Leucine, Glutamine, and Tyrosine Reciprocally Modulate the Translation Initiation Factors eIF4F and eIF2B in Perfused Rat Liver
1999; Elsevier BV; Volume: 274; Issue: 51 Linguagem: Inglês
10.1074/jbc.274.51.36168
ISSN1083-351X
AutoresO. Jameel Shah, David A. Antonetti, Scot R. Kimball, Leonard S. Jefferson,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoLeucine, glutamine, and tyrosine, three amino acids playing key modulatory roles in hepatic proteolysis, were evaluated for activation of signaling pathways involved in regulation of liver protein synthesis. Furthermore, because leucine signals to effectors that lie distal to the mammalian target of rapamycin, these downstream factors were selected for study as candidate mediators of amino acid signaling. Using the perfused rat liver as a model system, we observed a 25% stimulation of protein synthesis in response to balanced hyperaminoacidemia, whereas amino acid imbalance due to elevated concentrations of leucine, glutamine, and tyrosine resulted in a protein synthetic depression of roughly 50% compared with normoaminoacidemic controls. The reduction in protein synthesis accompanying amino acid imbalance became manifest at high physiologic concentrations and was dictated by the guanine nucleotide exchange activity of translation initiation factor eIF2B. Paradoxically, this phenomenon occurred concomitantly with assembly of the mRNA cap recognition complex, eIF4F as well as activation of the 70-kDa ribosomal S6 kinase, p70S6k. Dual and reciprocal modulation of eIF4F and eIF2B was leucine-specific because isoleucine, a structural analog, was ineffective in these regards. Thus, we conclude that amino acid imbalance, heralded by leucine, initiates a liver-specific translational failsafe mechanism that deters protein synthesis under unfavorable circumstances despite promotion of the eIF4F complex. Leucine, glutamine, and tyrosine, three amino acids playing key modulatory roles in hepatic proteolysis, were evaluated for activation of signaling pathways involved in regulation of liver protein synthesis. Furthermore, because leucine signals to effectors that lie distal to the mammalian target of rapamycin, these downstream factors were selected for study as candidate mediators of amino acid signaling. Using the perfused rat liver as a model system, we observed a 25% stimulation of protein synthesis in response to balanced hyperaminoacidemia, whereas amino acid imbalance due to elevated concentrations of leucine, glutamine, and tyrosine resulted in a protein synthetic depression of roughly 50% compared with normoaminoacidemic controls. The reduction in protein synthesis accompanying amino acid imbalance became manifest at high physiologic concentrations and was dictated by the guanine nucleotide exchange activity of translation initiation factor eIF2B. Paradoxically, this phenomenon occurred concomitantly with assembly of the mRNA cap recognition complex, eIF4F as well as activation of the 70-kDa ribosomal S6 kinase, p70S6k. Dual and reciprocal modulation of eIF4F and eIF2B was leucine-specific because isoleucine, a structural analog, was ineffective in these regards. Thus, we conclude that amino acid imbalance, heralded by leucine, initiates a liver-specific translational failsafe mechanism that deters protein synthesis under unfavorable circumstances despite promotion of the eIF4F complex. eukaryotic initiation factor 4E eukaryotic initiation factor 4G eukaryotic initiation factor 2B α subunit of eukaryotic initiation factor 2 eIF4E binding protein 1 branched chain amino acid mammalian target of rapamycin phosphatidylinositol 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid 3-(N-morpholino)propanesulfonic acid mitogen-activated protein glycogen synthase kinase 3 analysis of variance The amino acids represent a class of biologic molecules exerting dynamic and complex influences on highly disparate physiologic processes including pancreatic insulin and glucagon secretion, protein degradation and synthesis, hepatic gluconeogenesis, and sensitization of tissues to the anabolic effects of insulin (1Harper A.E. Miller R.H. Block K.P. Annu. Rev. Nutr. 1984; 4: 409-454Crossref PubMed Scopus (849) Google Scholar, 2Smith R. Elia M. Proc. Nutr. Soc. 1983; 42: 473-487Crossref PubMed Scopus (16) Google Scholar, 3Buse M.G. Reid S.S. J. Clin. Invest. 1975; 56: 1250-1261Crossref PubMed Scopus (544) Google Scholar, 4Mortimore G.E. Poso A.R. Kadowaki M. Wert Jr., J.J. J. Biol. Chem. 1987; 262: 16322-16327Abstract Full Text PDF PubMed Google Scholar, 5Mortimore G.E. Poso A.R. Lardeux B. Diab. Metab. Rev. 1989; 5: 49-70Crossref PubMed Scopus (177) Google Scholar). The effects of amino acids are somewhat enigmatic but often involve the interplay of hormones and other factors intrinsic to the cellular environment. Recently, the branched chain amino acid, leucine, has been demonstrated to modulate pathways of signal transduction and may indeed contribute importantly to the cellular interpretation of integrated signals. With regard to protein homeostasis, several reports now exist supporting the hypothesis that leucine impacts protein turnover through mechanisms beyond those of protein synthetic substrates (1Harper A.E. Miller R.H. Block K.P. Annu. Rev. Nutr. 1984; 4: 409-454Crossref PubMed Scopus (849) Google Scholar, 2Smith R. Elia M. Proc. Nutr. Soc. 1983; 42: 473-487Crossref PubMed Scopus (16) Google Scholar, 3Buse M.G. Reid S.S. J. Clin. Invest. 1975; 56: 1250-1261Crossref PubMed Scopus (544) Google Scholar, 4Mortimore G.E. Poso A.R. Kadowaki M. Wert Jr., J.J. J. Biol. Chem. 1987; 262: 16322-16327Abstract Full Text PDF PubMed Google Scholar). In an elegant series of experiments, Mortimore et al. (4Mortimore G.E. Poso A.R. Kadowaki M. Wert Jr., J.J. J. Biol. Chem. 1987; 262: 16322-16327Abstract Full Text PDF PubMed Google Scholar, 5Mortimore G.E. Poso A.R. Lardeux B. Diab. Metab. Rev. 1989; 5: 49-70Crossref PubMed Scopus (177) Google Scholar) demonstrated that the amino acids leucine, glutamine, and tyrosine, individually as well as cooperatively and in a manner that is concentration-dependent, attenuate hepatic macroautophagic proteolysis induced by deprivation of amino acids. Furthermore, insulin functions additively and synergistically with these amino acids, thereby enhancing the efficacy of proteolytic inhibition by leucine, glutamine, and/or tyrosine. Inherent in their potency as modulators of protein homeostasis, amino acids generally exert reciprocal control of hepatic protein degradation and synthesis. The latter process is governed primarily at the level of translation initiation through the regulative affinities and activities of several eukaryotic initiation factors (eIFs).1 Components of the translational apparatus demonstrated to play particularly important roles include the guanine nucleotide exchange factor, eIF2B, the eIF4F heterocomplex, and the 70-kDa 40 S ribosomal protein S6 kinase (p70S6k). The guanine nucleotide exchange factor, eIF2B, is a heteropentameric enzyme that performs a function critical for the successive and cyclic nature of initiation. After a ribosome is loaded onto the mRNA, the initiation complex is disassembled in a process requiring hydrolysis of eIF2-associated GTP. Because the resultant GDP bound to eIF2 dissociates very slowly and because eIF2·GTP is necessary for recruitment of Met-tRNAi, the eIF2B-catalyzed exchange of eIF2-bound GDP for GTP is essential for ternary complex formation and subsequent rounds of initiation (6Webb B.L.J. Proud C.G. Int. J. Biochem. Cell Biol. 1997; 29: 1127-1131Crossref PubMed Scopus (77) Google Scholar). The activity of eIF2B is negatively affected by phosphorylation of the α subunit of eIF2, which consequently competitively inhibits eIF2-targeted nucleotide exchange. Also, allosteric-like effects as well as direct phosphorylation of eIF2B are implicated in regulating its activity. Amino acid deprivation has been shown to suppress protein synthesis concomitant with inhibition of eIF2B activity. However, the involvement of the phosphorylation state of eIF2α remains debatable (7Kimball S.R. Horetsky R.L. Jefferson L.S. J. Biol. Chem. 1998; 273: 30945-30953Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 8Rowlands A.G. Montine K.S. Henshaw E.C. Panniers R. Eur. J. Biochem. 1988; 175: 93-99Crossref PubMed Scopus (81) Google Scholar, 9Gross M. Rubino M.S. J. Biol. Chem. 1989; 264: 21879-21884Abstract Full Text PDF PubMed Google Scholar). Although contentious, the association of eIF4A (an ATP-dependent RNA helicase), eIF4E (an mRNA cap-binding protein), and eIF4G (a scaffolding protein) has been suggested to be the rate-limiting event in the initiation of translation (10Sonenberg N. Herskey J.W.B. Mathews M.B. Sonenberg N. Translational Control. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 245-269Google Scholar). These factors, collectively referred to as eIF4F, bind to the m7GTP cap structure of mRNA and facilitate the recruitment of other eIFs as well as the 40 S ribosomal subunit, culminating in the formation of the 48 S preinitiation complex. The assembly of eIF4F is determined by the phosphorylation state of a family of competitive inhibitors of eIF4G, the eIF4E binding proteins (4E-BPs). Hypophosphorylated 4E-BPs exhibit strong affinity for eIF4E and as such, restrict access of eIF4G to eIF4E. Thus, aggregation of integral components of the cap-binding holocomplex is hindered. However, phosphorylation on multiple residues neutralizes the inhibitive properties of 4E-BPs and facilitates eIF4E·eIF4G interaction. The signal transduction pathway responsible for 4E-BP phosphorylation is common to p70S6k, a cell cycle-regulated kinase implicated in expression of mRNAs of the TOP (terminal oligopyrimidine) family (11Wang X. Campbell L.E. Miller C.M. Proud C.G. Biochem. J. 1998; 334: 261-267Crossref PubMed Scopus (298) Google Scholar, 12Hara K. Yonezawa K. Weng Q.-P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1148) Google Scholar). Deprivation of amino acids results in dephosphosphorylation of 4E-BP1 and p70S6k in several cell types (7Kimball S.R. Horetsky R.L. Jefferson L.S. J. Biol. Chem. 1998; 273: 30945-30953Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 11Wang X. Campbell L.E. Miller C.M. Proud C.G. Biochem. J. 1998; 334: 261-267Crossref PubMed Scopus (298) Google Scholar); these effects are reversed upon readdition of amino acids, and this reversal is rapamycin-sensitive, underscoring the involvement of the mammalian target of rapamycin (mTOR) in mediation of these signals. Amino acids influence hepatic protein turnover, at least in part, by reciprocal regulation of protein synthesis and protein degradation. Because a distinct group of amino acids, namely the regulatory group, and in particular, leucine, glutamine, and tyrosine, exert most of the observed inhibition of deprivation-induced proteolysis in the liver, we sought to characterize the modulatory role(s), if any, of these amino acids on hepatic protein synthesis. Furthermore, this study was designed to address the hepatic response to physiological changes in amino acid concentration in an effort to isolate potentially important amino acid signaling events; particularly, those induced by leucine. Thus, the specific effects of leucine, glutamine, and tyrosine on eIF4F assembly, eIF2B activity, p70S6k activation, and the relative contribution of these events in determination of overall protein synthesis was evaluated. Male Sprague-Dawley rats weighing approximately 100–125 g were maintained on a 12 h light/dark cycle and were provided food (Harlan-Teklad Rodent Chow) and water ad libitum. ECL detection reagents and horseradish peroxidase-conjugated sheep anti-mouse and donkey anti-rabbit immunoglobulins were purchased from Amersham Pharmacia Biotech. Polyvinylidene difluoride membranes were acquired from Bio-Rad. Insulin was purchased from Eli Lilly and Co. Rapamycin was purchased from Calbiochem. Livers were perfused in situessentially as described previously (13Flaim K.E. Peavy D.E. Everson W.V. Jefferson L.S. J. Biol. Chem. 1982; 257: 2932-2938Abstract Full Text PDF PubMed Google Scholar) with the following modifications. Perfusate was delivered at flow rate of 7 ml/min under nonrecirculating conditions. Following an initial 5 min washout, livers were perfused and radiolabeled for 15 min in the presence of 5 mm valine. The amino acid composition differed from that previously reported; the 1× designation is described in the legend of Fig. 1. Addition of this mixture has been shown to yield perfusate concentrations that closely approximate those reported for rat plasma (14Lunn P.G. Whitehead R.G. Baker B.A. Br. J. Nutr. 1976; 36: 219-230Crossref PubMed Google Scholar). The amino acid compositions of the perfusate utilized throughout this study were multiples of 1× as described in the figures. Moreover, under some circumstances, 10 nm insulin was added to the perfusing medium. For determination of protein synthesis,l-[3,4-3H]valine (NEN Life Science Products) was added at 1 μCi/ml to the perfusate. Rates of protein synthesis were determined essentially as described previously with slight modification (15Kimball S.R. Jurasinski C.V. Lawrence Jr., J.C. Jefferson L.S. Am. J. Physiol. 1997; 272: C754-C759Crossref PubMed Google Scholar) by measuring the incorporation of [3H]valine into newly synthesized protein. Quantitation of the respective factors and complexes were performed essentially as defined elsewhere (16Yoshizawa F. Kimball S.R. Vary T.C. Jefferson L.S. Am. J. Physiol. 1998; 275: E814-E820PubMed Google Scholar). eIF4E, 4E-BP1·eIF4E, and eIF4G·eIF4E complexes were immunoprecipitated from 10,000 × g supernatants of whole liver homogenate using a mouse anti-eIF4E monoclonal antibody. The antibody was raised against recombinant human eIF4E as described previously (20Kimball S.R. Everson W.V. Flaim K.E. Jefferson L.S. Am. J. Physiol. 1989; 256: C28-C34Crossref PubMed Google Scholar). The antibody-antigen complex was isolated by incubation with goat anti-mouse Biomag immunoglobulin G beads (PerSeptive Diagnostics). Prior to incubation with antigen-antibody complexes, the beads were washed in 1% nonfat, dry milk in buffer B (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 0.1% β-mercaptoethanol, 0.5% Triton X-100, 50 mm NaF, 50 mm β-glycerophosphate, 0.1 mmphenylmethylsulfonyl fluoride, 1 mm benzamidine, and 0.5 mm sodium vanadate). The beads were captured using a magnetic stand, washed twice with buffer B, and washed once with buffer B containing 500 mm NaCl rather than 150 mm. Immune complexes bound to the beads were eluted by resuspension of the beads in SDS sample buffer and then boiling for 5 min. The beads were pelleted by centrifugation, and the supernatants were subjected to SDS-polyacrylamide gel electrophoresis. Separated proteins were then electrophoretically transferred to polyvinylidene difluoride membranes. Following transfer, the membranes were incubated with a mouse monoclonal anti-eIF4E antibody, a rabbit polyclonal anti-4E-BP1 antibody, or a rabbit polyclonal anti-eIF4G antibody overnight at 4 °C. The immunoblots were then developed using an ECL Western blotting kit as described previously (20Kimball S.R. Everson W.V. Flaim K.E. Jefferson L.S. Am. J. Physiol. 1989; 256: C28-C34Crossref PubMed Google Scholar). Quantitation of the phosphorylation state of 4E-BP1 was carried out exactly as described previously (16Yoshizawa F. Kimball S.R. Vary T.C. Jefferson L.S. Am. J. Physiol. 1998; 275: E814-E820PubMed Google Scholar). Briefly, the phosphorylated and unphosphorylated forms of 4E-BP1 were collected by immunoprecipitation of 4E-BP1 from 10,000 × gsupernatants of whole liver homogenate. For this purpose a mouse monoclonal anti-4E-BP1 antibody was incubated with the 10,000 ×g supernatant. The immunoprecipitated proteins were collected and separated as outlined above. The migration of 4E-BP1 on SDS-polyacrylamide gels is inversely proportional to the degree of phosphorylation of the protein (17Lin T.-A. Kong X. Haystead T.A.J. Pause A. Belsham G. Sonenberg N. Lawrence J.C. Science. 1994; 266: 653-656Crossref PubMed Scopus (608) Google Scholar, 18Haystead T.A.J. Haystead C.M.M. Hu C. Lin T.-A. Lawrence J.C. J. Biol. Chem. 1994; 269: 23185-23191Abstract Full Text PDF PubMed Google Scholar). Therefore, multiple phosphorylation forms were separable following SDS-polyacrylamide gel electrophoresis as described above. The proportion of eIF2α in phosphorylated and unphosphorylated forms was determined using slab gel isoelectric focusing electrophoresis followed by protein immunoblotting. Aliquots of post-mitochondrial supernatants were heated for 3 min in SDS sample buffer at 100 °C, cooled to room temperature, and then mixed with 0.8 volume of isoelectric focusing gel buffer (0.1 g of dithiothreitol, 0.4 g of CHAPS, 5.4 g of urea, and 1 ml of Ampholytes (pH 3.5–9.5 from Pharmacia/LKB) in 6 ml of water). Proteins were resolved and detected using a rabbit monoclonal anti-eIF2α antibody as described elsewhere (19Kimball S.R. Jefferson L.S. Biochem. Biophys. Res. Commun. 1991; 177: 1082-1086Crossref PubMed Scopus (14) Google Scholar) and detected with ECL. Determination of eIF2B activity in liver was performed exactly as outlined elsewhere (20Kimball S.R. Everson W.V. Flaim K.E. Jefferson L.S. Am. J. Physiol. 1989; 256: C28-C34Crossref PubMed Google Scholar) by measuring the rate of exchange of [3H]GDP, which is present in an exogenous eIF2·[3H]GDP complex, for free, nonradiolabeled GDP. Briefly, following excision of the liver, the tissue was rinsed in ice-cold saline, weighed, and homogenized in a Polytron in four volumes of buffer consisting of 20 mmtriethanolamine, pH 7.0, 2 mm magnesium acetate, 150 mm potassium chloride, 0.5 mm dithiothreitol, 0.1 mm EDTA, 250 mm sucrose, 5 mmEGTA, and 50 mm β-glycerophosphate. Homogenates were then centrifuged for 10 min at 12,000 × g at 4 °C. Supernatants were then assayed for guanine nucleotide exchange activity. Essentially, 35 μl of a prepared binary complex, which was assembled by incubation of purified eIF2 with 1.3 μm[3H]GDP (10.7 Ci/mmol), was combined with a mixture consisting of 35 μl of liver homogenate, 87.5 μl of water, and 140 μl of buffer A (50 mm MOPS, pH 7.4, 209 μmGDP, 2 mm magnesium acetate, 100 mm potassium chloride, 1 mm dithiothreitol, and 200 μg/ml bovine serum albumin). The reaction was initiated by combination of these reactants and transfer to a 30 °C water bath. At five time points (0, 2, 4, 6, and 8 min), a 75-μl aliquot was removed and placed into tubes containing 2.5 ml of ice-cold wash buffer (buffer A devoid of bovine serum albumin). The contents were mixed and immediately filtered through a nitrocellulose filter disc. The guanine nucleotide exchange activity was measured as a decrease in eIF2·[3H]GDP complex bound to the filters. The activity of eIF2Bε kinase(s) was performed as described previously (21Jefferson L.S. Fabian J.R. Kimball S.R. Int. J. Biochem. Cell Biol. 1999; 31: 191-200Crossref PubMed Scopus (90) Google Scholar) except that 0.5 μg of purified, recombinant eIF2Bε was used as substrate in the reaction instead of the purified eIF2B holoenzyme. The amino acids leucine, glutamine, and tyrosine are known to hinder proteolysis; therefore, the role(s) of these regulatory amino acids in modulating protein synthesis was evaluated in the perfused liver. Perfusion with a complete, 10× amino acid mixture resulted in a 25% elevation in overall protein synthesis compared with livers administered a mixture of amino acids at 1× (Fig. 1 A). Intriguingly, perfusion with an imbalanced amino acid mixture comprised of leucine, glutamine, and tyrosine at 10×, whereas all other amino acids were maintained at 1× depressed protein synthesis by almost 50% relative to control. Thus, whereas heightened levels of a total amino acid mixture had an anabolic effect in the liver, equimolar amounts of the three regulatory amino acids impaired protein synthesis. This regulatory amino acid-mediated phenomenon became manifest at approximately 4× concentrations (Fig. 1 B). Although raising the concentration of leucine, glutamine, and tyrosine to 4× depressed protein synthesis, this hepatic response was exacerbated by a further increment in amino acid concentration to 10×. To determine the mechanism(s) by which leucine, glutamine, and tyrosine affects overall protein synthesis, their influence on particular translational control points were evaluated. Surprisingly, raising the concentration of leucine, glutamine, and tyrosine while maintaining all other amino acids at 1× potently disrupted the inhibitory eIF4E·4E-BP1 complex (Fig. 2 A) concomitant with hyperphosphorylation of 4E-BP1 (Fig. 2 B) and p70S6k (Fig. 2 D). Activation of hyperphosphorylated p70S6k was evidenced by hyperphosphorylation of endogenous S6 with increasing concentration of leucine, glutamine, and tyrosine (data not shown). Furthermore, eIF4E preferentially associated with eIF4G under these conditions (Fig. 2 C). Although doubling the concentrations of the regulatory amino acids (that is, 2×) relative to others exerted little effect on these factors, the phosphorylation states of 4E-BP1 and p70S6k were marked enhanced at 4×. Collectively, these results suggest that elevations in circulating levels of leucine, glutamine, and tyrosine above 2× promote assembly of eIF4F and activation of p70S6k despite a simultaneous concentration-dependent inhibition of global translation. Insulin potently modulates the phosphorylation states and activities of components of the eIF4F system as well as p70S6k (15Kimball S.R. Jurasinski C.V. Lawrence Jr., J.C. Jefferson L.S. Am. J. Physiol. 1997; 272: C754-C759Crossref PubMed Google Scholar, 22Xu G. Marshall C.A. Lin T.-A. Kwon G. Munivenkatappa R.B. Hill J.R. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 4485-4491Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,23Kimball S.R. Jefferson L.S. Fadden P. Haystead T.A. Lawrence Jr., J.C. Am. J. Physiol. 1996; 270: C705-C709Crossref PubMed Google Scholar). Therefore, we sought to characterize the role of insulin on these processes under conditions of mild amino acid imbalance. However, in the presence of either 0.5× (which was not statistically different from 1×; not shown) or 2× concentrations of leucine, glutamine, and tyrosine, insulin was without effect on the liver's protein synthetic response (Fig. 3 A). Whereas it appears that, in the hepatocyte, the anabolic effects of insulin are secondary to those of amino acids, insulin and amino acids synergistically promote assembly of eIF4F and activation of p70S6k (12Hara K. Yonezawa K. Weng Q.-P. Kozlowski M.T. Belham C. Avruch J. J. Biol. Chem. 1998; 273: 14484-14494Abstract Full Text Full Text PDF PubMed Scopus (1148) Google Scholar, 24Patti M.-E. Brambilla E. Luzi L. Landaker E.J. Kahn R.C. J. Clin. Invest. 1998; 101: 1519-1529Crossref PubMed Google Scholar). Our findings in the perfused liver corroborate these reports. In combination with insulin, the three regulatory amino acids additively promoted the disunion of the eIF4E·4E-BP1 complex and the association of eIF4E with eIF4G (Fig. 3,B and C). Although phosphorylation of p70S6k was enhanced slightly in the presence of insulin at 0.5× leucine, glutamine, and tyrosine, an additive influence of insulin was masked at higher amino acid concentrations perhaps because p70S6k was maximally activated in the presence of 2× amino acids (Fig. 3 D). In essence, insulin promoted eIF4F assembly within the physiologic realm but remained an impotent determinant of global protein synthesis. Proteolytic studies conducted in perfused rat liver have revealed that of the seven individual amino acids with inherent regulatory properties, macroautophagic proteolytic inhibition by leucine was unrivaled, although glutamine and tyrosine enhanced this inhibition when added in combination (25Mortimore G.E. Kadowaki M. Ciechanover A.J. Schwartz A.L. Cellular Proteolytic Systems. Wiley-Liss, Inc., New York1994: 65-87Google Scholar, 26Miotto G. Venerando R Khurana K.K. Siliprandi N. Mortimore G. J. Biol. Chem. 1992; 267: 22066-22072Abstract Full Text PDF PubMed Google Scholar). These three amino acids appear to reciprocally modulate autophagic proteolysis and the signal transduction pathway(s) leading to eIF4F complex formation and activation of p70S6k. Therefore, the contribution of leucine to the activating properties of the three regulatory amino acids was examined in the context of eIF4F assembly and p70S6k activation. As shown previously, concentrations of the regulatory amino acid trio mimicking the upper physiologic threshold (that is, 4×) inhibited hepatic protein synthetic activity approximately 25% (Fig. 4 A). However, replacing leucine with equivalent amounts of the structurally similar BCAA, isoleucine, prevented the observed defect in protein synthesis. Furthermore, a perfusate containing 4× leucine elicited a synthetic response intermediate between that observed with a complete, 1× mixture of amino acids and that of 4× leucine, glutamine, and tyrosine. These data suggest not only that these phenomena are leucine-specific but also that glutamine and tyrosine are only marginally influential in the absence of leucine. Leucine, glutamine, and tyrosine, when present at 4× concentrations, effectively destabilized the eIF4E·4E-BP1 complex (Fig. 4 B). However, substitution of leucine with isoleucine in the perfusate was virtually without effect on the stability of the complex, implying that leucine is required for maximal effect. Moreover, leucine appears to play a dominant role in this process because leucine alone nearly reduplicated the uncoupling of eIF4E·4E-BP1 observed with leucine, glutamine, and tyrosine. Furthermore, eIF4E and eIF4G were conjoined in the presence of the three regulatory amino acids, whereas exchange of leucine with isoleucine in the perfusing medium attenuated formation of this complex (Fig. 4 C). Again, leucine alone harbored the bulk of the influence of the regulatory triplet. Finally, optimal hyperphosphorylation of p70S6k was achieved in the presence of leucine, glutamine, and tyrosine, whereas the combination of isoleucine, glutamine, and tyrosine produced a pattern of phosphorylation similar to that of the control amino acid mixture (Fig. 4 D). Phosphorylation of p70S6k by leucine alone was interjacent to the cumulative effect of leucine, glutamine, and tyrosine and that of control. Thus, it appears that the influences of the three regulatory amino acids seen here are largely attributable to the weighty contribution(s) of leucine. However, the effect of leucine on the phosphorylation states of both 4E-BP1 and p70S6k, although augmented, remained submaximal, suggesting that the presence of glutamine and tyrosine serves to enhance these leucine-induced responses. Alterations in eIF4F complex assembly and/or eIF2B activity are often sufficient to account for corresponding changes in total protein synthesis. Because, in this inquiry, protein synthesis and regulatory amino acid concentrations were inversely correlated, and the diminution of total protein synthetic activity was independent of eIF4F assembly, an examination of the activity of eIF2B was undertaken. The guanine nucleotide exchange activity of eIF2B was inversely related to ambient concentrations of leucine, glutamine, and tyrosine. In fact, the percentage of decline in eIF2B activity virtually mirrored the depression in protein synthesis observed at identical regulatory amino acid concentrations (c.f. Fig. 1 B and Fig. 5 A) and was independent of modified expression of eIF2B subunits (data not shown). Hence, the dose-dependent attenuation of eIF2·GTP regeneration was sufficient to account for the suppression of global protein synthesis conduced by leucine, glutamine, and tyrosine. The mechanism triggering these changes in the exchange activity of eIF2B was not attributable to phosphorylation of eIF2α because the proportion of the phosphorylated species did not change appreciably as a function of regulatory amino acid concentration (data not shown). As expected, these changes in eIF2B activation were dictated by the superior influence of leucine. The rate of guanine nucleotide exchange diminished in the presence of a 4× mixture of leucine, glutamine, and tyrosine relative to that of a complete, 1× mixture (Fig. 5 B). Not unexpectedly, the catalytic activity of eIF2B was unaffected when isoleucine was substituted for leucine in the perfusing medium. Once again, however, the observed leucine-specific depression in eIF2B activity was independent of a change in eIF2α phosphorylation (Fig. 5 C). Taken together, the findings not only demonstrate that leucine, glutamine, and tyrosine, when present at concentrations of 4×, minimize the rate of eIF2B-mediated guanine nucleotide exchange, but also that leucine is indispensible for this effect. Because substantial evidence has accumulated to place mTOR downstream of amino acid-induced signals, we sought to address the requirement of mTOR as a mediator of translational regulation by leucine, glutamine, and tyrosine. Rapamycin is a macrolide with immunosuppressive properties that binds with high affinity to endogenous FK506-binding proteins. Although several members of this protein family have been demonstrated to interact with rapamycin, only the FK506-binding protein 12·rapamycin complex directly interacts and thereby inhibits the kinase activity associated with or intrinsic to mTOR. Thus, rapamycin has proven to be a powerful agent in elucidation of signal transduction pathways downstream of mTOR. Addition of this compound to the perfusing medium had little effect under basal, normoacidemic conditions, suggesting that at concentrations of 1×, the eIF4 system and p70S6k are largely inactive (Fig. 6). However, whereas 10× concentrations of leucine, glutamine, and tyrosine robustly induced the appearance of 4E-BP1-γ (Fig. 6 B) as well as slower electrophoretic species of p70S6k (Fig. 6 D), rapamycin completely abolished the
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