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Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, PA 17033
3To 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: • BCAA • mammalian target of rapamycin • protein synthesis
Amino acids serve an essential function in regulating protein turnover in various tissues including skeletal muscle. Previous reports using in vitro perfused or incubated skeletal muscle preparations with amino acids added to the medium at 5–10 times the normal plasma concentrations showed that the stimulatory effect on protein synthesis is due in part to enhanced mRNA translation (1–3). The exact way in which amino acids regulate mRNA translation and protein synthesis is not known. Furthermore, not all amino acids are required for the stimulation of protein synthesis. Of the amino acids, the BCAA (isoleucine, leucine, and valine) in particular play a regulatory role in stimulating protein synthesis in skeletal muscle (4). Indeed, the magnitude of stimulation of protein synthesis is the same after provision of a mixture of BCAA as that observed with a mixture containing all amino acids (2,3). Among BCAA, leucine appears unique in its ability to augment key events in translation initiation and stimulate protein synthesis (3,5,6). Indeed, omitting leucine from the perfusate while maintaining other plasma amino acid concentrations at 10 times (10X) the concentration found in the plasma of food-deprived rats did not stimulate protein synthesis in skeletal muscle (7).
Leucine stimulates protein synthesis through activation of intracellular signaling pathways that ultimately accelerate mRNA translation initiation. Examining the effect of leucine on mRNA translation is noteworthy given that translation initiation involves several regulatory steps that mediate the more immediate control of protein synthesis and are acutely responsive to metabolic perturbations of amino acid availability. Two of these regulatory steps are represented by the binding of the initiator methionyl-tRNA (met-tRNAi)4 to the 40S ribosomal subunit and the binding of the mRNA to the 43S preinitiation complex. Met-tRNAi binds with eukaryotic initiation factor (eIF)2 and GTP to form a ternary complex that subsequently binds to the 40S ribosomal subunit. Next, the GTP bound to eIF2 is hydrolyzed to GDP, and eIF2 is released from the ribosomal subunit with GDP. eIF2 must then exchange GDP for GTP to participate in a subsequent round of initiation and form a new ternary complex. eIF2B, a second translation initiation factor, controls guanine nucleotide exchange on eIF2, and inhibition of eIF2B activity reduces the amount of eIF2 · GTP available for ternary complex formation. Although this regulatory step is affected by severe amino acid limitation, no significant differences in eIF2B activity were observed when the perfusate amino acid concentration was either 1X or 10X amino acids in a perfused gastrocnemius preparation (7) or when rats were administered leucine orally (5). Consequently, enhanced eIF2B activity does not appear to be responsible for the leucine-induced stimulation of mRNA translation initiation.
Another key regulatory step in translation initiation involves the reversible binding of the eukaryotic initiation factor eIF4E · mRNA complex to eIF4G, a protein that mediates association of the eIF4E · mRNA complex with the 40S ribosomal subunit to allow translation of mRNA. Stimulation of protein synthesis by leucine is associated with an enhanced eIF4E to eIF4G assembly in skeletal muscle (5,7). eIF4E to eIF4G complex assembly is regulated in part by the association of eIF4E with a protein termed eIF4E binding protein-1 (4E-BP1). Sequestering of eIF4E by 4E-BP1 is controlled by phosphorylation of 4E-BP1 whereby hyperphosphorylation of 4E-BP1 prevents it from binding to eIF4E. Phosphorylation of 4E-BP1 is under control of a protein kinase referred to as the mammalian target of rapamycin (mTOR), which serves as a convergence point for signaling by growth factors and amino acids to the mRNA binding step of translation initiation.
In addition to regulation by phosphorylation of 4E-BP1, several studies (7–9) reported alterations in eIF4G · eIF4E association independent of changes in 4E-BP1 binding to eIF4E. Thus, another potential mechanism for enhanced eIF4E to eIF4G assembly involves phosphorylation of eIF4G (10,11). Increased phosphorylation of eIF4G correlates with conditions known to stimulate protein synthesis (11). Similarly, decreased eIF4G phosphorylation in the presence of mTOR activation is observed in skeletal muscle of septic rats, a condition that manifests resistance to stimulation of protein synthesis by insulin (2,12).
Previous investigations established that the leucine-dependent stimulation of mRNA translation is in part, rapamycin insensitive (6). This is supported by the fact that increases in skeletal muscle protein synthesis and eIF4E · eIF4G assembly are observed with leucine administration in the presence of rapamycin (a specific inhibitor of mTOR) (6). These earlier observations suggest that increases in skeletal muscle protein synthesis and eIF4E · eIF4G assembly with leucine can be regulated independently of mTOR-mediated signaling. Thus, closer examination of eIF4F complex formation with enhanced leucine availability may serve to identify alternate signaling pathways.
The cellular mechanisms by which amino acids modulate protein synthesis are beginning to be elucidated. Amino acids, and leucine in particular, consistently activate the 70-kDa ribosomal protein S6 kinase (S6K1) and the translation repressor 4E-BP1 using in vitro (13–15) and in vivo models (6,16–18). In contrast, amino acids do not stimulate PI3-kinase or protein kinase B (PKB) (Akt) signaling pathways activated by insulin and growth factors (15,19,20). Consequently, amino acids appear to activate mTOR signaling via a PKB-independent pathway.
The present investigation was designed to evaluate the extent to which signaling through mTOR is required for stimulation of mRNA translation and protein synthesis when leucine is administered directly to the skeletal muscle of postabsorptive rats. In this metabolic context, basal mTOR signaling is maintained, and the model system allows for partitioning the effects of increased leucine vs. circulating hormones (i.e., insulin) on modulating distinct events of translation initiation. By understanding the biochemical loci affected, we hypothesized that leucine alone would dramatically change key regulatory steps of translation initiation as well as upregulate yet undefined signaling pathways that can enhance translation initiation.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1000 h. Leucine was present at either 1X (i.e., approximately the concentration found in the plasma of a food-deprived rat) or 10 times (10X) the plasma concentration. 1X leucine concentration served as the control group for the experiments; 10X leucine was selected because previous studies showed it to be a maximally effective concentration for stimulation of skeletal muscle protein synthesis (3). All other amino acids were present at 1X concentration in the perfusate. Hindlimb perfusions. Hindlimb perfusions were carried out according to previously described methods (7,21). In some experiments, perfusions were executed with a specific inhibitor of PI3-kinase, LY 294002 (100 µmol/L) (Cayman Chemical). The inhibitor was present throughout the perfusion period. This concentration of LY 294002 was shown previously to inhibit insulin-stimulated protein synthesis in skeletal muscle (22).
Measurement of mixed muscle protein synthesis. The rate of protein synthesis was estimated in gastrocnemius and soleus by the incorporation of [3H]phenylalanine into mixed muscle proteins as described previously (2,23). Soleus muscles were used only for protein synthesis determination because tissue size precluded additional analysis of initiation factors.
Analysis of eukaryotic initiation factors (eIF). Gastrocnemius muscle was weighed and homogenized in 7 volumes of buffer containing 20 mmol/L HEPES (pH 7.4), 100 mmol/L potassium chloride, 0.2 mmol/L EDTA, 2 mmol/L EGTA, 50 mmol/L sodium fluoride, 50 mmol/L ß-glycerophosphate, 0.1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 1 mmol/L benzamidine, 1 mmol/L dithiothreitol (DTT), and 0.5 mmol/L sodium vanadate. The homogenate was centrifuged at 10,000 x g for 10 min at 4°C. The resulting supernatant was combined with an equal volume of SDS sample buffer and then subjected to protein immunoblot analysis as described previously (23). Samples were analyzed for the phosphorylation status of 4E-BP1, S6K1, eIF4G on Ser-1108 (Cell Signaling Technology), or mTOR on Ser-2448 (Cell Signaling Technology). Ser-2448 of mTOR is a site directly phosphorylated by PKB (24). Total mTOR, PKB, and eIF4G were measured by Western blot analysis using antibodies that recognize both the phosphorylated and unphosphorylated proteins. No change in the total mTOR, PKB, or eIF4G content was observed under any of the experimental conditions.
Quantification of eIF4G · eIF4E complex. For quantitation of the amount of eIF4G present in the eIF4G · eIF4E complex, eIF4E was immunoprecipitated from 10,000 x g supernatants using a monoclonal antibody. Samples were subjected to immunoblot analysis using a polyclonal antibody to eIF4G to assess the association of eIF4G with eIF4E (23). Results were normalized to the amount of eIF4E in the immunoprecipitate.
Determination of phosphorylation status of 4E-BP1. An aliquot of the 10,000 x g supernatant was boiled for 10 min and then centrifuged at 10,000 x g for 30 min at 4°C. The resulting supernatant was mixed with an equal volume of 2X sample buffer and then subjected to protein immunoblot analysis as described previously (23).
Immunoprecipitation of mTOR. Gastrocnemius muscle was homogenized on ice in homogenization buffer, which contained 2 mmol/L EGTA, 100 mmol/L NaCl, 50 mmol/L Tris · HCl (pH 7.4), 2 mmol/L ß-mercaptoethanol, 1 mmol/L PMSF, 10 mg/L leupeptin, 10 mg/L aprotinin, 1 mg/L microcystin, 5 mg/L pepstatin, 10% glycerol (v:v), and 0.1% Tween-20 (v:v). The homogenate was centrifuged at 1000 x g for 3 min at 4°C. All tissue samples were processed fresh and not previously frozen. Protein concentrations were determined using a detergent compatible protein assay kit (Bio-Rad).
mTOR was affinity purified for the immunoprecipitation kinase assays using an anti-peptide antibody (Bethyl Laboratories). Antibody was attached to protein A agarose beads (Sigma) as follows. First, 4 mg of beads/sample were hydrated in homogenizing buffer. Then beads were washed 2 times by gentle microcentrifugation for 1 min followed by resuspension in homogenizing buffer. Antibody (20 µL) was allowed to bind to the beads [10 µL in homogenization buffer (HB)] by rocking at 4°C overnight. The unbound protein was removed from the beads by 2 washes with Tris-buffered saline (TBS; 100 mmol/L NaCl and 50 mmol/L Tris · HCl, pH 7.4). Beads were then incubated with muscle supernatant containing 500 µg protein/sample and HB (normalized to 500 µL/sample) at 4°C for 2 h. Subsequently, beads were washed sequentially by centrifugation followed by resuspension in buffer A [100 mmol/L NaCl, 100 µmol/L zinc acetate, 50 mmol/L Tris · HCl, 1 mmol/L DTT, 10% glycerol (v:v), and 0.1% Tween-20 (v:v; pH 7.4)], buffer B (50 mmol/L NaCl, 100 µmol/L zinc acetate, 1 mmol/L DTT, and 10 mmol/L HEPES, pH 7.4), and ultimately, MnCl2 kinase assay buffer (55.5 mmol/L NaCl, 100 µmol/L zinc acetate, 1 mmol/L vanadate, 1.1 mmol/L DTT, 11.1 mmol/L HEPES, 55.5 mmol/L ß-glycerophosphate, 222.2 nmol/L microcystin LR, and 11.1 mmol/L MnCl2). After the final washing, beads were resuspended in kinase assay buffer (10 µL MnCl2 kinase assay buffer plus 200 µmol/L zinc acetate).
mTOR protein kinase assay.
The protein kinase activity of mTOR was determined by measuring the incorporation of 32Pi from [
-32P]ATP into 4E-BP1 with the use of mTOR immunoprecipitates. Kinase assay buffer (10 µL) was added to microcentrifuge tubes containing mTOR immunoprecipitates. Tubes were then incubated for 15 min on ice before the kinase assays were initiated by the addition of 5 µL each of recombinant 4E-BP1 (0.75 µg, Calbiochem) and [-32P]ATP (100 µmol/L, 9.25 kBq/µL) dissolved in the same kinase assay buffer. The tubes were then placed in a rotator (ATR), and the reaction mixture was incubated at 30°C with constant mixing. The kinase reactions were stopped after 10 min by placing the tubes in ice, then adding 20 µL of SDS-PAGE sample buffer and heating at 100°C for 5 min. Tubes were then centrifuged for 3–4 s at maximum speed in a microcentrifuge to pellet the agarose beads. Proteins in a 40-µL aliquot of the sample buffer were then resolved by electrophoresis on a 15% SDS-polyacrylamide gel. Gels were stained with Coomassie blue and dried. Radioactivity incorporated into 4E-BP1 was determined via autoradiography and then quantitated by densitometry using NIH Imaging software.
Determination of phosphorylation status of eIF4G in eIF4E immunoprecipitate. eIF4E was immunoprecipitated as described above from 10,000 x g supernatants using a monoclonal antibody. Samples were subjected to immunoblot analysis using a polyclonal antibody to eIF4G on Ser-1108. Results were normalized to the amount of eIF4G in the immunoprecipitate.
Statistical analysis.
Data are presented as means ± SEM. Results were compared using a two-tailed, two-sample (unequal variance) Student’s t test to assess differences between treatment groups. Differences were considered significant at an 
level of P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, ß, and ) with the -form representing the highest phosphorylated form. Approximately 25% of the total 4E-BP1 was present in the -form in muscle perfused with 1X leucine (Fig. 3A). This value is greater than previously reported values exhibited in food-deprived rats (6,25,26) and is most likely a consequence of the postabsorptive state of rats in the present investigation. Consequently, the extent of phosphorylation of 4E-BP1 did not change between 1X and 10X conditions (Fig. 3A).
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50% under both conditions. Again, this is most likely a consequence of the postabsorptive state of the rats in the present investigation. Furthermore, as confirmation of the lack of change with S6K1, site-specific phosphorylation of S6K1 on Thr-389 (a phosphorylation site necessary for full activation of the protein) was examined as well. No bands were observed with this antibody with either 1X or 10X leucine (data not shown).
Assembly of the active eIF4E · eIF4G complex was examined to determine the extent to which the complex assembly correlated with the stimulatory effect of leucine on rates of protein synthesis. Previous reports provided evidence that a strong correlation exists between rates of protein synthesis and assembly of the eIF4E · eIF4G complex in gastrocnemius with enhanced leucine availability (7). In the present investigation, eIF4E · eIF4G association was markedly increased (80%) solely by increasing leucine to 10X concentrations (Fig. 4A).
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70% above controls) on Ser-1108 (Fig. 4C). |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Previous experiments demonstrated that increasing the perfusate amino acid concentration from 1X to 10X enhances protein synthesis, translation efficiency, and assembly of the active eIF4E · eIF4G complex (2,3,7). Alternatively, these particular studies suggest that 4E-BP1 and S6K1 remain refractory to increasing leucine from 1X to 10X concentration utilizing the perfusion technique in postabsorptive rats (7). These experiments also revealed that when leucine was omitted from the perfusate, a 10X mixture of amino acids did not stimulate either protein synthesis or the binding of eIF4E to eIF4G (7). Collectively, the previous results underscore the importance of leucine in specifically regulating eIF4E · eIF4G assembly in skeletal muscle.
In earlier work with the perfusion model used in the present experiments, total tissue water was measured to account for potential changes in protein synthesis due to the increased cell volume (7). Cell swelling secondary to elevated amino acids was postulated to promote anabolism (32,33). It was determined that increasing amino acids from 1X to 10X plasma concentrations did not significantly alter the total tissue water content in gastrocnemius. Therefore, it is unlikely that the observed increase in protein synthesis in the present investigation resulted from changes in tissue water content, given the similar perfusion conditions.
We reported previously that leucine-dependent stimulation of mRNA translation initiation in vivo occurs in part through a rapamycin-insensitive pathway (6). When mTOR was inhibited through rapamycin treatment (an inhibitor of the protein kinase mTOR), the association of eIF4E with eIF4G was reduced as well as 4E-BP1 and S6K1 phosphorylation. However, despite inhibition of mTOR, leucine administration stimulated rates of skeletal muscle protein synthesis and eIF4E · eIF4G assembly without affecting 4E-BP1 phosphorylation (6). Thus, mTOR-mediated signaling could not completely account for the leucine-dependent stimulation of protein synthesis or eIF4F assembly. In other words, tonic input from mTOR signaling is required for leucine’s effect on protein synthesis, but mTOR is not the sole regulator of this response. An additional study examining oral leucine administration in diabetic rats demonstrated enhanced rates of protein synthesis in skeletal muscle without corresponding changes in the association of eIF4E · eIF4G or 4E-BP1 and S6K1 phosphorylation (25). Similarly, we demonstrated in the present study that rates of skeletal muscle protein synthesis can be enhanced in the absence of phosphorylation changes with 4E-BP1 and S6K1.
Further evidence suggesting that leucine may signal through alternate proteins or in conjunction with a parallel pathway includes the present findings with LY 294002 (specific PI3-kinase inhibitor), which did not inhibit the stimulation of protein synthesis via leucine. This directly contrasts with insulin- or insulin-like growth factor-1–mediated stimulation of protein synthesis whereby PI3-kinase input is indispensable (22). Moreover, PKB signaling remained unchanged with leucine stimulation in this study and agrees with previous findings (15,19). Overall, the evidence highlights the way in which leucine modulates translation initiation and protein synthesis through mechanisms that appear independent of mTOR signaling.
Nonetheless, an obvious question that arises with the present investigation is why leucine did not activate 4E-BP1 and S6K1, given that this had been reported to occur in vivo (5) and in cell culture models (14,34). One possible answer may be the lack of sustained insulin concentrations in the muscle perfusate because insulin likely provides permissive input for mTOR-mediated signaling. Oral administration of leucine in vivo results in transient increases in circulating insulin, but blocking this response with somatostatin prevents the leucine-induced hyperphosphorylation of 4E-BP1 and S6K1; yet eIF4E · eIF4G association is augmented (26). The results provided herein suggest that residual insulin signaling is maintained in postabsorptive rats providing basal phosphorylation of 4E-BP1 and S6K1, yet is not sufficient to induce hyperphosphorylation regardless of leucine availability. In contrast, eIF4F assembly may be uniquely regulated by leucine while requiring only basal insulin signaling. Thus, insulin or leucine alone may preferentially enhance select components of translation initiation, but the combined effect of leucine and insulin provides an integrated response.
eIF4F is considered the essential factor in selecting mRNAs for translation. Once eIF4E is released from 4E-BP1, it is then accessible to combine with eIF4G and eIF4A to form the heterotrimeric complex, eIF4F. eIF4G is a large polypeptide that serves as a scaffold for eIF4E, eIF4A, the mRNA, and the ribosome. eIF4G is intimately involved with regulating the translation apparatus during the initiation phase of mRNA translation. eIF4G, in addition to other translation functions, recruits the 40S ribosomal subunit to the 5' end of mRNA, coordinates the circularization of mRNA through eIF4E and poly(A)-binding protein interactions (35), and assists in mitogen-activated protein kinase signal-integrating kinase-1 and eIF4E association (36,37).
Enhanced eIF4F complex assembly is generally thought to occur through increased eIF4E availability subsequent to 4E-BP1 phosphorylation. However, several conditions exist (7–9) whereby changes in eIF4E · eIF4G association do not completely parallel 4E-BP1 phosphorylation alterations, suggesting that eIF4E availability is not coordinated solely by 4E-BP1. Nonetheless, previous findings in vivo demonstrated hyperphosphorylation of 4E-BP1 and corresponding increases in eIF4E · eIF4G association when starved rats are refed a nutritionally complete diet (16,38). Subsequent studies utilizing leucine alone confirmed the stimulatory effect on protein synthesis and eIF4F complex assembly (5).
In the present study, examining the regulation of eIF4G phosphorylation served as a targeted approach for exploring divergent signaling pathways induced by leucine and its subsequent effect on biomarkers of mRNA translation initiation. One potential mechanism for enhanced binding of eIF4E · eIF4G may involve phosphorylation of eIF4G (10,11). Increased phosphorylation of eIF4G correlates with conditions known to stimulate protein synthesis (11,39). In the present set of experiments, phosphorylation of eIF4G was significantly augmented by perfusion of the hindlimb with buffer containing elevated leucine concentrations. Leucine enhanced phosphorylation of eIF4G even though there was no appreciable hyperphosphorylation of S6K1 or 4E-BP1, suggesting that leucine may stimulate protein synthesis independently of S6K1 and 4E-BP1 activation. Furthermore, this study represents the first investigation to demonstrate increased eIF4G phosphorylation on Ser-1108 in skeletal muscle, specifically within the eIF4E immunoprecipitate. This finding provides evidence that the enhanced eIF4E · eIF4G assembly noted with increased leucine may be modulated through increased eIF4G phosphorylation directly in the eIF4E immunoprecipitated complex. Consequently, eIF4G phosphorylation may serve to recruit existing eIF4E not sequestered by 4E-BP1 and promote eIF4E · eIF4G association with elevated leucine.
Raught et al. (11) identified 3 distinct serum-stimulated phosphorylation sites (Ser 1108, 1148, and 1192) in the C-terminal one third of eIF4G. These phosphorylation sites were sensitive to specific inhibitors of PI3-kinase and mTOR signaling. On the other hand, the N-terminal truncation mutants used in that study were shown to be rapamycin- and PI3-kinase–insensitive. Thus, eIF4G phosphorylation on Ser-1108 may likely be regulated through both mTOR-dependent and -independent mechanisms. Consequently, phosphorylation of the N-terminal region on eIF4G through an mTOR-dependent signal could induce conformational changes to expose the C-terminal residues. These C-terminal residues would then be phosphorylated through an mTOR-independent mechanism, allowing for complete activation of eIF4G.
The signal transduction pathway responsible for stimulation of eIF4G phosphorylation is not known. In the present study, we observed an increase in phosphorylation of mTOR (Ser-2448). Although phosphorylation of Ser-2448 on mTOR was significantly increased with 10X leucine, previous work showed that mutation of this phosphorylation site to a nonphosphorylatable alanine residue does not prevent the insulin or PKB stimulation of S6K1 and 4E-BP1 phosphorylation in rapamycin-treated human embryonic kidney 293 cells (40). Additionally, amino acids can enhance Ser-2448 phosphorylation on mTOR without corresponding activation of PKB (41). Thus, the extent to which this site-specific phosphorylation affects mTOR activity is unclear, and this event may simply reflect a response to upstream kinases functioning in a PKB-independent fashion.
The results presented herein provide new insights into how leucine alone regulates key steps of translation initiation and protein synthesis directly in skeletal muscle without direct input from other regulatory stimuli. Furthermore, this model may provide a context wherein eIF4G phosphorylation and protein synthesis are enhanced independently of fiber type; this regulation may involve input from both mTOR-dependent and mTOR-independent signaling. Although the effects of eIF4G phosphorylation on modulating translation initiation are not presently understood, our results strongly support phosphorylation on Ser-1108 as an essential event associated with enhanced eIF4E to eIF4G association and eventual active complex assembly. Future investigations will focus on identifying alternate signaling pathways that may contribute to leucine-mediated, S6K1 and 4E-BP1-independent stimulation of skeletal muscle protein synthesis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported in part by National Institutes of Health grant DK15658 (L.S.J.) and grant GM 39722 (T.C.V.). D.R.B. was supported by a Mentor Based Postdoctoral Fellowship grant from the American Diabetes Association.
4 Abbreviations used: DTT, dithiothreitol; eIF, eukaryotic initiation factor; 4E-BP1, eIF4E binding protein-1; HB, homogenization buffer; met-tRNAi, initiator methionyl-tRNA; mTOR, mammalian target of rapamycin; PKB, protein kinase B; S6K1, ribosomal protein S6 kinase-1.
Manuscript received 10 March 2004. Initial review completed 6 April 2004. Revision accepted 30 April 2004.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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