Initiation factor 2B activity is regulated by protein phosphatase 1, which is activated by the mitogen-activated protein kinase-dependent pathway in insulin-like growth factor 1-stimulated neuronal cells.

We have previously demonstrated that insulin-like growth factor 1 (IGF1) induces eukaryotic initiation factor 2B (eIF2B) activation in neuronal cells through the phosphatidylinositol 3 kinase/glycogen synthase kinase 3 pathway as well as by activation of the mitogen-activated protein kinase (MAPK)-activating kinase (MEK)/MAPK signaling pathway (Quevedo, C., Alcázar, A., and Salinas, M. (2000) J. Biol. Chem. 275, 19192-19197). This paper addresses the mechanism involved in IGF1-induced eIF2B activation via the MEK/MAPK cascade in cultured neurons treated with IGF1 and demonstrates that extracellular signal-regulated MAP kinase 1 and 2 (ERK1 and -2) immunoprecipitates of IGF1-treated neuronal cells promote this activation. This effect did not directly result from eIF2B phosphorylation by ERK immunoprecipitates. In addition, recombinant ERK1 and -2 neither activate eIF2B nor phosphorylate it. Endogenous protein phosphatase 1 and 2A catalytic subunits (PP1C and PP2AC, respectively) were co-immunoprecipitated with ERK1 and -2, and the association of ERK with PP1C was stimulated by IGF1 treatment, resulting in increased PP1 activity. ERK immunoprecipitates incubated with PP1 inhibitors did not activate eIF2B, indicating that PP1C activates eIF2B. In vitro experiments with phosphorylated eIF2B showed that recombinant PP1C (alpha isoform) dephosphorylates and activates eIF2B. Paralleling eIF2B activation, IGF1 treatment induced PP1 activation in a MEK/MAPK-dependent fashion. Moreover, the treatment of neurons with the PP1 inhibitor tautomycin inhibited PP1 activation and prevented IGF1-induced eIF2B activation. These findings strongly suggest that IGF1-induced eIF2B activation in neurons is effected by PP1, the activation of which is mediated by the MEK/MAPK signaling pathway.

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. Transla-tional 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-tRNA i ) to form the ternary complex eIF2⅐GTP⅐Met-tRNA i . In this way, eIF2 factor recruits Met-tRNA i 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)(4)(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 (eIF2⅐GDP). Eukaryotic initiation factor 2B (eIF2B) is a heteropentameric protein that catalyzes the exchange of bound GDP from eIF2⅐GDP for GTP (4,6,7). The eIF2⅐GTP form is then available to undergo further interaction with Met-tRNA i , 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)(22)(23)(24)(25)(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 noncatalytic 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.
Primary Neuronal Cultures-Primary cultures of cells from cerebral cortex were prepared from 16 day-old fetuses removed from timedpregnant 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 ϫ 10 5 cells/cm 2 and cultured at 37°C with 7.5% CO 2 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 g/ml transferrin, 100 M putrescine, 20 nM progesterone, and 30 nM sodium selenite. Sixto 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 phosphatebuffered 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 g/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 ϫ g, and saved as cell extract. All steps were carried out at 4°C. A binary complex, eIF2⅐[ 3 H]GDP, was formed as described (17). The eIF2B activity of purified eIF2B (0.1-0.35 g) and of cell extracts (40 g of protein) were measured by the capacity to exchange eIF2-bound [ 3 H]GDP for free GDP during 3-or 5-min incubations, respectively (17). The substrate used was 1 pmol of eIF2⅐[ 3 H]GDP. eIF2B activity was expressed as a percentage of pmol of [ 3 H]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 g) from both untreated and IGF1-treated cells were immunoprecipitated with either 5 l of anti-ERK2 antibody or 3 l of anti-diphospho-ERK1 and -2 antibody, respectively, and with 25 l 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 ϫ g 3 times in buffer A (20 mM Hepes-NaOH, pH7.4, 10 mM MgCl 2 , 1 mM EDTA, and 1 mM dithiothreitol) and then incubated in 40 l of buffer A with purified eIF2B (3 g), 30 M ATP, and 7 Ci of [␥-32 P]ATP. After 20 min at 30°C, a 25-l aliquot was taken, and the reaction was stopped with 12.5 l of SDS sample buffer. It was then analyzed by SDS-PAGE. The dried gel was stained and exposed to film, and the 32 P 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 g) 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 g 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 g), 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 g 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 32 P-labeled phosphorylase a as the substrate. 32 P-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 g) 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 l. The phosphatase reaction was initiated by the addition of 10 l of 32 P-labeled phosphorylase a (60,000 cpm). After 20 min of incubation at 30°C, the reaction was stopped by the addition of 180 l of 20% (wt/v) trichloroacetic acid. The tubes were left on ice for 10 min and then centrifuged at 12,000 ϫ g for 5 min at 4°C. Aliquots (180 l) of the clear supernatant were counted to determine the phosphatase activity as the amount of 32 P 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 g of cell extracts.
In other experiments purified eIF2B factor (3 g) was phosphorylated with GSK3␤ immunoprecipitates using 30 M ATP and 4.5 Ci of [␥-32 P]ATP as described above. An aliquot of 32 P-labeled eIF2B (1.35 g) was then used instead of the peptide in the PP1C␣ activity assay. The reaction was stopped by adding 12.5 l 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 g) 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 g 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 antidiphospho-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 Ϯ 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.

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 Ϯ 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 Ϯ 0.53 in arbitrary units) was greater than that seen with immunoprecipitates from untreated control cells (6.0 Ϯ 2.1 in arbitrary units, p Ͻ 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).
eIF2B Activation by ERK1 and -2 Immunoprecipitates-To eIF2B Regulation by Protein Phosphatase 1 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 IGF1treated cells (168 Ϯ 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 Ϯ 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 IGF1treated 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).
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 Ϯ 21%) compared with immunoprecipitates from untreated control cells (100%, p Ͻ 0.05; Fig. 2C). On the contrary, PP2A phosphatase activity, although present, underwent no change upon IGF1 treatment in these immunoprecipitates (94 Ϯ 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 antidiphospho-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 potas-sium 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 Ϯ 2.5 to 114 Ϯ 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 Ϯ 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 eIF2B Regulation by Protein Phosphatase 1 protein, is associated with IGF1-activated MAP kinases (mainly ERK2) and activates eIF2B factor in vitro.
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 Ser 535 ) as a substrate. 0.05 units of PP1C␣ released more than 50% of 32 P (6,480 cpm) from the 32 P-labeled peptide, whereas 1.0 units of PP1C␥ were necessary to release a similar amount of 32 P (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 Ϯ 4.9 and 94 Ϯ 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 eIF2⅐[ 3 H]GDP is not affected by PP1C␣. 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 Ϯ 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.
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 IGF1treated cells (54 Ϯ 9.3 and 63 Ϯ 6.5%, respectively, versus 100% of control cells) without affecting PP2A activity (Fig. 6A). Interestingly, IGF1-induced eIF2B activation (138 Ϯ 8.1% versus 100% of control cells) was abolished by treatment with tautomycin (106 Ϯ 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 Ϯ 1.5, 25.5 Ϯ 0.5, 20 Ϯ 1.5, and 23 Ϯ 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. 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/ 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 Ϯ 0.2 pmol/g), was considered as 100%. *, p Ͻ 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 (Ϫ) or RIPA buffer (ϩ) 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 Ϯ 0.1 pmol/ g) was considered as 100%.
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 eIF2B Regulation by Protein Phosphatase 1 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 IGF1induced 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 sig-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 M) 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 Ϯ 654 and 3530 Ϯ 680 cpm/g, 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. ϩ, p Ͻ 0.05 versus control; **, p Ͻ 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 Ϯ 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. ϩ, p Ͻ 0.05 versus control; *, p Ͻ 0.05 versus cells treated with IGF1 alone. C, cell extracts (75 g) 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 g) 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.
eIF2B Regulation by Protein Phosphatase 1 naling 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 IGF1stimulated 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 Ser 535 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 Ser 535 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 coimmunoprecipitated 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.