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• W2149443458 abstract "The tuberous sclerosis gene products Tsc1 and Tsc2 behave as tumor suppressors by restricting cell growth, a function conserved among metazoans. Recent evidence has indicated that hyperactivation of S6 kinase 1 (S6K1) may represent an important biochemical change in the development of tuberous sclerosis-associated lesions. We show here that deletion of either Tsc1 or Tsc2 or expression of the Rheb (Ras homolog enriched in brain) GTPase leads to hyperphosphorylation of S6K1 at a subset of regulatory sites, particularly those of two essential residues functionally conserved among AGC superfamily serine/threonine kinases, i.e. the activation loop (T-loop; Thr-229) and the hydrophobic motif (H-motif; Thr-389). These sites are reciprocally and dose-dependently regulated when S6K1 is coexpressed with the Tsc1-Tsc2 complex. Mutations that render S6K1 mTOR (mammalian target of rapamycin)-resistant also protect S6K1 activity and phosphorylation from down-regulation by Tsc1/2. We demonstrate that two disease-associated mutations in Tsc2 fail to negatively regulate S6K1 activity concomitant with a failure to modify T-loop and H-motif phosphorylation. Finally, we identify one pathological Tsc2 mutation that retains its ability to negatively regulate S6K1, suggesting that, in some cases, tuberous sclerosis may develop independently of S6K1 hyperactivation. These results also highlight the importance of dual control of T-loop and H-motif phosphorylation of S6K1 by the Tsc1-Tsc2 complex. The tuberous sclerosis gene products Tsc1 and Tsc2 behave as tumor suppressors by restricting cell growth, a function conserved among metazoans. Recent evidence has indicated that hyperactivation of S6 kinase 1 (S6K1) may represent an important biochemical change in the development of tuberous sclerosis-associated lesions. We show here that deletion of either Tsc1 or Tsc2 or expression of the Rheb (Ras homolog enriched in brain) GTPase leads to hyperphosphorylation of S6K1 at a subset of regulatory sites, particularly those of two essential residues functionally conserved among AGC superfamily serine/threonine kinases, i.e. the activation loop (T-loop; Thr-229) and the hydrophobic motif (H-motif; Thr-389). These sites are reciprocally and dose-dependently regulated when S6K1 is coexpressed with the Tsc1-Tsc2 complex. Mutations that render S6K1 mTOR (mammalian target of rapamycin)-resistant also protect S6K1 activity and phosphorylation from down-regulation by Tsc1/2. We demonstrate that two disease-associated mutations in Tsc2 fail to negatively regulate S6K1 activity concomitant with a failure to modify T-loop and H-motif phosphorylation. Finally, we identify one pathological Tsc2 mutation that retains its ability to negatively regulate S6K1, suggesting that, in some cases, tuberous sclerosis may develop independently of S6K1 hyperactivation. These results also highlight the importance of dual control of T-loop and H-motif phosphorylation of S6K1 by the Tsc1-Tsc2 complex. Tuberous sclerosis is a hyperproliferative disorder resulting in the appearance of benign tumors in multiple organ systems including brain, skin, lungs, heart, eyes, kidneys, pancreas, and the skeleton (1Gomez M.R. Tuberous Sclerosis. 2nd Ed. Raven Press, New York1988Google Scholar, 2Hyman M.H. Whittemore V.H. Ach. Neurol. 2000; 57: 662-665Crossref PubMed Scopus (157) Google Scholar). Initially, linkage analysis of affected families suggested that two distinct loci participated in the manifestation of the disease (3Povey S. Burley M.W. Attwood J. Benham F. Hunt D. Jeremiah S.J. Franklin D. Gillet G. Malas S. Robson E.B. Tippett P. Edwards J.H. Kwiatkowski D.J. Super M. Mueller R. Fryer A. Clarke A. Webb D. Osborne J. Ann. Hum. Genet. 1994; 58: 107-127Crossref PubMed Scopus (234) Google Scholar). Meanwhile, TSC1 and TSC2 were identified by positional cloning as the genes whose mutations cause tuberous sclerosis (4The European Chromosome 16 Tuberous Sclerosis ConsortiumCell. 1993; 75: 1305-1315Abstract Full Text PDF PubMed Scopus (1500) Google Scholar, 5van Slegtenhorst M. de Hoogt R. Hermans C. Nellist M. Janssen B. Verhoef S. Lindhout D. van den Ouweland A. Halley D. Young J. Burley M. Jeremiah S. Woodward K. Nahmias J. Fox M. Ekong R. Osborne J. Wolfe J. Povey S. Snell R.G. Cheadle J.P. Jones A.C. Tachataki M. Ravine D. Sampson J.R. Reeve M.P. Richardson P. Wilmer F. Munro C. Hawkins T.L. Sepp T. Ali J.B.M. Ward S. Green A.J. Yates J.R.W. Kwiatkowska J. Henske E.P. Short M.P. Haines J.H. Jozwiak S. Kwiatkowski D.J. Science. 1997; 277: 805-808Crossref PubMed Scopus (1381) Google Scholar). In metazoans, Tsc1 and Tsc2 form a signaling complex that performs a cell growth suppressive function. Studies in Drosophila have demonstrated that loss-of-function mutations in either dTsc1 or dTsc2 increase cell size and do so cell autonomously (6Potter C.J. Huang H. Xu T. Cell. 2001; 105: 357-368Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 7Gao X. Pan D. Genes Dev. 2001; 15: 1383-1392Crossref PubMed Scopus (388) Google Scholar). Conversely, combined overexpression of dTsc1 and dTsc2 reduces cell size, whereas expression of either product individually is without effect (6Potter C.J. Huang H. Xu T. Cell. 2001; 105: 357-368Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar), which is consistent with a model in which the Tsc1-Tsc2 heterodimer is the functional configuration. Homozygous disruption of either Tsc1 or Tsc2 in mice is embryonically lethal, whereas mice heterozygous for either allele display increased organ size and a propensity for tumor development (8Kwiatkowski D.J. Zhang H. Bandura J.L. Heiberger K.M. Glogauer M. el-Hashemite N. Onda H. Hum. Mol. Genet. 2002; 11: 525-534Crossref PubMed Scopus (533) Google Scholar, 9Rennebeck G. Kleymenova E.V. Anderson R. Yeung R.S. Artzt K. Walker C.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15629-15634Crossref PubMed Scopus (86) Google Scholar). Recently, it has been demonstrated that a deficiency in either of the tuberous sclerosis gene products leads to hyperactivation of S6K1. 1The abbreviations used are: S6K1, S6 kinase 1; AID, autoinhibitory domain; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid; CT, carboxyl terminus; HA, hemagglutinin A; HEK, human embryonic kidney; MEF, mouse embryo fibroblast; mTOR, mammalian target of rapamycin; PDK, phosphoinositide-dependent kinase 1; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; Rheb, Ras homolog enriched in brain; rpS6, ribosomal protein 6. 1The abbreviations used are: S6K1, S6 kinase 1; AID, autoinhibitory domain; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid; CT, carboxyl terminus; HA, hemagglutinin A; HEK, human embryonic kidney; MEF, mouse embryo fibroblast; mTOR, mammalian target of rapamycin; PDK, phosphoinositide-dependent kinase 1; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol 3,4,5-trisphosphate; Rheb, Ras homolog enriched in brain; rpS6, ribosomal protein 6. In the absence of an S6K1 crystal structure, the model of S6K1 activation is based on extensive mutational and structure-function studies and by inferences from the structure and analysis of other AGC kinases, including Akt (10Yang J. Cron P. Thompson V. Good V.M. Hess D. Hemmings B.A. Barford D. Mol. Cell. 2002; 9: 1227-1240Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 11Yang J. Cron P. Good V.M. Thompson V. Hemmings B.A. Barford D. Nat. Struct. Biol. 2002; 9: 940-944Crossref PubMed Scopus (428) Google Scholar). It is postulated that the unphosphorylated autoinhibitory domain (AID) folds upon and occludes the amino-terminally positioned kinase domain, perhaps performing the following two functions: 1) limiting access of the substrate to the catalytic site; and 2) burying additional activating phosphorylation sites within the protein's interior (see Fig. 1A). Activation is achieved through a coordinated and sequential series of phosphorylations beginning with phosphorylation of the carboxyl-terminal AID. Up to six serine/threonine and proline sites, designated (S/T)P, are localized to the AID and undergo phosphorylation in response to activating stimuli. Substitution of four of these sites with alanine compromises the serum-induced activation of S6K1 (12Han J.W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), whereas exchange of these sites with phosphomimic acidic residues only slightly increases the basal activity (12Han J.W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 13Pearson R.B. Dennis P.B. Han J.-W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 14: 5279-5287Crossref PubMed Scopus (387) Google Scholar). It is therefore plausible that phosphorylation of the AID is necessary but not sufficient for S6K1 activation. Subsequently, two sites conserved among kinases of the AGC superfamily of serine/threonine kinases are phosphorylated, namely the activation loop (T-loop) site at position Thr-229 and the hydrophobic motif (H-motif) site at position Thr-389. Mutation of Thr-229 to either alanine or glutamate abolishes kinase activity (13Pearson R.B. Dennis P.B. Han J.-W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 14: 5279-5287Crossref PubMed Scopus (387) Google Scholar, 14Weng 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, 15Sugiyama H. Papst P. Gelfand E.W. Terada N. J. Immunol. 1996; 157: 656-660PubMed Google Scholar). However, whereas substitution of Thr-389 with alanine abolishes kinase activity (13Pearson R.B. Dennis P.B. Han J.-W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 14: 5279-5287Crossref PubMed Scopus (387) Google Scholar, 16Weng 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 (338) Google Scholar, 17Dennis P.B. Pullen N. Pearson R.B. Kozma S.C. Thomas G. J. Biol. Chem. 1998; 273: 14845-14852Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), glutamate substitution of the H-motif increases basal kinase activity (13Pearson R.B. Dennis P.B. Han J.-W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 14: 5279-5287Crossref PubMed Scopus (387) Google Scholar, 16Weng 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 (338) Google Scholar, 17Dennis P.B. Pullen N. Pearson R.B. Kozma S.C. Thomas G. J. Biol. Chem. 1998; 273: 14845-14852Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Additional phosphorylation sites have been mapped to Thr-367, Ser-371, and Ser-404 (13Pearson R.B. Dennis P.B. Han J.-W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 14: 5279-5287Crossref PubMed Scopus (387) Google Scholar, 18Dennis P.B. Pullen N. Kozma S.C. Thomas G. Mol. Cell. Biol. 1996; 16: 6242-6251Crossref PubMed Scopus (222) Google Scholar). However, it is less clear just how the phosphorylation of these sites participates in the collective regulation of S6K1. For S6K1, T-loop and H-motif phosphorylation is the net result of the integration of two major input pathways, nutrient sufficiency and growth factor adequacy. The nutrient sufficiency pathway senses the availability of glucose and amino acids as well as mitochondrial function and requires a complex comprised of mTOR, the regulatory associated protein of mTOR (Raptor), and GβL (19Kim D.-H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2328) Google Scholar, 20Hara K. Maruki Y. Long X. Yoshino K. Oshiro N. Hidayat S. Tokunaga C. Avruch J. Yonezawa K. Cell. 2002; 110: 177-189Abstract Full Text Full Text PDF PubMed Scopus (1434) Google Scholar). Raptor appears to physically recognize the TOS (target of rapamycin signaling) motif in S6K1 and another mTOR substrate, the eIF4E-binding protein (4EBP), and may function to present such substrates to the mTOR kinase (21Nojima H. Tokunaga C. Eguchi S. Oshiro N. Hidayat S. Yoshino K. Hara K. Tanaka N. Avruch J. Yonezawa K. J. Biol. Chem. 2003; 278: 15461-15464Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar). mTOR has been shown to phosphorylate S6K1 in vitro both at the H-motif site, Thr-389, and at the AID sites, Ser-411 and Thr-421/Ser-424, although Thr-389 appears to be the major site of phosphorylation (22Isotani S. Hara K. Tokunaga C. Inoue H. Avruch J. Yonezawa K. J. Biol. Chem. 1999; 274: 34493-34498Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Small interfering RNA depletion of mTOR, Raptor, or GβL compromises nutrient stimulation of S6K1 (19Kim D.-H. Sarbassov D.D. Ali S.M. King J.E. Latek R.R. Erdjument-Bromage H. Tempst P. Sabatini D.M. Cell. 2002; 110: 163-175Abstract Full Text Full Text PDF PubMed Scopus (2328) Google Scholar), demonstrating a requirement for each component of the complex in productive signaling. It is unclear precisely how nutrient availability is sensed by the mTOR-Raptor-GβL complex. Regulation of S6K1 by the growth factor adequacy pathway involves activation of phosphatidylinositol 3-kinase (PI3K) and, thus, the generation of phosphatidylinositol-3,4,5-trisphosphate (PIP3) in cellular membranes. S6K1 is activated by ectopic expression of PI3K (14Weng 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) and is inhibited by the PI3K inhibitor wortmannin (12Han J.W. Pearson R.B. Dennis P.B. Thomas G. J. Biol. Chem. 1995; 270: 21396-21403Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 14Weng 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), indicating that PI3K is both necessary and sufficient for S6K1 activity. In response to growth factors, PI3K phosphorylates phosphoinositides at the D3 position of the inositol ring, producing PIP3. The PH (pleckstrin homology) domains of phosphoinositide-dependent kinase 1 (PDK1) and Akt bind PIP3, resulting in the membrane localization and activation of these proteins (reviewed in Ref. 23Vanhaesebroeck B. Alessi D.R. Biochem. J. 2000; 346: 561-576Crossref PubMed Scopus (1389) Google Scholar). In mitogen-stimulated cells, S6K1 is phosphorylated at the T-loop site, Thr-229, by PDK1 (24Pullen N. Dennis P. Andjelkovic M. Dufner A. Kozma S.C. Hemmings B.A. Thomas G. Science. 1998; 279: 707-710Crossref PubMed Scopus (723) Google Scholar, 25Alessi D.R. Kozlowski M.T. Weng Q.-P. Morrice N. Avruch J. Curr. Biol. 1997; 8: 69-81Abstract Full Text Full Text PDF Scopus (512) Google Scholar). A phosphopeptide modeled after the phosphorylated H-motif of S6K1 binds much more efficiently to PDK1 than the unphosphorylated peptide (26Biondi R.M. Komander D. Thomas C.C. Lizcano J.M. Deak M. Alessi D.R. van Aalten D.M.F. EMBO J. 2002; 21: 4219-4228Crossref PubMed Scopus (166) Google Scholar), potentially explaining the strongly synergistic nature of H-motif and T-loop phosphorylation of S6K1. Moreover, when coexpressed in cells, PDK1 binds much more efficiently to an S6K1 variant bearing a phosphomimic H-motif substitution (27Biondi R.M. Kieloch A. Currie R.A. Deak M. Alessi D.R. EMBO J. 2001; 20: 4380-4390Crossref PubMed Scopus (304) Google Scholar), suggesting that the phosphorylated H-motif provides a surface with which PDK1 makes physical contact. Akt is also phosphorylated and activated by PDK1. In response to growth factors, Akt phosphorylates Tsc2 on as many as five sites (28Manning 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 (1265) Google Scholar, 29Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2376) Google Scholar), thereby inhibiting the intrinsic GTP exchange activity of Tsc2 for the small GTP-binding protein Rheb (30Zhang Y. Gao X. Saucedo L.J. Ru B. Edgar B.A. Pan D. Nat. Cell Biol. 2003; 5: 578-581Crossref PubMed Scopus (712) Google Scholar, 31Garami A. Zwartkruis F.J. Nobukuni T. Joaquin M. Roccio M. Stocker H. Kozma S.C. Hafen E. Bos J.L. Thomas G. Mol. Cell. 2003; 11: 1457-1466Abstract Full Text Full Text PDF PubMed Scopus (838) Google Scholar, 32Inoki K. Li Y. Xu T. Guan K.L. Genes Dev. 2003; 17: 1829-1834Crossref PubMed Scopus (1400) Google Scholar). Thus, in response to growth factors, Rheb accumulates in the GTP-liganded and, thus, active configuration. Although Rheb activation is sufficient to activate S6K1 even in the absence of amino acids (33Tee 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 (938) Google Scholar), whether or not Rheb signals to S6K1 indirectly through mTOR remains a debated issue. Given the requirement of T-loop and H-motif phosphorylation for S6K1 activation and given the finding that Tsc1 or Tsc2 loss-of-function results in hyperactivation of S6K1, we set out to determine what role individual phosphorylations play in the collective control of S6K1 by the Tsc1-Tsc2 complex. Antibodies and Reagents—Anti-S6K1 antibodies were raised in rabbits immunized with a synthetic peptide corresponding to amino acids 476–487 (476RQPNSGPYKKQA487), which are close to the carboxyl terminus of rat S6K1 and are used for immunoblotting. For immunoprecipitation of endogenous S6K1, anti-S6K1 antiserum was from Santa Cruz Biotechnology (catalog number sc-230). Anti-rpS6 (Ser(P)-240/Ser(P)-244) and anti-S6K1 (Thr-421(P)/Ser(P)-424) were from Cell Signaling Technology, anti-S6K1 (Thr(P)-229) and anti-S6K1 (Ser(P)-371) were from BIOSOURCE, anti-S6K1 (Ser(P)-411) was from Santa Cruz Biotechnology, anti-S6K1 (Thr(P)-389) was from Upstate Biotechnology, and anti-FLAG (M2) was from Sigma. Anti-HA (12CA5) and anti-Myc (9E10) monoclonal antibodies were purified from mouse ascites. Insulin and rapamycin were purchased from Sigma. LY294002 and wortmannin were purchased from Calbiochem. Plasmid Constructs—Amino-terminally HA-tagged, wild type rat S6K1, E389D3E-S6K1, D4E-S6K1, ΔCT-S6K1, and ΔNTΔCT-S6K1 cloned into pRK7 were generously provided by John Blenis (Harvard University) and Jim Jefferson (Pennsylvania State University College of Medicine). Human Tsc1 and Tsc2 cloned into pEFP2 were provided by Jeff DeClue (NCI, National Institutes of Health). Amino-terminally FLAG-tagged Tsc1 was constructed by site directed mutagenesis (QuikChange site-directed mutagenesis kit, Stratagene) using pEFP2-Tsc1 as a template. Sense and antisense primers were designed to incorporate the FLAG epitope as a loop structure, flanked on either side by sequence that annealed to the template. FLAG-tagged Tsc2 was constructed similarly using pEFP2-Tsc2 as a template. Amino-terminally Myc-tagged human Rheb was supplied by Paul Worley (Johns Hopkins University). Cell Culture and Transient Transfection—Mouse embryo fibroblasts (MEFs) from Tsc1- and Tsc2-null embryos and wild type MEFs were generously provided by David Kwiatkowski (Harvard University). MEFs were cultured in Dulbecco's modified Eagle's medium supplemented with fetal calf serum (10%, v/v) and penicillin, streptomycin, and ciprofloxacin. For experiments requiring insulin stimulation, MEFs were cultured in Dulbecco's modified Eagle's medium containing low serum (0.5% fetal calf serum, v/v) overnight and stimulated with insulin as indicated the following morning. HEK293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with fetal calf serum (10%, v/v) and penicillin, streptomycin, and ciprofloxacin. Cells were transfected by lipofection using Effectene transfection reagent (Qiagen). Cell Extraction and Immunoprecipitation—Cell lysates were extracted in lysis buffer (40 mm HEPES, pH 7.5, 0.3% CHAPS (v/v), 120 mm NaCl, 1 mm EDTA, 10 mm pyrophosphate, 10 mm glycerophosphate, 50 mm NaF, 1 μg/ml leupeptin, 1 mm Na3VO4, 500 μm phenylmethyl-sulfonyl fluoride, 1 mm dithiothreitol, and 1 μg/ml aprotinin). Lysates were rotated end-over-end at 4 °C for 20 min and clarified by centrifugation at 15,000 × g for 5 min. Protein concentrations were determined spectrophotometrically using the Bio-Rad DC protein assay kit. Lysates were either mixed 3:1 with 4× sample buffer and heated to 100 °C for 5 min or subjected to immunoprecipitation. For immunoprecipitation, lysates normalized for cell protein were incubated with 4–8 μg of anti-S6K1 antibody or with 2 μl of 12CA5 ascites and mixed end-over-end for 1–2 h at 4 °C. Protein A-agarose beads were added for an additional hour, and immune complexes were isolated by centrifugation. Immunoprecipitates were washed twice with lysis buffer and heated for 5 min to 100 °C in 1× sample buffer. Assay of S6K1 Kinase Activity—The assay of S6K1 activity has been described elsewhere (34Shah O.J. Kimball S.R. Jefferson L.S. Biochem. J. 2000; 347: 389-397Crossref PubMed Scopus (62) Google Scholar). In brief, immunopurified endogenous or exogenous S6K1 was incubated with a synthetic peptide (AKRRRLSSLRA), and 32P incorporation into the peptide substrate was measured by liquid scintillation counting. In Drosophila, loss of Tsc1 or Tsc2 leads to increased activity of dS6K and, as a result, a significant increase in the size of affected cells. To establish whether or not Tsc1 and Tsc2 regulation of S6K1 is conserved in mammalian cells, we assayed the kinase activity of S6K1 in vitro in MEFs isolated from Tsc1- or Tsc2-null mice or their wild type counterparts. Whereas treatment of wild type MEFs with insulin induced a marked increase in S6K1 activity, the basal activity of S6K1 isolated from Tsc1- and Tsc2-null MEFs was high and was not increased further by insulin (Fig. 1B). In fact, basal S6K1 activity in the knock-out MEFs was equivalent to the insulin-stimulated activity measured in wild type MEFs, suggesting that deletion of either Tsc1 or Tsc2 renders S6K1 constitutively activated. Nevertheless, the high basal activity of S6K1 in Tsc1- and Tsc2-null MEFs remained sensitive to rapamycin (40 nm) and to LY294002 (20 μm), which inhibits mTOR and PI3K equally efficiently at this concentration (35Brunn G.J. Williams J. Sabers C. Wiederrecht G. Abraham R.T. EMBO J. 1996; 15: 5256-5267Crossref PubMed Scopus (620) Google Scholar). In whole cell extracts from Tsc1- and Tsc2-null MEFs, basal phosphorylation of ribosomal protein S6 (rpS6), an endogenous S6K1 substrate, was equivalent to the insulin-stimulated levels observed in wild type MEFs (Fig. 1C, cf. lane 4 versus lanes 7 and 13). These data are consistent with genetic epistasis analyses conducted in Drosophila, indicating that mTOR lies downstream of the Tsc1-Tsc2 complex in the regulation of S6K1 (6Potter C.J. Huang H. Xu T. Cell. 2001; 105: 357-368Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 7Gao X. Pan D. Genes Dev. 2001; 15: 1383-1392Crossref PubMed Scopus (388) Google Scholar). Whereas overexpression of Tsc1-Tsc2 reduces H-motif phosphorylation of coexpressed S6K1 (29Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2376) Google Scholar), Tsc2-null cells display augmented H-motif phosphorylation of S6K1 relative to wild type cells (36Jaeschke A. Hartkamp J. Saitoh M. Roworth W. Nobukuni T. Hodges A. Sampson J. Thomas G. Lamb R. J. Cell Biol. 2002; 159: 217-224Crossref PubMed Scopus (182) Google Scholar). It remains to be resolved, however, whether T-loop phosphorylation (Thr-229) or phosphorylation of other regulatory sites in the AID is influenced by the loss of Tsc1 or Tsc2. To address this question, S6K1 was immunoprecipitated from Tsc1- or Tsc2-null MEFs as well as from wild type MEFs under different treatment conditions. The phosphorylation status of S6K1 was assessed using antiphosphopeptide antibodies specific for the phosphorylation sites Thr-229, Ser-371, Thr-389, Ser-411, and Thr-421/Ser-424 (see Fig. 1A). In all cell types, the pattern of basal and insulin-stimulated S6K1 activity (Fig. 1B) tightly correlated with H-motif phosphorylation at Thr-389 (Fig. 2). Importantly, whereas phosphorylation of Thr-389 in Tsc1- and Tsc2-null MEFs was inhibited by rapamycin, phosphorylation of this site was strongly resistant to inhibition by wortmannin. In contrast, S6K1 prepared from wild type MEFs displayed a reduction in H-motif phosphorylation when treated with either rapamycin or wortmannin. The wortmannin resistance of H-motif phosphorylation in MEFs devoid of Tsc1 or Tsc2 suggests that PI3K regulates S6K1 upstream of Tsc1-Tsc2 and that the effect of the loss of Tsc1-Tsc2 is dominant in the stimulation of S6K1 by PI3K. T-loop phosphorylation of S6K1 at Thr-229 under these conditions mirrors phosphorylation of the H-motif in that this phosphorylation was largely rapamycin-sensitive and wortmannin-resistant in Tsc1- and Tsc2-null cells. A similar correlation was observed for phosphorylation of the AID sites Ser-411 and Thr-421/Ser-424. Unlike the regulation of the sites described above, the phosphorylation of Ser-371 was neither robustly stimulated by insulin nor inhibited by rapamycin or wortmannin. Thus, phosphorylation of Ser-371 is unregulated under these conditions and appears to some extent constitutive. Analysis of the phosphorylation status of rpS6 (Fig. 2B) indicates that the cumulative effect of this multisite regulation was constitutive activation of S6K1 when Tsc1 or Tsc2 is functionally lost. Clearly, deletion of the genes encoding Tsc1 or Tsc2 induces maximal phosphorylation of S6K1 at several regulatory sites, including the T-loop and H-motif sites. Consequently, in Tsc1- and Tsc2-null cells S6K1 is constitutively activated and refractory to conditions that would otherwise be inactivating, e.g. serum-deprivation. Overexpression of the Tsc1-Tsc2 complex in cells has the converse effect, i.e. S6K1 inhibition (28Manning 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 (1265) Google Scholar, 29Inoki K. Li Y. Zhu T. Wu J. Guan K.L. Nat. Cell Biol. 2002; 4: 648-657Crossref PubMed Scopus (2376) Google Scholar, 36Jaeschke A. Hartkamp J. Saitoh M. Roworth W. Nobukuni T. Hodges A. Sampson J. Thomas G. Lamb R. J. Cell Biol. 2002; 159: 217-224Crossref PubMed Scopus (182) Google Scholar) (see below). We therefore sought to address whether or not this inhibition is associated with dephosphorylation of the same subset of Tsc1-Tsc2-regulated sites defined by the aforementioned studies. HEK293T cells exhibit high basal S6K1 activity (data not shown) and were therefore chosen for the subsequent experiments. Cells were transfected with increasing amounts of vectors expressing FLAG-tagged Tsc1 and FLAG-tagged Tsc2 together with HA-tagged S6K1, and S6K1 kinase activity was assayed in vitro at two different ectopic S6K1 gene dosages. Expression of Tsc1-Tsc2 led to a dosage-dependent reduction in cotransfected S6K1 activity (Fig. 3A). This effect was irrespective of the level of ectopically expressed S6K1 used in these experiments. Overexpression of Tsc1-Tsc2 reduced H-motif phosphorylation (Fig. 3B), paralleling the decrease in S6K1 kinase activity. In contrast, little change in the phosphorylation of the AID sites, Thr-421/Ser-424, was observed upon Tsc1-Tsc2 expression. This was somewhat unexpected, given the robust stimulation of Thr-421/Ser-424 phosphorylation observed in Tsc1- and Tsc2-null MEFs (Fig. 2A). Additionally, the phosphorylation of S6K1 at Ser-371 also was not significantly altered upon Tsc1-Tsc2 overexpression, which is consistent with this site remaining relatively unregulated by the Tsc1–2 complex. These data suggest that overexpression of Tsc1-Tsc2 inhibits S6K1 primarily through regulation of the H-motif and, potentially, T-loop phosphorylation, with a minimal effect on AID phosphorylation. We reasoned that the S6K1 mutations that artificially preserve or mimic H-motif phosphorylation should render the activity of the corresponding variant resistant to inhibition in cells overexpressing of Tsc1-Tsc2. Unfortunately, any modification of the T-loop site in S6K1, whether it be alanine or glutamate substitution, abolishes kinase activity (13Pearson R.B. Dennis P.B. Han J.-W. Williamson N.A. Kozma S.C. Wettenhall R.E.H. Thomas G. EMBO J. 1995; 14: 5279-5287Crossref PubMed Scopus (387) Google Scholar, 14Weng 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, 16Weng 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 (338) Google Scholar). Therefore, the necessity and sufficiency of regulation of the T-loop site could not be directly tested in such an assay. A series of HA-tagged truncation and phosphomimic S6K1 mutants were expressed in the presence or absence of FLAG-tagged Tsc1 and FLAG-tagged Tsc2, and the kinase activity of each mutant was assayed in vitro. Because the specific activities of each S6K1 construct differ as a result of each unique mutation, the activity measurements for each construct assayed in the absence of Tsc1-Tsc2 was assigned a value of 100% to allow comparison between constructs (Fig. 4A). The unadjusted kinase activity measurements are presented for reference in Fig. 4B, as is the rel" @default.
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• W2149443458 title "Critical Role of T-Loop and H-Motif Phosphorylation in the Regulation of S6 Kinase 1 by the Tuberous Sclerosis Complex" @default.
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• W2149443458 doi "https://doi.org/10.1074/jbc.m400957200" @default.
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