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Biochemical, Molecular, and Genetic Mechanisms

Rapamycin Limits Formation of Active Eukaryotic Initiation Factor 4F Complex Following Meal Feeding in Rat Hearts^1,2

Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033

^* To whom correspondence should be addressed. E-mail: tvary{at}psu.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
Feeding promotes protein synthesis in cardiac muscle through a stimulation of the messenger RNA translation initiation phase of protein synthesis by enhancing assembly of active eukaryotic initiation factor (eIF)4F complex. The experiments reported herein examined the potential role for a rapamycin-sensitive signaling pathway in increasing formation of active eIF4G-eIF4E complex during meal feeding. Hearts from male Sprague-Dawley rats fed a meal consisting of rat nonpurified diet were sampled prior to and 3 h following the meal in the presence or absence of treatment with rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) complex 1. Rapamycin prevented the meal feeding-induced stimulation of myocardial protein synthesis. Inhibition of mTOR with rapamycin decreased the association of rapamycin-associated TOR protein with mTOR and prevented the feeding-induced assembly of eIF4G-eIF4E complex. In contrast, the abundance of eIF4E binding protein-1 (4E-BP1)-eIF4E complex was unaffected by either meal feeding or rapamycin. Pretreatment with rapamycin completely prevented the feeding-induced phosphorylation of eIF4G(Ser¹¹⁰⁸), whereas the inhibitor only partially attenuated meal feeding-induced 70-kDa ribosomal protein S6 kinase1(Thr³⁸⁹) phosphorylation and extent of 4E-BP1 in the -form. Meal feeding-induced phosphorylation of protein kinase B on either Ser⁴⁷³ or Thr³⁰⁸ was unaffected by rapamycin. These findings suggest the extent of phosphorylation of eIF4G following meal feeding occurs by a rapamycin-sensitive mechanism in cardiac muscle. Furthermore, the rapamycin-sensitive reductions in phosphorylation of eIF4G may also lead to decreased formation of active eIF4G-eIF4E complex.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

Meal feeding presumably enhances mRNA translation initiation through an upsurge in the assembly of the active eukaryotic initiation factor (eIF)4G-eIF4E complex (1). Formation of the active eIF4E-eIF4G complex regulates mRNA translation initiation in cardiac muscle under a variety of conditions, including meal feeding(1–3). Enhanced assembly of this complex following meal feeding occurred through 2 mechanisms: an increased availability of eIF4E secondary to reduced abundance of the inactive eIF4E- eIF4E binding protein-1 (4E-BP1) complex and an augmented phosphorylation of eIF4G.

Phosphorylation of eIF4G on residues in the C-terminal region of the eIF4G protein including Ser¹¹⁰⁸ results in a fully active eIF4G (4). Increased phosphorylation of eIF4G on Ser¹¹⁰⁸ is associated with enhanced formation of active eIF4G-eIF4E complex in cells in culture (4,5), leading to an increased rate of protein synthesis. Likewise, elevated phosphorylation of eIF4G correlates with increased formation of active eIF4G-eIF4E and rates of mRNA translation following meal feeding (1) or infusion of Leu (6) in cardiac muscle.

Increased formation of active eIF4G-eIF4E complex may also rely on the availability of eIF4E. eIF4E availability is dependent in part on the translation repressor protein 4E-BP1. 4E-BP1 is a small molecular weight protein that, in the hypophosphorylated state, tightly binds eIF4E in an inactive eIF4E-4E-BP1 complex and blocks the ability of eIF4E to bind to eIF4G, thereby limiting cap-dependent translation. 4E-BP1 phosphorylation correlates with an increase translational stimulation.

Presently, there is little information regarding the modulation of regulatory mechanisms of mRNA translation in the heart during meal feeding. The mammalian target of rapamycin (mTOR) is a protein kinase that integrates nutritional and mitogenic signals to regulate mRNA translation in the heart. mTOR is a Ser/Thr kinase that phosphorylates 4E-BP1 (7–9), suggesting a role for mTOR (10) in mediating in part the effects of meal feeding to increase release of eIF4E from inactive eIF4E-4E-BP1 complex (1,6). mTOR exists in 2 complexes: mTOR complex1 (TORC1) and TORC2. TORC1 is sensitive to inhibition by rapamycin, a bacterial macrolide. Rapamycin inhibits mTOR through a gain-of-function mechanism in which it binds to the intracellular protein FK506 binding protein 12 to generate a drug-receptor complex that then binds to mTOR, thereby limiting its kinase activity. Because rapamycin can bind to free mTOR, TORC2 can also be inhibited by rapamycin, but the inhibition is dependent upon the cell line used and rapamycin sensitivity as new mTOR becomes synthesized or mTOR complexes turn over (11). By inhibiting the activity of TORC1, rapamycin either abolishes or severely inhibits the ability of cells to progress through the G1 phase of the cell cycle (12,13). As such, rapamycin remains a useful tool to probe the role of TORC1 in modulating signaling pathways. We studied the role of mTOR in mediating the upshot on effector molecules controlling translation initiation following meal feeding through inhibiting mTOR's activity with rapamycin.

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Meal feeding. For the meal-feeding experiments, male Sprague-Dawley rats (Charles River Laboratories) were caged in pairs and adapted to a reverse light cycle (the dark cycle began at 0700 and the light cycle began at 1900) as described previously (1,14,15). Rats were trained over a period of at least 12 d to be fed a meal when presented. Food (Teklad Rodent Diet no. 8604) was provided in 2 ceramic food cups for 3 h beginning 30 min after the beginning of the dark cycle (1,14,15). The concentration of Leu in Teklad Rodent Diet 8604 was 2.04% and overall protein content was 24.48%. Rats consumed water ad libitum. Experiments were approved by the Institutional Animal Care and Use Committee of Pennsylvania State University College of Medicine and adhered to NIH guidelines for the use of experimental animals.

After the training, rats were divided into 3 groups: unfed, fed, and fed + rapamycin. Rats received an intraperitoneal injection of either vehicle (unfed and fed groups) (0.75 mL/kg body weight) or rapamycin (fed + rapamycin) (1 g/L mixed in ethanol; 0.75 mL/kg body weight) at the beginning of the dark cycle 15 min before the usual presentation of the meal. The unfed group received no food in the morning prior to sampling the heart. The effects of meal feeding and Leu gavage have on translation initiation regulatory proteins are curtailed with the dose of rapamycin chosen (16,17). We previously reported that acute rapamycin administration reduces (15 g/d) food intake compared with rats consuming ad libitum (16). Therefore, the fed rats were pair-fed with the fed + rapamycin group. The heart was sampled 3 h after the presentation of the meal.

    Rates of protein synthesis. Rates of myocardial protein synthesis were determined in vivo using the flooding-dose technique, as originally described by Garlick et al. (18), and modified in our laboratory (19–23). Rats in each of 3 groups were injected with L-[2,3,4,5,6-³H]Phe (150 mmol/L, 30 mCi/L; 1 mL/100 g body weight) via the jugular vein. Samples (1 mL) were withdrawn via syringe from the carotid artery 2, 6, and 10 min after injection of radioisotope for measurement of the specific radioactivity in the plasma via HPLC as described in our laboratory (24). After the 10-min blood sample, the heart was excised and weighed. Cardiac muscle lysates were prepared as described below from the myocardium that was frozen between blocks precooled to the temperature of liquid nitrogen for measurement of incorporation of radioactive Phe into muscle proteins. Rates of protein synthesis were measured as the incorporation of [³H]Phe into protein as described previously (19–23).

    Preparation of cardiac muscle extracts. Frozen powdered tissue was homogenized in 7 volumes of buffer A (20 mmol/L HEPES, pH 7.4, 100 mmol/L potassium chloride, 0.2 mmol/L EDTA, 2 mmol/L EGTA, 1 mmol/L dithiothreitol, 50 mmol/L sodium fluoride, 50 mmol/L ß-glycerolphosphate, 0.1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L benzamidine, 0.5 mmol/L sodium vanadate, and 1 µmol/L microcystin LR) using a Polytron PT 10 homogenizer set at 60% of maximum power. The homogenate was centrifuged at 10,000 x g; 10 min at 4°C and the pellet was discarded. An aliquot of the 10,000 x g supernatant was mixed with an equal volume of 2x Laemmli SDS sample buffer (65°C) and then subjected to protein immunoblot analysis. Another aliquot was used to measure the protein concentration by the Biuret method with crystalline bovine serum albumin serving as a standard. A third aliquot was used for immunoprecipitation for measurement of the association of eIF4E with 4E-BP1 or eIF4G. For immunoprecipitation of rapamycin-associated TOR protein (RAPTOR), frozen powdered heart was homogenized in 7 volumes of buffer B (40 mmol/L HEPES, pH 7.5, 0.3% {3-[(3-cholamidopropyl)dimethylammonia]-1-propanesulfonate}, 120 mmol/L sodium chloride, 1 mmol/L EDTA, 50 mmol/L sodium fluoride, 10 mmol/L ß-glycerolphosphate, 10 mmol/L sodium pyrophosphate, 1.5 mmol/L sodium vanadate, and 1 tablet Roche Pharmaceuticals Complete EDTA-free Protease Inhibitor Cocktail/50 mL) using a Polytron PT 10 homogenizer set at 60% of maximum power. The homogenate was centrifuged at 1000 x g; 3 min at 4°C and the pellet was discarded.

    Determination of phosphorylation state of eIF4G, mTOR, 4E-BP1, 70-kDa ribosomal protein S6b kinase1, and protein kinase B. The relative extent of phosphorylation of eIF4G, mTOR, 4E-BP1, 70-kDa ribosomal protein S6 kinase1 (S6K1), and protein kinase B (PKB) proteins in the cardiac muscle extracts was measured using immunoblot techniques as previously described following electrophoresis using either a 5% Criterion gel (Bio-Rad Laboratories) for eIF4G and mTOR or 12.5% SDS-polyacrylamide gel electrophoresis Criterion gel for 4E-BP1, S6K1, and PKB proteins (Bio-Rad Laboratories) (1,25–27). The phosphorylated eIF4G, mTOR, 4E-BP1, S6K1, and PKB signal densities were normalized to the respective total eIF4G signal to reflect the relative ratio of phosphorylated eIF4G, mTOR, 4E-BP1, S6K1, and PKB to total eIF4G, mTOR, 4E-BP1, S6K1, and PKB, respectively.

    Quantification of eIF4G-eIF4E and eIF4E-4E-BP1 complexes. The association of eIF4G with eIF4E and eIF4E with 4E-BP1 was determined following immunoprecipitation with eIF4E (gift of Dr. Leonard Jefferson, Penn State University College of Medicine, Hershey, PA) in cardiac muscle using immunoblot techniques as previously described in our laboratory (1,25–27). The abundance of 4E-BP1 or eIF4G was normalized to the amount of eIF4E in the immunoprecipitate.

    Determination of RAPTOR associated with mTOR. The association of RAPTOR with mTOR in cardiac muscle was determined by immunoprecipitating RAPTOR from aliquots of 1000 x g homogenate supernatants using an anti-human RAPTOR antibody (Bethyl Laboratories). RAPTOR and mTOR proteins in the antibody-antigen complex were subjected to electrophoresis using a 5% Criterion gel (Bio-Rad Laboratories) as described above for mTOR (Determination of phosphorylation state of eIF4G mTOR, 4E-BP1, S6K1, and PKB). Proteins were then electrophoretically transferred to a polyvinylidene fluoride membrane (Biotrace, PALL). The polyvinylidene fluoride membranes were incubated with a rabbit anti-human RAPTOR antibody or a rabbit anti-rat mTOR antibody (Bethyl Laboratories). The blots were quantitated as described above. The abundance of mTOR was normalized to the amount of RAPTOR in the immunoprecipitate.

    Statistical analysis. Values are presented as means ± SEM of multiple densitometric analyses for each group. Data were analyzed using InStat statistical software package (GraphPad Software) by ANOVA analysis. Standard parametric (ANOVA) or Kruskal-Wallis nonparametric ANOVA tests were used to test for overall differences depending upon assessment of data sets by the InStat program. When ANOVA indicated a significant overall effect, differences among individual means were assessed using the post hoc Sidak test for multiple comparisons. Differences were considered significant at P < 0.05.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Rapamycin prevents the meal feeding-induced stimulation of myocardial protein synthesis. Meal feeding caused a 60% increase in protein synthesis (Table 1). Pretreatment with rapamycin prevented the meal-feeding-induced stimulation of protein synthesis.

    Phosphorylation of 4E-BP1. 4E-BP1 when phosphorylated resolves into distinct electrophoretic forms (, ß, and ), with the -form representing the highest phosphorylated form (31). Meal feeding induced a pronounced retardation in the mobility of 4E-BP1 on SDS-polyacrylamide gel electrophoresis, indicative of increased phosphorylation of the protein, such that 54% of the protein now migrated as the most highly phosphorylated -form (Table 3) compared with only 21% in hearts from unfed rats. Pretreatment of rats with rapamycin reduced the extent of 4E-BP1 in -form following meal feeding from 54 to 38%. The extent of 4E-BP1 in -form following rapamycin and meal feeding remained elevated (80%) compared with unfed controls.

    Association of eIF4E with 4E-BP1. The changes in phosphorylation of 4E-BP1 with meal feeding would be expected to modify the association of eIF4E with 4E-BP1. The association of 4E-BP1 with eIF4E did not differ in any of the conditions examined (Table 4).

    Phosphorylation of eIF4G. Having established that rapamycin prevents the meal feeding-induced association of eIF4G with eIF4E, we next investigated the effect of the inhibitor on eIF4G(Ser¹¹⁰⁸) phosphorylation following meal feeding (Table 4). Meal feeding increased eIF4G(Ser¹¹⁰⁸) phosphorylation 6.2-fold, an effect completely prevented by pretreatment with rapamycin.

    Association of RAPTOR with mTOR. Meal feeding did not alter the abundance of RAPTOR associated with mTOR (Table 2). In contrast, the amount of RAPTOR associated with mTOR was significantly reduced by 70% following pretreatment with rapamycin compared with values in fed rats.

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
In this study, we tested the hypothesis that pathways modified by mTOR were responsible for the myocardial response of translation initiation effector proteins to meal feeding by inhibiting its activity with rapamycin. Acceleration of cardiac muscle protein synthesis in response to feeding occurs in part through a stimulation of mRNA translation initiation. The key regulatory step responsible for accelerating mRNA translation initiation following meal feeding involves the recognition, unwinding, and binding of mRNA to the 43S preinitiation complex (1,25,32,33). A multi-subunit complex of eukaryotic factors referred to as eIF4F catalyzes these processes. The eIF4F complex is composed of eIF4A (an RNA helicase that unwinds secondary structure in the 5'-untranslated region of mRNA), eIF4E (a protein that binds directly to the 7-methyl GTP cap structure present at the 5'-end of most eukaryotic mRNAs), and eIF4G (a protein that functions as a scaffold for eIF4E, eIF4A, and the mRNA and the ribosome) (34–36). eIF4G links the mRNA cap structure, the poly(A)⁺ tail, and ribosomal subunit, because it has binding sites not only for eIF4E but also for eIF4A and eIF3 (37). To our knowledge, we were the first to report a positive linear relationship between rates of protein synthesis and amount of eIF4G associated with eIF4E in vivo (1,27,38). Consistent with this observation, meal feeding was associated with an 4-fold increase in the abundance of eIF4G associated with eIF4E, which was completely curtailed by administration of rapamycin.

Assembly of active eIF4G-eIF4E complex is dependent upon both the availability of eIF4E and the extent of eIF4G phosphorylation. Availability of eIF4E to bind with eIF4G and form active eIF4F complex is regulated by a family of low-molecular weight acid- and heat-stable proteins termed 4E-BPs (31,39). The activity of 4E-BPs is controlled through covalent modification whereby phosphorylation reduces the binding affinity for eIF4E, allowing eIF4E to bind to eIF4G and promote mRNA translation (40,41). The signaling pathway leading to changes in phosphorylation of 4E-BP1 following meal feeding occurs in part through mTOR. The activity of this Ser/Thr kinase is regulated by nutrients when it is part of a complex called TORC1, which includes not only mTOR but also RAPTOR and GßL as well (42,43). However, the regulation of 4E-BP1-eIF4E appears more complex, as we have reported that Leu stimulates protein synthesis in perfused hind limb preparations through a mechanism that appears independent of 4E-BP1 phosphorylation (8,26,33,44). In this study, rapamycin did not affect the dissociation of the inactive eIF4E-4E-BP1 complex. Indeed, the association of eIF4E with 4E-BP1 did not differ in any of the conditions examined despite an elevated level of 4E-BP1 in the -form during meal feeding in the presence or absence of the inhibitor. Hence, factors in addition to 4E-BP1 phosphorylation can modify the extent of formation of eIF4E-4E-BP1 complex in cardiac muscle in vivo.

Another potential mechanism modulating the assembly of eIF4G-eIF4E complex involves phosphorylation of eIF4G (26). eIF4G possesses 3 phosphorylation sites located in the C-terminal one-third of the protein corresponding to Ser residues 1108, 1148, and 1192 (4). Numerous reports have linked phosphorylation of eIF4G with corresponding changes in protein synthesis. Increased phosphorylation of eIF4G on Ser¹¹⁰⁸ is associated with enhanced assembly of eIF4E-eIF4G complex following exposure of cells in culture to serum (4) or gonadotrophin-releasing hormone (5). In vivo, insulin-like growth factor-I-induced stimulation of phosphorylation of eIF4G correlates with accelerated rates of protein synthesis in muscle (38,45). Likewise, enhanced phosphorylation of eIF4G correlated with stimulation of protein synthesis and assembly of the eIF4G-eIF4E complex following perfusion of hind limb muscle with buffer supplemented with Leu (26,46). On the other hand, thermal injury reduces the phosphorylation of eIF4G and assembly of eIF4E-eIF4G complex in heart (3). Hence, phosphorylation of eIF4G is a potentially important mechanism controlling protein synthesis through assembly of the eIF4G-eIF4E complex in striated muscle. The extent of phosphorylation of eIF4G was elevated 6-fold during meal feeding, but the rise was abrogated by pretreatment with rapamycin. Thus, rapamycin can completely prevent the meal feeding-induced phosphorylation of eIF4G, indicating this process is dependent upon TORC1.

Another major target of mTOR kinase activity appears to be 40S ribosomal protein S6 (47). Increased S6 phosphorylation has been implicated in the translational upregulation of mRNA transcripts containing a polypyrimidine tract at their 5' transcriptional start site (48). Mutational studies imply that phosphorylation Thr³⁸⁹, which is located in an unusual hydrophobic sequence outside the catalytic domain, plays a critical role in S6K1 activation (49). Mutation of Thr³⁸⁹ to Ala ablates kinase activity, whereas mutation to Glu confers constitutive kinase activity and rapamycin resistance (49). More importantly, phosphorylation of Thr³⁸⁹ is the principal target of rapamycin-induced S6K1 inactivation. Indeed, increased mTOR activity enhances phosphorylation of S6K1 on residue Thr³⁸⁹ (50,51). In this study, the observation that rapamycin only partially inhibits the phosphorylation of Thr³⁸⁹ suggests that mTOR may not be the only kinase responsible for phosphorylation of S6K1 following meal feeding, consistent with a conclusion reached by Pearson et al. (49) examining the regulation of S6K1 phosphorylation. Besides mTOR, at least 2 other upstream pathways through either phosphoinositol 3-kinase or phosphoinositol-dependent kinase-1 can modulate S6K1 phosphorylation (52). Furthermore, activation of protein synthesis by the 1-adrenergic agonist phenylephrine requires activation of Ca²⁺-independent protein kinase C (PKC), namely PKC and PKC (53). It is interesting to note that PKC is activated by meal feeding in cardiac muscle, although its exact role in S6K1 phosphorylation remains unresolved (1).

mTORC2 is posited to phosphorylate PKB(Ser⁴⁷³), resulting in its activation (43,54). A 1- or 24-h incubation with rapamycin eliminates S6K1 phosphorylation, consistent with inhibition of mTORC1 in transformed cells in culture (55). Because S6K1 normally suppresses the PI3K/PKB pathway (56,57), inhibition of S6K1 by rapamycin should lead to an increase in PKB(Ser⁴⁷³) phosphorylation. Whereas hearts from rats pretreated with the inhibitor showed a reduced phosphorylation of S6K1, they also showed an increased extent of PKB(Ser⁴⁷³) phosphorylation, which did not differ from meal-fed control rats, consistent with the above-mentioned scenario.

TORC1 and TORC2 complexes may share the same pool of TOR proteins. mTOR interacts with the RAPTOR and GßL proteins (58–60) to form a complex (TORC1) that is sensitive to inhibition by rapamycin. In addition, mTOR is also part of another distinct complex (TORC2) composed of the rapamycin-insensitive companion mTOR and GßL proteins. The rapamycin-insensitive companion mTOR protein-containing mTOR complex contains GßL but not RAPTOR and it neither regulates the mTOR effector kinase, S6K1, nor is it bound by FK506 binding protein 12-rapamycin (61). One scenario to account for the enhanced stimulation of downstream effector molecules of mTOR in cardiac muscle following meal feeding is through an increased association of RAPTOR with mTOR. In this study, the interaction of RAPTOR with mTOR was not modified by meal feeding. Hence, the ability of meal feeding to enhance changes in effector molecules involved in translation control does not appear dependent upon association of RAPTOR with mTOR during meal feeding. In contrast, pretreatment with rapamycin reduced the interaction of RAPTOR with mTOR by two-thirds. This latter observation is consistent with reports showing that mTORC1 becomes destabilized after addition of rapamycin, because binding of FKBP12-rapamycin to it weakens the RAPTOR-mTOR interaction (58). Thus, the decreased association of RAPTOR with mTOR may limit the ability of meal feeding to affect translational control via S6K1 and assembly of active eIF4G-eIF4E complex.

In summary, these findings suggest the extent of phosphorylation of eIF4G following meal feeding occurs through a rapamycin-sensitive mechanism in cardiac muscle. Furthermore, the rapamycin-sensitive reductions in phosphorylation of eIF4G may also lead to decreased formation of active eIF4G-eIF4E complex and, hence, a reduced rate of protein synthesis.

	ACKNOWLEDGMENTS

 
The authors thank Beth Gern and Rachel Eicher for their expert technical contributions. We thank Dr. Leonard S. Jefferson from our institution for kindly providing the eIF4E antibodies used in this study.

	FOOTNOTES

 
¹ Supported in part by National Institute of Alcohol Abuse and Alcoholism grant AA-12814 (T.C.V.), National Institute of General Medical Services GM-39722 (T.C.V.), and National Institute of Diabetes, Digestive Disease and Kidney grants DK-053843 and DK-062880 (C.H.L.).

² Author disclosures: T. C. Vary, G. Deiter, and C. J. Lynch, no conflicts of interest.

³ Abbreviations used: eIF, eukaryotic initiation factor; 4E-BP1, eIF4E binding protein-1; mRNA, messenger RNA; mTOR, mammalian target of rapamycin; PKB, protein kinase B; PKC, protein kinase C; RAPTOR, rapamycin-associated protein of mTOR; S6K1, 70-kDa ribosomal protein S6 kinase1; TORC1, mTOR complex1.

Manuscript received 1 March 2007. Initial review completed 26 March 2007. Revision accepted 14 June 2007.

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