Amino Acid-dependent Control of p70s6k
1999; Elsevier BV; Volume: 274; Issue: 2 Linguagem: Inglês
10.1074/jbc.274.2.1092
ISSN1083-351X
AutoresYasuhiko Iiboshi, Philip J. Papst, Hideki Kawasome, Hajime Hosoi, Robert T. Abraham, Peter J. Houghton, Naohiro Terada,
Tópico(s)Protein Kinase Regulation and GTPase Signaling
ResumoIn human T-lymphoblastoid cells, downstream signaling events of mammalian target of rapamycin (mTOR), including the activity of p70s6k and phosphorylation of eukaryotic initiation factor 4E-binding protein 1, were dependent on amino acid concentration in the culture media, whereas other growth-related protein kinases were not. Amino acid-induced p70s6kactivation was completely inhibited by rapamycin but only partially inhibited by wortmannin. Moreover, amino acid concentration similarly affected the p70s6k activity, which was dependent on a rapamycin-resistant mutant (S2035I) of mTOR. These data indicate that mTOR is required for amino acid-dependent activation of p70s6k. The mechanism by which amino acids regulate p70s6k activity was further explored: 1) amino acid alcohols, which inhibit aminoacylation of tRNA by their competitive binding to tRNA synthetases, suppressed p70s6k activity; 2) suppression of p70s6k by amino acid depletion was blocked by cycloheximide or puromycin, which inhibit utilization of aminoacylated tRNA in cells; and 3) in cells having a temperature-sensitive mutant of histidyl tRNA synthetase, p70s6k was suppressed by a transition of cells to a nonpermissible temperature, which was partially restored by addition of high concentrations of histidine. These results indicate that suppression of tRNA aminoacylation is able to inhibit p70s6k activity. Deacylated tRNA may be a factor negatively regulating p70s6k. In human T-lymphoblastoid cells, downstream signaling events of mammalian target of rapamycin (mTOR), including the activity of p70s6k and phosphorylation of eukaryotic initiation factor 4E-binding protein 1, were dependent on amino acid concentration in the culture media, whereas other growth-related protein kinases were not. Amino acid-induced p70s6kactivation was completely inhibited by rapamycin but only partially inhibited by wortmannin. Moreover, amino acid concentration similarly affected the p70s6k activity, which was dependent on a rapamycin-resistant mutant (S2035I) of mTOR. These data indicate that mTOR is required for amino acid-dependent activation of p70s6k. The mechanism by which amino acids regulate p70s6k activity was further explored: 1) amino acid alcohols, which inhibit aminoacylation of tRNA by their competitive binding to tRNA synthetases, suppressed p70s6k activity; 2) suppression of p70s6k by amino acid depletion was blocked by cycloheximide or puromycin, which inhibit utilization of aminoacylated tRNA in cells; and 3) in cells having a temperature-sensitive mutant of histidyl tRNA synthetase, p70s6k was suppressed by a transition of cells to a nonpermissible temperature, which was partially restored by addition of high concentrations of histidine. These results indicate that suppression of tRNA aminoacylation is able to inhibit p70s6k activity. Deacylated tRNA may be a factor negatively regulating p70s6k. The p70 S6 kinase (p70s6k) 1The abbreviations used are: s6k, S6 kinase; 4E-BP1, eIF-4E-binding protein 1; PI3K, phosphatidyl inositol-3 kinase; MeAIB, methyl 2-aminoisobutyric acid; eIF, eukaryotic (or eucaryotic) initiation factor; TOR, target of rapamycin; mTOR, mammalian TOR; FCS, fetal calf serum; WT, wild type; ts, temperature-sensitive. is a serine/threonine kinase that is ubiquitously activated at the G0/G1 transition of the cell cycle in mammalian cells (1Reinhard C. Thomas G. Kozma S.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4052-4057Crossref PubMed Scopus (98) Google Scholar, 2Susa M. Olivier A.R. Fabbro D. Thomas G. Cell. 1989; 57: 817-824Abstract Full Text PDF PubMed Scopus (80) Google Scholar, 3Terada N. Franklin R.A. Lucas J.J. Blenis J. Gelfand E.W. J. Biol. Chem. 1993; 268: 12062-12068Abstract Full Text PDF PubMed Google Scholar). This protein kinase phosphorylates 40 S ribosomal S6 protein at five serine residues near the carboxyl terminus in vitro (4Bandi H.R. Ferrari S. Krieg J. Meyer H.E. Thomas G. J. Biol. Chem. 1993; 268: 4530-4533Abstract Full Text PDF PubMed Google Scholar). Studies using targeted disruption of thep70 s6k gene (5Kawasome H. Papst P. Webb S. Keller G.M. Johnson G.L. Gelfand E.W. Terada N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5033-5038Crossref PubMed Scopus (161) Google Scholar) and using transfection of a dominant negative or a rapamycin-resistant mutant ofp70 s6k (6Jefferies H.B.J. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (813) Google Scholar, 7von Manteuffel S.R. Dennis P.B. Pullen N. Gingras A.-C. Sonenberg N. Thomas G. Mol. Cell. Biol. 1997; 17: 5426-5436Crossref PubMed Scopus (212) Google Scholar) have shown that p70s6kmediates S6 phosphorylation in vivo. S6 phosphorylation has been proposed to be a factor regulating initiation of mRNA translation (see review in Ref. 8Proud C.G. Curr. Top. Cell Regul. 1992; 32: 243-369Crossref PubMed Scopus (165) Google Scholar), and the studies described above (5Kawasome H. Papst P. Webb S. Keller G.M. Johnson G.L. Gelfand E.W. Terada N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5033-5038Crossref PubMed Scopus (161) Google Scholar,6Jefferies H.B.J. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (813) Google Scholar) concluded that the role of p70s6k in cell proliferation resides in specific regulation of translation of mRNAs encoding ribosomal proteins. Thus, p70s6k activity is considered to be a factor required for up-regulation of ribosomal biogenesis, which facilitates G1 progression during the cell cycle (9.Terada, N., Lucas, J. J., Szepesi, A., Franklin, R. A., Takase, K., and Gelfand, E. W. (1992) 186,1315–1321.Google Scholar). In regards to the activation of p70s6k, it appears there are at least two upstream regulatory pathways; one is through phosphatidyl inositol-3 kinase (PI3K) and the other is through mammalian target of rapamycin (mTOR; also termed FRAP and RAFT). Many growth factors, such as platelet-derived growth factor and epidermal growth factor, activate PI3K through their binding to specific receptors, and previous studies using chemical inhibitors of PI3K, mutant platelet-derived growth factor receptors, and mutant PI3Ks, all indicated that PI3K is involved in the activation of p70s6kby growth factors (10Cheatham B. Vlahos C.L. Cheatham L. Wang L. Blenis J. Kahn C.R. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (1002) Google Scholar, 11Chung J. Grammer T.C. Lemon K.P. Kazlauskas A. Blenis J. Nature. 1994; 370: 71-75Crossref PubMed Scopus (656) Google Scholar, 12Weng Q.-P. Andrabi K. Klippel A. Kozlowski M.T. Williams L.T. Avruch J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5744-5748Crossref PubMed Scopus (202) Google Scholar, 13Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1884) Google Scholar). Indeed, it has been demonstrated recently that PDK1, a downstream kinase of PI3K, phosphorylates p70s6k at Thr229 and activates the kinase (14Alessi D.R. Kozlowski M.T. Weng Q.-P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar,15Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (731) Google Scholar). The involvement of mTOR in the regulation of p70s6k has been initially demonstrated by studies using the immunosuppressive drug rapamycin (see reviews in Refs. 16Abraham R.T. Wiederrecht G.J. Annu. Rev. Immunol. 1996; 14: 483-510Crossref PubMed Scopus (575) Google Scholar and 17Brown E.J. Schreiber S.L. Cell. 1996; 86: 517-520Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Rapamycin associates with a cellular protein FKBP12 in cells, and the rapamycin-FKBP12 complex then binds to mTOR. It has also been demonstrated that mTOR is required for the inhibitory action of rapamycin on p70s6k (18Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (619) Google Scholar). Moreover, mTOR has an intrinsic protein serine/threonine kinase activity and regulates the activity and phosphorylation of p70s6k in vivo in a manner that is dependent on the kinase activity of mTOR (18Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (619) Google Scholar). Although initial reports concluded that mTOR did not directly phosphorylate p70s6k, it has been demonstrated that mTOR phosphorylates p70s6k at Thr389 in vitro (19Burnett P.E. Barrow R.K. Cohen N.A. Snyder S.H. Sabatini D.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1432-1437Crossref PubMed Scopus (947) Google Scholar). In addition to p70s6k, mTOR regulates another translation regulatory molecule, eIF-4E-binding protein 1 (4E-BP1) (20Lin T.-A. Kong X. Saltiel A.R. Blackshear P.J. Lawrence Jr., J.C. J. Biol. Chem. 1995; 270: 18531-18538Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 21Beretta L. Gingras A.-C. Svitkin Y.V. Hall M.N. Sonenberg N. EMBO J. 1996; 15: 658-664Crossref PubMed Scopus (604) Google Scholar, 22von Manteuffel S.R. Gingras A.-C. Ming X.-F. Sonenberg N. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4076-4080Crossref PubMed Scopus (223) Google Scholar, 23Hara K. Yonezawa K. Kozlowski M.T. Sugimoto T. Andrabi K. Weng Q.-P. Kasuga M. Nishimoto I. Avruch J. J. Biol. Chem. 1997; 272: 26457-26463Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar), independent of p70s6k activity (5Kawasome H. Papst P. Webb S. Keller G.M. Johnson G.L. Gelfand E.W. Terada N. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5033-5038Crossref PubMed Scopus (161) Google Scholar, 6Jefferies H.B.J. Fumagalli S. Dennis P.B. Reinhard C. Pearson R.B. Thomas G. EMBO J. 1997; 16: 3693-3704Crossref PubMed Scopus (813) Google Scholar). mTOR is demonstrated to phosphorylate the translation repressor protein 4E-BP1 at its serine and threonine residues (24Brunn G.J. Hudson C.C. Sekulic A. Williams J.M. Hosoi H. Houghton P.J. Lawrence Jr., J.C. Abraham R.T. Science. 1997; 277: 99-101Crossref PubMed Scopus (814) Google Scholar). The hypophosphorylated species of 4E-BP1 binds tightly to eIF-4E (an N7-methylguanosine cap-binding subunit of eIF-4F complex) and prevents eIF-4E from associating with eIF-4G (a scaffolding protein in eIF-4F complex). The phosphorylation of 4E-BP1 by mTOR is thought to release eIF-4E and facilitate translational initiation of capped mRNA (21Beretta L. Gingras A.-C. Svitkin Y.V. Hall M.N. Sonenberg N. EMBO J. 1996; 15: 658-664Crossref PubMed Scopus (604) Google Scholar). Thus, mTOR regulates 1) ribosomal protein synthesis at the level of mRNA translation through p70s6k activity, and 2) overall protein synthesis by controlling translational initiation of capped mRNA through 4E-BP1. In contrast to the PI3K pathway, which is activated by various growth factors, it was unclear what factor physiologically regulated mTOR. A study on proteolytic responses to amino acid deprivation demonstrated that ribosomal S6 phosphorylation was induced by supplementation of amino acids (25Blommaart E.F.C. Luiken J.J.F.P. Blommaart P.J.E. van Woerkom G.M. Meijer A.J. J. Biol. Chem. 1995; 270: 2320-2326Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar), suggesting that amino acid concentration in culture media may be a regulatory factor for p70s6k. Indeed, Foxet al. (26Fox H.L. Kimball S.R. Jefferson L.S. Lynch C.J. Am. J. Physiol. 1998; 274: C206-C213Crossref PubMed Google Scholar) demonstrated that amino acids stimulate phosphorylation of p70s6k in rat adipocytes. More recently, Hara et al. (27Hara 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 (1132) Google Scholar) have reported that amino acid concentration regulates p70s6k activity and phosphorylation of 4E-BP1 in Chinese hamster ovary cells. The study demonstrated that a rapamycin-resistant mutant (p70Δ2–46/ΔCT104) of p70s6kwas resistant to amino acid deprivation, indicating that amino acid sufficiency and mTOR both signal to p70s6k through a common effector, which could be mTOR itself or an mTOR-controlled downstream element. In this study, we further explored the mechanism by which amino acid concentration regulates p70s6k. Human T-lymphoblastoid Jurkat cells were obtained from ATCC. Human alveolar rhabdomyosarcoma Rh30 cells were established from the bone marrow of a patient with metastatic tumor (28Douglass E.C. Valentine M. Etcubanas E. Parham B.L. Weber P.J. Houghton P.J. Houghton J.A. Green A.A. Cytogenet. Cell. Genet. 1987; 45: 148-155Crossref PubMed Scopus (296) Google Scholar). Rh30 cells constitutively expressing wild type mTOR (WT-mTOR) or a rapamycin-resistant mTOR (S2035I) were obtained by transfection of cells with pcDNA3-AU1mTORwt or pcDNA3-AU1mTORSI. BHK21 cells and their temperature-sensitive mutant temperature-sensitive (ts) BN250 were obtained from RIKEN cell bank (Tsukuba, Japan). Rapamycin and wortmannin were obtained from Calbiochem (San Diego, CA), and stored in ethanol (1 mg/ml) and dimethyl sulfoxide (10 mm), respectively. Cycloheximide (Sigma) was dissolved in ethanol and stored as a 10 mg/ml stock solution. Puromycin (Sigma) was stored in distilled water (2 mg/ml). Amino acid alcohols (Sigma) l-leucinol,l-phenylalaninol, l-alaninol,l-histidinol, l-tyrosinol,l-methioninol, and d-leucinol were stored in distilled water (5 m). Methyl 2-aminoisobutyric acid (MeAIB) and 2-amino-2-norbornane-carboxylic acid were obtained from Sigma and stored in distilled water at 200 and 100 mm, respectively. Jurkat cells and Rh30 cells were maintained in RPMI 1640 medium (Life Technologies, Inc.) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) (HyClone, Logan, UT), 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies). For amino acid deprivation, exponentially growing cells were washed twice with amino acid-free RPMI 1640 medium (RPMI 1640 select-amine kit, catalog no. 17402, Life Technologies) and resuspended at 5 × 105 cells/ml in the same amino acid-free RPMI 1640 medium supplemented with 10% dialyzed FCS (catalog no. 26300, Life Technologies) for the indicated times at 37 °C in a 5%-CO2 incubator. In some experiments, individual amino acids were deprived instead of total amino acid deprivation. For amino acid supplementation, cells (5 × 105 cells/ml) were incubated in the amino acid-free RPMI 1640 medium supplemented with 10% dialyzed FCS for 3–16 h. The same volume of RPMI 1640 medium containing 2× concentrations of amino acids +10% dialyzed FCS were added to the culture, and cells were incubated for the indicated times. The concentrations (in μm) of amino acids (1×) in RPMI 1640 (Life Technologies) were as follows: Gln, 2050; Asn, 380; His, 100; Trp 24; Pro, 170; Cyst, 410; Gly, 130; Val, 170; Leu, 380; Ile, 380; Ser, 290; Thr, 170; Phe, 90; Tyr, 110; Met, 100; Glu, 140; Asp 150; Arg, 1150; and Lys, 220. BHK21 and tsBN250 cells were maintained in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 10% (v/v) heat-inactivated FCS (HyClone) at 33 °C in a 5%-CO2 incubator. For transition of cells to nonpermissible temperature, culture plates were transferred to a 5%-CO2 incubator at 39 °C. Cells (2 × 106) were harvested, and the activities of the kinases were measured as we described previously (3Terada N. Franklin R.A. Lucas J.J. Blenis J. Gelfand E.W. J. Biol. Chem. 1993; 268: 12062-12068Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were lysed in a buffer containing 10 mm potassium phosphate, 1 mmEDTA, 5 mm EGTA, 10 mm MgCl2, 50 mm β-glycerophosphate, 1 mmNa3VO4, 2 mm dithiothreitol, 40 μg/ml phenylmethylsulfonyl fluoride, and 0.1% Nonidet P-40. The protein kinases in 250 μg of total cellular proteins were immunoprecipitated using specific antibodies: for p70s6k, a rabbit polyclonal antibody raised against the carboxyl-terminal 18 amino acids of p70s6k (sc-230, Santa Cruz Biotechnology, Santa Cruz, CA); for Akt, a goat polyclonal antibody against an epitope corresponding to amino acids 461–480 of human Akt1 (sc-1618, Santa Cruz Biotechnology); for p90rsk, a rabbit polyclonal antibody raised against an epitope 682–724 of mouse Rsk1 (06–185, Upstate Biotechnology, Lake Placid, NY); and for Cdk2, a rabbit polyclonal antibody raised against an epitope 283–298 of human Cdk2 (sc-163, Santa Cruz Biotechnology). The kinase activity was measured by incorporation of 32P into specific substrate peptides (for p70s6k and p90rsk, an S6 peptide, RRRLSSLRA; for Akt, a Gsk3 peptide, GRPRTSSFAEG; for Cdk2, a histone H1 peptide, AVAAKKSPKKAKKPA). Cells (5 × 106) were washed with phosphate-buffered saline and lysed at 4 °C with 25 mm Tris-HCl, pH 7.4, 50 mm NaCl, 0.5% sodium deoxycholate, 2% Nonidet P-40, 0.2% SDS, 1 μmphenylmethylsulfonyl fluoride, 50 μg/ml aprotinin, 50 μm leupeptin. Lysates were resolved by SDS 7.5% (for p70s6k) or 15% (for 4E-BP1) polyacrylamide gels and transferred to nitrocellulose filters. After blocking of the filters with a solution containing 1% bovine serum albumin, the filters were incubated with primary antibodies. The antibodies used for p70s6k are the same as described above. For 4E-BP1, a rabbit polyclonal antibody raised against a recombinant His-tagged rat PHAS1 (4E-BP1) was used (24Brunn G.J. Hudson C.C. Sekulic A. Williams J.M. Hosoi H. Houghton P.J. Lawrence Jr., J.C. Abraham R.T. Science. 1997; 277: 99-101Crossref PubMed Scopus (814) Google Scholar). Specific reactive proteins were detected by the ECL method, employing a donkey anti-rabbit Ig antibody linked to horseradish peroxidase (Amersham Pharmacia Biotech). Cells (2 × 106) were harvested, centrifuged at 1500 rpm for 5 min, and resuspended in 100 μl of a buffer containing 140 mm NaCl, 5.4 mmKCl, 1.8 mm CaCl2, 0.8 mmMgSO4, 5 mmd-glucose, 25 mm HEPES (pH 7.5), 25 mm Tris (pH 7.5). The cell suspension was loaded on 100 μl of a buffer containing 140 mm NaCl, 5.4 mm KCl, 1.8 mmCaCl2, 0.8 mm MgSO4, 5 mmd-glucose, 25 mm HEPES (pH 7.5), 25 mm Tris (pH 7.5), 50 μm nonradioactive His, and 1 μl (1 μCi) of [3H]His (1 mCi/ml) (Amersham Pharmacia Biotech) with or without 10 mm ofl-histidinol, layered on 300 μl of silicone oil/paraffin oil (92:8) in microcentrifuge tubes (29Kumakura T. Takase T. Terada N. Gelfand E.W. Life Sci. 1995; 57: 75-81Crossref Scopus (18) Google Scholar). After the indicated time (30 s, 2 min, 5 min, 30 min, or 60 min), cells were centrifuged for 2 min at 14,000 rpm, and water phase was discarded. After washing surface of oil twice with 750 μl of phosphate-buffered saline, oil phases were discarded also. Cell pellets were then lysed in 50 μl of saline with 2% Triton X-100. Radioactivity of the total lysates was measured using a liquid scintillation counter. The human T-lymphoblastoid cell line, Jurkat cells, were transferred to an amino acid-free medium supplemented with 10% dialyzed FCS and incubated for the indicated times (amino acid deprivation). In addition, after culturing cells in the amino acid-free medium with 10% dialyzed FCS for 16 h, cells were transferred to the regular RPMI 1640 medium containing amino acids with 10% dialyzed FCS and incubated at the indicated times (amino acid supplementation). Initially, the activity of p70s6k was measured in the system, as well as the activities of other growth-related serine/threonine kinases, Akt, p90rsk, and Cdk2 (Fig.1). p70s6k activity decreased within 15 min after deprivation of amino acids and became undetectable within 30 min; levels remained undetectable throughout the time course. Additionally, supplementation of amino acids increased p70s6k activity within 15 min, and the activity reached a plateau level within 60 min. In contrast, the activity of Akt, which is a downstream kinase of PI3K (13Burgering B.M.T. Coffer P.J. Nature. 1995; 376: 599-602Crossref PubMed Scopus (1884) Google Scholar), was not inhibited by amino acid deprivation, nor did it increase following amino acid supplementation. p90rsk is a downstream kinase of extracellular signal-regulated kinases and has the highest homology with p70s6k in the catalytic domains (30Banerjee P. Ahmad M.F. Grove J.R. Kozlosky C. Price D.J. Avruch J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8550-8554Crossref PubMed Scopus (145) Google Scholar). In contrast to p70s6k, the activity of p90rsk transiently increased by transferring cells to an amino acid-free medium, and then decreased gradually. However, p90rsk remained partially active throughout the time course of the experiments. Additionally, amino acid supplementation transiently decreased p90rskactivity, but overall it did not dramatically alter kinase activity for 360 min. Cdk2 is active especially at the late G1 and S phases of the cell cycle. Cdk2 activity was partially decreased by both amino acid deprivation and by supplementation within 30 min, but the kinase remained partially active throughout the time course. The hyperphosphorylated species of p70s6k, which correspond to the active form of the kinase, is determined as a band(s) with a lower mobility in immunoblots (9.Terada, N., Lucas, J. J., Szepesi, A., Franklin, R. A., Takase, K., and Gelfand, E. W. (1992) 186,1315–1321.Google Scholar). On the other hand, the hypophosphorylated p70s6k, which corresponds to the inactive form of the kinase, is determined as a band with the highest mobility. Cells treated with an amino acid-starved medium demonstrated only the hypophosphorylated species of p70s6k, and supplementation of amino acids increased the hyperphosphorylated species of p70s6k within 5–15 min (Fig. 2). In contrast to p70s6k, amino acid deprivation/supplementation essentially did not alter the phosphorylation status of p90rsk, similarly evaluated by a mobility shift in an immunoblot analysis (data not shown). The phosphorylation status of 4E-BP1, another downstream event of mTOR, was also examined by a gel mobility shift in an immunoblot assay (20Lin T.-A. Kong X. Saltiel A.R. Blackshear P.J. Lawrence Jr., J.C. J. Biol. Chem. 1995; 270: 18531-18538Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 21Beretta L. Gingras A.-C. Svitkin Y.V. Hall M.N. Sonenberg N. EMBO J. 1996; 15: 658-664Crossref PubMed Scopus (604) Google Scholar, 22von Manteuffel S.R. Gingras A.-C. Ming X.-F. Sonenberg N. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4076-4080Crossref PubMed Scopus (223) Google Scholar, 23Hara K. Yonezawa K. Kozlowski M.T. Sugimoto T. Andrabi K. Weng Q.-P. Kasuga M. Nishimoto I. Avruch J. J. Biol. Chem. 1997; 272: 26457-26463Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). Cells treated with an amino acid-starved medium demonstrated bands with higher mobilities, corresponding to the hypophosphorylated species of 4E-BP1. Amino acid supplementation induced bands with lower mobilities, which correspond to hyperphosphorylated 4E-BP1, within 15 min. These hyperphosphorylated bands became more predominant within 1–3 h. Additionally, hyperphosphorylated species of p70s6kand 4E-BP1 were eliminated by amino acid deprivation within 5–15 min and 30 min, respectively (data not shown). Because amino acid concentrations affected p70s6k without affecting Akt activity, it does not seem that amino acids regulate p70s6k through PI3K. Furthermore, similar effects of amino acids on p70s6k and 4E-BP1 suggested that mTOR, or an unidentified factor that mediates mTOR signals to both p70s6k and 4E-BP1, is involved in amino acid-induced activation of p70s6k. In order to further investigate these observations, the effects of wortmannin and rapamycin on amino acid-induced p70s6k activation were examined. Jurkat cells were treated in an amino acid-free medium for 16 h and then supplemented with total amino acids or none for 1 h in the presence or absence of specific inhibitors of PI3K and mTOR, wortmannin (100 nm) and rapamycin (10 ng/ml), respectively. As shown in Fig. 3, p70s6k activation induced by amino acid addition was inhibited by rapamycin. In contrast, wortmannin at the concentration that is known to inhibit PI3K activity over 95% inhibited p70s6k only by ∼25%. In contrast, Akt, which was already active in the cell culture regardless of amino acid concentrations, was inhibited by wortmannin by about 60%. Similar to the p70s6k activity, 4E-BP1 phosphorylation induced by amino acid addition was inhibited by rapamycin but not by wortmannin (data not shown). Rapid activation of p70s6k (within 15 min) after re-addition of total amino acids suggested that this may be independent of newly synthesized proteins. In order to confirm this, cycloheximide was added to the culture before addition of total amino acids. Cycloheximide (100 μm), which inhibits protein synthesis over 95%, did not inhibit amino acid-induced p70s6kactivation (data not shown). These data indicate that amino acids activate p70s6k independent of newly synthesized proteins. In order to further investigate mechanisms by which amino acids regulate the mTOR pathway, we utilized cells constitutively expressing a rapamycin-resistant mTOR. Human rhabdomyosarcoma Rh30 cells constitutively expressing either WT-mTOR or a rapamycin-resistant mutant (S2035I-mTOR) were obtained by co-transfection of the mTOR expression vectors and a neo-resistant gene expression vector, followed by G418 selection as described previously (31Sugiyama H. Papst P. Fujita M. Gelfand E.W. Terada N. Oncogene. 1997; 15: 443-452Crossref PubMed Scopus (10) Google Scholar). Parental Rh30 cells, WT-mTOR Rh30 cells, or S2035I-mTOR Rh30 cells were first incubated for 3 h in an amino acid-deprived medium. Rapamycin (10 ng/ml) was then added to the culture. Thirty min after addition of rapamycin, total amino acids were added, and cells were incubated for the indicated times. As shown in Fig.4, amino acid supplementation was not able to induce p70s6k in the presence of rapamycin in parental Rh30 cells or WT-mTOR Rh30 cells, as shown in Jurkat cells above. In contrast, amino acids did induce p70s6k in the presence of rapamycin in S2035I-mTOR Rh30 cells. The mechanism by which amino acids regulate p70s6k activity was further explored. Fox et al. (26Fox H.L. Kimball S.R. Jefferson L.S. Lynch C.J. Am. J. Physiol. 1998; 274: C206-C213Crossref PubMed Google Scholar) reported that supplementation of a set of neutral amino acids, which were preferentially transported by system L transporter, was able to induce p70s6kphosphorylation. In contrast, another set of neutral amino acids preferentially transported by system A or ASC, or a set of charged amino acids, was less effective. A potential explanation for these results is that a specific amino acid transporter, such as system L, is linked to activation of p70s6k. In order to investigate this possibility, we examined the effects of specific inhibitors of various amino acid transporter systems on amino acid-induced p70s6k activation. There are three major amino acid transporter systems known for uptake of neutral amino acids; system A, system L, and system ASC. It is reported that over 90% of neutral amino acids are transported through these three systems in mammalian cells (32Christensen H.N. Physiol. Rev. 1990; 70: 43-77Crossref PubMed Scopus (963) Google Scholar). Each of the three systems has a model substrate that has a high affinity to the system. MeAIB has a high affinity to system A and competitively inhibits transport of other amino acids through the system. In contrast, MeAIB does not significantly affect transport of amino acids through other transporters, including system L and system ASC. Similarly, 2-amino-2-norbornane-carboxylic acid and cysteine can competitively inhibit amino acids-transport through system L and system ASC, respectively. Jurkat cells were initially incubated with amino acid-deprived medium for 16 h, and then 116 concentrations of total amino acids contained in regular RPMI 1640 medium were added to the culture in the presence or absence of these competitive model substrates (10 mm). This concentration of total amino acids was able to induce approximately one-half the activity of p70s6k when compared with addition of 1× total amino acids of RPMI 1640 medium. As shown in Fig. 5, all of the model substrates partially inhibit p70s6kactivation induced by amino acid supplementation. This is inconsistent with the idea that a specific neutral transporter is linked to p70s6k activation. If amino acid transporters are signaling mediators that sense extracellular amino acid concentration and subsequently transduce signals to p70s6k, then the data suggest that all three systems should link to p70s6kactivation. Alternatively, the data may be explained by altered intracellular amino acid concentration. The mechanism by which the cell recognizes the lack of an amino acid seems reasonably well understood in bacteria and yeast, and basically it is through increased concentration of intracellular deacylated tRNA or, in some cases, through reduced availability of aminoacylated tRNA (see under "Discussion"). In order to investigate the involvement of the tRNA aminoacylation in the regulation of p70s6k in mammalian cells, we initially utilized amino acid alcohols. The alcohol derivatives of amino acids inhibit their corresponding tRNA synthetases and thus prevent aminoacyl-tRNA formation (33Hansen B.S. Vaughan M.H. Wang L. J. Biol. Chem. 1972; 247: 3854-3857Abstract Full Text PDF PubMed Google Scholar, 34Kilberg M.S. Hutson R.G. Laine R.O. FASEB J. 1994; 8: 13-19Crossref PubMed Scopus (91) Google Scholar). For example,l-histidinol inhibits l-His binding to tRNAHis synthetase and thereby increases deacylated tRNAHis. Various amino acid alcohols were added to exponentially growing Jurkat cells. As shown in Fig.6 A, addition of 2 mm of either leucinol, phenylalaninol, alaninol, histidinol, tyrosinol, o
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