As in Fig. 1, the
modified form of GRP78 and the phosphorylated form of eIF-2 migrated to
slightly more acidic positions.
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DISCUSSION |
Any factor coupling protein processing to mRNA translation
would be anticipated to respond dynamically to altered rates of protein
flow through the ER. In the present report, ADP-ribosylation of the ER
chaperone, GRP78, was analyzed in conjunction with eIF-2 phosphorylation during challenge with agents that inhibit mRNA translation by various mechanisms. Phosphorylation/dephosphorylation of
eIF-2 provided a sensitive index for monitoring rapid changes in
translational initiation capabilities even when amino acid incorporation was strongly inhibited. This parameter also provided a
functional marker for assessing whether alterations in the availability of GRP78 affected translational initiation. Direct-acting inhibitors of
mRNA translation such as pactamycin, cycloheximide, and emetine that depressed protein flow to the ER produced ADP-ribosylation of
GRP78 without the phosphorylation of eIF-2 (Fig. 1). This observation is explicable in terms of the ADP-ribosylation and inactivation of
GRP78 following its release from proteins completing processing in the
ER. Under conditions where new protein influx is suppressed at mRNA
translation, ER processing requirements for GRP78 are sharply reduced.
Sodium arsenite, which modifies the sulfhydryl groups of cytoplasmic
proteins such that binding to heat shock proteins occurs, facilitated
eIF-2 phosphorylation in conjunction with the ADP-ribosylation of
GRP78. This observation is in accord with the finding that arsenite
does not directly affect ER protein processing other than by depressing
the flow of protein to the organelle (28). Ca2+-mobilizing
agents and dithiothreitol, however, prevented ADP-ribosylation of GRP78
while producing eIF-2 phosphorylation. These changes were consistent
with the inhibition of mRNA translation secondary to the inhibition
of protein processing. Under this circumstance free GRP78 would be
anticipated to decline in response to increased binding opportunities
on protein folding intermediates. ADP-ribosylated GRP78 values fell
sharply during application of ER stressors.
In assessing the potential role of GRP78 in regulating eIF-2
phosphorylation, several time-dependent parameters must be
considered. Cycloheximide maximally inhibits amino acid incorporation
within 1-2 min. Clearing the ER of processible protein, however,
proceeds relatively slowly and varies from protein to protein. Small
proteins such as
1-antitrypsin are largely cleared
within 30 min, whereas large proteins such as thyroglobulin require
several hours (32, 33). ADP-ribosylation of GRP78 would depend upon the
release of the active form of the chaperone from proteins completing ER processing. ADP-ribosylation of GRP78 in response to cycloheximide treatment increased rapidly for 60 min (Fig. 3) rising by 4 h to
maximal values of approximately 60% (Fig. 8). This value provides an
indication of the fraction of the GRP78 that was engaged catalytically in protein folding at the start of the incubation. The ADP-ribosylation of GRP78 in cells treated for 90 min with cycloheximide was readily reversible upon removal of the inhibitor by washout as monitored by
complete restoration of rates of amino acid incorporation (Fig. 3). The
onset and offset of the modification, which respectively reflect the
depletion and repletion of ER protein folding intermediates, appeared
to proceed at approximately the same rate in cells that were not
treated further with ER stressors. Deribosylation was accelerated by
the addition of either ionomycin or dithiothreitol and was complete by
90 min. In contrast, the eIF-2 phosphorylation that occurred in
response to these agents achieved maximal values within 15 min that
were subsequently sustained throughout the incubation. Phosphorylation
of eIF-2 did not appear to be directly dependent upon either
deribosylation (Fig. 3) or ADP-ribosylation (Fig. 5) of GRP78.
The relatively slow rate of ADP-ribosylation of GRP78 during
cycloheximide treatment raised the possibility that the free active,
monomeric form of the chaperone was accumulating prior to its
modification. In the event that this form of GRP78 functions in a mass
action manner to suppress eIF-2 kinase activity, alterations would be
expected to occur in the sensitivity of eIF-2 phosphorylation in
response to ER stressors. Such changes were indeed observed. Cells
adjusted to graded rates of amino acid incorporation with cycloheximide
and then exposed for 15 min to ionomycin or dithiothreitol displayed
remarkable reductions in eIF-2 phosphorylation as amino acid
incorporation was increasingly inhibited (Fig. 4). Phosphorylation was
largely unaffected by 50% inhibitions of protein synthesis, markedly
reduced at 70% inhibitions, and abolished by inhibitions exceeding
90%. These changes were accompanied by relatively marginal alterations
in the content of the ADP-ribosylated form of GRP78. The sensitivity of
eIF-2 phosphorylation in response to stressors also varied as a
function of increasing pretreatment times with cycloheximide at full
inhibitory doses (Fig. 5). Phosphorylation declined sharply within 15 min reaching minimal values at 30 min of pretreatment. At longer
incubation periods sensitivity to stressors recovered and approached
control values by 90 min. These later periods were associated with
increasing contents of ADP-ribosylated GRP78. Collectively these
results appear to reflect an early increase followed by a decline in
the active form of GRP78 during cycloheximide treatment.
ADP-ribosylation of GRP78 occurred spontaneously during longer term
incubations without concurrent eIF-2 phosphorylation. Cells were
normally provided with fresh growth medium 16 h before harvesting.
Under this condition basal contents of the ribosylated protein were low
and frequently negligible. Spontaneous ADP-ribosylation of GRP78 began
to appear after 1-2 h in incubations without serum. High cell density
facilitated the fractional modification of GRP78 presumably via a more
rapid exhaustion of nutrients. The ADP-ribosylated form of GRP78
invariably accumulated as rates of protein synthesis declined relative
to ER processing capability. In effect, the ADP-ribosylation of the
chaperone appeared to provide a buffer system by which the rate of
protein processing could be balanced with that of protein synthesis.
The induction of grp78 mRNA only occurs when protein
processing capacity is lower than synthetic capability, as would be
exemplified in cells provided with fresh medium (31). The ability of
cycloheximide and other direct inhibitors of mRNA translation to
block grp78 mRNA induction (34, 35) therefore becomes
readily explicable. GRP78 appears to differ from those rapidly
inducible proteins that are also rapidly degraded upon removal of the
inducer. GH3 cells exposed to Ca2+-mobilizing
agents induce grp78 mRNA to nearly maximal degrees within 3-4 h in conjunction with expression of the chaperone and the
development of resistance to the stressor (31, 36). This resistance is
maintained for at least 4 h following removal of the stressor
without a perceptible fall in total GRP78. The results of the present
report indicate that ADP-ribosylation provides an alternative to
degradation for rapidly decommissioning excess chaperone. This
alternative is reversible for several hours. It is also apparent that
ADP-ribosylation of excess GRP78 would complicate efforts to develop
mutants that overexpress the active form of GRP78 in response to a promoter.
During extended incubations with cycloheximide, approximately 40% of
the total GRP78 pool was resistant to modification by ADP-ribosylation
(Fig. 8). The modified chaperone was selectively lost, either through
degradation or secretion, over 24 h. These observations imply that
a large fraction of the total GRP78 in GH3 cells is not
subject to catalytic recycling. The GRP78 of this fraction was
indistinguishable from the catalytically active form of the chaperone.
The long term stability of this pool of GRP78 is consistent with the
existence of a large pool of high affinity binding sites for the
chaperone. Conceivably such sites could be created by interactions of
GRP78 with misfolded proteins incapable of ER to Golgi transport, by
complexing of GRP78 to translocation intermediates during elongation
arrest, or by strong interactions with ER resident proteins. The
stability of this pool of GRP78 appears to exceed that of ER protein
folding intermediates reported to undergo proteasomal degradation.
Cells that were treated for several hours with brefeldin A, which
expands the protein content of the ER without inhibiting processing,
displayed reduced ADP-ribosylation of GRP78 and increasing eIF-2
phosphorylation when challenged with cycloheximide (Fig. 7). These
results are compatible with the conclusion that GRP78 binds well to
proteins that have largely completed maturation.
The diagram displayed in Fig. 10 models
the various mass-action interactions of GRP78 that are supported by
this report in conjunction with the literature. In this scheme the free
monomeric, active form of GRP78 serves as a multifunctional modulator
of various ER-supported processes including regulation of eIF-2 kinase and mRNA translation, regulation of grp78 expression,
and the catalysis of protein folding, as well as, potentially, the
targeting of misfolded proteins for degradation (37). In cells with
high rates of protein synthesis, the bulk of the GRP78 in the
non-modified form (approximately 60%) is complexed with protein
folding intermediates. Any slowing of protein synthesis relative to
protein processing capability would result in the short term
accumulation of the free, active form of GRP78 which is subject to
subsequent inactivation by ADP-ribosylation. The ADP-ribosylated form
of the chaperone provides a buffering system permitting rates of
protein processing to be balanced with protein synthesis. The remaining
40% of the non-modified GRP78 recycles poorly, and its function is
unknown. An accumulation of protein folding intermediates would lower
the free, monomeric pool of GRP78 while facilitating deribosylation of
the modified chaperone and the dissociation of the chaperone from
transmembrane components that are responsible for feedback regulation
of mRNA translation and grp78 induction. Induction of
GRP78 would be anticipated to foster eIF-2 dephosphorylation and
resumption of amino acid incorporation, in accord with experimental observation.

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Fig. 10.
The dynamics of GRP78/BiP. Solid
arrows indicate the immediate interactions of free unmodified
chaperone, and dashed arrows indicate consequences of these
interactions. The and + symbols indicate suppressive
and stimulatory actions, respectively, of the free unmodified form of
GRP78/BiP. For further details, see "Discussion."
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