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Originally published In Press as doi:10.1074/jbc.M111782200 on March 20, 2002

J. Biol. Chem., Vol. 277, Issue 21, 18718-18727, May 24, 2002
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Nerve Growth Factor Specifically Stimulates Translation of Eukaryotic Elongation Factor 1A-1 (eEF1A-1) mRNA by Recruitment to Polyribosomes in PC12 Cells*

Emmanuel PetroulakisDagger and Eugenia Wang§

From the Dagger  McGill University, Department of Neurology and Neurosurgery and Sir Mortimer B. Davis Jewish General Hospital, Lady Davis Institute for Medical Research, Bloomfield Centre for Research in Aging, Montréal, Québec H3T 1E2, Canada and the § Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky 40292

Received for publication, December 10, 2001, and in revised form, March 8, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

During postnatal brain development the level of peptide elongation factor-1A (eEF1A-1) expression declines and that of the highly homologous isoform, eEF1A-2, increases in neurons. eEF1A-1 is implicated in cytoskeletal interactions, tumorigenesis, differentiation, and the absence of eEF1A-2 is implicated in neurodegeneration in the mouse mutant, wasted. The translation of eEF1A-1 mRNA is up-regulated via mitogenic stimulation. However, it is not known if eEF1A-1 mRNA translation is regulated by neurotrophins or if its synthesis is differentially regulated than that of the neuronal isoform, eEF1A-2. Regulated translation of these factors by neurotrophins, particularly by the Trk class of neurotrophin receptors, would implicate them in differentiation, survival, and neuronal plasticity. In this study, we investigated the effect of nerve growth factor (NGF) stimulation on the synthesis of eEF1A-1 and eEF1A-2. We found that NGF stimulation causes a preferential synthesis of eEF1A-1 over eEF1A-2 in PC12 cells. We analyzed the co-sedimentation of eEF1A-1 mRNA with polyribosome fractions in sucrose gradients, and found that NGF stimulation enriched the presence of eEF1A-1 mRNA in polyribosomes, indicating that the translation of eEF1A-1 mRNA is regulated by NGF. Inhibitors of phosphatidylinositol 3-kinase (LY 294002), mammalian target of rapamycin (rapamycin), and the NGF receptor, TrkA (K-252a), but not of mitogen-activated protein kinase (PD 98059), prevented the recruitment of eEF1A-1 mRNA to polyribosomes. The mobilization of eEF1A-1 mRNA to polyribosomes was rapamycin-sensitive in both proliferating and differentiated PC12 cells, indicating the importance of this pathway during differentiation. Our data shows that after growth factor withdrawal, an NGF-signaling pathway stimulates eEF1A-1 mRNA translation in proliferating and differentiated PC12 cells. Therefore, eEF1A-1 mRNA is a specific translational target of TrkA signaling.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Eukaryotic elongation factor 1A (eEF1A-1)1 is a ubiquitously expressed protein responsible for delivering aminoacyl tRNAs to the A-site of the small ribosomal subunit during the elongation phase of protein synthesis (reviewed in Ref. 1). eEF1A-1 expression is largely regulated at the translational level (2). A terminal oligopyrimidine (TOP) tract in the 5'-untranslated region (UTR) of eEF1A-1 mRNA is a key regulatory cis-element implicated in the shifting of eEF1A-1 mRNA from ribonucleoprotein complexes to polyribosomes, where mRNAs are actively engaged in translation (3, 4). This shift is tightly linked with the activation of ribosomal S6 kinase 1 (S6K1) and is inhibited by rapamycin, an immunosuppressant that potently inhibits mammalian target of rapamycin (mTOR) (3, 5). The TOP mRNA family of mRNAs also includes ribosomal RNAs, elongation factor 2 (eEF2), and insulin-like growth factor II (reviewed in Ref. 4). The La autoantigen and cellular nucleic acid binding protein are trans-acting factors involved in translational regulation of TOP mRNAs (6-8). In the absence of growth factors, the majority of TOP mRNA translation is repressed and a small proportion is associated with polyribosomes.

eEF1A-1 mRNA and protein levels decline specifically in brain, heart, and skeletal muscle during development (9-11). The decline of eEF1A-1 expression is compensated by expression of eEF1A-2, a highly homologous (92%) isoform whose expression level during development is inversely correlated with that of eEF1A-1 (9, 11, 12). In muscle, eEF1A-2 substitutes functionally for eEF1A-1 in translation (13), and the absence of eEF1A-2 has been implicated in neurodegeneration in the mutant mouse, wasted (11, 14). Importantly, eEF1A-2 is specifically expressed in terminally differentiated cells such as neurons and differentiated myotubes (10). Recently, isoform-specific antibodies made it possible to discriminate between eEF1A-1 and eEF1A-2 protein expression during normal development and in response to injury (11, 15). However, the mechanisms that are involved during expression switching have not been characterized.

Neurotrophins play a variety of roles during neuronal differentiation, development, and survival (reviewed in Refs. 16-18). Signaling by nerve growth factor (NGF), the prototypical neurotrophin, has been studied and characterized extensively in pheochromocytoma cells (PC12) and mouse primary sympathetic neurons (18). NGF signaling is mediated by the TrkA receptor in addition to the low affinity neurotrophin receptor, p75 (19). In PC12 cells, NGF activates the ribosomal S6 kinases (20-22) and induces protein synthesis by activation of phosphatidylinositol 3-kinase (23), implying the involvement of TOP mRNA translation. Also, translational regulation of gene expression by NGF is implicated in the protein synthesis that is required for neuritogenesis (24, 25).

In this study, we show that both eEF1A-1 and eEF1A-2 are expressed in PC12 cells, and that the eEF1A-1 isoform is preferentially synthesized in response to NGF. We characterized the preferential synthesis of eEF1A-1, and we show that NGF regulates the translation of eEF1A-1 mRNA by its shifting to polyribosomes, as indicated by sucrose gradient sedimentation assays.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- PC12 cells were maintained on collagen-coated Petri dishes (1.5 µg/cm2; rat tail type VII, Sigma) in Dulbecco's modified eagle medium (ICN Biochemicals, Montreal, Quebec, Canada) supplemented with antibiotics (ICN) and 10% heat-inactivated fetal bovine serum (Wisent Inc., St. Bruno, Quebec, Canada). Cells were subcultured by treatment with 0.1% trypsin/1 mM EDTA and reseeded at 1:3 or 1:4 dilutions every 6 days. For differentiation, 1.5 × 106 cells were seeded in 60-mm dishes for 24 h, then switched to differentiation medium (Dulbecco's modified Eagle's medium containing 2% fetal bovine serum and NGF (2.5 S; 100 ng/ml) (Invitrogen Canada Inc., Burlington, ON)), and changed every 48 h. After 6 days, the cells were switched to serum-free Dulbecco's modified Eagle's medium, supplemented with NGF (100 ng/ml), and replaced every 24 h.

Western Blot Analysis-- PC12 cells were washed twice with ice-cold phosphate-buffered saline, harvested by scraping in 1 ml of phosphate-buffered saline, pelleted by centrifugation for 20 s at 10 000 × g, and stored at -80 °C. Pellets were resuspended in extraction buffer: 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% IGEPAL CA-630 (Sigma), 0.25% sodium deoxycholate, aprotinin (1 µg/ml), leupeptin (2 µg/ml), and pepstatin (1 µg/ml) (inhibitors were from Roche (Laval, PQ, Canada)), and phenylmethylsulfonyl fluoride (1 mM) (ICN). The lysate was cleared by centrifugation, and the resulting supernatant was used as the cell extract. Protein concentrations were determined using the BioRad protein assay detection kit (26). Extracts were diluted no more than 2-fold with SDS-PAGE loading buffer (1 M Tris-HCl, (pH 6.8), 10% SDS, 20% glycerol, 10% beta -mercaptoethanol), heated at 95 °C for 10 min, and subjected to SDS-PAGE (27). Proteins were transferred to PROTRAN nitrocellulose (Xymotech Biosystems, Mount Royal, PQ, Canada) and blocked in 5% non-fat milk in Tris-buffered saline/0.05% Tween 20 for 2 h at room temperature. Primary antibodies were used according to the manufacturer's conditions or as previously described (11). Bound antibodies were detected using horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies (CAPPEL) for 1 h and visualized by enhanced chemiluminescence (Amersham Biosciences) after exposure to Kodak X-OMAT-AR film. Monoclonal antibodies specific for neurofilament 68 and 200 kDa were from Sigma. Monoclonal anti-actin and anti-eEF1A were from Calbiochem (San Diego, CA) and Upstate Biotechnologies (Lake Placid, NY), respectively. Polyclonal antibodies specific for p42/p44 MAP kinase, phospho-p42/p44 MAP kinase, p70 S6K (S6K1), phospho-p70 S6K (phospho-S6K1), ribosomal protein S6, phospho-S6 (Ser-235/-236), phospho-S6 (Ser-240/-244) were from New England Biolabs Ltd. (Mississauga, ON, Canada). To analyze NGF-induced protein phosphorylation by Western blot analysis using phosphospecific antibodies, cell extracts were prepared using the manufacturer's recommendations.

Metabolic Labeling with [35S]Methionine and Immunoprecipitation-- PC12 cells (2 × 106) were seeded in 60-mm Petri dishes for 48 h. To withdraw growth factors, cells were washed once and incubated for 3 h in serum-free medium. To deplete intracellular methionine, cells were washed once and incubated for 1 h in methionine-free medium. Fresh methionine-free medium (1.5 ml) containing [35S]methionine (100 µCi/ml) (PerkinElmer Life Sciences) was then added for 10 min before stimulation with NGF (100 ng/ml) (Invitrogen) or dialyzed fetal bovine serum (10%) (Invitrogen). LY 294002 (20 µM), PD 98059 (50 µM), and K-252a (200 nM) were purchased from Calbiochem. Rapamycin (100 ng/ml) was purchased from Sigma. Inhibitor stock solutions were prepared at 1000-fold dilutions in dimethyl sulfoxide, Me2SO); control cultures were treated only with Me2SO. Inhibitors were included during the methionine-depletion and [35S]methionine-labeling steps. To immunoprecipitate eEF1A-1/eEF1A-2, extracts were prepared (as described above), diluted to equal concentrations (1-5 µg/µl), and precleared with 20 µl of protein G-Sepharose, Fast Flow (Amersham Biosciences) for 1 h, and then incubated overnight at 4 °C with the anti-eEF1A antibody (1:100 dilution). Immune complexes were collected on 30 µl of protein G-Sepharose for 3 h and then washed three times (10 min each) with 1 ml of 10 mM Tris-HCl (pH 8.6), 600 mM NaCl, 0.1% SDS, 0.1% IGEPAL CA-630. The protein G-Sepharose was finally resuspended in SDS-PAGE buffer and heated at 95-100 °C for 10 min, and an aliquot was subjected to PAGE with some modifications (28, 29). Essentially, gels (1.5 mm thickness) consisted of a 4% stacking gel (70 mM Tris-HCl (pH 6.8), 4 mM EDTA, 0.4% SDS, 5% glycerol), and a 20-cm separating gel (100 mM Tris-HCl, 300 mM glycine, 0.4% SDS, 5% glycerol). Proteins were separated for 13 h at 20 mA. Gels were fixed, dried under vacuum, and exposed to a phosphorimaging screen or to Kodak X-OMAT AR film at -80 °C. In the latter case, gels were treated with Amplify Fluorographic Reagent (Amersham Biosciences). Protein band intensities were quantified using ImageQuant (Molecular Dynamics, Sunnyvale, CA).

Isolation of Polyribosomal RNA and Northern Blot Analysis-- Approximately 5 × 106 cells, grown in 100-mm dishes, were treated with cycloheximide (100 µg/ml) (Sigma) for 10 min at 37 °C. Cells were then washed twice with ice-cold phosphate-buffered saline containing cycloheximide (100 µg/ml), harvested in 1 ml of phosphate-buffered saline, and pelleted by centrifugation for 2 min at 6000 rpm in a microcentrifuge. The pellet was resuspended and incubated for 10 min in 1 ml of ice-cold lysis buffer: 10 mM Tris-HCl (pH 7.4), 0.5% IGEPAL CA-630, 150 mM NaCl, 10 mM MgCl2, 2 mM dithiothreitol, 100 µg/ml cycloheximide, and 120 units/ml RNAGuard (Amersham Biosciences). The lysate was cleared by centrifugation, and 800 µl of the supernatant was applied to a 11.5-ml sucrose gradient (12-45%) (50 mM Tris-HCl (pH 7.4), 50 mM KCl, 10 mM MgCl2, 3 mM dithiothreitol, 15 units/ml RNAGuard). Gradients were subjected to centrifugation at 38,000 rpm for 2 h at 4 °C in a Beckman SW40Ti rotor, fractionated from the top (1.3 ml/min) using an auto densi-flow density gradient fractionator (Labconco), and pumped through a flowcell cuvette (Beckman). The absorbance at 260 nm was recorded every 3 s using a Beckman DU-65 spectrophotometer. Fifteen fractions (800-850 µl each) were collected, and 300 µl of each were treated with proteinase K (100 µg/ml) (Roche) and 0.2% SDS for 60 min at 56 °C. RNA was extracted with an equal volume of water-saturated phenol:chloroform (1:1) (Invitrogen) and precipitated with 2.5 volumes of 95% ethanol in the presence of yeast tRNA (30 µg) (Roche). The RNA was pelleted by centrifugation at 10,000 × g for 30 min at 4 °C, washed once with 70% ethanol and centrifuged again. RNA was dissolved in loading buffer (30), fractionated in a 1% agarose/formaldehyde gel (30), transferred to Hybond-N+ membrane (Amersham Biosciences), and cross-linked by UV irradiation using a GS gene linker (BioRad). cDNA fragments of beta -actin and the 3'-UTR of eEF1A-1 mRNAs were generated by PCR (14), and used to produce [alpha -32P]dCTP (PerkinElmer Life Sciences)-labeled probes using a random primer labeling kit (Roche). Membranes were prehybridized for 2 h at 68 °C in ExpressHyb (CLONTECH) containing herring sperm DNA (250 µg/ml) (Roche). The denatured probe was added and incubated for 14 h at 68 °C. Membranes were washed for 40 min at room temperature in 2× SSC (sodium-saline-citrate)/0.1% SDS with one change after 20 min. Membranes were finally washed three times (15 min each) at 60 °C in 0.1× SSC/0.1% SDS and exposed to a phosphorimaging screen. Band intensities were determined using ImageQuant.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

eEF1A-1 Is Preferentially Synthesized in Response to NGF Stimulation-- Growth factor stimulation induces protein synthesis and a specific activation of eEF1A-1 translation (31). Because eEF1A-1 and eEF1A-2 proteins are already expressed at high levels in PC12 cells and because their predicted molecular masses differ by only 0.5 kDa, we used modified SDS-PAGE procedures to detect quantifiable changes in expression. First, we have previously used eEF1A-1- and eEF1A-2-specific antibodies to demonstrate that the pan-eEF1A-specific monoclonal antibody used in this study recognizes both isoforms (11). Secondly, immunoprecipitation assays were conducted using extracts from [35S]methionine-labeled PC12 cells, allowing us to accurately quantify the levels of eEF1A-1 and eEF1A-2 protein expression. We quantified the eEF1A-1/eEF1A-2 ratio, and we used this ratio as a specific indicator of the relative level of eEF1A-1 synthesis. Thirdly, we achieved a highly resolved separation of immunoprecipitated [35S]methionine-labeled eEF1A-1 and eEF1A-2 using a modified SDS-PAGE system (see "Materials and Methods").

To determine the effect of NGF stimulation on the synthesis of eEF1A-1 and eEF1A-2, non-differentiated PC12 cells were subjected to serum deprivation for 3 h and then exposed to NGF (100 ng/ml) for 15, 30, 60, 120, and 180 min in the presence of [35S]methionine. Newly synthesized, 35S-labeled eEF1A-1 and eEF1A-2 proteins at each time point were immunoprecipitated from cell extracts using the pan-eEF1A-specific antibody and analyzed by SDS-PAGE (Fig. 1A). In untreated PC12 cells, eEF1A-2 protein was found to be the prominent isoform at each time point in growth factor-free conditions (untreated, Fig. 1A). In NGF-treated PC12 cells, the overall rate of eEF1A-2 synthesis was slightly increased as judged by the immunoprecipitated levels of protein. In comparison, there was an abundance of eEF1A-1 detected after 120 and 180 min of NGF stimulation (Fig. 1A). By comparing the levels of eEF1A-1 and eEF1A-2 proteins after 60, 120, and 180 min, eEF1A-1 was preferentially synthesized after NGF stimulation (Fig. 1A). To demonstrate the distinction between cells that express eEF1A-2 and those that express only eEF1A-1, we also labeled non-differentiated P19 embryonic teratocarcinoma cells that express only the eEF1A-1 isoform. Only a single band corresponding to eEF1A-1 was detected in P19 cells, and a doublet of eEF1A-1 and eEF1A-2 was detected in PC12 cell extracts (Figs. 1A, lanes 14 and 15). No additional bands were detected when immunoprecipitations were conducted in the absence of antibody (-ab, Fig. 1A, lane 13).


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  		Fig. 1.   NGF stimulates preferential synthesis of eEF1A-1 protein in PC12 cells. A, PC12 cells were incubated in serum-free medium for 3 h and then in methionine-free medium for 1 h. Cells were then incubated in methionine-free medium containing 35S-methonine (100 µCi/ml) for 10 min (indicated as 0 min) and then stimulated with NGF (100 ng/ml) for 15, 30, 60, 120, and 180 min. Carrier-treated (untreated, lanes 1-6) and NGF-treated (NGF, lanes 7-12) cells were harvested at each indicated time point. eEF1A-1 and eEF1A-2 proteins (indicated by arrows) were immunoprecipitated from corresponding cell extracts using the anti-eEF1A antibody. As controls, immunoprecipitations were done without antibody (-ab, lane 13), from P19 cells (lane 14), and PC12 cells (lane 15). B, an equal number of PC12 cells were incubated in serum-free medium for 3 h prior to stimulation with NGF (100 ng/ml) for 5, 15, 20, 60, 90, 120, and 180 min. Cells were extracted at 0 min as untreated controls. Extracts were subjected to Western blot analysis for the expression of p42/p44 MAP kinase, phospho-p42/p44 MAP kinase, S6K1 (p70 S6K), phospho-S6K1 (p70 S6K), S6, phospho-S6 (Ser-235/-235), and phospho-S6 (Ser-240/-244). These results were similar in three independent experiments.

In parallel experiments, we verified that NGF signaling was sustained during stimulation with NGF for 3 h. Phosphorylated p42/p44 MAP kinase was detected within 5 min of NGF exposure (Fig. 1B). A maximum level of phosphorylated p44/p42 was maintained for 60 min and was still detectable after 180 min. We could not detect phosphorylated p44/p42 MAPK in untreated cells. Similarly, the maximum levels of phosphorylated S6K1 and S6 were detected after 15 min of NGF exposure (Fig. 1B). Phosphorylation of S6 was maintained at its maximum level during NGF treatment. Therefore, the expected signaling pathways were activated and sustained by exposure to NGF (reviewed in Ref. 18).

NGF-induced Synthesis of eEF1A-1 Is Mediated by PI3K, mTOR, and TrkA-specific Signaling-- Mitogen-induced synthesis of eEF1A-1 is mediated by through PI3K and mTOR (3, 32). We quantified the effect of NGF stimulation on the eEF1A-1/eEF1A-2 ratio and determined the signaling pathways involved in altering the eEF1A-1/eEF1A-2 ratio by using pharmacological inhibitors.

In the absence of growth factors, the amount of incorporated [35S]methionine was ~2.5 times more [35S]methionine was incorporated in eEF1A-2 than eEF1A-1 in PC12 cells as indicated by the immunoprecipitated levels (Fig. 2A, lane 1). Consistent with the result of Fig. 1A, there was a specific increase in the level of eEF1A-1 in NGF-stimulated cells resulting in a statistically significant increase (1.62 ± 0.18-fold; p < 0.001) in the eEF1A-1/eEF1A-2 ratio (Fig. 2A, lane 2). A similar effect was observed in serum-stimulated PC12 cells (Fig. 2B, lane 2). Again, we used P19 control cell extracts that contain only eEF1A-1 in order to distinguish its mobility from eEF1A-2 that is also present in PC12 cells (Fig. 2, A and B, lanes 8 and 9). Again, no additional bands were detected when immunoprecipitations were conducted in the absence of antibody (-ab) (Fig. 2, A and B, lane 7). These results show that serum deprivation causes a specific decrease, while NGF stimulation induced a specific increase in eEF1A-1 synthesis.


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  		Fig. 2.   Effect of signaling inhibitors on eEF1A-1 and eEF1A-2 synthesis upon stimulation with NGF and serum. PC12 cells were serum-deprived, metabolically labeled with [35S]methionine, and stimulated with NGF (100 ng/ml) or dialyzed serum (10%) as described under "Materials and Methods." A, the anti-eEF1A antibody was used to immunoprecipitate newly synthesized [35S]methionine-labeled eEF1A-1 and eEF1A-2 from control (serum-deprived; Me2SO carrier) (C, lane 1), NGF-stimulated (N, lane 2), and NGF-stimulated cells that were pretreated with LY 294002 (20 µM) (L, lane 3), rapamycin (100 nM) (R, lane 4), PD 98059 (50 µM) (P, lane 5), or K-252a (200 nM) (K, lane 6). Immunoprecipitations were also conducted from serum-stimulated cells in the absence of antibody (-ab, lane 7). Immunoprecipitates from P19 cells (lane 8) and PC12 cells (lane 9) were used as negative and positive controls for eEF1A-2 synthesis, respectively. Protein levels were analyzed by SDS-PAGE and band densities were determined by phosphorimaging analysis. eEF1A-1/eEF1A-2 ratios were quantified, and the data represents the average (mean ± S.D.) of at least three experiments conducted in triplicate. B, immunoprecipitations from serum-stimulated PC12 cells. All other conditions were identical to those in A. C, PC12 cells were serum deprived and pretreated for 2 h with the inhibitors specified in A prior to stimulation with NGF for 30 min. Cells were extracted as described under "Material and Methods" and subjected to Western blot analysis for the expression of p42/p44 MAP kinase, phospho-p42/p44 MAP kinase, S6K1 (p70 S6K), phospho-S6K1 (p70 S6K), S6, phospho-S6 (Ser-235/-235). The result was identical in three independent experiments.

To determine the signaling pathways involved in NGF-mediated increases in the eEF1A-1/eEF1A-2 ratio by NGF, PC12 cells were pretreated with PI3K inhibitor (LY 294002), the mTOR inhibitor (rapamycin), or K-252a, an inhibitor of the high affinity NGF-receptor (TrkA). The MAP kinase kinase (MEK) inhibitor, PD 98059, was used in order to assess the involvement of the Ras-MAPK pathway. Pretreatment with LY 294002 completely inhibited the increase of the eEF1A-1/eEF1A-2 ratio in NGF and serum-stimulated PC12 cells (Fig. 2, A and B, lane 3). Rapamycin also inhibited the increase of eEF1A-1, although there was a small inhibitory effect on eEF1A-2 synthesis that resulted in a slight increase of the eEF1A-1/eEF1A-2 ratio in NGF-stimulated cells (Fig. 2, A and B, lane 4). In comparison, PD 980509 had relatively no effect on the eEF1A-1/eEF1A-2 ratio after NGF stimulation, although the up-regulation of eEF1A-1 synthesis by NGF was partly inhibited (Fig. 2A, lane 5). We verified that this was due to the effect of PD98059 alone (data not shown). Pretreatment with K-252a prevented the increase of eEF1A-1/eEF1A-2 only in NGF-stimulated cells (Fig. 2A, lane 5). Based on the inhibitory effects of LY 294002, rapamycin, and K-252a, we concluded that NGF stimulation caused a preferential increase in eEF1A-1 synthesis that is mediated by PI3K, mTOR, and through the TrkA receptor, respectively.

To verify the specificity of the inhibitors, the phosphorylation status of p44/p42 MAPK, S6K1, and S6 was determined in parallel experiments (Fig. 2C). In NGF-treated cells the maximum level of phosphorylated MAPK, S6K1, and S6 at serine 235/236 was detected after 30 min. In PD 98059-pretreated cells, the phosphorylation of MAPK by NGF, but not that of S6K1 or S6, was inhibited. Pretreatment with LY294002 or rapamycin prevented maximum phosphorylation of S6K1 and S6. However, very low levels of hypo-phosphorylated S6K1 and S6 were present in our preparations. Pretreatment with K-252a resulted in complete inhibition of MAPK, S6K1, and S6 phosphorylation. The total amount of MAPK, S6K1, and S6 was similar in all samples, as judged by analyzing the total level of each protein (Fig. 2C). These results indicated that the intended signaling pathways were inhibited.

NGF Stimulates Polyribosome Assembly in PC12 Cells-- NGF stimulation increases the rate of protein synthesis in PC12 cells (23). To demonstrate that this increase is associated with an increase in polyribosome assembly, we prepared polyribosome profiles by sucrose gradient centrifugation from serum-deprived, NGF-stimulated, and serum-stimulated PC12 cells. In serum-deprived PC12 cells, we observed distinct 40 S, 60 S, and 80 S ribosome peaks in fractions 4, 6, and 8, respectively (Fig. 3A). In NGF-stimulated PC12 cells, these peaks disappeared as a result of shifting to polyribosome fractions (12-15) (Fig. 3B). These peaks also shifted in serum-stimulated PC12 cells, as expected (Fig. 3C). To substantiate the 40 S, 60 S, 80 S, and polyribosome peaks as those we describe here, profiles were prepared in the absence of MgCl2 and in the presence of 30 mM EDTA in order to disrupt polyribosomes. In EDTA-disrupted polyribosome preparations we observed a peak in fraction 4, corresponding to the accumulation of disrupted ribosomes (Fig. 3D). These results indicated that NGF stimulates polyribosome assembly in PC12 cells.


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  		Fig. 3.   NGF stimulation induces polyribosome assembly and rapamycin-sensitive shifting of eEF1A-1 mRNA to polyribosomes in PC12 cells. PC12 cell extracts were subjected to sucrose gradient (12-45%) centrifugation followed by fractionation and monitoring of the absorbance at 260 nm as described under "Material and Methods." Polyribosome profiles were prepared from untreated (serum deprived)(A), NGF-stimulated (B), and serum-stimulated (C) PC12 cells. D, profiles from serum-stimulated PC12 cells prepared in the absence of MgCl2 and in the presence of 30 mM EDTA. The sedimentation of 40 S, 60 S, and 80 S ribosome peaks in growth factor-free conditions is indicated (arrows in A). The distribution of eEF1A-1 and beta -actin mRNAs was analyzed in polyribosome profiles from untreated (Me2SO carrier) (E), NGF-stimulated (F), serum-stimulated (G), NGF-stimulated, rapamycin pretreated (H), serum stimulated, rapamycin pretreated (I), and rapamycin-treated (J) PC12 cells. Rapamycin (100 ng/ml) was included in the medium for 1.5 h prior to stimulation with NGF (100 ng/ml). Gradients were fractionated into 15 fractions, and RNA was extracted and subjected to Northern blot analysis for the expression of eEF1A-1 and beta -actin mRNAs. The levels of eEF1A-1 ([circf) and beta -actin (open circle ) mRNAs are shown for each fraction, and the relative density of expression in each fraction is indicated in the corresponding graphs. The results are representative of at least two independent experiments.

Inhibition of mTOR, but Not Inhibition of MAPK, Prevents the NGF-mediated Redistribution of eEF1A-1 mRNA to Polyribosomes in PC12 Cells-- The above results suggested that eEF1A-1 synthesis was specifically up-regulated by NGF stimulation (Fig. 2A). Because NGF-mediated stimulation of eEF1A-1 synthesis was inhibited by rapamycin, we hypothesized that eEF1A-1 translation was increased by shifting eEF1A-1 mRNA from subpolyribosome to polyribosome fractions of sucrose gradients (31, 33). We analyzed the distribution of eEF1A-1 mRNA in polyribosome profiles from NGF-stimulated PC12 cells by Northern blot analysis using a probe specific for the 3'-UTR of eEF1A-1 mRNA.

In serum-deprived PC12 cells, the majority of eEF1A-1 mRNA co-sedimented with subpolyribosome fractions (Fig. 3E). Peaks of eEF1A-1 mRNA expression co-sedimented with fractions 4, 6, and at a maximum level in fraction 8. In comparison, the co-sedimentation of beta -actin, which is known to be associated with polyribosomes, was enriched in polyribosome fractions 12 and 13. The distribution of beta -actin mRNA was clearly distinct from that of eEF1A-1 mRNA. These results were consistent with the accumulation of eEF1A-1 mRNA in subpolyribosome fractions in the absence of growth factors (31).

In NGF-stimulated PC12 Cells, eEF1A-1 mRNA Was Redistributed to Polyribosomes (Fig. 3F). Small peaks of eEF1A-1 mRNA expression remained in subpolyribosomes (fractions 4 and 8), and an increase was found in polyribosome fractions with a maximum level of eEF1A-1 co-sedimentation in fraction 14 (Fig. 3F). Serum stimulation also resulted in a similar pattern of eEF1A-1 mRNA redistribution (Fig. 3G). In comparison, the distribution of beta -actin mRNA was not altered by stimulation with NGF or serum. Taken together, eEF1A-1 mRNA redistributed from subpolyribosomes to polyribosomes in NGF-stimulated PC12 cells.

In rapamycin-treated PC12 cells, NGF and serum stimulation failed to induce shifting of eEF1A-1 mRNA to polyribosomes (Fig. 3, H and I). Treatment with rapamycin alone had no effect on the polyribosome distribution of eEF1A-1, 18 S and 28 S rRNAs (not shown), or beta -actin (Fig. 3J). Therefore, the NGF-mediated shift of eEF1A-1 mRNA to polyribosomes was rapamycin-sensitive.

The Ras-MAPK pathway is rapidly activated, sustained, and leads to activation of p90 ribosomal S6 kinase by NGF (reviewed in Ref. 18). To assess the role of the Ras-MAPK pathway in NGF-mediated shifting of eEF1A-1 mRNA to polyribosomes, we examined NGF-stimulated PC12 cells that were pretreated with the MAPK inhibitor, PD 98059 (50 µM). Densitometric analysis of eEF1A-1 distribution in polyribosome profiles is summarized in Table I, part I. In untreated PC12 cells, eEF1A-1 mRNA co-sedimented primarily with subpolyribosomes (fractions 1-11), as expected. In comparison, the majority of eEF1A-1 mRNA was enriched in polyribosomes (fractions 12-15) after NGF stimulation. This resulted in a 5-fold increase in the polyribosome:subpolyribosome (P:SP) ratio of eEF1A-1, indicating that its translation was increased (Table I, part I). Pretreatment with PD98059 did not prevent the recruitment of eEF1A-1 mRNA or ribosomal RNAs (data not shown) to polyribosome fractions by NGF stimulation (Table I, part I). Treatment with PD 98059 alone had a small effect that reduced the P:SP ratio (Table I, part I), and this was consistent with the immunoprecipitation results (Fig. 2A and data not shown). Taken together, these data demonstrate that activation of the Ras-MAPK pathway did not lead to shifting of eEF1A-1 mRNA to polyribosomes. These results were also consistent with the immunoprecipitation assays and the inhibition of MAPK phosphorylation by PD 98059 (Fig. 2, A and C).

                              
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Table I
Recruitment of eEF1A-1 mRNA to polyribosome fractions by NGF stimulation is inhibited by PD98059 and K252a
Polyribosome profiles were examined by Northern blot analysis, and the results were summarized for three experiments (indicated as parts I, II, and III), which are discussed in the text. Part I, eEF1A-1 mRNA profiles from untreated (M2SO carrier-treated), NGF-treated, NGF + PD98059-treated, and PD98059-treated. Part II, eEF1A-1 mRNA profiles from untreated (M2SO carrier-treated), NGF-treated, NGF + K252a-treated, and K252a-treated. Part III, eEF1A-1 mRNA profiles from untreated (carrier-treated), BDNF-, NGF-, and serum-stimulated PC12 cells. RNA was extracted from each polyribosome profile (15 fractions) and subjected to Northern blot analysis to determine the distribution of eEF1A-1 mRNA among subpolyribosome (SP) and polyribosome (P) fractions. The relative amount of eEF1A-1 mRNA in each fraction was determined by densitometry (Image Quant) and was expressed in arbitrary densitometric units as the density relative to that determined for eEF1A-1 in fraction 4. The sum of the relative densities in each fraction was determined for each RNA pool (P and SP) in order to obtain the P:SP ratio (indicated as the value in the far right column).

NGF-mediated Redistribution of eEF1A-1 to Polyribosomes Is TrKA-dependent-- K-252a is a potent inhibitor of TrkA signaling in PC12 cells (34). We evaluated the receptor specificity for NGF-induced redistribution of eEF1A-1 to polyribosomes by pretreatment with K-252a (summarized in Table I, part II). As expected for untreated PC12 cells, the majority of eEF1A-1 mRNA co-sedimented with subpolyribosome fractions, and NGF stimulation induced redistribution of eEF1A-1 to polyribosomes (Table I, part II), resulting in an increased P:SP ratio for eEF1A-1. Pretreatment with K-252a completely inhibited the shifting of eEF1A-1 mRNA to polyribosome fractions by NGF (Table I, part II), and treatment with K-252a alone did not affect the distribution of ribosomal RNA (not shown), eEF1A-1, and beta -actin mRNAs (Table I, part II and data not shown). Taken together, TrkA-specific signaling leads to rapamycin-sensitive shifting of eEF1A-1 to polyribosomes upon NGF stimulation.

Because PC12 cells express both p75 and TrkA neurotrophin receptors, we wanted to determine whether a p75-specific (i.e. TrkA-independent) signaling cascade contributes to the NGF-induced shifting of eEF1A-1 mRNA to polyribosomes. Brain-derived neurotrophic factor (BDNF), which elicits signaling through TrkB, a neurotrophin receptor that is not expressed in PC12 cells, is also a ligand for the p75 receptor (35-37). PC12 cells were serum-deprived and then treated with BDNF (100 ng/ml) for 3 h. In untreated cells, the majority of eEF1A-1 mRNA co-sedimented with subpolyribosome fractions, as expected (Table I, part III). In BDNF-treated PC12 cells, the distribution of rRNAs (not shown) and eEF1A-1 mRNA was not affected (Table I, part III). As positive controls, the characteristic shift of eEF1A-1 mRNA to polyribosomes was observed following treatment with NGF or serum, as indicated by the 7-fold increase in the P:SP ratio for eEF1A-1 (Table I, part III). In comparison, beta -actin mRNA was always enriched in polyribosome fractions (data not shown). Because PC12 cells did not respond to BDNF-treatment, the biological activity of the BDNF used in our study was assessed by its efficiency in inducing phosphorylation of MAPK in neuronal P19 embryonic teratocarcinoma cells. P19 cells were differentiated for 10 days (38), subjected to serum deprivation for 24 h and treated with BDNF (100 ng/ml) for 30 min. We found increased phosphorylation of MAPK in BDNF-treated P19 neuronal cells (Fig. 4). As a positive control, we induced MAPK phosphorylation in PC12 cells with NGF (Fig. 4). We could not detect phosphorylated MAPK, S6K1, or S6 in BDNF-treated PC12 cells (data not shown). These results indicated that unlike NGF, BDNF does not induce the redistribution of eEF1A-1 mRNA to polyribosomes in PC12 cells. Taken together with the previous result (Table I, part II), we conclude that the shifting of eEF1A-1 mRNA to polyribosomes is mediated by specific activation of TrkA by NGF.


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  		Fig. 4.   Assay for BDNF signaling activity. p44/p42 MAPK and phospho-p44/p42 MAPK expression levels in BDNF-treated (+) and carrier-treated (-) neuronally differentiated P19 embryonic teratocarcinoma cells and NGF-treated (+) and carrier-treated (-) PC12 cells. The results are representative of two independent experiments.

NGF Induces Shifting of eEF1A-1 mRNA to Polyribosomes in Differentiated PC12 Cells-- In the above experiments, the NGF-induced translation of eEF1A-1 was studied in non-differentiated PC12 cells. In vivo there is a predominance of eEF1A-2 protein expression in postdevelopmental neurons in the brain (11). To explore the possibility that NGF has an alternative effect on the eEF1A-1/eEF1A-2 ratio in differentiated cells, we investigated the synthesis of eEF1A-1 and eEF1A-2 in differentiated PC12 cells. After 7 days of differentiation with NGF (100 ng/ml), PC12 cells exhibited extensive neurite outgrowth, which correlated to the increased expression of the neurofilament proteins, NF-68 and NF-200 (Fig. 5A). Differentiated PC12 cells were subjected to NGF-deprivation, and then stimulated with NGF for 20, 40, 60, 90, 120, and 180 min in the presence of [35S]methionine. By immunoprecipitation, we found that both eEF1A-1 and eEF1A-2 were synthesized in differentiated PC12 cells in growth factor-free conditions (untreated, Fig. 5B). In NGF-stimulated cells, the synthesis of eEF1A-2 was not significantly affected (NGF, Fig. 5B). In comparison, we observed an increase in the rate of eEF1A-1 protein synthesis during NGF exposure (NGF, Fig. 5B). We quantified the newly synthesized levels of eEF1A-1 and eEF1A-2 and found that upon re-exposure to NGF, the eEF1A-1/eEF1A-2 increased by almost 2-fold, while it remained relatively unchanged in untreated cells (Fig. 5C).


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  		Fig. 5.   NGF stimulates eEF1A-1 protein synthesis in differentiated PC12 cells subjected to NGF withdrawal. PC12 cells were differentiated with NGF (100 ng/ml) for 7 days prior to growth factor deprivation and metabolic labeling with [35S]methionine as described under "Material and Methods." A, Western blot analysis of neurofilament proteins, NF68 and NF200, and actin during differentiation of PC12 cells for 0-7 days. B, cells were incubated with [35S]methionine (100 µCi/ml) for 10 min before treatment with NGF (100 ng/ml) for 20, 40, 60, 90, 120, and 180 min; eEF1A-1 and eEF1A-2 proteins were immunoprecipitated from cell extracts using the monoclonal anti-eEF1A antibody that detects both isoforms. Immunoprecipitates were separated by SDS-PAGE and exposed to X-OMAT AR film. C, band densities were quantified to determine the eEF1A-1/eEF1A-2 ratio at each time point in control () and NGF-stimulated (open circle ) PC12 cells.

Because the synthesis of eEF1A-1 was preferentially stimulated by re-exposure to NGF in differentiated PC12 cells, we used sucrose sedimentation assays to determine whether the increase of eEF1A-1 synthesis was regulated at the translational level by the shifting of eEF1A-1 mRNA to polyribosomes. In NGF-deprived (untreated) PC12 cells, the majority of eEF1A-1 mRNA co-sedimented with subpolyribosome fractions (Fig. 6A). After NGF- stimulation, there was a substantial increase of eEF1A-1 mRNA in polyribosomes (Fig. 6B). eEF1A-1 mRNA became enriched in polyribosomes (fraction 13) by NGF stimulation, consistent with the abundance of beta -actin mRNA (Fig. 6B). These results indicated that restimulation with NGF induced the distribution of eEF1A-1 mRNA to polyribosomes, resulting in increased translation of eEF1A-1. The shifting of eEF1A-1 mRNA to polyribosomes in differentiated PC12 cells was also inhibited by rapamycin and K-252a (Fig. 6, C and D). Treatment with rapamycin or K-252a alone resulted in a small decrease in the polyribosome-associated proportion of eEF1A-1 mRNA (Fig. 6, E and F). In comparison, beta -actin was always enriched in polyribosome fractions. Taken together, NGF-withdrawal followed by restimulation with NGF caused a rapamycin- and K-252a-sensitive increase of eEF1A-1 translation in differentiated PC12 cells.


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  		Fig. 6.   Rapamycin and K-252a inhibit the shift of eEF1A-1 mRNA to polyribosomes upon NGF stimulation in differentiated, NGF-deprived PC12 cells. PC12 cells were differentiated in the presence of NGF for 7 days as described under "Material and Methods." Differentiated PC12 cells were rinsed once and incubated with serum-free medium for 3 h. Rapamycin (100 ng/ml) or K-252a (200 nM) was included in the medium for 1.5 h prior to stimulation with NGF (100 ng/ml). Cell extracts and polyribosome profiles were prepared as described under "Material and Methods" from untreated (A), NGF-stimulated (B), rapamycin-pretreated, NGF-stimulated (C), K-252a pretreated, NGF-stimulated (D), rapamycin-treated (E), and K-252a-treated (F) PC12 cells. Each gradient was fractionated into 15 fractions, and RNA was extracted and subjected to Northern blot analysis for the expression of eEF1A-1 and beta -actin mRNAs. The levels of eEF1A-1 () and beta -actin (open circle ) mRNAs are shown for each fraction, and the relative density of expression in each fraction is indicated in the corresponding graphs.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Growth factor-mediated mRNA translation induces rapid changes of protein expression (39). In quiescent cells, mitogens stimulate the mobilization of TOP mRNAs from translationally inactive ribonucleoprotein pools to polyribosome pools, where they are efficiently translated (4). In this study, we show that NGF, which promotes neuronal differentiation and survival (18), also stimulates TOP mRNA translation (exemplified by eEF1A-1). Though the role of eEF1A-1 is in protein synthesis (40), growth factors also mediate its interactions with actin (41) and its nuclear localization (42). Thus, the NGF-mediated increase of eEF1A-1 synthesis implicates this isoform in its non-translational roles (1), during neuronal differentiation.

During postnatal brain development, eEF1A-1 expression is replaced by eEF1A-2 (11). Though both eEF1A isoforms are expressed in neurons (10, 43), it is not known if growth factors differentially regulate their synthesis. Importantly, we show that NGF-induced translation of eEF1A-1 mRNA is TrkA-mediated and involves PI3K/mTOR signaling (summarized in Fig. 7). In comparison, eEF1A-2 was synthesized efficiently even in the absence of growth factors. Because eEF1A-2 mRNA does not contain a TOP element in its 5'-UTR (12, 44), the abundance of eEF1A-2 in PC12 cells is consistent with its status as a non-TOP mRNA, such as beta -actin, that is primarily polyribosome-associated (4). However, at this time we cannot explain the presence of eEF1A-2 in non-differentiated PC12 cells because its expression in neurons is terminal differentiation-dependent (10).


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  		Fig. 7.   Regulation of eEF1A-1 mRNA translation by NGF. Schematic showing the activation of eEF1A-1 translation by NGF through the TrkA-PI3K signaling pathway leading to shifting of eEF1A-1 mRNA from ribonucleoproteins to polyribosome fractions. Inhibition by K-252a, LY 294002, and rapamycin is indicated (perp ). Inhibition (perp ) of MEK by PD 98059 does not prevent shifting of eEF1A-1 mRNA to polyribosomes. The involvement of S6 phosphorylation in mediating TOP mRNA translation is likely not required (50).

In PC12 cells, NGF increases protein synthesis, leads to phosphorylation of S6K1 and the eIF4E-binding protein (4EBP) (45, 46), and increases eIF2B activity (23). Importantly, rapamycin only partially inhibits NGF-induced protein synthesis (23), indicating that mTOR regulates translation of a distinct subset mRNAs that includes TOP mRNAs. However, previous studies examined these mechanisms only in non-differentiated PC12 cells. We subjected NGF-differentiated PC12 cells to NGF withdrawal followed by NGF restimulation to demonstrate that a eEF1A-1 is also preferentially translated in differentiated PC12 cells. This result implicates the importance of Trk-mediated TOP mRNA translation in differentiated neurons. We emphasized the involvement of TrkA by showing that K-252a, a Trk-inhibitor (34), inhibits NGF-mediated eEF1A-1 mRNA translation in both PC12 states. BDNF, whose receptor (TrkB) is not expressed in PC12 cells (36) did not affect eEF1A-1 mRNA translation. This also suggested that the p75 neurotrophin receptor (37), that is also bound by BDNF (35), does not contribute to Trk-mediated TOP mRNA translation. Taken together, NGF induces polyribosome assembly via TrkA-mediated signaling and involves distinct translation of eEF1A-1 mRNA (Fig. 7).

NGF-mediated eEF1A-1 mRNA translation is inhibited by rapamycin, a specific inhibitor of mTOR (47, 48). S6K1, a major target of mTOR and thought to be important for TOP mRNA translation (49), is not required (50). mTOR is also regulated by nutrients and amino acids (47). Importantly, amino acid-mediated regulation of TOP mRNAs is only partially regulated by mTOR, requires PI3K, but does not require S6K1 activity (50). The importance of mTOR in neurotrophin-mediated translation in neurons has been described. For example, BDNF regulates translation initiation in cortical neurons (51), and the effect of BDNF on long term synaptic plasticity in hippocampal neurons is mediated by mTOR (52). Neurotrophin-mediated translational control also involves MAPK (45, 51), though this pathway does not appear to regulate TOP mRNAs (Table I, part I). TOP mRNA translation and the involvement of S6K1 in these other systems have not been studied. Other factors such as neurabin, which localizes with S6K1 in nerve terminals (53), and the interaction between mTOR and gephyrin (54), are likely important for neurotrophin-mediated TOP mRNA translation.

Despite the abundant expression of eEF1A-2 in postdevelopmental brain (11), the eEF1A-1 isoform is implicated in translational control by NGF. Future studies are required to determine whether Trk-mediated mTOR activity regulates TOP mRNA translation in in vivo. Importantly, it is not known if the eEF1A isoforms share their translational and non-translational functions in neurons.

    ACKNOWLEDGEMENTS

We thank Dr. Antonis Koromilas, Dr. Kostas Pantopoulos, and Richard Marcotte for comments and suggestions during the preparation of the manuscript.

    FOOTNOTES

* This work was supported by Research Operating Grant AG10461 from NIA, National Institutes of Health (to E. W.)The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current Address and to whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Louisville School of Medicine, 570 S. Preston St., Rm. 304, Louisville, KY 40292. Tel.: 502-852-2554; Fax: 502-852-2555; E-mail: eugenia.wang@louisville.edu.

Published, JBC Papers in Press, March 20, 2002, DOI 10.1074/jbc.M111782200

    ABBREVIATIONS

The abbreviations used are: eEF1A, eukaryotic elongation factor-1A; TOP, terminal oligopyrimidine; UTR, untranslated region; mTOR, mammalian target of rapamycin; NGF, nerve growth factor; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; P:SP, polyribosome:subpolyribosome ratio; BDNF, brain-derived neutrophic factor.

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