Phosphorylation of Mammalian Target of Rapamycin (mTOR) at Ser-2448 IsMediated by p70S6 Kinase
The mammalian target of rapamycin (mTOR) coordinates cell growth with thegrowth factor and nutrient/energy status of the cell. The phosphatidylinositol3-kinase-AKT pathway is centrally involved in the transmission of mitogenicsignals to mTOR. Previous studies have shown that mTOR is a direct substratefor the AKT kinase and identified Ser-2448 as the AKT target site in mTOR. Inthis study, we demonstrate that rapamycin, a specific inhibitor of mTORfunction, blocks serum-stimulated Ser-2448 phosphorylation and that this drugeffect is not explained by the inhibition of AKT. Furthermore, thephosphorylation of Ser-2448 was dependent on mTOR kinase activity, suggestingthat mTOR itself or a protein kinase downstream from mTOR was responsible forthe modification of Ser-2448. Here we show that p70S6 kinase phosphorylatesmTOR at Ser-2448 in vitro and that ectopic expression ofrapamycin-resistant p70S6 kinase restores Ser-2448 phosphorylation inrapamycin-treated cells. In addition, we show that cellular amino acid status,which modulates p70S6 kinase (S6K1) activity via the TSC/Rheb pathway,regulates Ser-2448 phosphorylation. Finally, small interfering RNA-mediateddepletion of p70S6 kinase reduces Ser-2448 phosphorylation in cells. Takentogether, these results suggest that p70S6 kinase is a major effector of mTORphosphorylation at Ser-2448 in response to both mitogen- and nutrient-derivedstimuli. The mammalian target of rapamycin (mTOR) coordinates cell growth with thegrowth factor and nutrient/energy status of the cell. The phosphatidylinositol3-kinase-AKT pathway is centrally involved in the transmission of mitogenicsignals to mTOR. Previous studies have shown that mTOR is a direct substratefor the AKT kinase and identified Ser-2448 as the AKT target site in mTOR. Inthis study, we demonstrate that rapamycin, a specific inhibitor of mTORfunction, blocks serum-stimulated Ser-2448 phosphorylation and that this drugeffect is not explained by the inhibition of AKT. Furthermore, thephosphorylation of Ser-2448 was dependent on mTOR kinase activity, suggestingthat mTOR itself or a protein kinase downstream from mTOR was responsible forthe modification of Ser-2448. Here we show that p70S6 kinase phosphorylatesmTOR at Ser-2448 in vitro and that ectopic expression ofrapamycin-resistant p70S6 kinase restores Ser-2448 phosphorylation inrapamycin-treated cells. In addition, we show that cellular amino acid status,which modulates p70S6 kinase (S6K1) activity via the TSC/Rheb pathway,regulates Ser-2448 phosphorylation. Finally, small interfering RNA-mediateddepletion of p70S6 kinase reduces Ser-2448 phosphorylation in cells. Takentogether, these results suggest that p70S6 kinase is a major effector of mTORphosphorylation at Ser-2448 in response to both mitogen- and nutrient-derivedstimuli. The mammalian target of rapamycin(mTOR) 1The abbreviations used are: mTOR, mammalian target of rapamycin; PI,phosphatidylinositol; FKBP, FK506-binding protein; siRNA, small interferingRNA; HA, hemagglutinin; HEK, human embryonic kidney; DMEM, Dulbecco's modifiedessential medium; FCS, fetal calf serum; WT, wild type; SI,rapamycin-resistant; GST, glutathione S-transferase; AMPK,AMP-activated protein kinase; TSC, tuberous sclerosis complex. is a member ofthe phosphatidylinositol 3 (PI-3)-kinase-related kinase family (PIKK), whichincludes ATM, ATR, hSMG-1, and DNA-PK(1Hay N. Sonenberg N. GenesDev. 2004; 18: 1926-1945Crossref PubMed Scopus (3484) Google Scholar, 2Abraham R.T. GenesDev. 2001; 15: 2177-2196Crossref PubMed Scopus (1676) Google Scholar, 3Abraham R.T. DNA Repair(Amst.). 2004; 3: 919-925Crossref PubMed Scopus (42) Google Scholar).These large Ser/Thr protein kinases play essential roles in cellular responsesto growth factors and stress. In particular, mTOR plays a critical role incoordinating cell growth with growth factor inputs as well as cellularnutrient and energy status. The bacterially derived macrolide ester, rapamycin, is clinically approvedas an immunosuppressant and shows promising anti-tumor activity. Rapamycin,when complexed with FKBP-12 (FK506-binding protein, 12 kDa), bindsspecifically to mTOR at a conserved stretch of ∼100 amino acids termed theFKBP-12·rapamycin binding domain(4Chen J. Zheng X.F. Brown E.J. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4947-4951Crossref PubMed Scopus (450) Google Scholar). Mutation of a criticalserine residue (Ser-2035) in the FKBP-12·rapamycin binding domain to amore bulky amino acid, such as isoleucine, abrogates FKBP-12·rapamycinbinding to mTOR, and generates a rapamycin-resistant form of mTOR(4Chen J. Zheng X.F. Brown E.J. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4947-4951Crossref PubMed Scopus (450) Google Scholar,5Hosoi H. Dilling M.B. Shikata T. Liu L.N. Shu L. Ashmun R.A. Germain G.S. Abraham R.T. Houghton P.J. Cancer Res. 1999; 59: 886-894PubMed Google Scholar). The mechanism by whichrapamycin inhibits mTOR function remains poorly understood. Recent studies have provided significant insights into the growth factorand nutrient signaling pathway(s) upstream of mTOR. Stimulation of many growthfactor receptors leads to activation of the PI-3 kinase-AKT pathway, and itappears that this pathway is centrally involved in the coupling of mitogenicstimuli to mTOR. Moreover, loss of the tumor suppressor PTEN provides apowerful signal for tumor progression and simultaneously confers increasedsensitivity to the anti-proliferative effect of rapamycin(6Neshat M.S. Mellinghoff I.K. Tran C. Stiles B. Thomas G. Petersen R. Frost P. Gibbons J.J. Wu H. Sawyers C.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10314-10319Crossref PubMed Scopus (913) Google Scholar,7Podsypanina K. Lee R.T. Politis C. Hennessy I. Crane A. Puc J. Neshat M. Wang H. Yang L. Gibbons J. Frost P. Dreisbach V. Blenis J. Gaciong Z. Fisher P. Sawyers C. Hedrick-Ellenson L. Parsons R. Proc. Natl. Acad. Sci. U. S.A. 2001; 98: 10320-10325Crossref PubMed Scopus (565) Google Scholar). Nutrient/energy status alsoregulates mTOR signaling. Although it has been suggested that mTOR directlysenses ATP levels in the cell(8Dennis P.B. Jaeschke A. Saitoh M. Fowler B. Kozma S.C. Thomas G. Science. 2001; 294: 1102-1105Crossref PubMed Scopus (807) Google Scholar), a more plausible mechanismof mTOR regulation involves the ATP-regulated LKB1/AMPK pathway(9Inoki K. Zhu T.Q. Guan K.L. Cell. 2003; 115: 577-590Abstract Full Text Full Text PDF PubMed Scopus (3034) Google Scholar,10Shaw R.J. Bardeesy N. Manning B.D. Lopez L. Kosmatka M. DePinho R.A. Cantley L.C. CancerCell. 2004; 6: 91-99Scopus (886) Google Scholar). Signaling through boththe PI-3 kinase/AKT and LKB1/AMPK pathways as well as nutrient cues allconverge at the level of the TSC1/2 complex, which genetic and biochemicalevidence establish as a negative regulator of mTOR(11Gao X. Zhang Y. Arrazola P. Hino O. Kobayashi T. Yeung R.S. Ru B. Pan D. Nat. Cell.Biol. 2002; 4: 699-704Crossref PubMed Scopus (577) Google Scholar, 12Inoki K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1439) Google Scholar, 13Tee A.R. Manning B.D. Roux P.P. Cantley L.C. Blenis J. Curr. Biol. 2003; 13: 1259-1268Abstract Full Text Full Text PDF PubMed Scopus (952) Google Scholar, 14Stocker H. Radimerski T. Schindelholz B. Wittwer F. Belawat P. Daram P. Breuer S. Thomas G. Hafen E. Nat. Cell. Biol. 2003; 5: 559-565Crossref PubMed Scopus (435) Google Scholar).TSC2 is a direct target of both AKT and AMPK. Phosphorylation of TSC2 by AKTresults in the inactivation of the TSC1/2 complex(15Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell. Biol. 2002; 4: 648-657Crossref PubMed Scopus (2421) Google Scholar, 16Manning B.D. Tee A.R. Logsdon M.N. Blenis J. Cantley L.C. Mol. Cell. 2002; 10: 151-162Abstract Full Text Full Text PDF PubMed Scopus (1286) Google Scholar, 17Potter C.J. Pedraza L.G. Xu T. Nat. Cell Biol. 2002; 4: 658-665Crossref PubMed Scopus (782) Google Scholar),whereas phosphorylation of TSC2 by AMPK results in enhanced activity(9Inoki K. Zhu T.Q. Guan K.L. Cell. 2003; 115: 577-590Abstract Full Text Full Text PDF PubMed Scopus (3034) Google Scholar). In addition to the regulation of mTOR by the TSC/Rheb pathway, ourlaboratory and others have provided evidence that mTOR may be a directsubstrate for AKT (18Sekulic A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. CancerRes. 2000; 60: 3504-3513Google Scholar,19Nave B.T. Ouwens D.M. Withers D.J. Alessi D.R. Shepherd P.R. Biochem. J. 1999; 344: 427-431Crossref PubMed Scopus (785) Google Scholar). The proposed AKTphosphorylation site (Ser-2448) in mTOR lies within a C-terminal regulatoryregion, which, when deleted, results in elevated mTOR activity invitro and in cells (18Sekulic A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. CancerRes. 2000; 60: 3504-3513Google Scholar,20Edinger A.L. Thompson C.B. Oncogene. 2004; 23: 5654-5663Crossref PubMed Scopus (69) Google Scholar). In this study, we demonstrate that mTOR phosphorylation at Ser-2448 isblocked by rapamycin, and this effect is independent of the AKT activationstatus, which suggests that Ser-2448 phosphorylation is catalyzed by a proteinkinase other than AKT. We show that Ser-2448 is directly phosphorylated byp70S6 kinase in vitro and that expression of a rapamycin-resistantp70S6 kinase in cells maintains Ser-2448 phosphorylation in the presence ofrapamycin. Furthermore, changes in amino acid availability, which affectsp70S6 kinase activity, also modulates Ser-2448 phosphorylation. Finally,Ser-2448 phosphorylation is reduced in cells by siRNA-mediated depletion ofp70S6 kinase. Taken together, these data suggest that p70S6 kinase is themajor protein kinase responsible for Ser-2448 phosphorylation in mammaliancells. Antibodies and Reagents—Rapamycin was obtained from the NCI,National Institutes of Health (Bethesda, MD). Wortmannin, the anti(α)-FLAG M2 antibody, α-FLAG M2-agarose, and the FLAG peptide wereobtained from Sigma. The monoclonal α-AU1 and α-HA (12CA5)antibodies were purchased from Covance (Berkeley, CA). α-AKT andα-phospho-AKT (Ser-473) antibodies were purchased from Cell SignalingTechnology (Beverly, MA), and α-p70S6 kinase antibody was purchased fromSanta Cruz Biotechnology (Santa Cruz, CA). The α-mTOR andα-phospho-Ser-2448 mTOR (α-mTORp2) antibodies have been describedpreviously (18Sekulic A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. CancerRes. 2000; 60: 3504-3513Google Scholar,21Chiang G.G. Abraham R.T. Methods Mol. Biol. 2004; 281: 125-141PubMed Google Scholar). Cell Culture, DNA Constructs, siRNA, and Transfections—HEK293 cells, MCF-7 cells, and HeLa cells were maintained in high glucoseDulbecco's modified essential medium (DMEM) (Irvine Scientific, Santa Ana, CA)supplemented with 10% FCS (Hyclone, Logan, UT). HEK 293 cells stablyexpressing AU1 epitope-tagged, rapamycin-resistant (SI) mTOR have beendescribed (22Edinger A.L. Linardic C.M. Chiang G.G. Thompson C.B. Abraham R.T. Cancer Res. 2003; 63: 8451-8460PubMed Google Scholar). Forexperiments involving serum starvation, cells were washed twice withphosphate-buffered saline and then serum-starved for 16–18 h in DMEMsupplemented with 0.1% FCS. For serum stimulation, cells were incubated for 15min with 10% FCS prior to cell harvest. For amino acid deprivation, cells werewashed twice with phosphate-buffered saline and incubated for 50 min in highglucose DMEM minus amino acids. Cells were restimulated for 10 min withserum-free, high glucose DMEM prior to cell harvest. Where indicated, cellswere pretreated for 30 min with rapamycin or wortmannin before stimulationwith amino acids or serum. The wild type (WT), SI phosphorylation site mutants (Ser-2448 → Alaand Ser-2448 → Glu), and rapamycin-resistant, kinase-inactive mTOR cDNAscloned into the expression vector pcDNA3 (Invitrogen) have been described(18Sekulic A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. CancerRes. 2000; 60: 3504-3513Google Scholar,22Edinger A.L. Linardic C.M. Chiang G.G. Thompson C.B. Abraham R.T. Cancer Res. 2003; 63: 8451-8460PubMed Google Scholar). The HA-tagged wild-typep70S6 kinase and the FLAG-tagged p70S6 kinase were provided by Dr. NaohiroTerada (University of Florida, Gainesville, FL). The HA-tagged,kinase-inactive (K100R) p70S6K mutant in the expression vector pRK7 and theHA-tagged, rapamycin-resistant ΔN/C p70S6K in the expression vector pBJwere provided by Dr. John Blenis (Harvard Medical School, Boston, MA). The luciferase siRNA control and the p70S6K siRNA SMARTpool were purchasedfrom Dharmacon (Lafayette, CO). siRNAs were transfected into HeLa cells withOligofectamine (Invitrogen) according to the manufacturer's protocol. Cellswere harvested 48 h posttransfection. HEK 293 cells were transfected with FuGENE 6 (Roche Applied Science)according to the manufacturer's protocol. Cells were used in experiments at36–48 h posttransfection. Immunoprecipitations, in Vitro Kinase Reactions, andImmunoblotting—For mTOR immunoprecipitations, cells were suspendedin lysis buffer (50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 50mm β-glycerophosphate, 10% glycerol 10 10 mm mm with α-mTOR antibody or α-AU1 were on protein washed with lysis in For p70S6 kinase transfected HEK 293 cells were were with were on washed in lysis by in buffer (50 mm pH 7.4, 10 mm NaCl, 10% mm were in of p70S6 kinase were by addition of of acids and 100 for min at 30 and with For phosphorylation of mTOR by p70S6 HEK transfected with a FLAG-tagged p70S6 kinase expression vector were were and p70S6 kinase was with p70S6 FLAG from serum-starved HEK 293 cells were in 30 ofp70S6 kinase and kinase were by the addition of of kinase and 100 incubated for min at 30 and with were by and to was as G.G. Abraham R.T. Methods Mol. Biol. 2004; 281: 125-141PubMed Google Scholar). antibodies with protein for antibodies or antibody for monoclonal by enhanced that mTOR in vitro by AKT at a residue (Ser-2448) a C-terminal of A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. CancerRes. 2000; 60: 3504-3513Google Scholar,19Nave B.T. Ouwens D.M. Withers D.J. Alessi D.R. Shepherd P.R. Biochem. J. 1999; 344: 427-431Crossref PubMed Scopus (785) Google Scholar). In that phosphorylation at the Ser-2448 site in cells with to rapamycin not these results suggested phosphorylation was by a protein an with AKT. we MCF-7 cells in serum The cells were restimulated with 10% FCS in or presence of 100 rapamycin or 100 serum Ser-2448 a in AKT phosphorylation at ofthe cells with 100 a that not mTOR or other PI-3 kinase family R.S. Abraham R.T. CancerRes. Scholar), phosphorylation of mTOR, as well as the modification of with the for PI-3 kinase in AKT D.R. M. B. P. N. P. 15: PubMed Scopus Google Scholar). phosphorylation was also by rapamycin; to rapamycin effect on the phosphorylation at changes in the phosphorylation of mTOR at Ser-2448 with the activation of AKT in these cells. We binding of FKBP-12·rapamycin to mTOR for the in Ser-2448 phosphorylation. used HEK 293 cells that a mTOR → in the FKBP-12·rapamycin binding The SI mTOR protein reduced binding and confers rapamycin when mammalian cells H. Dilling M.B. Shikata T. Liu L.N. Shu L. Ashmun R.A. Germain G.S. Abraham R.T. Houghton P.J. Cancer Res. 1999; 59: 886-894PubMed Google Scholar). the experiments this with serum-starved HEK mTOR cells In these cells, serum a in the phosphorylation of the SI mTOR Ser-2448. The in Ser-2448 phosphorylation was rapamycin or effect on the the SI mTOR results as is the mTOR phosphorylation of the Ser-2448 site in SI dependent on in to the phosphorylation of SI mTOR at Ser-2448 is to that the effect of rapamycin on is dependent on the inhibition of signaling mTOR Kinase for mTOR kinase activity was Ser-2448 we transfected HEK 293 cells epitope-tagged, WT, SI mTOR, or a rapamycin-resistant, 48 the transfected cells were with rapamycin, and the phosphorylation of Ser-2448 was in the mTOR reduced the phosphorylation of both the kinase-inactive mTOR of the rapamycin-resistant SI mTOR was by the SI mTOR, the rapamycin-resistant, kinase-inactive mTOR mutant this kinase-inactive mTOR rapamycin when transfected into mammalian A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. CancerRes. 2000; 60: 3504-3513Google Scholar). thephosphorylation of the Ser-2448 site on the expression mTOR. Ser-2448 is not a mTOR (18Sekulic A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. CancerRes. 2000; 60: 3504-3513Google Scholar), this that a downstream target of mTOR directly or the phosphorylation of Ser-2448 in cells. Phosphorylation of Ser-2448 by p70S6 Kinase in mTOR target p70S6 kinase a for Ser-2448 kinase. p70S6 mTOR at Ser-2448 in we transfected HEK with an expression a HA-tagged of wild-typep70S6 kinase or a p70S6 kinase mutant to in the domain L. M. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). were with α-HA as a and a protein mTOR amino acids as into the with the antibody, the p70S6 not phosphorylation of the Ser-2448 a control for the of the we the kinase with a protein a Ser-2448 → as the of this with p70S6 to the of the with the antibody not We also p70S6 kinase phosphorylated this we serum-starved HEK 293 Ser-2448 phosphorylation to levels and mTOR We then the of a kinase to the mTOR into Ser-2448 with the incubated with ATP a Ser-2448 phosphorylation. The phosphorylation of mTOR was when the kinase were in the that the Ser-2448 phosphorylation prior to of mTOR from the cells not with with protein, of mTOR was ATP and kinase. Taken together, these results with AKT (18Sekulic A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. CancerRes. 2000; 60: 3504-3513Google Scholar,19Nave B.T. Ouwens D.M. Withers D.J. Alessi D.R. Shepherd P.R. Biochem. J. 1999; 344: 427-431Crossref PubMed Scopus (785) Google the mTOR at at in vitro p70S6 Kinase p70S6 kinase as an in cells, then phosphorylation of mTOR at this residue to rapamycin in cells transfected with a kinase this we mTOR with p70S6 kinase or a p70S6 kinase mutant is to L. M. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: PubMed Scopus Google Scholar). of mTOR at Ser-2448 to rapamycin in with p70S6 kinase In rapamycin to in the ΔN/C p70S6 cells. suggest that p70S6 kinase is a major effector of in serum-stimulated cells. Ser-2448 Phosphorylation by of p70S6 kinase is by amino as well growth K. K. C. J. J. Biol. Full Text Full Text PDF PubMed Scopus Google C. Thomas G. Kozma S.C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). p70S6 kinase is to the phosphorylation of mTOR at Ser-2448 in we that of amino acids to cells to in p70S6 kinase activity and this we HEK 293 cells acids and then restimulated the cells with amino acids in the of rapamycin acid Ser-2448 phosphorylation to a level that in cells of amino acids to these a in Ser-2448 which was the of of p70S6 of kinase thephosphorylation of p70S6 kinase and mTOR by cellular that both were dependent on mTOR amino acid rapamycin the activation of as by changes in thephosphorylation of this protein kinase at these results p70S6 than plays the role in the mTOR phosphorylation at Ser-2448 by amino acids. of p70S6 Kinase in Cells p70S6 kinase was responsible phosphorylation in cells, we used siRNA to p70S6 of HeLa cells with p70S6 siRNA reduced expression with a in Ser-2448 control cells were In both control kinase cells, AKT phosphorylation was evidence that p70S6 kinase is responsible for in cells. Phosphorylation of mTOR at Ser-2448 has a for of the PI-3 kinase pathway as well as the activation Wang K.L. A. Oncogene. 2004; 23: PubMed Scopus Google Scholar, G. S. K. D. A. L. Sawyers C.L. Cancer Res. 2003; 63: Google Scholar, X. M. V. Yang Y. Yang D. D. Cancer Res. 2004; 10: PubMed Scopus Google Scholar, J. Y. R. A. Wang H. M. 2004; PubMed Scopus Google on the that AKT the Ser-2448 is that changes in Ser-2448 phosphorylation in PI-3 kinase activity by factors or The that the regulation phosphorylation is more than proposed and phosphorylation is not to AKT. We that Ser-2448 phosphorylation (18Sekulic A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. CancerRes. 2000; 60: 3504-3513Google Scholar,19Nave B.T. Ouwens D.M. Withers D.J. Alessi D.R. Shepherd P.R. Biochem. J. 1999; 344: 427-431Crossref PubMed Scopus (785) Google Scholar). Although the with the that AKT is an effector an plausible is that is to wortmannin K. J. Mol. Cell. 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Biol. 2002; 4: 648-657Crossref PubMed Scopus (2421) Google Scholar). of phosphorylation of p70S6 kinase and results in elevated K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1439) Google of cells with which glucose the pathway via activity, and results in a in mTOR K. Zhu T.Q. Guan K.L. Cell. 2003; 115: 577-590Abstract Full Text Full Text PDF PubMed Scopus (3034) Google not Furthermore, the depletion of or not mTOR in T. Full Text Full Text PDF PubMed Scopus Google Scholar). that other of regulation by p70S6 kinase it has been that phosphorylation ofthe by p70S6 kinase leads to A. T. S. H. J. S. Shepherd P.R. I. Biol. 2004; PubMed Scopus Google Scholar). of the mammalian p70S6 kinase pathway via loss of or in the inactivation and of and leads Wang Z. T. Curr. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). results to the that a mTOR Ser-2448 mutant the to signal to p70S6 A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. CancerRes. 2000; 60: 3504-3513Google Scholar). AKT as the of Ser-2448 it was to loss ofthe Ser-2448 site to signaling through the AKT → p70S6 kinase The results that is a than a of p70S6 the of with to mTOR signaling remains we not in mTOR with mTOR Ser-2448 → or Ser-2448 → mutants when mTOR not Furthermore, ectopic expression of Ser-2448 → or Ser-2448 → mutants that also rapamycin-resistant SI cell activity in HEK 293 cells we of this rapamycin is to a of (22Edinger A.L. Linardic C.M. Chiang G.G. Thompson C.B. Abraham R.T. Cancer Res. 2003; 63: 8451-8460PubMed Google D. H. P. D.M. Curr. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google E. R. A. S. A. M.N. Nat. Cell.Biol. 2004; 6: PubMed Scopus Google Scholar). that ectopic expression of mutant in the cells confers rapamycin to p70S6 kinase the phosphorylation of Ser-2448 on the not of the of Ser-2448 that the mTOR Ser-2448 and Ser-2448 → be in cells that mTOR. is that the mTOR regulation through phosphorylation of is more than In addition phosphorylation catalyzed by p70S6 kinase and by AMPK L.G. D. Shepherd P.R. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google of and Ser-2448 to be of AMPK phosphorylation within this of mTOR remains L.G. D. Shepherd P.R. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In we that mTOR phosphorylation at Ser-2448 is directly by AKT in a the downstream target of mTOR, p70S6 kinase. The presence of phosphorylation in the mTOR (18Sekulic A. Hudson C.C. Homme J.L. Yin P. Otterness D.M. Karnitz L.M. Abraham R.T. CancerRes. 2000; 60: 3504-3513Google L.G. D. Shepherd P.R. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google that roles in studies to phosphorylation of mTOR by p70S6 kinase or in or growth cells. We Dr. John Blenis and Dr. for p70S6 We also of the Abraham for