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Intracellular Sensing of Amino Acids in Xenopus laevis Oocytes Stimulates p70 S6 Kinase in a Target of Rapamycin-dependent Manner

2002; Elsevier BV; Volume: 277; Issue: 12 Linguagem: Inglês

10.1074/jbc.m107694200

ISSN

1083-351X

Autores

Graham Christie, Éric Hajduch, Harinder S. Hundal, Christopher G. Proud, Peter M. Taylor,

Tópico(s)

Mass Spectrometry Techniques and Applications

Resumo

Amino acids exert modulatory effects on proteins involved in control of mRNA translation in animal cells through the target of rapamycin (TOR) signaling pathway. Here we use oocytes ofXenopus laevis to investigate mechanisms by which amino acids are "sensed" in animal cells. Small (∼48%) but physiologically relevant increases in intracellular but not extracellular total amino acid concentration (or Leu or Trp but not Ala, Glu, or Gln alone) resulted in increased phosphorylation of p70S6K and its substrate ribosomal protein S6. This response was inhibited by rapamycin, demonstrating that the effects require the TOR pathway. Alcohols of active amino acids substituted for amino acids with lower efficiency. Oocytes were refractory to changes in external amino acid concentration unless surface permeability of the cell to amino acids was increased by overexpression of the System L amino acid transporter. Amino acid-induced, rapamycin-sensitive activation of p70S6K was conferred when System L-expressing oocytes were incubated in extracellular amino acids, supporting intracellular localization of the putative amino acid sensor. In contrast to lower eukaryotes such as yeast, which possess an extracellular amino acid sensor, our findings provide the first direct evidence for an intracellular location for the putative amino acid sensor in animal cells that signals increased amino acid availability to TOR/p70S6K. Amino acids exert modulatory effects on proteins involved in control of mRNA translation in animal cells through the target of rapamycin (TOR) signaling pathway. Here we use oocytes ofXenopus laevis to investigate mechanisms by which amino acids are "sensed" in animal cells. Small (∼48%) but physiologically relevant increases in intracellular but not extracellular total amino acid concentration (or Leu or Trp but not Ala, Glu, or Gln alone) resulted in increased phosphorylation of p70S6K and its substrate ribosomal protein S6. This response was inhibited by rapamycin, demonstrating that the effects require the TOR pathway. Alcohols of active amino acids substituted for amino acids with lower efficiency. Oocytes were refractory to changes in external amino acid concentration unless surface permeability of the cell to amino acids was increased by overexpression of the System L amino acid transporter. Amino acid-induced, rapamycin-sensitive activation of p70S6K was conferred when System L-expressing oocytes were incubated in extracellular amino acids, supporting intracellular localization of the putative amino acid sensor. In contrast to lower eukaryotes such as yeast, which possess an extracellular amino acid sensor, our findings provide the first direct evidence for an intracellular location for the putative amino acid sensor in animal cells that signals increased amino acid availability to TOR/p70S6K. amino acid(s) aminooxy acetic acid 2-amino-bicyclo[2,2,1]hepta-2-carboxylic acid modified Barth's medium (mammalian) target of rapamycin p70 S6 kinase mitogen activated protein kinase phorbol 12-myristate 13-acetate eukaryotic initiation factor 4E-binding protein messenger RNA complementary RNA α-ketoisocaproic acid extracellular signal-regulated kinase There is growing evidence that nutrients, in particular amino acids (AAs),1 exert powerful modulatory effects on proteins involved in cell signaling, mRNA translation, and amino acid transport (for review, see Refs. 1van Sluijters D.A. Dubbelhuis P.F. Blommaart E.F. Meijer A.J. Biochem. J. 2000; 351: 545-550Crossref PubMed Scopus (123) Google Scholar, 2Shah O.J. Anthony J.C. Kimball S.R. Jefferson L.S. Am. J. Physiol. 2000; 279: E715-E729Crossref PubMed Google Scholar, 3Christie G.R. Hyde R. Hundal H.S. Curr. Opin. Clin. Nutr. Metab. Care. 2001; 4: 425-431Crossref PubMed Scopus (15) Google Scholar). Recent studies indicate that signaling pathways responsive to AAs in mammalian cells show a similarity to those that have already been well characterized in lower organisms such as yeast and bacteria (4Ferenci T. Curr. Opin. Microbiol. 1999; 2: 208-213Crossref PubMed Scopus (108) Google Scholar, 5Gingras A.C. Raught B. Sonenberg N. Genes Dev. 2001; 15: 807-826Crossref PubMed Scopus (1193) Google Scholar, 6Rohde J. Heitman J. Cardenas M.E. J. Biol. Chem. 2001; 276: 9583-9586Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). The serine-threonine protein kinase mammalian target of rapamycin ((m)TOR) is proposed to act as a "nutrient-dependent gatekeeper" (5Gingras A.C. Raught B. Sonenberg N. Genes Dev. 2001; 15: 807-826Crossref PubMed Scopus (1193) Google Scholar) for several cell functions. Two key translational regulators, p70 S6 kinase (p70S6K) and eukaryotic initiation factor 4E-binding protein (4E-BP1) lie downstream of (m)TOR in a cell signaling pathway responsive to AA supplementation (7Wang X. Campbell L.E. Miller C.M. Proud C.G. Biochem. J. 1998; 334: 261-267Crossref PubMed Scopus (295) Google Scholar, 8Hara 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 (1139) Google Scholar, 9Iiboshi Y. Papst P.J. Kawasome H. Hosoi H. Abraham R.T. Houghton P.J. Terada N. J. Biol. Chem. 1999; 274: 1092-1099Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 10Campbell L.E. Wang X. Proud C.G. Biochem. J. 1999; 344: 433-441Crossref PubMed Scopus (70) Google Scholar). Increased phosphorylation of p70S6K and 4E-BP1 in response to AA supplementation is inhibited by the immunosuppressant rapamycin, a specific inhibitor of (m)TOR (5Gingras A.C. Raught B. Sonenberg N. Genes Dev. 2001; 15: 807-826Crossref PubMed Scopus (1193) Google Scholar, 6Rohde J. Heitman J. Cardenas M.E. J. Biol. Chem. 2001; 276: 9583-9586Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). The activation of p70S6K by phosphorylation enables it to phosphorylate its downstream target ribosomal protein S6 and increase translation of so-called 5′-terminal tract of pyrimidines mRNAs, which generally encode ribosomal proteins and elongation factors (2Shah O.J. Anthony J.C. Kimball S.R. Jefferson L.S. Am. J. Physiol. 2000; 279: E715-E729Crossref PubMed Google Scholar, 11Meyuhas O. Eur. J. Biochem. 2000; 267: 6321-6330Crossref PubMed Scopus (436) Google Scholar). The activation of p70S6K is thought to lead to up-regulation of ribosome biosynthesis and thus increase the translational capacity of the cell. Phosphorylation of 4E-BP1 increases protein synthesis by releasing eukaryotic initiation factor 4E, which can then form the eukaryotic initiation factor 4F complex as a prerequisite for translation of the capped mRNAs (reviewed in Ref. 2Shah O.J. Anthony J.C. Kimball S.R. Jefferson L.S. Am. J. Physiol. 2000; 279: E715-E729Crossref PubMed Google Scholar). Although potential upstream roles of phosphoinositide 3-kinase (8Hara 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 (1139) Google Scholar, 9Iiboshi Y. Papst P.J. Kawasome H. Hosoi H. Abraham R.T. Houghton P.J. Terada N. J. Biol. Chem. 1999; 274: 1092-1099Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 12Kleijn M. Proud C.G. Biochem. J. 2000; 347: 399-406Crossref PubMed Scopus (33) Google Scholar), protein kinase B (7Wang X. Campbell L.E. Miller C.M. Proud C.G. Biochem. J. 1998; 334: 261-267Crossref PubMed Scopus (295) Google Scholar, 8Hara 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 (1139) Google Scholar, 12Kleijn M. Proud C.G. Biochem. J. 2000; 347: 399-406Crossref PubMed Scopus (33) Google Scholar, 13Nave B.T. Ouwens M. Withers D.J. Alessi D.R. Shepherd P.R. Biochem. J. 1999; 344: 427-431Crossref PubMed Scopus (787) Google Scholar, 14Armstrong J.L. Bonavaud S.M. Toole B.J. Yeaman S.J. J. Biol. Chem. 2001; 276: 952-956Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), and MAPK (8Hara 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 (1139) Google Scholar, 12Kleijn M. Proud C.G. Biochem. J. 2000; 347: 399-406Crossref PubMed Scopus (33) Google Scholar, 14Armstrong J.L. Bonavaud S.M. Toole B.J. Yeaman S.J. J. Biol. Chem. 2001; 276: 952-956Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) in the AA-induced stimulatory effects on 4E-BP1 and p70S6K have all been investigated, their relative contributions remain unclear and may vary depending on cell type and experimental conditions used. Despite recent attention to the processes of nutrient-related signaling in animal cells (1van Sluijters D.A. Dubbelhuis P.F. Blommaart E.F. Meijer A.J. Biochem. J. 2000; 351: 545-550Crossref PubMed Scopus (123) Google Scholar), a major unanswered question is how these nutrient stimuli are "sensed" by cells to initiate signaling. Does sensing of amino acids occur extracellularly, e.g. via a membrane bound receptor or amino acid transporter or intracellularly? This question has important implications for the way in which animal cells respond to changes in nutrient availability, because cytosolic and extracellular amino acid pools may be far from equilibrium. In an attempt to address these key questions, we have investigated AA sensing in the X. laevis oocyte. X. laevisoocytes are large single cells of up to 1 mm in diameter, which possess active translational and transcriptional machinery, and are suitable for microinjection of substances (e.g. of nutrients in solution) and overexpression of proteins (e.g. nutrient transporters). In comparison to typical mammalian cell culture systems, which are necessarily grown in nutrient-rich media making it more difficult to study AA-sensing mechanisms, stage VI oocytes can survive in nutrient-deprived media for several days if necessary and are arrested in meiotic prophase. Nevertheless, they receive a maternal nutrient supply in vivo and may be predicted to respond to nutrients including AAs in a manner similar to mammalian cells. Therefore, X. laevis oocytes represent a useful experimental system in which we could accurately manipulate intracellular as well as extracellular AA concentrations within physiologically relevant limits to study AA sensing mechanisms using p70S6K activity as a downstream reporter. The high sequence identity between X. laevis p70S6K and mammalian (rat) p70S6K(93%) plus conservation of all the phosphorylation sites and regulatory motifs present in the mammalian enzyme (15Schwab M.S. Kim S.H. Terada N. Edfjall C. Kozma S.C. Thomas G. Maller J.L. Mol. Cell. Biol. 1999; 19: 2485-2494Crossref PubMed Scopus (56) Google Scholar) supported the use of antibodies developed for mammalian p70S6K. These features of the Xenopus oocyte coupled with its relative impermeability to AAs (16Peter G.J. Davidson I.G. Ahmed A. McIlroy L. Forrester A.R. Taylor P.M. Biochem. J. 1996; 318: 915-922Crossref PubMed Scopus (20) Google Scholar, 17Taylor P.M. Kaur S. Mackenzie B. Peter G.J. J. Exp. Biol. 1996; 199: 923-931PubMed Google Scholar) have allowed us to distinguish between effects of intracellular and extracellular nutrients in an animal cell. Unless otherwise specified, chemicals were obtained from Sigma. d-[3H]sucrose, [3H]tryptophan, [3H]glutamine, and [3H]phenylalanine were all purchased from PerkinElmer Life Sciences. Oocytes were isolated by collagenase treatment of ovarian tissue obtained from mature femaleX. laevis toads (South African Xenopus facility) using methods described previously (16Peter G.J. Davidson I.G. Ahmed A. McIlroy L. Forrester A.R. Taylor P.M. Biochem. J. 1996; 318: 915-922Crossref PubMed Scopus (20) Google Scholar). Defolliculated, stages V–VI (prophase-arrested) oocytes were selected and maintained at 18 °C in modified Barth's medium (MBM) containing (in mm) 88 NaCl, 1 KCl, 2.4 NaHCO3, 0.82 MgSO4·72O, 0.66 NaNO3, 0.75 CaCl2·2H2O, and 5.0 Hepes, pH 7.6 (adjusted with Tris base), and 10 mg/liter gentamycin sulfate. Solutions of specified composition (see Figs. Figure 1, Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7) were microinjected into the cytoplasm of individual oocytes (10–20 oocytes/condition). Composition of the AA mix used is described below (for review see Ref. 17Taylor P.M. Kaur S. Mackenzie B. Peter G.J. J. Exp. Biol. 1996; 199: 923-931PubMed Google Scholar and TableI). In parallel, separate oocytes were injected with an identical volume of d-[3H]sucrose solution, and the retained injectate volume was calculated from measurement of oocyte radioactivity using methods described previously (16Peter G.J. Davidson I.G. Ahmed A. McIlroy L. Forrester A.R. Taylor P.M. Biochem. J. 1996; 318: 915-922Crossref PubMed Scopus (20) Google Scholar). This volume was 27.4 ± 4.4 nl/oocyte (mean ± S.E., n = 9 separate typical experiments). Following injection, cells were incubated in MBM at 18 °C for 30 min unless stated otherwise prior to lysis in extraction buffer (10 μl/oocyte) containing a mixture of protein phosphatase and proteinase inhibitors (18Herbert T.P. Kilhams G.R. Batty I.H. Proud C.G. J. Biol. Chem. 2000; 275: 11249-11256Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar) plus 1 μm microcystin LR (Calbiochem).Table IConcentrations of representative AAs in AA mix solutions and calculated changes in intracellular AA concentrations following microinjection of 20× AA mixAAOocyte [AA]i1-aCalculated from data (17) using an estimated oocyte volume of 0.75 μl. Mean increase in individual intracellular AA concentration following microinjection of 27.4 nl of 20× AA/oocyte = 48.5 ± 14.0% (calculated for the 15 AAs where intracellular concentration of AA is known (17)). Complete composition of 1× AA mix is given in text. i, intracellular.1× AA1-bComparable to X. laevisplasma AA concentrations (17).5× AA20× AAIncrease in [AA]i after injection of 20× AA mixmmmmmmmm%Ser0.640.241.214.8627.7Gln0.210.673.3313.34228.4Glu4.800.0750.381.521.2Gly0.550.412.048.1654.5Ala0.850.472.359.4240.3Leu0.250.160.803.2246.4Phe0.170.0550.271.0822.8His0.270.070.341.3620.3Arg0.470.130.662.6420.71-a Calculated from data (17Taylor P.M. Kaur S. Mackenzie B. Peter G.J. J. Exp. Biol. 1996; 199: 923-931PubMed Google Scholar) using an estimated oocyte volume of 0.75 μl. Mean increase in individual intracellular AA concentration following microinjection of 27.4 nl of 20× AA/oocyte = 48.5 ± 14.0% (calculated for the 15 AAs where intracellular concentration of AA is known (17Taylor P.M. Kaur S. Mackenzie B. Peter G.J. J. Exp. Biol. 1996; 199: 923-931PubMed Google Scholar)). Complete composition of 1× AA mix is given in text. i, intracellular.1-b Comparable to X. laevisplasma AA concentrations (17Taylor P.M. Kaur S. Mackenzie B. Peter G.J. J. Exp. Biol. 1996; 199: 923-931PubMed Google Scholar). Open table in a new tab In experiments to determine effects of extracellular AAs, all oocyte incubations (10–20 oocytes/condition) were in MBM-based solutions at 18 °C for 30 min unless stated otherwise prior to cell lysis in extraction buffer as described above. In experiments in which the effects of rapamycin were investigated, cells were preincubated with 100 nm rapamycin (Calbiochem) in MBM for 15 min prior to oocyte microinjection or incubation. Rapamycin (100 nm) was also present during both post-injection and incubation periods. In certain experiments, oocytes were treated with phorbol 12-myristate 13-acetate (PMA, 1 μm) for 30 min to stimulate cell signaling pathways linked to phosphorylation of p70S6K. A 20× AA stock solution was prepared, which when diluted to 1× gave a composition that was comparable to the plasma concentrations found in X. laevis. Concentrations (1×) were (in μm) 38 Asp, 270 Thr, 243 Ser, 63 Asn, 667 Gln, 186 Pro, 76 Glu, 408 Gly, 471 Ala, 173 Val, 46 Met, 90 Ile, 161 Leu, 83 Tyr, 54 Phe, 418 Lys, 68 His, 132 Arg, 38 Cys, and 70 Trp (for review see Ref. 17Taylor P.M. Kaur S. Mackenzie B. Peter G.J. J. Exp. Biol. 1996; 199: 923-931PubMed Google Scholar and Table I). AA solutions (20×) used for intracellular microinjections were prepared in water, whereas extracellular incubation solutions (20, 5, and 1×) were prepared in MBM. Plasmids containing cDNA for the System L transporter subunits 4F2hc (kindly provided by Dr. L.C. Kuhn, Institute of Physiology, University of Zurich, Switzerland) and IU12 (kindly provided by Dr. Y-B Shi, NIH, Rockville, MD) were linearized prior to cRNA synthesis (mMessage mMachine, Ambion). Individual oocytes were co-injected with 4F2hc and IU12 cRNA (50 ng of each cRNA in 50 nl of injected cRNA/oocyte). Oocytes were incubated in MBM for 2–4 days to allow expression of injected cRNA before experimentation. AA transport in oocytes at 2–4 days post-injection was measured at 22 °C as influx of radiolabeled AA tracer. Experiments were carried out in MBM buffer, and uptake of3H-labeled Phe, Gln, or Trp was measured using a method described previously (19Peter G.J. Davies A. Watt P.W. Birrell J. Taylor P.M. Biochem. J. 1999; 343: 169-176Crossref PubMed Google Scholar). Total oocyte lysates were centrifuged at 14,000 × g for 2 min, and the clear supernatant was retained (cleared lysate). The protein concentrations of cleared lysates were determined using the BCA method (Pierce). Proteins from cleared lysates (40 μg of protein/lane) were resolved by SDS-PAGE (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207856) Google Scholar) and transferred electrophoretically onto nitrocellulose membranes (Amersham Biosciences, Inc.) as described previously (21Hyde R. Christie G.R. Litherland G.J. Hajduch E. Taylor P.M. Hundal H.S. Biochem. J. 2001; 355: 563-568Crossref PubMed Scopus (72) Google Scholar). Membranes were probed using antibodies specific for p70S6K (Santa Cruz Biotechnology, Inc.), phosphorylated p70S6K (Thr-389, fromCell Signaling Technologies), phosphorylated p44/42 MAPK (Thr-202/Tyr-204, from Cell Signaling Technologies), and phosphorylated S6 ribosomal protein (Ser-235, kindly supplied by the Division of Signal Transduction Therapy, School of Life Sciences, University of Dundee) and were immunodetected by ECL (Amersham Biosciences, Inc.) using appropriate horseradish peroxidase-conjugated secondary antibodies (Diagnostics Scotland, Carluke, Lanarkshire, Scotland). Blots were quantified using a Bio-Rad GS-670 imaging densitometer. Microinjection of oocytes with 20× total AAs that raises intracellular AA concentrations by small but physiologically relevant amounts (see Table I), produced a mobility shift in p70S6K as detected by immunoblot analysis. This well characterized feature reflects phosphorylation of the p70S6K protein (Fig.1 A). This increase in p70S6K phosphorylation was an AA-specific event and not merely due to microinjection of cells or to osmotic effects of the injectate, because it was not observed in oocytes injected with either water or solutions of l-glucose at concentrations of up to 100 mm in the injectate (Fig. 1 A and Fig. 3). The intracellular AA-induced increase in p70S6Kphosphorylation was rapid, being detected within 20 min after injection (Fig. 1 B), and was sustained up to 3 h post-injection (Figs. 1, A and B). In contrast, the incubation of oocytes with excess extracellular total AAs (5× plasma concentration) for either 1 or 24 h resulted in no significant increase in phosphorylation of p70S6K, visualized by the lack of any significant mobility shift of p70S6K following immunoblot analysis (Fig. 1 C). Phosphorylation of p70S6K following microinjection of AAs was confirmed using a phospho-specific p70S6K antibody (Fig.2), and as an index of intracellular p70S6K activity, we studied the phosphorylation of its substrate S6 using an appropriate phospho-specific (Ser-235) antibody (Fig. 2). The phospho-specific p70S6K antibody we have used detects the phosphorylation of Thr-389, which has previously been demonstrated to be a rapamycin-sensitive/mTOR-dependent phosphorylation site (22Dennis P.B. Pullen N. Kozma S.C. Thomas G. Mol. Cell. Biol. 1996; 16: 6242-6251Crossref PubMed Scopus (225) Google Scholar, 23Weng Q.P. Kozlowski M. Belham C. Zhang A. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). The incubation of oocytes with rapamycin prior to and following microinjection with AAs prevented the AA-induced mobility shift of the p70S6K protein. In line with this finding, rapamycin also prevented the AA-induced phosphorylation of p70S6K, detected using the phospho-specific antibody, and substantially reduced the concomitant phosphorylation of S6 (Fig. 2). Therefore, it appears that a direct increase in intracellular AA concentrations to ∼48% above that normally present within the oocyte (see Table I) results in a TOR-dependent increase in phosphorylation of p70S6K and that of its downstream target ribosomal protein S6, whereas exposure to relatively high concentrations of extracellular AAs (5× plasma levels) had no such effect on this pathway. The observation that the X. laevisoocyte is acutely sensitive to increases in internal but not external AA concentration suggests that this cell type possesses an intracellular AA sensor.Figure 3Effect of microinjection of AA, AA analogues, and l-glucose on phosphorylation of p70S6K inX. laevis oocytes. X. laevis oocytes were microinjected with solutions of indicated composition. Cell lysates were prepared 30 min post-injection and subjected to immunoblot analysis using an antibody specific for phosphorylated p70S6K (Thr-389). A, leucine (L-isomer and D-isomer) andl-leucinamide were all injected at 3.2 mm(equivalent to 20× Leu). B, all amino acids were injected at their respective 20× AA mix concentrations (3.2 mm Leu, 1.36 mm His, 3.72 mm Pro, 1.52 mm Glu in upper panel; 9.42 mmAla, 8.16 mm Gly, 13.34 mm Gln, 2.64 mm Arg, 8.36 mm Lys in lower panel. See Table I for additional details).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2Microinjection of AAs or Leu alone inX. laevis oocytes results in increased phosphorylation of p70S6K and S6 ribosomal protein. X. laevis oocytes (uninjected, U) were microinjected with water (W) 20× AAs or 3.2 mm Leu (20× Leu,L) ± rapamycin (R). Cell lysates were prepared 30 min post-injection and subjected to immunoblot analysis using antibodies specific for total p70S6K (top panel), phosphorylated p70S6K (Thr-389) (middle panel), or phosphorylated ribosomal protein S6 (Ser-235) (bottom panel). In top panel, upward bandshift (p70S6K (PP) shown byarrowheads) indicates phosphorylated forms of this protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Microinjection of l-Leu (20× plasma concentration) alone was sufficient to induce p70S6K phosphorylation and pathway activation over the same timescale (Fig. 2). l-Leucinamide was able to produce a similar effect to l-Leu, butd-Leu was ineffective (Fig.3 A). Certain otherl-AAs were able to significantly increase the phosphorylation of p70S6K when microinjected into X. laevis oocytes alone at 20× plasma concentration such as Trp (see below), Phe, Arg, Lys, and Gly but not Gln, Glu, Pro, or Ala (for example, see Fig. 3 B). As Leu by itself induced increased phosphorylation of p70S6K, we explored the dose dependence of these effects by microinjecting various concentrations of Leu (Fig.4). Relatively small increases in intracellular Leu concentration resulted in increased phosphorylation of p70S6K (e.g. injecting 1 mm Leu results in an ∼7% increase in intracellular Leu concentration and appears to induce nearly 50% maximal increase in p70S6Kphosphorylation, see Fig. 4 and Table I). The large size and low surface area/volume ratio of the X. laevis oocyte compared with mammalian cells in addition to a relatively low surface permeability to AAs means that intracellular AA concentrations rise relatively slowly as a result of any increase in extracellular AA concentrations (17Taylor P.M. Kaur S. Mackenzie B. Peter G.J. J. Exp. Biol. 1996; 199: 923-931PubMed Google Scholar). Overexpression of the System L AA transport system (24Christensen H.N. Physiol. Rev. 1990; 70: 43-77Crossref PubMed Scopus (965) Google Scholar) in the oocyte plasma membrane (25Ritchie J.W. Peter G.J. Shi Y.B. Taylor P.M. J. Endocrinol. 1999; 163: R5-R9Crossref PubMed Scopus (60) Google Scholar) markedly increases surface permeability to many neutral AAs including Leu, Trp, and Phe, causing for example an 11× increase in [3H]Trp uptake (Fig.5 C) and a 16× increase in [3H]Phe uptake (results not shown) at 1× AA mix concentrations compared with control oocytes. For System L-overexpressing oocytes incubated in 1× AA mix, we estimate that this represents a potential to increase intraoocyte concentrations of AAs such as Phe by up to 75% after 30 min (values approaching those estimated for typical mammalian cells, for review see Ref. 26Taylor P.M. Rennie M.J. Low S.Y. Van Winkle L. Biomembrane Transport. Academic Press, San Diego, CA1999: 295-325Crossref Google Scholar). Thus, this possibility offers an opportunity to test further the idea that AA sensing is intracellular. When cells overexpressing System L were incubated for 30 min in either plasma AA mix (1 or 20×, Fig. 5,A and B) or 1× Leu (Fig. 5 D), there was now an increase in p70S6K phosphorylation that was not seen in similarly treated control cells or in System L-expressing cells incubated in MBM alone (Fig. 5, A and B). As observed for cells microinjected directly with AAs, this increase in p70S6K phosphorylation was also inhibited by rapamycin, again implicating TOR-dependent signaling in the AA-induced response (Fig. 5, A and D). Rapamycin (Fig.5 C) or Me2SO (vehicle for rapamycin stock, results not shown) had no significant effect upon Trp uptake by oocytes overexpressing System L. To confirm that the effects of overexpressed System L AA-dependent p70S6K phosphorylation were a response to cellular AA entry, oocytes were incubated with 1× plasma AAs plus 5 mm 2-amino-bicyclo[2,2,1]hepta-2-carboxylic acid (BCH), a specific inhibitor of System L-dependent transport (24Christensen H.N. Physiol. Rev. 1990; 70: 43-77Crossref PubMed Scopus (965) Google Scholar). 5 mm BCH almost completely inhibited the activity of overexpressed System L transport measured as uptake of [3H]Trp at physiological concentration (Fig.5 C). BCH abolished the ability of external AA to increase p70S6K phosphorylation (Fig. 5 B), presumably because it is blocking AA entry into the cells through System L (Fig.5 C) and preventing any subsequent sensing and signaling to TOR/p70S6K (Fig. 5 B). BCH microinjected into oocytes had no effect itself upon the phosphorylation of p70S6K (Fig.6 A). Yeast cells are believed to sense a decrease in intracellular AA concentration as a consequence of a decrease in tRNA charging (27Wek S.A. Zhu S. Wek R.C. Mol. Cell. Biol. 1995; 15: 4497-4506Crossref PubMed Google Scholar, 28Hinnebusch A.G. Microbiol. Rev. 1988; 52: 248-273Crossref PubMed Google Scholar, 29Qiu H. Dong J. Hu C. Francklyn C.S. Hinnebusch A.G. EMBO J. 2001; 20: 1425-1438Crossref PubMed Scopus (69) Google Scholar). In this study, the injection of excess tRNA (460 ng/oocyte, type Y-SA from bakers' yeast in water) had no effect upon p70S6K phosphorylation (data not shown). To investigate further the possible involvement of tRNA aminoacylation in regulation of p70S6K, we microinjected three available AA alcohols (leucinol, tryptophanol, and alaninol) as competitive inhibitors of aminoacyl-tRNA charging by tRNA synthetases (30Vaughan M.H. Hansen B.S. J. Biol. Chem. 1973; 248: 7087-7096Abstract Full Text PDF PubMed Google Scholar). Surprisingly, increased intracellular concentrations of leucinol and tryptophanol resulted in increased phosphorylation of p70S6K, although their effects were less potent than those of the respective AAs (Fig.6 B). In contrast, alaninol like Ala itself was without effect (data not shown). As leucinamide proved an effective substitute for Leu as an activator of the p70S6K pathway in X. laevis oocytes (Fig.3 A), we also examined the effect of KIC, the natural aminotransferase product of Leu. Increasing the intracellular concentration of KIC by microinjection resulted in increased phosphorylation of p70S6K (Fig. 6 C). In an attempt to isolate any contribution of intracellular KIC-Leu interconversions to the observed response, the effects of microinjecting AOAA, an inhibitor of aminotransferases, were also investigated. Microinjection of AOAA alone (100 mm in injectate producing an estimated 3.6 mm final concentration in cytosol) had no effect on basal p70S6K phosphorylation, but it inhibited the stimulatory effects of co-injected KIC, Leu, or AA mix on p70S6K phosphorylation (Fig. 6 C). AOAA also unexpectedly blocked the stimulatory effects of injected leucinol and tryptophanol on p70S6K phosphorylation (Fig. 6,C and D). To exclude the possibility that AOAA was exerting a general toxic effect on oocytes, we demonstrated that the presence of AOAA had no effect on (a) activation of p70S6K by another stimulus (PMA, see Fig.7) or (b) activation of the p44/42 MAPK (Erk) pathway (Fig. 7). These results argue for a relatively specific effect of AOAA in our experiments and also show that Leu activation of p70S6K does not involve activation of the ERK pathway. Therefore, it is possible that the observed effects of AOAA result from it inhibiting the putative intracellular AA sensor rather than through inhibition of aminotransferases of which AA alcohols are not substrates to our knowledge. The data presented here show that the X. laevis oocyte is able to sense relatively small increases in intracellular AAs such as Leu, resulting in increased phosphorylation of both p70S6K and its physiological downstream target ribosomal protein S6. The activation of p70S6K is thought to up-regulate rapidly the translation of 5′-terminal tract of pyrimidines mRNAs encoding proteins involved in mRNA translation, thus increasing the overall capacity of the cell for protein synthesis in response to an increase in AA (substrate) availability. The ability of intracellular AA to activate p70S6K in oocytes is inhibited by rapamycin, indicating an important role for the TOR pathway in this response to AA as also reported for mammalian cells. In a number of mammalian cell types, (m)TOR has been demonstrated to play a pivotal role in AA-sensitive signaling responses, acting as a checkpoint for integration of detected changes in AA availability and signaling to proteins involved in regulation of mRNA translation such as 4E-BP1 and p70S6K (5Gingras A.C. Raught B. Sonenberg N. Genes Dev. 2001; 15: 807-826Crossref PubMed Scopus (1193) Google Scholar, 6Rohde J. Heitman J. Cardenas M.E. J. Biol. Chem. 2001; 276: 9583-9586Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). In X. laevis oocytes, it appears that intracellular rather than extracellular AA levels must increase to stimulate phosphorylation of p70S6K, implying the existence of an intracellular rather than extracellular AA-sensing mechanism. Oocytes became sensitive to elevated extracellular AA concentrations only when their surface permeability to AAs such as Leu was greatly increased by the overexpression of a System L transporter. It is the increased capacity to transport AAs into the cell via System L rather than the presence of the transporter protein itself that renders the cell more sensitive to extracellular AAs, because cells overexpressing the System L transport system incubated in MBM showed no detectable changes in p70S6K phosphorylation. Because of the low AA transport capacity in native X. laevis oocytes, extracellular AAs will enter the cell at a much slower rate than in cells overexpressing an AA transport system, or indeed cells in which AAs are microinjected directly into the cytoplasm. The increase in intracellular AA or Leu levels necessary to increase p70S6K phosphorylation in oocytes is relatively small, indicating that the sensing mechanism can detect small changes above basal values. Indeed, increasing intracellular Leu concentration by only ∼7% (Fig. 4) produces a detectable increase in the phosphorylation of p70S6K compared with uninjected cells or cells injected with water. Such increases in intracellular AA concentration are well within physiological limits. For example, in rats that have been fasted for 60 h, re-feeding with protein results in increases in muscle and liver Leu concentrations of 81 and 85%, respectively, within 1 h of the meal (26Taylor P.M. Rennie M.J. Low S.Y. Van Winkle L. Biomembrane Transport. Academic Press, San Diego, CA1999: 295-325Crossref Google Scholar). Our findings raise the question of the nature of the putative AA intracellular sensor(s). Are AAs detected directly by an AA "receptor," or are indirect indices of AA concentration(e.g. transaminase activity, tRNA charging, see below) monitored by the systems triggering nutrient-sensitive signaling pathways? A transmembrane AA receptor (Ssy1p) has been characterized in yeast (31Forsberg H. Ljungdahl P.O. Mol. Cell. Biol. 2001; 21: 814-826Crossref PubMed Scopus (131) Google Scholar), but no structural homologue has been identified to date in higher organisms. In rat hepatocytes, however, Miotto et al. (32Miotto G. Venerando R. Marin O. Siliprandi N. Mortimore G.E. J. Biol. Chem. 1994; 269: 25348-25353Abstract Full Text PDF PubMed Google Scholar) have shown that the antigen peptide derivative Leu-8-multiple antigen peptide, which is not transported into hepatocytes, was as effective as Leu at suppressing macroautophagy and proteolysis. On this basis, they proposed site(s) on the plasma membrane that can generate intracellular signals in response to stimulation by extracellular Leu or Leu analogues (32Miotto G. Venerando R. Marin O. Siliprandi N. Mortimore G.E. J. Biol. Chem. 1994; 269: 25348-25353Abstract Full Text PDF PubMed Google Scholar). More recently, Conigrave et al. (33Conigrave A.D. Quinn S.J. Brown E.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4814-4819Crossref PubMed Scopus (427) Google Scholar) have suggested that the functional properties of a Ca2+-receptor reveal a potential role as a sensor for AAs. However, the effects of Ca2+-receptor activation by AAs on downstream signaling targets (e.g. TOR, 4E-BP1, or p70S6K) have not been investigated (33Conigrave A.D. Quinn S.J. Brown E.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4814-4819Crossref PubMed Scopus (427) Google Scholar). Ca2+-receptor was found to be more sensitive to aromatic AAs than branched chain AAs. Leu, for example, was found to be ineffective in activating this sensor. This observation contrasts with our results and those of others (7Wang X. Campbell L.E. Miller C.M. Proud C.G. Biochem. J. 1998; 334: 261-267Crossref PubMed Scopus (295) Google Scholar, 8Hara 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 (1139) Google Scholar) showing that the branched chain AAs (particularly Leu) act as potent initiators of AA sensing and signaling effects. Our results are consistent with the presence of an intracellular mechanism that senses certain neutral and cationic AAs, also recognizing at least some of their respective AA alcohols, amides, and ketoacids, and activates the TOR pathway, resulting in downstream phosphorylation of p70S6K and S6. We were unable to exclude the possibility that the effects of the ketoacid KIC resulted from it being transaminated to Leu, because the transaminase inhibitor AOAA somehow appeared to inhibit the AA-sensing and/or signaling mechanism. This effect of AOAA was relatively specific to AAs and did not prevent the phorbol ester PMA from stimulating phosphorylation of p70S6K. We speculate that AOAA may act as a direct inhibitor of the AA sensor mechanism in oocytes, in line with previous work indicating that certain AA derivatives may block leucine-induced activation of p70S6K in H4IIE hepatoma cells (34Shigemitsu K. Tsujishita Y. Miyake H. Hidayat S. Tanaka N. Hara K. Yonezawa K. FEBS Lett. 1999; 447: 303-306Crossref PubMed Scopus (87) Google Scholar). The structural specificity of the sensor mechanism inXenopus oocytes appears broadly similar to that observed in mammalian cells (e.g. H4IIE hepatoma (34Shigemitsu K. Tsujishita Y. Miyake H. Hidayat S. Tanaka N. Hara K. Yonezawa K. FEBS Lett. 1999; 447: 303-306Crossref PubMed Scopus (87) Google Scholar)), favoring leucine over many other AAs and L-isomers over D-isomers, although it appears that the carboxylate group is not essential. Our data contrast with a previous study (9Iiboshi Y. Papst P.J. Kawasome H. Hosoi H. Abraham R.T. Houghton P.J. Terada N. J. Biol. Chem. 1999; 274: 1092-1099Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) where AA alcohols suppressed the activity of p70S6K in Jurkat cells. One possible explanation for this difference is that in the current study we are increasing intracellular AA alcohols directly by microinjection, whereas in previous studies, cells were incubated with high extracellular concentrations of AA alcohols (9Iiboshi Y. Papst P.J. Kawasome H. Hosoi H. Abraham R.T. Houghton P.J. Terada N. J. Biol. Chem. 1999; 274: 1092-1099Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Increased tRNA charging is most unlikely to be involved in the sensing mechanism because certain AA alcohols and leucinamide as well as AAs stimulated p70S6K. Other recent work (35Dennis P.B. Jaeschke A. Saitoh M. Fowler B. Kozma S.C. Thomas G. Science. 2001; 294: 1102-1105Crossref PubMed Scopus (813) Google Scholar) also indicates that AA pool size rather than the amount of aminoacylated tRNA is important for (m)TOR signaling, and furthermore it is reported (Jousse and Ron, quoted in (36Raught B. Gingras A-C Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7037-7044Crossref PubMed Scopus (511) Google Scholar)) that the mammalian GCN2 kinase, which is believed to sense tRNA charging levels, is not required for nutrient-dependent activation of the (m)TOR pathway. The possibility that signaling is initiated in response to altered general AA metabolism, or resulting changes in cell energy status also seems very unlikely, because metabolically active AAs such as Ala or Gln were without any stimulatory effect. In conclusion, we present evidence that X. laevis oocytes possess an intracellular AA sensor that detects increased intracellular concentrations of AAs and modulates signaling through the TOR pathway. Using X. laevis oocytes, we have demonstrated that the selectivity of this putative intracellular sensor for AAs is similar to that observed in mammalian cells. Although overexpression of an AA transporter does not itself affect TOR signaling, this does allow extracellular AAs to stimulate p70S6K. The AA transporter thus acts as a conduit rather than a sensing or signaling element. The ability to detect and respond appropriately to small changes in AA availability well within physiological ranges should enable a cell to fine-tune the energy-expensive process of protein synthesis in relation to nutritional circumstances. Therefore, the X. laevisoocyte provides a useful model system for further investigating AA sensing and signaling pathways in vertebrate cells.

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