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INTRODUCTION |
The pathway through which growth factors promote their
effects in protein synthesis is not fully understood at least in
neurons of the central nervous system. Protein synthesis is activated in different cell types by a variety of growth factors essential to
cell growth, differentiation, and survival. Translational control starts at the level of initiation (1, 2) and depends on eukaryotic
initiation factor 2 (eIF2).1
This binds to GTP and interacts with the initiator methionyl-tRNA (Met-tRNAi) to form the ternary complex
eIF2·GTP·Met-tRNAi. In this way, eIF2 factor recruits
Met-tRNAi to the 40 S ribosomal subunit. This together with
other initiation factors binds to mRNA, leading to the recognition
of the AUG start codon (3-5). Upon formation of the 80 S initiation
complex, GTP is hydrolyzed, and eIF2 is released from the ribosome as a
functionally inactive binary complex (eIF2·GDP). Eukaryotic
initiation factor 2B (eIF2B) is a heteropentameric protein that
catalyzes the exchange of bound GDP from eIF2·GDP for GTP (4, 6, 7).
The eIF2·GTP form is then available to undergo further interaction
with Met-tRNAi, leading to a new round of initiation. eIF2B
activity therefore plays a key role in regulating translation
initiation. elF2B factor can be mainly regulated by two mechanisms.
First, elF2 phosphorylation of the
subunit (eIF2
) inhibits eIF2B
because phosphorylated eIF2
is a competitive inhibitor of eIF2B (8,
9). Secondly, eIF2B activity can be regulated by phosphorylation of its
subunit. Four kinases have been described to phosphorylate the
subunit of eIF2B (eIF2B
); they are casein kinase (CK) 1 and 2, glycogen synthase kinase 3 (GSK3), and dual specificity
tyrosine-phosphorylated and -regulated kinase (10-13). elF2B
phosphorylation by CK1 and -2 enhances eIF2B activity, whereas
phosphorylation by GSK3 has an inhibitory effect (14-17). The
phosphorylation by GSK3 requires previous elF2B
phosphorylation,
which is catalyzed in vitro by dual specificity
tyrosine-phosphorylated and -regulated kinase (13).
The translational inhibition caused by eIF2B inhibition through eIF2
phosphorylation is a well known cellular mechanism that triggers in
response to different stress situations (18-20). However, in growth
factor-treated cells and in response to other different treatments,
changes in eIF2B activity independent of eIF2
phosphorylation have
been described in vivo (21-26). Increased eIF2B activity
paralleling GSK3 inactivation in response to nerve and epidermal growth
factors (26), insulin (27), and insulin-like growth factor 1 (IGF1) have been reported (17). IGF1 exerts its action by activating multiple
signal transduction pathways, notably the mitogen-activated protein
kinase (MAPK)-activating kinase (MEK)/MAPK and phosphatidylinositol 3 kinase (PI3K) pathways (28-31). In neuronal cells, we previously showed that elF2B activation by IGF1 depends on both GSK3 inactivation, via a mechanism mediated by PI3K, and MAPK activation (32). The link
between IGF1-induced GSK3 inactivation and PI3K activity is provided by
protein kinase B, which is located downstream of PI3K and
phosphorylates GSK3 at a conserved serine inhibitory site (33, 34).
Nevertheless, the signaling pathway or the mechanism through which
IGF1-induced MAPK activation leads to eIF2B activation remains unknown.
The phosphorylation status of proteins depends on the relative
activities of both kinases and phosphatases. However, the possible role
of protein phosphatases in eIF2B regulation has not been established.
Protein phosphatases 1 (PP1) and 2A (PP2A) are two major and
structurally related families of serine/threonine phosphatases that
regulate a large number of cellular processes, including neuronal
signaling (35). The regulation of PP1 and PP2A catalytic subunits by
extracellular signals seems to be mediated mainly by association with
non-catalytic regulatory subunits which inhibit, modulate, or target
catalytic subunits to various subcellular structures and
substrates (36, 37).
The aim of the present work was to investigate the mechanism of eIF2B
regulation by IGF1-induced MAPK activation in cultured neurons. By
studying extracellular signal-regulated kinase (ERK) 1 and 2, MAP
kinases, and PP1, a novel transduction pathway leading to eIF2B
activation in neurons was discovered. Evidence is provided that
IGF1-induced eIF2B activation, promoted via MAPK signaling, is exerted
by PP1 activation.
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EXPERIMENTAL PROCEDURES |
Materials--
IGF1, inhibitor 2 (I2), tautomycin, purified
recombinant PP1 catalytic subunit (PP1C)
isoform (PP1C
),
anti-ERK1 and -2 polyclonal antibody, and anti-diphospho-ERK1 and -2 (Thr183 and Tyr185 in ERK2) (the active forms
of the kinases) monoclonal antibodies were provided by Sigma. Purified
recombinant PP1C
, 4E-BP1, and fostriecin were from Calbiochem,
purified recombinant ERK1 and GSK3
and anti-PP2A catalytic subunit
(PP2AC) polyclonal antibody were purchased from Upstate Biotechnology,
and purified recombinant ERK2 and
anti-phospho-GSK3
/
(Ser21/Ser9) (the
inactive form of the kinases) polyclonal antibodies were provided by
New England Biolabs. PD98059 was obtained from Biomol, LY294002 was
from Alexis, anti-ERK2 and anti-eIF2
polyclonal antibodies were from
Santa Cruz Biotechnology, and anti-PP1C and anti-GSK3
monoclonal
antibodies were from Transduction Laboratories. Leibovitz L-15, Ham's
F-12 and high glucose Dulbecco's media were purchased from Invitrogen.
[3H]GDP and [
-32P]ATP were supplied by
Amersham Biosciences, and synthetic peptides were supplied by
Mimotopes. eIF2 and eIF2B were purified from calf brain (38).
Primary Neuronal Cultures--
Primary cultures of cells from
cerebral cortex were prepared from 16 day-old fetuses removed from
timed-pregnant Sprague-Dawley rats. The fetuses were placed in
Leibovitz L-15 medium for brain dissection. The cerebral cortex was
separated from the rest of the brain using iridectomy scissors, and the
meningeal membranes were carefully removed. The resulting pieces were
then dissociated using a Pasteur pipette and 20-21-gauge needles to
make a homogeneous cell suspension. Trypan blue exclusion was used to
count the living cells. Neurons were seeded on plastic multidishes
precoated with 0.05 mg/ml poly-D-lysine at a density of
2-2.5 × 105 cells/cm2 and cultured at
37 °C with 7.5% CO2 in air in high glucose Dulbecco's medium with 15% fetal calf serum. After 24 h, cultures were
placed and maintained in serum-free Dulbecco's/Ham's F-12 medium
(1:1, v/v, D:F medium) supplemented with 1.8 mg/ml glucose, 100 µg/ml transferrin, 100 µM putrescine, 20 nM
progesterone, and 30 nM sodium selenite. Six- to 7-day-old
cultured neurons were used in all experiments. The neuronal content, as
determined by immunocytochemistry with antibodies to neuron-specific
protein
-tubulin isotype III, was found to be more than 90%. Cells
were maintained in D:F medium without supplements for 16 h before
treatments and then placed in the same medium in the absence or
presence of additives. When inhibitors were used, cells were treated
with them for 1 h (or 2 h in the case of tautomycin) before
and during IGF1 treatment. Cells were washed with ice-cold
phosphate-buffered saline before harvesting.
eIF2B Activity Measurement--
Both untreated and treated cells
cultured on 35-mm multidishes were lysed for 10 min in hypotonic buffer
(10 mM Tris-HCl, pH 7.6, 10 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 1 mM benzamidine, 10 µg/ml
leupeptin, pepstatin and antipain, 2 mM
-glycerophosphate, 2 mM sodium molybdate, and 0.2 mM sodium orthovanadate). The lysate was made up to 4 mM magnesium acetate and 140 mM potassium
acetate, centrifuged for 10 min at 12,000 × g, and
saved as cell extract. All steps were carried out at 4 °C. A binary
complex, eIF2·[3H]GDP, was formed as described (17).
The eIF2B activity of purified eIF2B (0.1-0.35 µg) and of cell
extracts (40 µg of protein) were measured by the capacity to exchange
eIF2-bound [3H]GDP for free GDP during 3- or 5-min
incubations, respectively (17). The substrate used was 1 pmol of
eIF2·[3H]GDP. eIF2B activity was expressed as a
percentage of pmol of [3H]GDP released from the binary
complex with respect to controls.
eIF2B Phosphorylation and Activation by ERK Immunoprecipitates
and Recombinant ERK--
Cell extracts (500 or 75 µg) from both
untreated and IGF1-treated cells were immunoprecipitated with either 5 µl of anti-ERK2 antibody or 3 µl of anti-diphospho-ERK1 and -2 antibody, respectively, and with 25 µl of protein A-Sepharose or
protein G-Sepharose, respectively (Amersham Biosciences) following
previously described procedures (39). For the eIF2B
phosphorylation
assay, ERK immunoprecipitates obtained with the two different
antibodies were washed and centrifuged for 5 min at 2300 × g 3 times in buffer A (20 mM Hepes-NaOH, pH7.4, 10 mM MgCl2, 1 mM EDTA, and 1 mM dithiothreitol) and then incubated in 40 µl of buffer
A with purified eIF2B (3 µg), 30 µM ATP, and 7 µCi of
[
-32P]ATP. After 20 min at 30 °C, a 25-µl aliquot
was taken, and the reaction was stopped with 12.5 µl of SDS sample
buffer. It was then analyzed by SDS-PAGE. The dried gel was stained and
exposed to film, and the 32P was incorporated into the
eIF2B
protein quantified using an image analyzer (DiversityOne,
Bio-Rad).
To measure eIF2B activity, ERK immunoprecipitates were incubated with
eIF2B (0.5-1 µg) as described above for the phosphorylation assay
but in the absence of radioactive ATP. After incubation, the reaction
mixture was centrifuged, and an aliquot of the supernatant (corresponding to 0.1-0.35 µg of eIF2B) was taken for eIF2B activity analysis. In some cases, the immunoprecipitates were preincubated with
200 nM I2 or 20 nM tautomycin for 12 min before
incubation with eIF2B. In other experiments purified recombinant
ERK1 and ERK2 (3 and 10 units, respectively) instead of ERK
immunoprecipitates from cell extracts were used to phosphorylate
eIF2B
, and eIF2B activity was assayed following the same protocol as above.
As a positive control of ERK activity, ERK immunoprecipitates as well
as purified ERK1 and -2 were incubated with 4E-BP1 (3 µg), a known
substrate for these MAPKs in vitro, under the same conditions described for eIF2B phosphorylation. 4E-BP1 phosphorylation was quantified using the image analyzer as for the assessment of ERK activity.
Protein Phosphatase Detection in ERK Immunoprecipitates--
To
detect the presence of protein phosphatases that co-immunoprecipitate
with ERK1 and -2, both ERK2 and diphospho-ERK1 and -2 immunoprecipitates were analyzed by SDS-PAGE and Western blot. The
membranes were developed with antibodies against diphosphorylated ERK1
and -2, ERK1 and -2, PP1C, and PP2AC proteins. In other experiments, diphospho-ERK1 and -2 immunoprecipitates (from 75 µg cell extracts) were washed either with 0.4 M potassium acetate in buffer
A, 1% (v/v) Triton X-100 in buffer A, or RIPA buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 125 mM KCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS). After centrifugation, immunoprecipitates were washed again three times with buffer A, incubated with purified eIF2B, and eIF2B activity was assessed as
described above.
PP1 and PP2A Phosphatase Activity Assays--
PP1 and PP2A
phosphatase activity was determined according to the method of Cohen
et al. (40) using purified 32P-labeled
phosphorylase a as the substrate. 32P-Labeled
phosphorylase a was prepared by incubating phosphorylase b (10 mg/ml) with phosphorylase kinase (0.2 mg/ml) as
previously described (41). Cell extracts (0.5 µg) prepared as
described for eIF2B activity assessment in hypotonic buffer but in the
absence of phosphatase inhibitors were preincubated for 12 min at
30 °C without and with I2 or fostriecin in 50 mM
Tris-HCl, pH 7.6, 1 mM dithiothreitol, 0.1 mM
EDTA in a volume of 30 µl. The phosphatase reaction was initiated by
the addition of 10 µl of 32P-labeled phosphorylase
a (60,000 cpm). After 20 min of incubation at 30 °C, the
reaction was stopped by the addition of 180 µl of 20% (wt/v)
trichloroacetic acid. The tubes were left on ice for 10 min and then
centrifuged at 12,000 × g for 5 min at 4 °C.
Aliquots (180 µl) of the clear supernatant were counted to determine
the phosphatase activity as the amount of 32P released.
Phosphorylase a is a substrate for both PP1 and PP2A.
Therefore, to differentiate between these two protein phosphatases, assays were performed in the presence of either I2 or fostriecin, specific inhibitors of PP1 and PP2A, respectively. The doses of PP1 and
PP2 inhibitors used were predetermined by a set of dose-response experiments in which the effect of each inhibitor was independently measured. The PP1 activity inhibited by I2 (100-500 nM)
was comparable with the activity remaining in the presence of the PP2A
inhibitor fostriecin (400-1000 nM). Thus, the sum of the
PP1 and PP2A activities represents the total phosphorylase a
phosphatase activity of the cell extracts. Accordingly, a concentration
of 200 nM I2 was chosen to assess phosphatase activity in
further experiments. PP1 activity was defined as the phosphorylase
a phosphatase activity inhibited by I2. PP2A activity was
defined as the remaining activity. PP1 and PP2A phosphatase activities
were also assayed in ERK immunoprecipitates from 35 µg of cell extracts.
PP1C
and PP1C
Phosphatase Activity Assays--
PP1C
and
PP1C
phosphatase activities were assayed using the peptide
RRAAEELDSRAGS(P)PQL based on eIF2B
531-542 rat sequence
(42) phosphorylated by GSK3
. Purified GSK3
(100 milliunits) was
immunoprecipitated with 2 µl of anti-GSK3
and 25 µl of protein
G-Sepharose as described elsewhere (39). GSK3
immunoprecipitates
were incubated for 20 min at 30 °C with 15 µg of eIF2B
peptide
in 55 mM Tris-HCl, pH 7.6, 5 mM magnesium acetate, 0.1 mM ATP, and 0.5 µCi of
[
-32P]ATP in a volume of 25 µl. The radioactivity
incorporated into the peptide was determined by liquid scintillation.
32P-Labeled peptide (12,000 cpm) was incubated for 30 min
at 30 °C with PP1C
(1.16 units/µg) or PP1C
(2.0 units/µg)
in 25 µl of 50 mM Tris-HCl, pH 7.0, 0.1 mM
EDTA, 0.2 mg/ml bovine serum albumin, and 10 µM
MnCl2 (only for the PP1C
assay). After incubation, samples were spotted onto P81 phosphocellulose paper and rinsed three
times with 3% (v/v) phosphoric acid, and phosphatase activity was counted.
In other experiments purified eIF2B factor (3 µg) was phosphorylated
with GSK3
immunoprecipitates using 30 µM ATP and 4.5 µCi of [
-32P]ATP as described above. An aliquot of
32P-labeled eIF2B (1.35 µg) was then used instead of the
peptide in the PP1C
activity assay. The reaction was stopped by
adding 12.5 µl of SDS sample buffer and analyzed by SDS-PAGE and autoradiography.
To study the effect of recombinant PP1C on eIF2B activity, purified
eIF2B factor (0.5-1 µg) was incubated with PP1C
or PP1C
under
the same conditions as described for the peptide. After incubation, an
aliquot containing about 0.1-0.35 µg of eIF2B was used for assessing
eIF2B activity.
Determination of eIF2
, GSK3, and ERK1 and -2 Phosphorylation--
Cell extracts, prepared in the same way as for
the eIF2B assay, were analyzed using horizontal isoelectric focusing
slab gels to detect eIF2
phosphorylation and SDS-PAGE to detect GSK3
and ERK phosphorylation. After electrophoresis, the gels were
transferred to a polyvinylidene difluoride membrane (Amersham
Biosciences), and the blots were visualized by specific anti-eIF2
,
anti-phospho-GSK3
/
, and anti-diphospho-ERK1 and -2 antibodies.
The bands corresponding to eIF2
and phosphorylated eIF2
proteins
were quantified as described above.
Statistical Analysis--
Results are expressed as means ± S.E. for independent experiments. Statistical analysis was performed
using the t test for paired and unpaired data
versus control values or analysis of variance and Dunnett's
post-test for comparisons between treated groups.
 |
RESULTS |
ERK1 and -2 Do Not Phosphorylate eIF2B--
Recently we reported
that IGF1 induces MAPK activation, mainly of ERK2, and that this
signaling pathway is involved in eIF2B activation in neuronal cultures
(17). eIF2B
contains Pro-Leu-Thr-Pro and Ser-Pro consensus sequences
for recognition by ERK1 and -2 MAP kinases. To determine whether
eIF2B
was a substrate for ERK2 kinase in vitro, we
incubated eIF2B with ERK2 immunoprecipitates from untreated controls
and IGF1-treated cells in an eIF2B phosphorylation assay. The results
showed that ERK2 immunoprecipitates from IGF1-treated cells did not
increase eIF2B
phosphorylation and even slightly decreased eIF2B
phosphorylation (68.6 ± 6.5% versus 100% of control cells; Fig. 1A, top
panel). To test whether MAP kinases were active in the
immunoprecipitates, parallel experiments were performed with the known
substrate 4E-BP1. As shown, the observed 4E-BP1 phosphorylation with
ERK2 immunoprecipitates from IGF1-treated cells (15.5 ± 0.53 in
arbitrary units) was greater than that seen with immunoprecipitates
from untreated control cells (6.0 ± 2.1 in arbitrary units,
p < 0.05; Fig. 1A, bottom
panel). This finding confirms the previously reported MAPK
activation induced by IGF1 (17). Furthermore, and supporting the
results obtained with the immunoprecipitates, eIF2B was not
phosphorylated by either recombinant ERK1 or ERK2 in vitro
(data not shown).

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Fig. 1.
ERK1 and -2 immunoprecipitates from
IGF1-treated neuronal cells activate eIF2B factor activity without
promoting eIF2B phosphorylation.
A, ERK2 immunoprecipitates from untreated
(control) or IGF1-treated (100 ng/ml, 30 min) neuronal cells
were incubated with purified eIF2B or recombinant 4E-BP1 in a
phosphorylation assay. Representative experiments are shown of eIF2B
phosphorylation (eIF2B -P, top
panel) and 4E-BP1 phosphorylation (4E-BP1-P,
bottom panel) analyzed by SDS-PAGE and autoradiography. The
numbers express the quantification of eIF2B
phosphorylation and 4E-BP1 phosphorylation bands in arbitrary units or
in percentages (data shown in parentheses) and represent the
average of three independent experiments run in duplicate.
B, ERK2 immunoprecipitates (IPERK2) or
diphospho-ERK1 and -2 immunoprecipitates
(IPERK1/2-P2) were obtained from neuronal cells
either untreated (control) or treated with IGF1 (100 ng/ml) without or
with PD98059 (30 µM) or LY294002 (30 µM)
for 30 min. Immunoprecipitates were incubated with purified eIF2B and
assayed for eIF2B activity as described under "Experimental
Procedures." The results were obtained from three to six independent
experiments run in duplicate; error bars indicate S.E. eIF2B
activity, corresponding to immunoprecipitates from control cells
(1.05 ± 0.2 pmol/µg), was considered as 100%. *,
p < 0.05 versus control; **,
p < 0.01 versus control.
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eIF2B Activation by ERK1 and -2 Immunoprecipitates--
To study
the participation of MAP kinase in IGF1-induced eIF2B activation, we
tested the effect of ERK2 immunoprecipitates from IGF1-treated and
untreated neurons on purified eIF2B activity. Interestingly, eIF2B
activity was significantly increased after incubation with
immunoprecipitates from IGF1-treated cells (168 ± 20%; Fig.
1B). A similar result was found when diphospho-ERK1 and -2 immunoprecipitates, obtained with an antibody against diphospho-ERK1 and -2 (active form of the kinases), were incubated with eIF2B factor
(150 ± 13%; Fig. 1B). eIF2B activation induced by ERK
immunoprecipitates in IGF1-induced neurons was inhibited by cell
treatment with the MAPK inhibitor PD98059, whereas the treatment of
cells with the PI3K inhibitor LY294002 had no effect (Fig.
1B). Besides, incubation of purified eIF2B with purified
recombinant ERK1 or -2 did not change eIF2B activity (not shown). All
these findings demonstrate that ERK1 or -2 MAP kinases does not
directly modify eIF2B phosphorylation status or activity. Conversely,
the above findings suggest that an unknown factor that
co-immunoprecipitates with activated ERK1 and -2 may be responsible for
eIF2B activation in IGF1-stimulated neurons. Furthermore, when ATP was
omitted in incubations with immunoprecipitates, eIF2B activation
occurred (not shown), indicating that this effect might not be mediated
by kinase activity.
Detection of PP1C in IGF1-activated ERK1 and -2 Immunoprecipitates--
To further investigate the nature of the
unknown factor that activates eIF2B, we studied the potential
involvement of PP1 and PP2A, the two main protein phosphatases involved
in cell growth and signaling in eukaryotic cells (36, 37). Accordingly,
we searched for PP1 and PP2A catalytic subunits in ERK1 and -2 immunoprecipitates effected with four different antibodies: anti-ERK1
and -2 diphosphorylated, anti-ERK1 and -2, anti-PP1C, and anti-PP2AC.
As shown in Fig. 2A, both PP1C
and PP2AC proteins were detected. Interestingly, PP1C levels found in
ERK2 immunoprecipitates from IGF1-treated neurons were higher than
those from untreated cells, whereas PP2AC levels showed no difference
(Fig. 2A). Increased PP1C levels were also detected in
diphospho-ERK1 and -2 of IGF1-treated cells, whereas PP2AC was poorly
detected (Fig. 2B). These findings further support a close
relationship between PP1 and the diphosphorylated active form of the
kinases (mainly ERK2) in immunoprecipitates from IGF1-stimulated
neuronal cells (Fig. 2, A and B).

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Fig. 2.
PP1C co-immunoprecipitates with ERK1 and -2 in IGF1-treated neuronal cells. ERK2 immunoprecipitates
(A) or diphospho-ERK1 and -2 immunoprecipitates
(B) from untreated (control) or IGF1-treated (100 ng/ml, 30 min) neuronal cells were analyzed by SDS-PAGE and Western
blot. Immunoblots were consecutively probed and developed again after
stripping with four different antibodies, anti-diphospho-ERK1 and -2 (top panel), anti-ERK1 and -2 (middle top panel),
anti-PP1C (middle bottom panel) and anti-PP2AC (bottom
panel). Diphospho-ERK1 and -2 (ERK1/2-P2),
ERK1 and -2, PP1C, and PP2AC proteins are indicated by
arrows. A and B show representative
results from three-four experiments performed with different batches of
neuronal cultures. PP1 (C) and PP2A (D)
phosphatase activities were measured in diphospho-ERK1 and -2 immunoprecipitates from untreated (control) or IGF1-treated
(100 ng/ml, 30 min) neuronal cells as described under "Experimental
Procedures." PP1 and PP2A phosphatase activities corresponding to
immunoprecipitates from control cells (1803 ± 148 and 831 ± 215 cpm, respectively) were considered as 100% in each case. The
results were obtained from four independent experiments run in
duplicate; error bars indicate S.E. *, p < 0.05 versus control.
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To find out whether the presence of PP1C and PP2AC in the
immunoprecipitates correlated with phosphatase activity, we measured PP1 and PP2A activities in ERK immunoprecipitates. Although PP1 and
PP2A activities were found in the immunoprecipitates, only PP1
phosphatase activity was significantly increased in diphospho-ERK1 and
-2 immunoprecipitates from IGF1-treated cells (179 ± 21%) compared with immunoprecipitates from untreated control cells (100%,
p < 0.05; Fig. 2C). On the contrary, PP2A
phosphatase activity, although present, underwent no change upon IGF1
treatment in these immunoprecipitates (94 ± 8% from IGF1-treated
cells versus 100% of control cells; Fig. 2D).
Similar results were obtained when measuring PP1 and PP2A phosphatase
activities in ERK2 immunoprecipitates (not shown). For further
experiments, only ERK immunoprecipitates obtained with
anti-diphospho-ERK1 and -2 antibodies were used.
PP1C Associated with Diphosphorylated ERK1 and -2 Activates eIF2B
Factor--
To determine whether PP1C associated to ERK1 and -2 MAP
kinases was responsible for the eIF2B activation induced by ERK
immunoprecipitates, the diphospho-ERK1 and -2 immunoprecipitates from
IGF1-treated cells were washed before incubation with eIF2B. When the
immunoprecipitates were washed with buffer containing 0.4 M
potassium acetate or 1% (v/v) Triton X-100, no changes in eIF2B
activation were observed. However, when the immunoprecipitates were
washed in more stringent conditions using RIPA buffer, eIF2B activity
dropped from 156 ± 2.5 to 114 ± 5.3%, the latter being a
value close to that obtained with diphospho-ERK1 and -2 immunoprecipitates from untreated control cells (100%) (Fig.
3A). Western blot analysis of
washed diphospho-ERK1 and -2 immunoprecipitates revealed that only RIPA
buffer removed PP1C from immunoprecipitates from untreated control and
IGF1-treated neurons (Fig. 3B). To further assess the
involvement of PP1 in eIF2B activation, the eIF2B assay was performed
with the specific PP1 inhibitor I2 (200 nM). I2 blocked the
eIF2B activation induced by diphospho-ERK1 and -2 immunoprecipitates
from IGF1-treated neurons (104 ± 5.1% versus 100% of
untreated control cells; Fig. 3C). Similar results were
obtained using another specific PP1 inhibitor, tautomycin (20 nM, not shown). PP1 inhibitors I2 and tautomycin at
100-1000 and 20-100 nM concentrations, respectively, produced no effects on eIF2B activity in the absence of
immunoprecipitates (not shown). These findings provide evidence that
PP1C, either by itself or by forming a complex with some other
regulatory protein, is associated with IGF1-activated MAP kinases
(mainly ERK2) and activates eIF2B factor in vitro.

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Fig. 3.
PP1C in ERK1 and -2 immunoprecipitates activates eIF2B factor. A,
diphospho-ERK1 and -2 immunoprecipitates from IGF1-treated (100 ng/ml,
30 min) neuronal cells were washed with buffer A with or without
potassium acetate (AcK, 0.4 M) or Triton X-100
(1%) as well as with RIPA buffer. Immunoprecipitates were then
incubated with purified eIF2B and assayed for eIF2B activity as
described under "Experimental Procedures." The results were
obtained from three to seven independent experiments run in duplicate;
error bars indicate S.E. eIF2B activity, corresponding to
immunoprecipitates washed in buffer A from control cells (1.12 ± 0.2 pmol/µg), was considered as 100%. *, p < 0.05 versus immunoprecipitates washed in buffer A alone
(bar marked as none). B,
diphosphorylated ERK1 and -2 immunoprecipitates from untreated
(control) and IGF1-treated neurons were washed with buffer A
( ) or RIPA buffer (+) and analyzed by Western blot. The immunoblots
were probed and developed again consecutively after stripping with
anti-PP1 (top panel) and anti-diphospho-ERK1 and -2 (bottom panel) antibodies. The figure shows a representative
experiment of three different experiments run in duplicate.
C, diphospho-ERK1 and -2 immunoprecipitates from untreated
(control) or IGF1-treated neurons were incubated with
purified eIF2B in the presence of I2 (200 nM) and then
assayed for eIF2B activity as in A. The results were
obtained from three independent experiments run in duplicate;
error bars indicate the S.E. eIF2B activity, corresponding
to immunoprecipitates from control cells (0.57 ± 0.1 pmol/µg)
was considered as 100%.
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PP1C
Activates eIF2B Factor--
To test whether PP1 could
elicit eIF2B dephosphorylation, in vitro studies using
recombinant
and
PP1C isoforms were performed. The activities of
PP1C
and PP1C
phosphatases were assayed using a peptide based on
the eIF2B
rat sequence (containing the well characterized
GSK3-regulated phosphorylation site Ser535) as a substrate.
0.05 units of PP1C
released more than 50% of 32P (6,480 cpm) from the 32P-labeled peptide, whereas 1.0 units of
PP1C
were necessary to release a similar amount of 32P
(Fig. 4A). This suggests that
PP1C
dephosphorylates the peptide much more efficiently than did
PP1C
. In addition, as shown in Fig. 4B, eIF2B factor
phosphorylated by GSK3
was also efficiently dephosphorylated by
PP1C
, confirming that, at least in vitro, eIF2B is a PP1C
substrate. Interestingly, only PP1C
was able to stimulate purified
eIF2B activity; PP1C
had no effect (127 ± 4.9 and 94 ± 6.3%, respectively, versus 100% in the absence of
phosphatase; Fig. 4C). On the other hand, higher
concentrations of PP1C
inhibited eIF2B, suggesting that it might
dephosphorylate other residues required for optimal eIF2B activity
(Fig. 4D). At the concentrations tested, PP1C
had no
effect on eIF2B activity when the assay was performed without
preincubation of the two together and they were only incubated for 3 min in the eIF2B assay (not shown). This result indicates that the
binary complex eIF2·[3H]GDP is not affected by
PP1C
.

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Fig. 4.
eIF2B factor activation by
PP1C . A, PP1C and PP1C
phosphatase activities were measured using the peptide
RRAAEELDSRAGS(P)PQL based on the eIF2B 531-542 rat
sequence phosphorylated by GSK3 as described under "Experimental
Procedures." Data were obtained from two independent experiments
performed in duplicate. Error bars (less than 10%) have
been omitted to simplify the figure. B, immunoprecipitated
GSK3 was incubated with purified eIF2B in a phosphorylation assay,
and phosphorylated eIF2B was then incubated with PP1C as described
under "Experimental Procedures." The figure corresponds to a
representative experiment analyzed by SDS-PAGE and autoradiography of
three independent experiments run in duplicate. Lanes 1-4,
eIF2B phosphorylation by GSK3 (eIF2B -P);
lanes 3 and 4, eIF2B -P dephosphorylation by
PP1C (lane 3, 0.1 units; lane 4, 1.0 units).
C, PP1C (0.1 units) and PP1C (1.0 units) were
incubated with purified eIF2B and then assayed for eIF2B activity as
described under "Experimental Procedures." eIF2B activity
corresponding to control experiments performed in the absence of
phosphatase (1.08 ± 0.25 pmol/µg) was considered as 100%. Data
were obtained from four to seven independent experiments run in
duplicate; error bars indicate S.E. **, p < 0.01 versus control. D, PP1C (0.05, 0.1, and
0.2 units) was incubated with eIF2B and then assayed for eIF2B activity
as in C, eIF2B activity corresponding to control experiments
performed in the absence of PP1C (0.0 units; 1.02 ± 0.2 pmol/µg) was considered as 100%. Data were obtained from two
independent experiments run in duplicate; error bars
indicate S.E. *, p < 0.05 versus
control.
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IGF1 Induces PP1 Activation through the MEK/MAPK
Signaling Pathway in Vivo--
PP1 phosphatase activity was assayed in
untreated control and IGF1-treated neuronal cells using phosphorylase
a as substrate. In treated neurons, IGF1 induced PP1
activation (147 ± 16% versus 100% of control cells)
that was abolished by PD98059 but was not reduced by LY294002. PP2A
activity showed no change (Fig. 5). This
finding is fully consistent with the results described above and
demonstrates that IGF1-induced PP1 activation is dependent on the
MEK/MAPK signaling pathway and is independent of the PI3K pathway.

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Fig. 5.
IGF1 induces PP1 activation in
vivo. PP1 and PP2A phosphatase activities were measured
in cell extracts from untreated (control) or IGF1-treated
(100 ng/ml, 30 min) neuronal cells without or with LY294002 (30 µM) or PD98059 (30 µM) as described under
"Experimental Procedures." PP1 and PP2A phosphatase activities
corresponding to control cells (6356 ± 934 and 4902 ± 736 cpm/µg, respectively) were considered as 100% in each case. Data
were obtained from 3-10 independent experiments run in duplicate;
error bars indicate S.E. +, p < 0.05 versus control; *, p < 0.05 versus cells treated with IGF1 alone.
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PP1 Is Essential to IGF1-induced eIF2B Activation--
With the
above results in mind, we tested whether treatment of cells with a
specific inhibitor of PP1 would block IGF1-induced eIF2B activation.
Cultured neurons were preincubated with 100 nM tautomycin,
a cell-permeable specific PP1 inhibitor, for 2 h and then exposed
to IGF1 treatment. Tautomycin specifically inhibited PP1 activity in
both untreated control and IGF1-treated cells (54 ± 9.3 and
63 ± 6.5%, respectively, versus 100% of control cells) without affecting PP2A activity (Fig.
6A). Interestingly, IGF1-induced eIF2B activation (138 ± 8.1% versus
100% of control cells) was abolished by treatment with tautomycin
(106 ± 9.4% versus 138% of IGF1-treated cells; Fig.
6B), indicating that IGF1-iduced eIF2B activation is
dependent on PP1 activity. PP1 inhibition in vivo might
modify the phosphorylation status of other proteins involved in eIF2B
regulation, such us eIF2
, GSK3, or ERK1 and -2. The percentages of
phosphorylated eIF2
(with respect to total eIF2
) found in
untreated control, tautomycin-treated, IGF1-treated, and tautomycin
plus IGF1-treated cells were 23.5 ± 1.5, 25.5 ± 0.5, 20 ± 1.5, and 23 ± 1.0%, respectively, showing that
tautomycin does not modify eIF2
phosphorylation status (Fig.
6C). Furthermore, the IGF1-regulated phosphorylation
sites in GSK3 and ERK1 and -2 kinases were not modified by tautomycin
(Fig. 6D). Together, these findings support the idea that
PP1 activity is essential for eIF2B activation and that it does not
regulate either eIF2
, GSK3, or ERK1 and -2 phosphorylation in
IGF1-stimulated neuronal cells.

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Fig. 6.
PP1 inhibition abolishes IGF1-induced eIF2B
activation in vivo. A, neuronal cells,
either untreated (control) or treated with IGF1 (100 ng/ml)
in the absence or presence of tautomycin (100 µM) for 30 min. Cells were processed, and cell extracts were used to measure PP1
and PP2A phosphatase activities. PP1 and PP2A phosphatase activities
corresponding to control cells (5464 ± 654 and 3530 ± 680 cpm/µg, respectively) were considered as 100% in each case. Data
were obtained from four to six independent experiments run in
duplicate; error bars indicate S.E. +,
p < 0.05 versus control; **,
p < 0.01 versus cells treated with IGF1
alone. B, eIF2B activity was measured in cell extracts
prepared as in A. eIF2B activity corresponding to control
cells (19.53 ± 2.6 pmol/mg) was considered as 100%. Data were
obtained from four to six independent experiments run in duplicate;
error bars indicate S.E. +, p < 0.05 versus control; *, p < 0.05 versus cells treated with IGF1 alone. C, cell
extracts (75 µg) prepared as in A were analyzed using
isoelectric focusing slab gels, and the immunoblots were developed with
anti-eIF2 antibody. The bands corresponding to eIF2 and
phosphorylated eIF2 (eIF2 -P) proteins are
shown in a representative immunoblot. D, cell extracts (50 µg) prepared as in A were analyzed by SDS-PAGE, and
immunoblots were consecutively probed and developed after stripping
with anti-phospho-GSK3 / antibody (top panel) and
anti-diphospho-ERK1 and -2 antibody (ERK1/2-P2,
bottom panel). The figures shown in C and
D correspond to a representative experiment of three
independent experiments run in duplicate.
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DISCUSSION |
We have previously demonstrated that IGF1-induced eIF2B activation
in neurons is promoted through PI3K and GSK3 kinases (17). We also
reported that the IGF1-induced MEK/MAPK activation pathway was involved
in eIF2B activation. This mechanism has been found operative in other
cell types as well (43). The findings of the present investigation
suggest that IGF1-activated ERK1 and -2 MAP kinases are not directly
responsible for eIF2B activation and that IGF1 promotes eIF2B
activation through protein phosphatase PP1, which is activated by IGF1
in a MEK/MAPK-dependent fashion.
To determine whether IGF1-activated ERK1 and -2 are directly
responsible for eIF2B activation, purified eIF2B was incubated with ERK
immunoprecipitates from untreated control and IGF1-treated neurons.
eIF2B incubation with ERK immunoprecipitates from IGF1-treated cells
activates eIF2B, whereas preincubation of cells with MEK inhibitor
PD98059 abolished eIF2B activation. This suggests that MEK activation
is required for this to occur. Treatment of cells with PI3K inhibitor
LY294002 had no effect on eIF2B activation. This result is not in
disagreement with previously reported results (17) because these
studies used cell extracts, whereas the present study used ERK
immunoprecipitates. The fact that both ERK immunoprecipitates and
recombinant ERK failed to phosphorylate eIF2B together with the failure
of recombinant ERK to activate eIF2B reasonably supports the idea that
ERK1 and -2 are not directly involved in eIF2B activation. Besides,
eIF2B activation by ERK immunoprecipitates was also observed in the
absence of ATP, which discards any further co-immunoprecipitated kinase
activity as being responsible for this effect.
Because eIF2B activity is regulated by
phosphorylation/dephosphorylation reactions (44), it was considered
appropriate to study the potential role of phosphatase activity in
IGF1-induced eIF2B activation via MEK. The presence of PP1 and PP2A
catalytic subunits in ERK1 and -2 immunoprecipitates was investigated
(i) because dephosphorylation was found in eIF2B
subunit when
incubated with ERK immunoprecipitates, (ii) because of the
aforementioned ATP independence of eIF2B activation of the
immunoprecipitates, and (iii) because PP1 participates in glycogen
synthase regulation, a protein whose GSK3-recognized sequence is also
present in eIF2B
(45).
The results show that phosphatases PP1C and PP2AC are found in ERK1 and
-2 immunoprecipitates, suggesting a potential association of such
phosphatases with ERK1 and -2. PP1C was eliminated from ERK
immunoprecipitates only when they were subjected to a stringent wash,
supporting the idea of its specific interaction with ERK. A clear
relationship was seen between IGF1 stimulation, ERK1 and -2 phosphorylation on the one hand, and the amount of PP1C in the
immunoprecipitated complex on the other. Additionally, only PP1
activity in immunoprecipitates increased after IGF1 treatment. Furthermore, using specific PP1 inhibitors, co-immunoprecipitated PP1
was responsible for the eIF2B activation induced by ERK
immunoprecipitates. It is not surprising that PP2A is mostly present in
ERK2 immunoprecipitates because it has been identified as one of the
physiological ERK2 phosphatases (46). These findings suggest that IGF1
promotes eIF2B activation through PP1 protein phosphatase via its
association with phosphorylated ERK.
Several additional in vivo investigations were included in
this work that further clarify the role of PP1 in eIF2B regulation; (i)
PP1 activity was induced by IGF1 treatment, and using the specific MEK
inhibitor PD98059, PP1 activation was found to depend on MEK
activation, and (ii) experiments carried out with tautomycin, a
permeable-specific PP1 inhibitor, showed that specific IGF1-induced PP1
activation is essential for eIF2B factor activation by IGF1. The fact
that tautomycin did not modify the phosphorylation status of either
eIF2
or GSK3 suggests that eIF2B regulation by PP1 is independent of
these regulatory mechanisms in IGF1-stimulated neurons. These in
vivo findings together with those demonstrating in
vitro regulation of eIF2B by PP1 (specifically PP1C
) might also
suggest direct in vivo eIF2B regulation by PP1. Because
tautomycin did not modify ERK1 and -2 phosphorylation either,
indicating that PP1 does not regulate these kinases, and because PP1 is
activated in a MEK-dependent fashion, it might be concluded
that PP1 is likely to act downstream of the MEK/MAPK signaling pathway
in IGF1-stimulated neuronal cells.
12-O-Tetradecanoylphorbol-13-acetate and insulin have been
reported to activate PP1 in muscle cells and adipocytes by MAPK, which
regulates PP1 activity by promoting PP1 regulatory subunit
phosphorylation (41, 45, 47). The present work shows an association
between PP1C and ERK1 and -2 in response to IGF1 that elicits eIF2B
activation. Further experimental work is needed to elucidate the role
of ERK in PP1 regulation in IGF1-stimulated neuronal cells.
A potential role for PP1 in the regulation of eIF2B activity is, thus,
reported. It is generally accepted that rat eIF2B
is phosphorylated
in Ser535 by GSK3 and that this phosphorylation exerts an
inhibitory effect on eIF2B activity (44). Conversely, removing the
phosphate in this residue would activate eIF2B. In the present work,
using recombinant PP1C
, we demonstrate that PP1 is able to both
dephosphorylate eIF2B
phosphorylated by GSK3 and activate eIF2B
factor in vitro. When higher concentrations of PP1C
were
used, eIF2B inactivation was observed. Other phosphorylated residues in
eIF2B have been described in vivo, some of which are
phosphorylated in vitro by CK2 and are essential for eIF2B
activity (12). Excess PP1C
might also release the phosphates in
those essential residues required for eIF2B activity to effect its
inhibition. According to these findings eIF2B activation would require
both GSK3 inactivation and PP1 activation to maintain
Ser535 in a dephosphorylated form in response to IGF1 in
neuronal cells. A similar type of regulation involving both GSK3 and
PP1 has been described for glycogen synthase after insulin treatment in
rat skeletal muscle cells and adipocytes (48). However, an additional mechanism of eIF2B regulation by PP1 should not be discarded. Further
research will be of interest to address this issue in the future.
In summary, this paper reports two novel findings concerning eIF2B
regulation by IGF1 in neuronal cells; IGF1-induced eIF2B activation is
dependent on PP1 activity, and both co-immunoprecipitated endogenous
PP1C and recombinant PP1C
activate eIF2B factor. These findings
suggest that PP1 may be a physiological eIF2B phosphatase in neuronal
cells. An additional finding concerning PP1 regulation by IGF1 is that
IGF1 stimulates both PP1 activity (via MEK/MAPK-dependent pathway) and an association between activated MAPK (notably ERK2) and
PP1C. This complex results in enhanced PP1 activity, which is efficient
for eIF2B activation. All these processes are closely related to one
another and run in parallel with levels comparable with those of eIF2B
activation by IGF1 in neuronal cells.