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EMBO J. Nov 1, 1995; 14(21): 5279–5287.
PMCID: PMC394637

The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain.

Abstract

The immunosuppressive agent rapamycin induces inactivation of p70s6k with no effect on other mitogen-activated kinases. Here we have employed a combination of techniques, including mass spectrometry, to demonstrate that this effect is associated with selective dephosphorylation of three previously unidentified p70s6k phosphorylation sites: T229, T389 and S404. T229 resides at a conserved position in the catalytic domain, whose phosphorylation is essential for the activation of other mitogen-induced kinases. However, the principal target of rapamycin-induced p70s6k inactivation is T389, which is located in an unusual hydrophobic sequence outside the catalytic domain. Mutation of T389 to alanine ablates kinase activity, whereas mutation to glutamic acid confers constitutive kinase activity and rapamycin resistance. The importance of this site and its surrounding motif to kinase function is emphasized by its presence in a large number of protein kinases of the second messenger family and its conservation in putative p70s6k homologues from as distantly related organisms as yeast and plants.

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  • Alcorta DA, Crews CM, Sweet LJ, Bankston L, Jones SW, Erikson RL. Sequence and expression of chicken and mouse rsk: homologs of Xenopus laevis ribosomal S6 kinase. Mol Cell Biol. 1989 Sep;9(9):3850–3859. [PMC free article] [PubMed]
  • Ballou LM, Siegmann M, Thomas G. S6 kinase in quiescent Swiss mouse 3T3 cells is activated by phosphorylation in response to serum treatment. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7154–7158. [PMC free article] [PubMed]
  • Ballou LM, Jenö P, Thomas G. Protein phosphatase 2A inactivates the mitogen-stimulated S6 kinase from Swiss mouse 3T3 cells. J Biol Chem. 1988 Jan 25;263(3):1188–1194. [PubMed]
  • Ballou LM, Luther H, Thomas G. MAP2 kinase and 70K S6 kinase lie on distinct signalling pathways. Nature. 1991 Jan 24;349(6307):348–350. [PubMed]
  • Boyle WJ, van der Geer P, Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. [PubMed]
  • Brown EJ, Albers MW, Shin TB, Ichikawa K, Keith CT, Lane WS, Schreiber SL. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature. 1994 Jun 30;369(6483):756–758. [PubMed]
  • Chen J, Zheng XF, Brown EJ, Schreiber SL. Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci U S A. 1995 May 23;92(11):4947–4951. [PMC free article] [PubMed]
  • Chung J, Kuo CJ, Crabtree GR, Blenis J. Rapamycin-FKBP specifically blocks growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell. 1992 Jun 26;69(7):1227–1236. [PubMed]
  • Chung J, Grammer TC, Lemon KP, Kazlauskas A, Blenis J. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature. 1994 Jul 7;370(6484):71–75. [PubMed]
  • Dhand R, Hiles I, Panayotou G, Roche S, Fry MJ, Gout I, Totty NF, Truong O, Vicendo P, Yonezawa K, et al. PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity. EMBO J. 1994 Feb 1;13(3):522–533. [PMC free article] [PubMed]
  • Downward J. Signal transduction. Regulating S6 kinase. Nature. 1994 Sep 29;371(6496):378–379. [PubMed]
  • Evan GI, Lewis GK, Ramsay G, Bishop JM. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. [PMC free article] [PubMed]
  • Ferrari S, Bannwarth W, Morley SJ, Totty NF, Thomas G. Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7282–7286. [PMC free article] [PubMed]
  • Ferrari S, Pearson RB, Siegmann M, Kozma SC, Thomas G. The immunosuppressant rapamycin induces inactivation of p70s6k through dephosphorylation of a novel set of sites. J Biol Chem. 1993 Aug 5;268(22):16091–16094. [PubMed]
  • Grove JR, Banerjee P, Balasubramanyam A, Coffer PJ, Price DJ, Avruch J, Woodgett JR. Cloning and expression of two human p70 S6 kinase polypeptides differing only at their amino termini. Mol Cell Biol. 1991 Nov;11(11):5541–5550. [PMC free article] [PubMed]
  • Han JW, Pearson RB, Dennis PB, Thomas G. Rapamycin, wortmannin, and the methylxanthine SQ20006 inactivate p70s6k by inducing dephosphorylation of the same subset of sites. J Biol Chem. 1995 Sep 8;270(36):21396–21403. [PubMed]
  • Haribabu B, Dottin RP. Identification of a protein kinase multigene family of Dictyostelium discoideum: molecular cloning and expression of a cDNA encoding a developmentally regulated protein kinase. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1115–1119. [PMC free article] [PubMed]
  • Herman PK, Emr SD. Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Dec;10(12):6742–6754. [PMC free article] [PubMed]
  • Hiles ID, Otsu M, Volinia S, Fry MJ, Gout I, Dhand R, Panayotou G, Ruiz-Larrea F, Thompson A, Totty NF, et al. Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit. Cell. 1992 Aug 7;70(3):419–429. [PubMed]
  • Jefferies HB, Reinhard C, Kozma SC, Thomas G. Rapamycin selectively represses translation of the "polypyrimidine tract" mRNA family. Proc Natl Acad Sci U S A. 1994 May 10;91(10):4441–4445. [PMC free article] [PubMed]
  • Jones PF, Jakubowicz T, Pitossi FJ, Maurer F, Hemmings BA. Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4171–4175. [PMC free article] [PubMed]
  • Jones PF, Jakubowicz T, Hemmings BA. Molecular cloning of a second form of rac protein kinase. Cell Regul. 1991 Dec;2(12):1001–1009. [PMC free article] [PubMed]
  • Jones SW, Erikson E, Blenis J, Maller JL, Erikson RL. A Xenopus ribosomal protein S6 kinase has two apparent kinase domains that are each similar to distinct protein kinases. Proc Natl Acad Sci U S A. 1988 May;85(10):3377–3381. [PMC free article] [PubMed]
  • Kato JY, Matsuoka M, Polyak K, Massagué J, Sherr CJ. Cyclic AMP-induced G1 phase arrest mediated by an inhibitor (p27Kip1) of cyclin-dependent kinase 4 activation. Cell. 1994 Nov 4;79(3):487–496. [PubMed]
  • Kolodziej PA, Young RA. Epitope tagging and protein surveillance. Methods Enzymol. 1991;194:508–519. [PubMed]
  • Kozma SC, Thomas G. p70s6k/p85s6k: mechanism of activation and role in mitogenesis. Semin Cancer Biol. 1994 Aug;5(4):255–260. [PubMed]
  • Kozma SC, Ferrari S, Bassand P, Siegmann M, Totty N, Thomas G. Cloning of the mitogen-activated S6 kinase from rat liver reveals an enzyme of the second messenger subfamily. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7365–7369. [PMC free article] [PubMed]
  • Kunz J, Henriquez R, Schneider U, Deuter-Reinhard M, Movva NR, Hall MN. Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell. 1993 May 7;73(3):585–596. [PubMed]
  • Kuo CJ, Chung J, Fiorentino DF, Flanagan WM, Blenis J, Crabtree GR. Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature. 1992 Jul 2;358(6381):70–73. [PubMed]
  • Land M, Islas-Trejo A, Freedman JH, Rubin CS. Structure and expression of a novel, neuronal protein kinase C (PKC1B) from Caenorhabditis elegans. PKC1B is expressed selectively in neurons that receive, transmit, and process environmental signals. J Biol Chem. 1994 Mar 25;269(12):9234–9244. [PubMed]
  • Lane HA, Thomas G. Purification and properties of mitogen-activated S6 kinase from rat liver and 3T3 cells. Methods Enzymol. 1991;200:268–291. [PubMed]
  • Lane HA, Morley SJ, Dorée M, Kozma SC, Thomas G. Identification and early activation of a Xenopus laevis p70s6k following progesterone-induced meiotic maturation. EMBO J. 1992 May;11(5):1743–1749. [PMC free article] [PubMed]
  • Lane HA, Fernandez A, Lamb NJ, Thomas G. p70s6k function is essential for G1 progression. Nature. 1993 May 13;363(6425):170–172. [PubMed]
  • Marshall CJ. Signal transduction. Hot lips and phosphorylation of protein kinases. Nature. 1994 Feb 24;367(6465):686–686. [PubMed]
  • Maurer RA. Isolation of a yeast protein kinase gene by screening with a mammalian protein kinase cDNA. DNA. 1988 Sep;7(7):469–474. [PubMed]
  • Ming XF, Burgering BM, Wennström S, Claesson-Welsh L, Heldin CH, Bos JL, Kozma SC, Thomas G. Activation of p70/p85 S6 kinase by a pathway independent of p21ras. Nature. 1994 Sep 29;371(6496):426–429. [PubMed]
  • Mischak H, Bodenteich A, Kolch W, Goodnight J, Hofer F, Mushinski JF. Mouse protein kinase C-delta, the major isoform expressed in mouse hemopoietic cells: sequence of the cDNA, expression patterns, and characterization of the protein. Biochemistry. 1991 Aug 13;30(32):7925–7931. [PubMed]
  • Mukhopadhyay NK, Price DJ, Kyriakis JM, Pelech S, Sanghera J, Avruch J. An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase. J Biol Chem. 1992 Feb 15;267(5):3325–3335. [PubMed]
  • Nourse J, Firpo E, Flanagan WM, Coats S, Polyak K, Lee MH, Massague J, Crabtree GR, Roberts JM. Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature. 1994 Dec 8;372(6506):570–573. [PubMed]
  • Ohno S, Kawasaki H, Konno Y, Inagaki M, Hidaka H, Suzuki K. A fourth type of rabbit protein kinase C. Biochemistry. 1988 Mar 22;27(6):2083–2087. [PubMed]
  • Chen CA, Okayama H. Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. Biotechniques. 1988 Jul-Aug;6(7):632–638. [PubMed]
  • Ono Y, Fujii T, Ogita K, Kikkawa U, Igarashi K, Nishizuka Y. The structure, expression, and properties of additional members of the protein kinase C family. J Biol Chem. 1988 May 15;263(14):6927–6932. [PubMed]
  • Pearson RB, Kemp BE. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 1991;200:62–81. [PubMed]
  • Price DJ, Grove JR, Calvo V, Avruch J, Bierer BE. Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science. 1992 Aug 14;257(5072):973–977. [PubMed]
  • Reinhard C, Thomas G, Kozma SC. A single gene encodes two isoforms of the p70 S6 kinase: activation upon mitogenic stimulation. Proc Natl Acad Sci U S A. 1992 May 1;89(9):4052–4056. [PMC free article] [PubMed]
  • Reinhard C, Fernandez A, Lamb NJ, Thomas G. Nuclear localization of p85s6k: functional requirement for entry into S phase. EMBO J. 1994 Apr 1;13(7):1557–1565. [PMC free article] [PubMed]
  • Sabatini DM, Erdjument-Bromage H, Lui M, Tempst P, Snyder SH. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell. 1994 Jul 15;78(1):35–43. [PubMed]
  • Schaeffer E, Smith D, Mardon G, Quinn W, Zuker C. Isolation and characterization of two new drosophila protein kinase C genes, including one specifically expressed in photoreceptor cells. Cell. 1989 May 5;57(3):403–412. [PubMed]
  • Su L, Parissenti AM, Riedel H. Functional carboxyl terminal deletion map of protein kinase C alpha. Receptors Channels. 1993;1(1):1–9. [PubMed]
  • Susa M, Vulević D, Lane HA, Thomas G. Inhibition or down-regulation of protein kinase C attenuates late phase p70s6k activation induced by epidermal growth factor but not by platelet-derived growth factor or insulin. J Biol Chem. 1992 Apr 5;267(10):6905–6909. [PubMed]
  • Toda T, Cameron S, Sass P, Wigler M. SCH9, a gene of Saccharomyces cerevisiae that encodes a protein distinct from, but functionally and structurally related to, cAMP-dependent protein kinase catalytic subunits. Genes Dev. 1988 May;2(5):517–527. [PubMed]
  • Weng QP, Andrabi K, Klippel A, Kozlowski MT, Williams LT, Avruch J. Phosphatidylinositol 3-kinase signals activation of p70 S6 kinase in situ through site-specific p70 phosphorylation. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5744–5748. [PMC free article] [PubMed]
  • Weng QP, Andrabi K, Kozlowski MT, Grove JR, Avruch J. Multiple independent inputs are required for activation of the p70 S6 kinase. Mol Cell Biol. 1995 May;15(5):2333–2340. [PMC free article] [PubMed]
  • Wettenhall RE, Aebersold RH, Hood LE. Solid-phase sequencing of 32P-labeled phosphopeptides at picomole and subpicomole levels. Methods Enzymol. 1991;201:186–199. [PubMed]
  • Zhang SH, Lawton MA, Hunter T, Lamb CJ. atpk1, a novel ribosomal protein kinase gene from Arabidopsis. I. Isolation, characterization, and expression. J Biol Chem. 1994 Jul 1;269(26):17586–17592. [PubMed]

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