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Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033
2To whom correspondence should be addressed. E-mail: jjefferson{at}psu.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • translation initiation • eukaryotic initiation factors 4E and 4G • mTOR • microarray analysis
The protein and RNA content of the liver decline during food deprivation, and both macromolecules are replenished upon commencement of feeding (1,2). In rat liver, the fasting to feeding transition results in a rapid aggregation of ribosomes on mRNA, an effect that is sustained for an extended period of time (2,3). The observed accumulation of polysomes suggests a stimulation of the process involving the initiation of mRNA translation, which would explain in part the subsequent increase in protein and RNA content.
Further evidence for the involvement of translation initiation in the feeding-induced increase in the protein and RNA content of the liver is provided by the reported increase in phosphorylation of eukaryotic initiation factor (eIF)3 4E-binding protein 1 (4E-BP1) and ribosomal protein (rp) S6 kinase 1 (S6K1) (4,5), 2 biomarkers of signaling through the mammalian target of rapamycin (mTOR), 1 h after food intake (4,6). Increased phosphorylation of 4E-BP1 and S6K1 in rat liver was also reported to occur in response to oral administration of leucine (7). Phosphorylation of 4E-BP1 on multiple residues promotes its dissociation from the 5'-7-methyl(m7)-GTP-cap binding protein (7,8), eIF4E, allowing eIF4E to associate with eIF4G to form the active eIF4F complex that mediates the binding of mRNA to the 40S ribosomal subunit (9). Thus, in response to food intake, increased 4E-BP1 phosphorylation typically enhances eIF4F complex assembly and thereby stimulates translation initiation (4,5).
Increased phosphorylation and the resulting activation of S6K1 enhance the phosphorylation of rpS6, which is implicated in mediating translational control of mRNAs containing a 5'-terminal oligopyrimidine tract (TOP) sequence (10). The mRNAs encoding most ribosomal proteins as well as several elongation factors contain a 5'-TOP sequence, suggesting that the 5'-TOP sequence permits translational regulation of proteins involved in the translation process itself. Therefore, translation of mRNAs containing a 5'-TOP sequence could contribute to the increase in protein and RNA content of the liver in response to food intake (11). An increase in S6K1 and rpS6 phosphorylation in rat liver parallels the increased polysome association of mRNAs containing a 5'-TOP sequence after oral administration of leucine (12,13). The effect of oral leucine administration is prevented by treatment with rapamycin, demonstrating a dependence on mTOR-signaling for the stimulation of 5'-TOP translation in rat liver (13). However, rapamycin does not alter the global rate of protein synthesis in the liver. Thus, mTOR signaling is activated in response to feeding or leucine administration; however, it is unlikely that activation of the pathway accounts for the stimulation of global rates of protein synthesis (12,13). On the other hand, stimulation of mTOR signaling may play an important role in the feeding-induced increase in RNA content of the liver through activation of ribosome biosynthesis (13).
The overall objective of the present study was to gain insight into the mechanism(s) involved in the regulation of protein synthesis and the translation of selected mRNAs in the liver in response to feeding. To maximize the chance of seeing novel changes, a time course analysis was performed to investigate both rapid and delayed changes in protein synthesis and mRNA translation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Sample preparation.
One portion (
0.5 g) of the liver was weighed and homogenized in 7 volumes of Buffer A consisting of (in mmol/L) 20 HEPES (pH 7.4), 100 KCl, 0.2 EDTA, 2 EGTA, 50 NaF, 50 ß-glycerophosphate, 0.1 phenylmethylsulfonyl fluoride, 1 benzamidine, and 0.5 sodium vanadate using a Polytron homogenizer. An aliquot of the homogenate was used for measurement of protein synthesis, as described below. The remaining homogenate was centrifuged at 10,000 x g for 10 min at 4°C. The resulting supernatant was used for analysis of the phosphorylation status of S6K1, rpS6, eIF4G, and 4E-BP1 as well as the association of eIF4G, 4E-BP1, and 4E-BP2 with eIF4E as described below. A second portion (1 g) of the liver was weighed and homogenized in 3 volumes of Buffer B consisting of (in mmol/L) 40 HEPES (pH 7.5), 100 KCl, and 5 magnesium chloride. The homogenate was centrifuged at 3000 x g for 15 min, and the resulting supernatant was used for analysis of polysome aggregation as described below. A third portion of liver (0.3 g) was homogenized in 5 volumes of Buffer C consisting of (in mmol/L) 45 HEPES (pH 7.4), 0.375 magnesium acetate, 0.075 EDTA, 95 potassium acetate, 2.03 digitonin, 10% (v:v) glycerol, and 0.003 microcystin. The homogenate was centrifuged at 10,000 x g for 10 min at 4°C, and the resulting supernatant was analyzed for eIF2B activity as described below.
Measurement of protein synthesis. The rate of synthesis of total mixed liver protein was measured as previously described (14). Briefly, the fractional rate of protein synthesis was estimated from the rate of incorporation of radioactive phenylalanine into total protein. The fractional rate of protein synthesis is the rate of incorporation of [3H]phenylalanine into protein using the serum phenylalanine specific radioactivity as representative of the precursor pool during the incorporation period, i.e., the time elapsed from injection of [3H]phenylalanine until tissue homogenization (5).
Polysome profiles. One volume of detergent [10% (v:v) Triton X-100, 0.24 mol/L deoxycholate sodium salt] was added to 9 volumes of the supernatant of livers homogenized in Buffer B; an aliquot of the sample was layered over a 10–70% linear sucrose density gradient. The gradients were centrifuged at 90,000 x g for 3 h at 4°C in a Beckman SW28 rotor. After centrifugation, the gradients were fractionated on an Isco gradient fractionator while the UV absorption at 254 nm was recorded continuously. Five fractions (5.0 mL each) and a final 2.5-mL fraction were collected for extraction of total RNA as described below.
Measurement of protein phosphorylation status. Phosphorylation of eIF4G was assessed by Western blot analysis using an anti-phospho-eIF4G (Ser 1108) antibody (Cell Signaling Technology) and normalized to total eIF4G using a polyclonal anti-eIF4G antibody. Phosphorylation of 4E-BP1 was assessed as a change in electrophoretic mobility during SDS-PAGE. The association of eIF4G, 4E-BP1, or 4E-BP2 with eIF4E was evaluated as described previously (15). eIF4E phosphorylation was evaluated using a polyclonal antibody to eIF4E (Ser 209) (Cell Signaling Technology) and normalized to total eIF4E. Hyperphosphorylation of S6K1 was assessed as decreased migration during SDS-PAGE using a polyclonal anti-S6K1 antibody; S6K1 phosphorylation was also analyzed using a polyclonal antibody that recognizes only the Thr 389 phosphorylated form of S6K1 (Cell Signaling Technology). Finally, rpS6 phosphorylation was evaluated using a 1:1 mixture of polyclonal antibodies that recognize either the Ser 235/236 or Ser 240/244 phosphorylated forms of rpS6 (Cell Signaling Technology).
Polysomal RNA extraction, purification, and microarray analysis. Total RNA was isolated from sucrose density gradient fractions by performing 2 phenol:chloroform (5:1) (Ambion) extractions. The 2nd phenol:chloroform extraction was followed by an RNA precipitation step using 0.1 volume 5 mol/L ammonium acetate (Ambion) and 2 volumes 100% ethanol. The precipitate was washed with 100% ethanol and dissolved in RNA Storage Solution (Ambion) containing Anti-RNase (Ambion) to prevent degradation. RNA from each condition was then pooled into 1 of 2 fractions termed subpolysomal, consisting of nonribosome-associated mRNA, 40S and 60S ribosomal subunits, and mRNA associated with a single 80S ribosome, or polysomal, consisting of mRNA associated with multiple ribosomes. Subpolysomal RNA from each condition was pooled with subpolysomal RNA from the same treatment group; polysomal RNA from each condition was pooled with polysomal RNA from the same treatment group. The RNA was further purified using the RNeasy Kit (Qiagen) and eluted in RNase free water (Ambion). DNase I treatment was then performed on the samples using the DNase Free Kit (Ambion). The quality, purity, and concentration of the RNA was assessed using a LabChip analyzed on an Agilent 2100 Bioanalyzer. Microarray analysis was performed in the Juvenile Diabetes Research Foundation Functional Genomics Core Facility at The Pennsylvania State University College of Medicine. Briefly, total RNA (50 µg) from each treatment group was indirectly labeled with CY5; total RNA (50 µg) from a rat reference library (Stratagene) was indirectly labeled with CY3. Then, the labeled cDNA was hybridized to a Rat Liver Array (MWG Biotech). The microarrays were analyzed on a Perkin Elmer ScanArray 4000XL, and the data were analyzed using GeneSpring version 6.0.
Total RNA extraction from liver. Total RNA was extracted from the liver using an RNeasy Kit (Qiagen). The quality and quantity of the RNA was assessed by measuring the A260 and A280 with a Beckman Coulter spectrophotometer.
Quantitative real-time PCR. Quantitative real-time PCR (QRT-PCR) was performed as previously described (16). Briefly, 1 µg total RNA was incubated with 1 µL Oligo (dT) (Invitrogen) for 10 min at 70°C, and the sample was then placed on ice. Next, 5 µL 2X reaction buffer, 2 µL 0.1 mmol/L dithiothreitol (Invitrogen), and 1 µL Superscript Platinum Taq Polymerase were added to the reaction mixture. The samples were then incubated at 42°C for 65 min and stored at –20°C until QRT-PCR was performed. Serial dilutions were performed on the samples to a final dilution of 1:16. QRT-PCR was performed using QuantiTect SYBR Green PCR following the manufacturer’s instructions. The primers used for each mRNA analyzed were as follows: albumin-upper-5'-TGGGCAGTAGCTCGTATGA-3' and lower-5'-CAACAGGTCGCCGTGACA-3'; eEF1A-upper-5'-CTAATATGCCGTGGTTCAAG-3' and lower-5'-CGCAGAGGCTTGTCAGTTG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-upper-5'-GGGCTGCCTTCTCTTGTGA-3' and lower-5'-TGAACTTGCCGTGGGTAGA-3'; Hsp8a-upper-5'-TGTCCTCATCAAGCGCAATA-3' and lower-5'-GGCCCTTTCACCTTCATAC-3'; rpS6-upper-5'-ACTGGCTGTCAGAAACTCAT-3' and lower-5'-CCACATAACCCTTCCACTCT-3'; rpS8 upper-5'-CGTGCTCTGAGATTGGATGT-3' and lower- 5'-CGGACAAGCTCGTTATTGG-3'; rpL26 upper-5'-TCTCACATTCGGAGGAAGAT-3' and lower-5'-TGTCCTCGAACAACCTGAAC-3'.
Statistical analysis. All data were analyzed by InStat 3.0 (GraphPad Software) using an ANOVA multiple comparisons test and further analyzed using Dunnet’s Multiple Comparison Test when the P-value for the ANOVA was <0.05. The level of significance was set at P < 0.05 for all statistical tests. All data are presented as means ± SEM.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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mTOR-mediated signaling was rapidly stimulated and sustained in response to feeding.
Signaling through mTOR is typically assessed by examining the phosphorylation status of various mTOR substrates including S6K1 and 4E-BP1 [reviewed in (7,8)]. S6K1 is phosphorylated on multiple residues and resolves into multiple electrophoretic bands when subjected to SDS-PAGE with the most highly phosphorylated form of the protein, the 
-band, having the slowest migration. S6K1 hyperphosphorylation, analyzed in this manner, was significantly elevated within 15 min and remained elevated through the remainder of the 180-min time course (Fig. 3A). Phosphorylation of S6K1 (Thr 389) results in maximal kinase activity and is catalyzed directly by mTOR (20). Examination of this site after 15 min demonstrated increased phosphorylation that was sustained for 180 min (Fig. 3B). Together, the data suggest that the activity of S6K1 was rapidly increased and sustained throughout the 180-min period. As further evidence of the activation of S6K1, phosphorylation of its substrate, rpS6, was examined. A significant increase in rpS6 phosphorylation was evident by 15 min and was also sustained for 180 min (Fig. 3C), further suggesting that S6K1 activity was rapidly stimulated after commencement of feeding and remained active for 180 min.
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-band, corresponds to the most highly phosphorylated form of the protein and is unable to associate with eIF4E. Examination of 4E-BP1 revealed a significant increase in phosphorylation within 15 min after commencement of feeding, an effect that was sustained throughout the remainder of the 180-min time course (Fig. 3D). Thus, unlike the changes in eIF4E (Ser 209) and eIF4G (Ser 1108) phosphorylation, the feeding-induced stimulation of protein synthesis was delayed relative to the stimulation of mTOR signaling. Dissociation of 4E-BP1 from eIF4E occurred more rapidly than 4E-BP2 dissociation in response to feeding. Because phosphorylation of 4E-BP1 typically results in alterations in the association of eIF4E with its various binding partners, the association of eIF4E with either 4E-BP1, 4E-BP2, or eIF4G was assessed. Hyperphosphorylation of 4E-BP1 typically results in its dissociation from eIF4E, which is one mechanism of augmenting eIF4F complex assembly and stimulating translation initiation. The amount of 4E-BP1 associated with eIF4E was significantly reduced within 15 min of the commencement of feeding compared with food-deprived controls and remained lower throughout the remainder of the time course (Fig. 4A). 4E-BP2, a second eIF4E binding protein, is also expressed in the liver (21), and its dissociation from eIF4E exposes the eIF4G binding site and permits eIF4G binding to eIF4E (22). Unlike the result obtained for 4E-BP1, a shift in mobility of 4E-BP2 on SDS-PAGE was not observed (data not shown). Moreover, no change in the association of 4E-BP2 with eIF4E occurred (Fig. 4B). Dissociation of 4E-BP1 allows eIF4G to bind to eIF4E (22), and therefore, the amount of eIF4G bound to eIF4E was assessed. The eIF4E · eIF4G association increased significantly within 15 min (Fig. 4C). The increased association of eIF4E and eIF4G was maintained for 60 min but then returned to the food-deprived control value by 180 min. Thus, eIF4E · eIF4G association was rapidly stimulated in parallel with increased phosphorylation and dissociation of 4E-BP1; however, unlike the changes in the association of 4E-BP1 with eIF4E, the response was not sustained for 180 min.
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, and Hsp8a, mRNAs whose polysomal contents were elevated, was assessed in both the subpolysomal fraction and the polysomal fraction by QRT-PCR. The ratio of mRNAs encoding rpL26, rpS6, rpS8, eEF1, and Hsp8a in the polysome compared with the subpolysome fraction increased after feeding, whereas GAPDH distribution was not affected (Fig. 7A). As further evidence that the increased abundance of particular mRNAs in the polysomal fraction was not due to increased expression, QRT-PCR analysis of mRNAs was performed on total RNA. The mRNA expression of GAPDH was not affected (Fig. 7B). Further, when normalized to GAPDH expression, rpL26, rpS6, rpS8, eEF1, and Hsp8a mRNA content was not altered (Fig. 7C), confirming the translational regulation of these mRNAs. Thus, after commencement of feeding, a rapid stimulation of 5'-TOP mRNA translation, as indicated by increased polysome association of rpS6, rpS8, rpL26, and eEF1A, and non-5'-TOP mRNA translation, as noted by increased polysome association of Hsp8a mRNA, were observed. Although the importance of increased translation of non-5'-TOP mRNAs remains unclear, the microarray data suggests that a novel mechanism(s) for translational regulation of gene expression may be altered in response to food intake in the liver.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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One of the earliest events in the response to feeding is a stimulation of intracellular signal transduction pathways that subsequently result in phosphorylation of a number of proteins that either mediate or control mRNA translation. Phosphorylation of these biomarkers of mRNA translation leads to an increase in the translation of most mRNAs, although some mRNAs are affected to a greater or lesser extent than others. One rate-controlling process in translation initiation is assembly of the eIF4F complex, which is regulated in part through an mTOR-mediated signaling pathway. Previous studies demonstrated an increase in phosphorylation of 4E-BP1 at 1 time point, i.e., 60 min after feeding (4,6,23). In the present study, phosphorylation of 2 proteins, 4E-BP1 and S6K1, lying downstream of mTOR in the phosphatidylinositol-3 kinase signal transduction pathway was maximally increased within 15 min after the start of feeding and remained elevated through the 180-min time point, suggesting that mTOR-mediated signaling was rapidly stimulated and sustained. Enhanced polysome formation was also observed within 15 min of feeding, but was not maximal until the 60-min time point, suggesting that the stimulation of polysome aggregation was delayed relative to the changes in 4E-BP1 and S6K1 phosphorylation. Increased incorporation of [3H]phenylalanine into total mixed liver proteins was further delayed and was not significant until 60 min after the commencement of feeding. Overall, the results support a scenario in which increased nutrient and hormone signaling in response to feeding induces a sequence of events beginning with activation of the mTOR-mediated signaling pathway and progressing through phosphorylation of biomarkers of mRNA translation (e.g., 4E-BP1, S6K1, and rpS6), increased association of ribosomes with mRNA, and upregulated translation of selected mRNAs. Feeding also induces a global stimulation of protein synthesis, but this response is delayed compared with mTOR-mediated signaling and may be due to activation of other signaling pathways, a suggestion that is consistent with the observation that administration of rapamycin does not inhibit global rates of protein synthesis in the liver (13).
The changes in mTOR-mediated signaling were accompanied by enhanced incorporation of mRNAs encoding a number of selected proteins into polysomes. In particular, a number of mRNAs that contain 5'-TOP sequences exhibited a preferential shift into polysomes after feeding, suggesting that phosphorylation of S6K1 and rpS6 might be involved in the process. Results of previous studies provide support for such a suggestion. For example, inhibition of mTOR signaling in cells in culture using rapamycin decreases both S6K1 activity and rpS6 phosphorylation and reduces the association of 5'-TOP mRNAs with polysomes (24,25). Similarly, a recent study reported that rapamycin prevents the leucine-induced redistribution of ribosomal protein mRNAs into polysomes in rat liver (13), although it does not prevent its shift into monosomes (i.e., mRNA associated with a single ribosome). The latter result agrees with reports suggesting that S6K1 activation and rpS6 phosphorylation may not be sufficient for enhanced translation of 5'-TOP mRNAs (26,27). Despite the uncertainty of the dependence of 5'-TOP mRNA translation on S6K1 activation or rpS6 phosphorylation, it is clear that feeding rapidly promotes an increase in the polysome association of ribosomal protein mRNAs in the liver. Moreover, the enhanced association of ribosomal protein mRNAs with polysomes observed in the present study, in combination with previous studies reporting an mTOR-mediated increase in rDNA transcription (28), provides a likely explanation for the subsequent increase in ribosome content in response to feeding (1,2).
Additionally, the results of the microarray analysis provided insight into the translational upregulation of 78 different mRNAs (Figs. 5and 6) and downregulation of 50 mRNAs (data not shown). Although 42 of the 78 mRNAs did contain or were likely to contain such a sequence, 36 mRNAs were identified that are not known to contain a 5'-TOP sequence. Of the non-5'-TOP mRNAs, Hsp8a was confirmed to shift from the subpolysomal fraction to the polysomal fraction by QRT-PCR. Thus, it is possible that some of the 36 non-5'-TOP mRNAs may be regulated by a novel mechanism(s). Interestingly, albumin mRNA incorporation into polysomes was observed to increase by microarray analysis. Because the rat albumin mRNA lacks a 5'-TOP sequence (accession #M16825), the results suggest that its translation as well as other mRNAs may be regulated through unique mechanisms that have not yet been identified.
As with S6K1 phosphorylation, 4E-BP1 hyperphosphorylation was rapidly stimulated and sustained for an extended period of time. The increase in phosphorylation of 4E-BP1 corresponded to a decrease in 4E-BP1 · eIF4E association. Thus, like S6K1 and rpS6 phosphorylation, the changes in 4E-BP1 paralleled the observed stimulation of 5'-TOP translation. In contrast, the association of 4E-BP2 with eIF4E was not altered at any time up to 180 min after feeding. 4E-BP2 has not been studied extensively although it shares 60% sequence identity with 4E-BP1, and the residues phosphorylated on 4E-BP1 are fairly well conserved (21). The conclusion from previous studies is that 4E-BP1 and 4E-BP2 are regulated in the same manner and serve the same function, i.e., to sequester eIF4E. However, the data presented in the present work and elsewhere (29) suggest that 4E-BP1 and 4E-BP2 are differentially regulated, raising the possibility that they may serve different functions in the regulation of the translational control of protein synthesis. It is interesting to speculate that 4E-BP1 may be involved primarily in the regulation of 5'-TOP translation, whereas 4E-BP2 may have other functions in regulating translation initiation. Future studies using mice lacking either 4E-BP1 or 4E-BP2 could address this issue.
Unlike the increased phosphorylation of mTOR substrates in response to feeding, a decrease in eIF4E (Ser 209) phosphorylation was observed within 60 min and maintained through the remainder of the time course. The result was not entirely unexpected because oral administration of leucine significantly reduces eIF4E (Ser 209) phosphorylation in skeletal muscle, despite a stimulation of protein synthesis and mTOR-mediated signaling (30). However, the results of the present work demonstrate once again an association between a decrease in eIF4E (Ser 209) phosphorylation and an increase in the global rate of total protein synthesis in rat liver, although the functional consequences of decreased eIF4E (Ser 209) phosphorylation are unclear.
eIF4G (Ser 1108) phosphorylation increases in cells in culture after serum stimulation (31) and parallels an increased rate of protein synthesis (32), but the functional consequence of the increase in phosphorylation at Ser1108 or a subsequent residue has not been elucidated. Phosphorylation of eIF4G (Ser1108) corresponded to the aggregation of ribosomes as indicated by the polysome profile analysis, and the observed maximal elevation in phosphorylation at 60 min paralleled an increase in the global rate of protein synthesis. Thus, although little is known about the function of eIF4G (Ser 1108) phosphorylation, the data support a model in which eIF4G (Ser 1108) phosphorylation functions in the regulation of the global rate of total protein synthesis. One possible function of eIF4G (Ser 1108) phosphorylation that has not been explored is that it may increase the affinity of eIF4G for other components of the eIF4F complex such as eIF4A. Another possibility might be to increase the association of eIF4E with eIF4G. However, in the present study eIF4E · eIF4G association decreased by 180 min, a time at which eIF4G (Ser 1108) phosphorylation was still elevated. The basis for the maintenance of protein synthesis at a time when the eIF4G · eIF4E association had returned to the food-deprived control value is unclear; however, it is possible that once mRNA is associated with polysomes, the requirement for the eIF4G · eIF4E complex in maintaining mRNA in polysomes is reduced, resulting in a sustained elevation of protein synthesis. Thus, further study of the functional consequence of eIF4G phosphorylation is required to gain insight into its importance in the regulation of protein synthesis. However, because a definitive role for eIF4G phosphorylation in modulating the function of the protein has not been delineated, the possibility that the changes in eIF4G phosphorylation observed in the present study represent a nonfunctional modification cannot be eliminated.
Another rate-controlling process in translation initiation is the assembly of the 43S preinitiation complex consisting of the eIF2 · GDP · initiator methionyl tRNAi complex bound to the 40S ribosomal complex. The rate-limiting enzyme for this process is eIF2B, which catalyzes the exchange of GDP bound to eIF2 for GTP. The activity of eIF2B is regulated by phosphorylation of eIF2 (Ser 51), which competitively inhibits the guanine nucleotide exchange activity of eIF2B. Previous work demonstrated a correlation between eIF2B activity and changes in the global rate of protein synthesis. Feeding animals a diet devoid of 1 or more essential amino acids stimulated an increase in eIF2 (Ser 51) phosphorylation, a decrease in eIF2B activity, and a decrease in the rate of global protein synthesis (12). However, 1 h after the feeding of a complete meal, Yoshizawa et al. (6) did not observe any significant change in eIF2B activity. The possibility remained that an increase in eIF2B activity occurred during the first 60 min of feeding. For this reason, eIF2B activity was assessed in the present work, but no change in its activity, as measured by an in vitro activity assay, or in eIF2 (Ser51) phosphorylation was observed at any time (data not shown). The results suggest that the changes in protein synthesis and polysome aggregation were not regulated through modulation of eIF2B activity.
Overall, a role for changes in phosphorylation of eIF4E and eIF4G must be considered because the time course for changes in these biomarkers is the same as that observed for alterations in the global rate of protein synthesis. Additionally, the results illustrate a rapid and sustained stimulation of mTOR-mediated signaling that parallels an increase in polysomal association of many ribosomal protein mRNAs, as well as other mRNAs that are not known to possess a 5'-TOP sequence, and emphasize the coordinated response to increase the rate of protein synthesis and ribosome biogenesis. Finally, after feeding, both hepatic protein synthesis and ribosomal protein mRNA translation undergo rapid and sustained stimulation, which is likely mediated by both mTOR-dependent and -independent signaling events.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: 4E-BP, eukaryotic initiation factor 4E binding protein; eIF, eukaryotic initiation factor; eEF, eukaryotic elongation factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Hsp8a, heat shock protein 8a; m7GTP, 7-methyl-GTP; mTOR, mammalian target of rapamycin; QRT-PCR, quantitative real-time PCR; rp, ribosomal protein; S6K1, ribosomal protein S6 kinase 1; TOP, terminal oligopyrimidine tract.
Manuscript received 29 June 2004. Initial review completed 9 August 2004. Revision accepted 22 December 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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