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Immunopurified Mammalian Target of Rapamycin Phosphorylates and Activates p70 S6 Kinase α in Vitro

1999; Elsevier BV; Volume: 274; Issue: 48 Linguagem: Inglês

10.1074/jbc.274.48.34493

1083-351X

Shuji Isotani, Kenta Hara, Chiharu Tokunaga, Hitomi Inoue, Joseph Avruch, Kazuyoshi Yonezawa,

PI3K/AKT/mTOR signaling in cancer

p70 S6 kinase α (p70α) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70α activation in a manner that can be overcome by coexpression of p70α with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70α polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70α phosphorylation in vitro is accompanied by a substantial restoration in p70α kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70α by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70α activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined. p70 S6 kinase α (p70α) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70α activation in a manner that can be overcome by coexpression of p70α with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70α polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70α phosphorylation in vitro is accompanied by a substantial restoration in p70α kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70α by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70α activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined. p70 S6 kinase α antibody mammalian target of rapamycin hemagglutinin phosphoinositide 3-kinase 3-phosphoinositide-dependent protein kinase 1 eukaryotic initiation factor-4E binding protein 1 glutathioneS-transferase radioimmune precipitation buffer 4-morpholinepropanesulfonic acid FK506-binding protein p70 S6 kinase α (p70α),1 whose major substrate is the 40 S ribosomal protein S6, plays a critical role in the translation of a subclass of mRNAs that contain a short oligopyrimidine sequence immediately following the transcriptional start site (1Meyuhas O. Avni D. Shama S. Hershey W.B. Mathews M.B. Sonenberg N. Translational Control of Ribosomal Protein mRNAs in Eukaryotes. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1996: 363-388Google Scholar). p70α is activated in response to insulin/mitogensin vivo through a multisite phosphorylation of serine and threonine residues (2Avruch J. Mol. Cell. Biochem. 1998; 182: 31-48Crossref PubMed Scopus (323) Google Scholar). Several sets of independently regulated p70α phosphorylation sites have been identified (3Weng Q.P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (211) Google Scholar, 4Weng 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, 5Pearson R.B. Dennis P.B. Han J.W. Williamson N.A. Kozma S.C. Wettenhall R.E. Thomas G. EMBO J. 1995; 14: 5279-5287Crossref PubMed Scopus (388) Google Scholar, 6Moser B.A. Dennis P.B. Pullen N. Pearson R.B. Williamson N.A. Wettenhall R.E. Kozma S.C. Thomas G. Mol. Cell. Biol. 1997; 17: 5648-5655Crossref PubMed Scopus (87) Google Scholar); one set consists of Ser/Thr-Pro motifs, five of which are clustered in a psuedosubstrate autoinhibitory domain in the noncatalytic carboxyl-terminal tail (Ser-434, Ser-441, Ser-447, Ser-452, and Thr-444 in p70α), and two others, Thr-390 and Ser-394, are located in a 65-amino acid segment immediately carboxyl-terminal to the kinase catalytic domain. A second set of regulated phosphorylation sites, Thr-412 and Ser-427, exhibit the sequence motif Phe-Ser/Thr-Phe/Tyr. Thr-252, located on the activation loop in catalytic subdomain VIII, is the site at which 3-phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates p70α (7Alessi 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 (516) Google Scholar, 8Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (727) Google Scholar). Among these, the phosphorylation of Thr-252, Ser-394, and Thr-412 is necessary for the activation of p70α kinase catalytic function; the attainment of physiologic levels of p70α activity results from a strongly synergistic, positive site-site interaction between the phosphorylated Thr-252 and Thr-412 residues (7Alessi 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 (516) Google Scholar). In addition to its regulation by insulin and mitogens through PI-3 kinase-dependent pathways, p70α can also be activated by increasing concentrations of extracellular amino acids in the absence of serum or mitogens to the level attained by maximal mitogen stimulation (9-11). Moreover, a threshold level of cellular amino acids is necessary for p70α to be susceptible to activation by mitogens. Withdrawal of amino acids from the nutrient medium results in a rapid, selective deactivation of p70α, which becomes unresponsive to mitogens; readdition of amino acids restores the mitogen responsiveness of p70α (9Hara 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 (1124) Google Scholar, 10Patti M.E. Brambilla E. Luzi L. Landaker E.J. Kahn C.R. J. Clin. Invest. 1998; 101: 1519-1529Crossref PubMed Google Scholar, 11Shigemitsu K. Tsujishita Y. Hara K. Nanahoshi M. Avruch J. Yonezawa K. J. Biol. Chem. 1999; 274: 1058-1065Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The immunosuppressant drug rapamycin inhibits p70α in vivo(12Price D.J. Grove J.R. Calvo V. Avruch J. Bierer B.E. Science. 1992; 257: 973-977Crossref PubMed Scopus (589) Google Scholar, 13Chung J. Kuo C.J. Crabtree G.R. Blenis J. Cell. 1992; 69: 1227-1236Abstract Full Text PDF PubMed Scopus (1026) Google Scholar). This is achieved indirectly by the ability of a rapamycin-FKBP12 complex to bind to the mTOR polypeptide and inhibit mTOR kinase activity; mTOR mutants unable to bind the rapamycin-FKBP12 complex can rescue p70α from rapamycin-induced dephosphorylation and inhibition but only if the mTOR catalytic domain is intact (14Brown E.J. Beal P.A. Keith C.T. Chen J. Shin T.B. Schreiber S.L. Nature. 1995; 377: 441-446Crossref PubMed Scopus (618) Google Scholar, 15Hara 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 (409) Google Scholar). As for the biochemical steps by which the mTOR kinase controls p70α phosphorylation and activity, evidence is available in support of two independent, but nonexclusive mechanisms. The possibility that mTOR inhibits an inactivating p70α-phosphatase is supported by both indirect and direct experiments. Thus, a doubly deleted p70α mutant (p70α-Δ2–46/ΔCT104) can be activated by mitogens and inhibited by low concentrations of wortmannin but is insensitive to inhibition by rapamycin (3Weng Q.P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (211) Google Scholar) or amino acid withdrawal (9Hara 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 (1124) Google Scholar); these features are most readily explained if mitogens and PI-3 kinase control p70α-kinases, whereas amino acid sufficiency and mTOR negatively regulate a p70α-phosphatase. A recent report provides direct evidence implicating protein phosphatase 2A in this role (16Peterson R.T. Desai B.N. Hardwick J.S. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4438-4442Crossref PubMed Scopus (429) Google Scholar). Conversely, Burnett et al. (17Burnett 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 (939) Google Scholar) reported that mTOR can directly phosphorylate prokaryotic recombinant fragments of p70α in vitro at sites important to activation, including Thr-412. The latter finding was surprising, inasmuch as all sites of mTOR-catalyzed phosphorylation on the eukaryotic initiation factor-4E binding protein 1 (eIF-4E BP1) reside in Ser/Thr-Pro motifs (18Brunn G.J. Fadden P. Haystead T.A.J. Lawrence Jr., J.C. J. Biol. Chem. 1997; 272: 32547-32550Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 19Gingras A.-C. Gygi S.P. Raught B. Polakiewicz R.D. Abraham R.T. Hoekstra M.F. Aebersold R. Sonenberg N. Genes Dev. 1999; 13: 1422-1437Crossref PubMed Scopus (1007) Google Scholar). We therefore inquired whether mTOR can phosphorylate and/or activate a precisely folded full-length p70α polypeptide expressed in mammalian cells and dephosphorylated and inactivated in vivo by the pretreatment with rapamycin. The anti-mTOR antibody was described previously (20Nishiuma T. Hara K. Tsujishita Y. Kaneko K. Shii K. Yonezawa K. Biochem. Biophys. Res. Commun. 1998; 252: 440-444Crossref PubMed Scopus (24) Google Scholar). The anti-phosphopeptide antibodies against Thr-412, Ser-434, and Thr-444/Ser-447 of p70α (the anti-412-P Ab, the anti-434-P Ab, and the anti-444/447-P Ab, respectively), the anti-FLAG antibody, and the anti-HA antibody were described previously (21Weng Q.P. Kozlowski M. Belham C. Zhang A.H. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). The antibody against carboxyl-terminal peptides of p70α (the anti-p70αC Ab) was purchased from Santa Cruz Biotechnology. The expression vectors of the wild-type (W-mTOR) and the kinase-negative mutant (NK-mTOR) of mTOR, of the wild-type p70α and the mutant p70α (p70α-ΔCT104), and of GST-PDK1 were described previously (3Weng Q.P. Andrabi K. Kozlowski M.T. Grove J.R. Avruch J. Mol. Cell. Biol. 1995; 15: 2333-2340Crossref PubMed Scopus (211) Google Scholar, 7Alessi 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 (516) Google Scholar, 15Hara 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 (409) Google Scholar). Site-specific mutants of p70α (p70α-Thr-412 → Ala, p70α-5A, and p70α-ΔCT104/Thr-412 → Ala) were generated using polymerase chain reaction based on the pcDNA1 plasmids. Recombinant p70α was prepared from HEK293 cells that were transfected with various p70α cDNAs and extracted after serum starvation for 16 h and the following treatment with 0.2 μm rapamycin for 30 min prior to harvest. Endogenous mTOR was extracted from HEK293 cells, and recombinant mTOR was extracted from HEK293 cells transfected with mTOR cDNAs. All cells were lysed in ice-cold buffer A (20 mmTris (pH 7.4), 20 mm NaCl, 1 mm EDTA, 20 mm β-glycerophosphate, 5 mm EGTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 1 μm leupeptin), and the supernatants were obtained after centrifugation at 10,000 ×g for 20 min at 4 °C. The anti-mTOR immunocomplex and the anti-recombinant p70α immunocomplex were each prepared using separate supernatants incubated 2 h at 4 °C with the specific antibodies. The two supernatants were then mixed together, and protein G-Sepharose beads were added immediately and incubated for 45 min at 4 °C. Normal mouse immunogloblin was added to cell supernatant to provide control for the anti-mTOR immunocomplex, whereas the anti-HA or anti-FLAG antibodies were added to cell supernatant prepared from mock-transfected cells to provide the controls corresponding to recombinant HA-tagged mTOR or FLAG-tagged p70α immunocomplexes. The protein G beads containing immunocomplexes were washed twice with buffer A containing 0.5 m NaCl (the high salt wash buffer) followed by two washes with either the same high salt wash buffer or the high salt wash buffer containing 1% Nonidet P-40 or the RIPA buffer. The RIPA buffer consisted of 20 mm Tris (pH = 7.4), 1% Triton X-100, 0.1% SDS, 0.1% deoxycholate, and 150 mm NaCl. The immunocomplexes were further washed twice with buffer B (10 mm Hepes (pH = 7.4), 50 mmβ-glycerophosphate, 50 mm NaCl) and subjected to the kinase assay. The kinase reaction was started by the addition of buffer C (10 mm Hepes (pH = 7.4), 50 mm NaCl, 50 mm β-glycerophosphate, 10 mm MnCl2, 100 μm ATP (10 μCi of [γ-32P] ATP). The reaction was incubated for 30 min at 30 °C and terminated by the addition of the SDS sample buffer. The ability of mTOR to stimulate the kinase activity of p70α toward S6 in vitro was measured using a two step kinase reaction. In the first step, after p70α was immunoprecipitated with mTOR on protein G-Sepharose beads, the immunoprecipitate was washed twice with the high salt wash buffer containing 1% Nonidet P-40 and twice with buffer B, and the immunoprecipitate was incubated in buffer C with nonradioactive ATP for the indicated times. The first kinase reaction was terminated by washing the beads twice with the ice-cold high salt wash buffer and twice with buffer D (20 mm MOPS (pH 7.4), 10 mm β-glycerophosphate, 1 mmdithiothreitol) and subjected to the second kinase assay. The samples were incubated in the S6 kinase assay mixture (50 mm MOPS (pH 7.2), 12 mm MgCl2, 2 mm EGTA, 10 mm β-glycerophosphate, 0.5 μm protein kinase inhibitor, 1 mm dithiothreitol, 0.5A 260 units of 40 S ribosomal subunit, and 60 μm ATP (5 μCi of [γ-32P] ATP) for 15 min at 30 °C, and the reaction was terminated by the addition of the SDS sample buffer. To measure the effects of GST-PDK1 on mTOR-catalyzed activation of p70α, a three step kinase assay was employed. The first kinase reaction was performed as described above, and the reaction was terminated by washing the beads with the ice-cold buffer E (50 mm Tris (pH 7.4), 0.1 mm EGTA). The second kinase reaction was initiated by adding buffer F (50 mmTris (pH 7.4), 0.1 mm EGTA, 1 mmdithiothreitol, 2 μm protein kinase inhibitor, 10 mm MgCl2, 1 mg/ml bovine serum albumin, 100 μm ATP) containing either purified GST-PDK1 (7Alessi 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 (516) Google Scholar) or control buffer and continued for 30 min at 30 °C. The second kinase reaction was terminated by washing the beads with the ice-cold high salt wash buffer twice and buffer D twice and subjected to the S6 kinase assay as described above. After the kinase reaction was terminated, the reaction mixtures were separated on SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membrane was analyzed by autoradiography using x-ray film and a BAS-2000 Bioimaging analyzer (Fuji). Then, the membrane was immunoblotted with the indicated antibody as the first antibody and visualized by using ECL method. To obtain a precisely folded full-length p70α polypeptide that was expressed in mammalian cells and dephosphorylated and inactivatedin vivo, HEK293 cells were transiently transfected with a full-length HA-tagged p70α, and cells were deprived of serum and treated with rapamycin (0.2 μm) for 30 min prior to harvest. HA-tagged p70α was immunopurified on protein G-Sepharose beads, together with either a control immunoglobulin or an anti-mTOR immunocomplex; each were prepared separately from extracts of HEK293 cells. To define optimal conditions for detection of mTOR-catalyzed p70α phosphorylation, the protein G-Sepharose beads were washed in several ways prior to the kinase assay (Fig.1 A). Although mTOR-catalyzed p70α phosphorylation was detectable with all washing conditions, the mTOR autophosphorylation and kinase activity toward p70α is substantially enhanced when the immunoprecipitate is washed with the high salt wash buffer containing 1% Nonidet P-40 or the RIPA buffer, compared with that prepared after washing with the high salt wash buffer without detergent. The result was unexpected, as we have previously shown that washing of mTOR immunoprecipitates with 1% Nonidet P-40 reduces greatly the ability of mTOR to catalyze eIF-4E BP1 phosphorylation (20Nishiuma T. Hara K. Tsujishita Y. Kaneko K. Shii K. Yonezawa K. Biochem. Biophys. Res. Commun. 1998; 252: 440-444Crossref PubMed Scopus (24) Google Scholar). These differences in mTOR-catalyzed p70α phosphorylation are not because of differences in the recovery of mTOR polypeptide, as demonstrated by the immunoblot with the anti-mTOR antibody (Fig. 1 A). mTOR-catalyzed 32P incorporation into p70α is detectable within 5 min after initiation of the kinase reaction and increases over 30 min (Fig. 1 B); no 32P incorporation into the wild-type p70α substrate (i.e. p70α autophosphorylation) is detectable in the absence of mTOR (Fig. 1 B, lanes 6 and7). Moreover, mTOR-catalyzed 32P incorporation into the kinase-inactive, ATP binding site mutant of p70α (p70α-Lys-123 → Met) is similar in extent to that seen with wild-type p70α substrate (data not shown). These results show that the 32P incorporation into p70α occurring in the presence of mTOR is catalyzed by a mTOR-associated kinase and is not because of a stimulation of p70α autophosphorylation. To confirm that the phosphorylation of p70α by mTOR is dependent on the intrinsic kinase activity of mTOR, we compared the ability of a recombinant wild-type or kinase-negative mutant of mTOR to phosphorylate p70α in vitro (Fig. 1 C). In contrast to the robust 32P incorporation into p70α catalyzed by recombinant wild-type mTOR, no phosphorylation of p70α is detectable on incubation with the kinase-negative mutant of mTOR. We next examined the effects of mTOR kinase inhibitors on mTOR-catalyzed p70α phosphorylation in vitro (Fig. 1 D). Incubation of mTOR immunoprecipitates with a rapamycin-FKBP complex severely inhibits mTOR autophosphorylation, as well as the phosphorylation of p70α, whereas neither rapamycin or FKBP singly have any effect. Wortmannin, at a concentration previously shown to inhibit mTOR kinase toward eIF-4E BP in vitro also inhibits mTOR-catalyzed p70α phosphorylation. These results indicate that the phosphorylation of p70α in vitro requires the intrinsic kinase activity of mTOR. To examine the effects of stimulation by mitogens and depletion of amino acids on the mTOR kinase activity, serum-deprived cells were treated with or without 10% serum in the presence of amino acids or incubated for up to 2 h in the amino acid-free buffer; mTOR was immunoprecipitated and assayed for kinase activity toward p70α. No significant alterations in the mTOR kinase toward p70α resulted from these treatments (data not shown); thus the effects of these perturbations on mTOR kinase activity toward p70α, if any, do not survive immunoprecipitation and washing. We employed a panel of anti-p70α phosphopeptide antibodies (21Weng Q.P. Kozlowski M. Belham C. Zhang A.H. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar) to examine whether mTOR catalyzed the phosphorylation in vitroof sites on p70α known to be phosphorylated in vivo. The phospho-specific immunoreactivity at all sites examined appeared to be increased by mTOR; however, this was most unmistakable with Thr-412 (Fig. 1, A and B), which exhibits no phospho-specific immunoreactivity prior to incubation with mTOR. Overall phospho-specific immunoreactivity at Thr-444/Ser-447 is also substantially increased over the initial level, whereas the response at Ser-434 is equivocal in that the modest apparent increase in overall phospho-specific immunoreactivity at Ser-434 may be entirely attributable to the upshift of a fraction of p70α polypeptides resulting from mTOR-catalyzed phosphorylation at other sites with a consequent spreading of Ser-434-P immunoreactivity over a greater area. In view of the limitation of immunoblot for quantitative analysis, we compared the extent of mTOR-catalyzed 32P incorporation into wild-type p70α with that observed using equal amounts of several p70α mutants as substrates. The p70α mutants examined were (i) p70α-5A in which five Ser/Thr-Pro sites (Ser-434, Ser-441, Thr-444, Ser-447, and Ser-452) in the carboxyl-terminal autoinhibitory domain are substituted by Ala; (ii) p70α-Thr-412 → Ala; (iii) p70α-ΔCT104 in which the carboxyl-terminal 104 amino acids are deleted and the protein terminates after Ser-421; (iv) p70α-ΔCT104/Thr-412 → Ala. The quantitative importance of Thr-412 as a site of mTOR-catalyzed p70α phosphorylation is clearly evident in Fig. 2; mutation of p70α Thr-412 to Ala reduces mTOR-catalyzed 32P incorporation into full-length p70α by about 80% and into p70α-ΔCT104 by a similar extent; the mTOR-catalyzed 32P incorporation into the p70α-ΔCT104/Thr-412 → Ala mutant is less than 10% of that seen with full-length p70α wild-type (Fig. 2 B). Thus, Thr-412 is a dominant site of mTOR-catalyzed p70α phosphorylation in vitro. Overall mTOR-catalyzed 32P incorporation into the p70α-5A mutant is diminished by about 25% compared with p70α wild-type, whereas 32P incorporation into the ΔCT104 mutant is diminished by 50–60%. These results indicate that a portion of mTOR-catalyzed p70α phosphorylation is directed to the carboxyl-terminal tail, at least half of which is into the Ser/Thr-Pro sites mutated in the 5A variant. This is consistent with the results of the anti-Thr-444/Ser-447-P immunoblots (Fig. 1, A andB, and Fig. 2 A). The lesser total 32P incorporation into p70α-ΔCT104 as compared with p70α-5A suggests that there might be phosphorylation site(s) other than the five Ser/Thr-Pro sites within the carboxyl-terminal 104 amino acids. One such site may be Ser-427, located in a Phe-Ser-Phe motif similar to that surrounding Thr-412. Another possible explanation is that the absence of the carboxyl-terminal 104 amino acids may impair the ability of mTOR to phosphorylate Thr-412. The phosphorylation of p70α Thr-412 is known to be critical for its S6 kinase activity and substitution of this residue with an acidic amino acid results in a substantial increase in “basal” S6 kinase activity (5Pearson R.B. Dennis P.B. Han J.W. Williamson N.A. Kozma S.C. Wettenhall R.E. Thomas G. EMBO J. 1995; 14: 5279-5287Crossref PubMed Scopus (388) Google Scholar, 21Weng Q.P. Kozlowski M. Belham C. Zhang A.H. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). We therefore inquired whether the p70α kinase activity is increased by mTOR-catalyzed phosphorylation in vitro. As shown in Fig. 3, the p70α kinase activity shows a time-dependent increase on incubation with mTOR (Fig. 3 B), which parallels the extent of mTOR-catalyzed phosphorylation at Thr-412 and Thr-444/Ser-447 detected by immunoblot (Fig. 3 A). In contrast, no S6 kinase activity is detectable in the absence of mTOR. To establish whether the activation of p70α in vitro requires the intrinsic kinase activity of mTOR, the recombinant wild-type and kinase-negative mutant of mTOR were employed for the assays (Fig. 3 C). As in Fig.3 A, incubation of p70α with wild-type mTOR significantly increased the S6 kinase activity, whereas no activation is detected on incubation of p70α with kinase-negative mTOR. These results clearly indicate that the kinase activity of p70α, which had been fully inactivated in vivo by the treatment of cells with rapamycin, was restored, at least in part, by the phosphorylationin vitro catalyzed by the kinase activity intrinsic to the mTOR catalytic domain. PDK1-catalyzed phosphorylation of Thr-252 on the p70α activation loop is a critical and probably final step in the physiologic activation of p70α in vivo (7Alessi 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 (516) Google Scholar, 8Pullen N. Dennis P.B. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (727) Google Scholar, 21Weng Q.P. Kozlowski M. Belham C. Zhang A.H. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). The ability of PDK1 to phosphorylate Thr-252 is regulated primarily by the accessibility of the p70α activation loop to PDK1, which in turn is controlled by a series of prior p70α phosphorylations. Phosphorylation of the multiple Ser/Thr-Pro motifs clustered in the psuedosubstrate autoinhibitory segment of the p70α carboxyl-terminal tail serves to disocclude the catalytic domain, greatly enhancing access to PDK1. A similar effect can be achieved by deletion of the p70α carboxyl-terminal tail (to give p70α-ΔCT104). At any level of PDK1 activity, the extent of Thr-252 phosphorylation of p70α-ΔCT104 is substantially greater than with a similar amount of full-length p70α polypeptide. In addition, the S6 kinase activity generated by any extent of PDK1-catalyzed Thr-252 phosphorylation is significantly higher for p70α-ΔCT104 as compared with full-length p70α. Displacement of the p70α carboxyl-terminal tail is also necessary for the phosphorylation of Thr-412 in vivo, and modification of Thr-412 itself significantly enhances the ability of PDK1 to phosphorylate Thr-252. In addition, the simultaneous phosphorylation of Thr-412 and Thr-252 appears to generate a synergistic activation of p70α. Thus, the substitution of Thr-412 by Glu in p70α-ΔCT104 alone gives a 6-fold increase in S6 kinase activity, and the PDK1 catalyzed phosphorylation of p70α-ΔCT104 Thr-252 alone gives a 15-fold increase, but the two modifications together give at least a 240-fold increase in S6 kinase activity over the unmodified p70α-ΔCT104 polypeptide (7Alessi 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 (516) Google Scholar). The importance of the strong positively cooperative effect of Thr-252 and Thr-412 phosphorylation for the physiologic activation of p70α is illustrated by the response of p70α to the inhibitors rapamycin and wortmannin; these agents each cause a rapid dephosphorylation of Thr-412 but a slower and lesser dephosphorylation of Thr-252. Despite the preservation of Thr-252 phosphorylation, S6 kinase activity in the presence of rapamycin or wortmannin declines in parallel to Thr-412 dephosphorylation (21Weng Q.P. Kozlowski M. Belham C. Zhang A.H. Comb M.J. Avruch J. J. Biol. Chem. 1998; 273: 16621-16629Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). In view of the ability of mTOR to catalyze the in vitrophosphorylation of p70α Thr-412 as well as sites within the autoinhibitory segment in the p70α carboxyl-terminal tail and the potential effects of such phosphorylations on the response of p70α to PDK1, we compared the p70α activation achieved in vitro by mTOR or PDK1 alone to that achieved by sequential phosphorylation by mTOR and PDK1 and to that achieved in vivo by stimulation of the cells with 10% serum. As shown in Fig.4, mTOR alone increased the S6 kinase activity of p70α in vitro by more than 10-fold, whereas PDK1 alone hardly activated p70α, presumably reflecting the relatively poor access of Thr-252 to PDK1 in full-length, inactive p70α as seen previously (7Alessi 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 (516) Google Scholar). In contrast, phosphorylation of p70α by PDK1 after a prior phosphorylation by mTOR increased the p70α activity by 10-fold over that engendered by mTOR alone, to a level roughly 70-fold greater than that generated by PDK1 acting alone. Moreover, the S6 kinase activity generated in vitro by the sequential action of mTOR and PDK1 is indistinguishable from that achieved in vivo by stimulation of cells with 10% serum (Fig. 4). The present results demonstrate that mTOR can catalyze directly the phosphorylation and activation of p70α in vitro. In addition, mTOR can activate p70α in a synergistic manner with PDK1in vitro, and it is likely that this occurs in vivo. Nevertheless, the nature of the physiologic inputs that control mTOR-catalyzed p70α phosphorylation and the relative contribution of mTOR-catalyzed p70α phosphorylation to overall p70α regulation in vivo are not clear. Pretreatment of 3T3-L1 cells with insulin has been reported to cause a modest (1.3–2-fold) increase in the ability of mTOR to catalyze eIF-4E BP1 phosphorylationin vitro. Moreover, coexpression with active protein kinase B may enhance mTOR kinase, although evidence for direct activation of mTOR by protein kinase B is lacking. On this basis, mTOR has been proposed to be an intermediate in the insulin/PI-3 kinase-dependent activation of p70α (22Scott P.H. Brunn G.J. Kohn A.D. Roth R.A. Lawrence Jr., J.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7772-7777Crossref PubMed Scopus (412) Google Scholar). To the contrary, the ability of the rapamycin-resistant p70α-Δ2–46/ΔCT104 mutant to undergo insulin-stimulated, wortmannin-inhibitable Thr-412 phosphorylation and activation in the presence of concentrations of rapamycin far in excess of those required for complete inhibition of endogenous wild-type p70α and mTOR indicates that insulin-responsive kinases exist that are capable of p70α (Thr-412) phosphorylation and activation, other than mTOR (9Hara 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 (1124) Google Scholar). Thus, the contribution of mTOR to insulin-stimulated p70α Thr-412 phosphorylation in vivo is unsettled but may be minor. Another possible role for the mTOR kinase is as the mediator of the amino acid-stimulated phosphorylation and activation of p70α; as yet however, direct evidence supporting amino acid regulation of the mTOR kinase activity is lacking (9Hara 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 (1124) Google Scholar). A plausible synthesis for the operation of the mTOR kinase in vivo is that insulin- and amino acid-induced signals each converge independently in the regulation of mTOR, although insulin also controls an alternative, mTOR-independent set of p70α-kinases. In turn, mTOR controls p70α through direct phosphorylation, as well as through the negative regulation of a p70α phosphatase. Gingras et al. (19Gingras A.-C. Gygi S.P. Raught B. Polakiewicz R.D. Abraham R.T. Hoekstra M.F. Aebersold R. Sonenberg N. Genes Dev. 1999; 13: 1422-1437Crossref PubMed Scopus (1007) Google Scholar) reported that mTOR phosphorylates eIF-4E BP1 in vitro primarily on Thr-37 and Thr-46; these phosphorylations do not themselves result in the release of eIF-4E but are required for the further phosphorylation on several carboxyl-terminal serum-sensitive sites, and these latter phosphorylations result in the release of eIF-4E. In the case of p70α, mTOR alone gives some activation, but by phosphorylating the Ser/Thr-Pro sites within the carboxyl-terminal 104 amino acids to improve access of PDK1 and by phosphorylating Thr-412, mTOR strongly promotes the ability of PDK1 to phosphorylate Thr-252. In both instances, mTOR phosphorylation acts primarily in a “priming” role rather than a sole activator. An intriguing and unresolved aspect of mTOR function is the apparent ability of its single catalytic domain to catalyze phosphorylation of Ser/Thr-Pro sites, such as those on eIF-4E BP1 and p70α Thr-444/Ser-447, as well as Phe-Ser/Thr-Phe/Tyr sites, such as p70α Thr-412. Although these two kinds of mTOR kinase activity appear differentially sensitive to detergent, they are both inhibited by rapamycin/FKBP-12 in vitro and by mutations in the mTOR kinase domain. Assuming both activities are physiologically meaningful, such a breadth in substrate specificity is relatively unprecedented among the protein kinases. Finally, it should be noted that Phe-Ser/Thr-Phe/Tyr motifs homologous to p70α Thr-412 are found in many kinases of the AGC (protein kinase A, G, and C) subclass, and it is of interest to note the recent report that PKCδ activation and phosphorylation at Ser-662 (in the context Phe-Ser-Phe) is inhibitable by rapamycin (23Ziegler W.H. Parekh D.B. Le Good J.A. Whelan R.D.H. Kelly J.J. Frech M. Hemmings B.A. Parker P.J. Curr. Biol. 1999; 9: 522-529Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). In conclusion, the present study demonstrates thein vitro activation of p70α by mTOR-catalyzed phosphorylation involving p70α Thr-412, a critical site conserved in the other AGC kinase subfamily members. We are grateful to Dr. Y. Nishizuka for encouragement. We thank Dr. D. R. Alessi for providing cDNA of PDK1. We also thank Dr. U. Kikkawa for valuable advice and H. Miyamoto for technical assistance. The skillful secretarial assistance of M. Kusu is cordially acknowledged.