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. 1995 Nov 1;14(21):5279–5287. doi: 10.1002/j.1460-2075.1995.tb00212.x

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

R B Pearson 1, P B Dennis 1, J W Han 1, N A Williamson 1, S C Kozma 1, R E Wettenhall 1, G Thomas 1
PMCID: PMC394637  PMID: 7489717

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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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alcorta D. A., Crews C. M., Sweet L. J., Bankston L., Jones S. W., Erikson R. L. Sequence and expression of chicken and mouse rsk: homologs of Xenopus laevis ribosomal S6 kinase. Mol Cell Biol. 1989 Sep;9(9):3850–3859. doi: 10.1128/mcb.9.9.3850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ballou L. M., 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] [Google Scholar]
  3. Ballou L. M., Luther H., Thomas G. MAP2 kinase and 70K S6 kinase lie on distinct signalling pathways. Nature. 1991 Jan 24;349(6307):348–350. doi: 10.1038/349348a0. [DOI] [PubMed] [Google Scholar]
  4. Ballou L. M., 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. doi: 10.1073/pnas.85.19.7154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boyle W. J., 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. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  6. Brown E. J., Albers M. W., Shin T. B., Ichikawa K., Keith C. T., Lane W. S., Schreiber S. L. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature. 1994 Jun 30;369(6483):756–758. doi: 10.1038/369756a0. [DOI] [PubMed] [Google Scholar]
  7. Chen C. A., 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] [Google Scholar]
  8. Chen J., Zheng X. F., Brown E. J., Schreiber S. L. 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. doi: 10.1073/pnas.92.11.4947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chung J., Grammer T. C., Lemon K. P., Kazlauskas A., Blenis J. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature. 1994 Jul 7;370(6484):71–75. doi: 10.1038/370071a0. [DOI] [PubMed] [Google Scholar]
  10. Chung J., Kuo C. J., Crabtree G. R., 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. doi: 10.1016/0092-8674(92)90643-q. [DOI] [PubMed] [Google Scholar]
  11. Dhand R., Hiles I., Panayotou G., Roche S., Fry M. J., Gout I., Totty N. F., Truong O., Vicendo P., Yonezawa K. 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. doi: 10.1002/j.1460-2075.1994.tb06290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Downward J. Signal transduction. Regulating S6 kinase. Nature. 1994 Sep 29;371(6496):378–379. doi: 10.1038/371378a0. [DOI] [PubMed] [Google Scholar]
  13. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ferrari S., Bannwarth W., Morley S. J., Totty N. F., 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. doi: 10.1073/pnas.89.15.7282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ferrari S., Pearson R. B., Siegmann M., Kozma S. C., 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] [Google Scholar]
  16. Grove J. R., Banerjee P., Balasubramanyam A., Coffer P. J., Price D. J., Avruch J., Woodgett J. R. 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. doi: 10.1128/mcb.11.11.5541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Han J. W., Pearson R. B., Dennis P. B., 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. doi: 10.1074/jbc.270.36.21396. [DOI] [PubMed] [Google Scholar]
  18. Haribabu B., Dottin R. P. 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. doi: 10.1073/pnas.88.4.1115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Herman P. K., Emr S. D. 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. doi: 10.1128/mcb.10.12.6742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hiles I. D., Otsu M., Volinia S., Fry M. J., Gout I., Dhand R., Panayotou G., Ruiz-Larrea F., Thompson A., Totty N. F. Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit. Cell. 1992 Aug 7;70(3):419–429. doi: 10.1016/0092-8674(92)90166-a. [DOI] [PubMed] [Google Scholar]
  21. Jefferies H. B., Reinhard C., Kozma S. C., 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. doi: 10.1073/pnas.91.10.4441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jones P. F., Jakubowicz T., Hemmings B. A. Molecular cloning of a second form of rac protein kinase. Cell Regul. 1991 Dec;2(12):1001–1009. doi: 10.1091/mbc.2.12.1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jones P. F., Jakubowicz T., Pitossi F. J., Maurer F., Hemmings B. A. 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. doi: 10.1073/pnas.88.10.4171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jones S. W., Erikson E., Blenis J., Maller J. L., Erikson R. L. 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. doi: 10.1073/pnas.85.10.3377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kato J. Y., Matsuoka M., Polyak K., Massagué J., Sherr C. J. 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. doi: 10.1016/0092-8674(94)90257-7. [DOI] [PubMed] [Google Scholar]
  26. Kolodziej P. A., Young R. A. Epitope tagging and protein surveillance. Methods Enzymol. 1991;194:508–519. doi: 10.1016/0076-6879(91)94038-e. [DOI] [PubMed] [Google Scholar]
  27. Kozma S. C., 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. doi: 10.1073/pnas.87.19.7365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kozma S. C., Thomas G. p70s6k/p85s6k: mechanism of activation and role in mitogenesis. Semin Cancer Biol. 1994 Aug;5(4):255–260. [PubMed] [Google Scholar]
  29. Kunz J., Henriquez R., Schneider U., Deuter-Reinhard M., Movva N. R., Hall M. N. Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell. 1993 May 7;73(3):585–596. doi: 10.1016/0092-8674(93)90144-f. [DOI] [PubMed] [Google Scholar]
  30. Kuo C. J., Chung J., Fiorentino D. F., Flanagan W. M., Blenis J., Crabtree G. R. Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase. Nature. 1992 Jul 2;358(6381):70–73. doi: 10.1038/358070a0. [DOI] [PubMed] [Google Scholar]
  31. Land M., Islas-Trejo A., Freedman J. H., Rubin C. S. 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] [Google Scholar]
  32. Lane H. A., Fernandez A., Lamb N. J., Thomas G. p70s6k function is essential for G1 progression. Nature. 1993 May 13;363(6425):170–172. doi: 10.1038/363170a0. [DOI] [PubMed] [Google Scholar]
  33. Lane H. A., Morley S. J., Dorée M., Kozma S. C., Thomas G. Identification and early activation of a Xenopus laevis p70s6k following progesterone-induced meiotic maturation. EMBO J. 1992 May;11(5):1743–1749. doi: 10.1002/j.1460-2075.1992.tb05226.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lane H. A., Thomas G. Purification and properties of mitogen-activated S6 kinase from rat liver and 3T3 cells. Methods Enzymol. 1991;200:268–291. doi: 10.1016/0076-6879(91)00146-n. [DOI] [PubMed] [Google Scholar]
  35. Marshall C. J. Signal transduction. Hot lips and phosphorylation of protein kinases. Nature. 1994 Feb 24;367(6465):686–686. doi: 10.1038/367686a0. [DOI] [PubMed] [Google Scholar]
  36. Maurer R. A. Isolation of a yeast protein kinase gene by screening with a mammalian protein kinase cDNA. DNA. 1988 Sep;7(7):469–474. doi: 10.1089/dna.1.1988.7.469. [DOI] [PubMed] [Google Scholar]
  37. Ming X. F., Burgering B. M., Wennström S., Claesson-Welsh L., Heldin C. H., Bos J. L., Kozma S. C., Thomas G. Activation of p70/p85 S6 kinase by a pathway independent of p21ras. Nature. 1994 Sep 29;371(6496):426–429. doi: 10.1038/371426a0. [DOI] [PubMed] [Google Scholar]
  38. Mischak H., Bodenteich A., Kolch W., Goodnight J., Hofer F., Mushinski J. F. 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. doi: 10.1021/bi00246a008. [DOI] [PubMed] [Google Scholar]
  39. Mukhopadhyay N. K., Price D. J., Kyriakis J. M., 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] [Google Scholar]
  40. Nourse J., Firpo E., Flanagan W. M., Coats S., Polyak K., Lee M. H., Massague J., Crabtree G. R., Roberts J. M. Interleukin-2-mediated elimination of the p27Kip1 cyclin-dependent kinase inhibitor prevented by rapamycin. Nature. 1994 Dec 8;372(6506):570–573. doi: 10.1038/372570a0. [DOI] [PubMed] [Google Scholar]
  41. 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. doi: 10.1021/bi00406a040. [DOI] [PubMed] [Google Scholar]
  42. 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] [Google Scholar]
  43. Pearson R. B., Kemp B. E. Protein kinase phosphorylation site sequences and consensus specificity motifs: tabulations. Methods Enzymol. 1991;200:62–81. doi: 10.1016/0076-6879(91)00127-i. [DOI] [PubMed] [Google Scholar]
  44. Price D. J., Grove J. R., Calvo V., Avruch J., Bierer B. E. Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. Science. 1992 Aug 14;257(5072):973–977. doi: 10.1126/science.1380182. [DOI] [PubMed] [Google Scholar]
  45. Reinhard C., Fernandez A., Lamb N. J., Thomas G. Nuclear localization of p85s6k: functional requirement for entry into S phase. EMBO J. 1994 Apr 1;13(7):1557–1565. doi: 10.1002/j.1460-2075.1994.tb06418.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Reinhard C., Thomas G., Kozma S. C. 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. doi: 10.1073/pnas.89.9.4052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sabatini D. M., Erdjument-Bromage H., Lui M., Tempst P., Snyder S. H. 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. doi: 10.1016/0092-8674(94)90570-3. [DOI] [PubMed] [Google Scholar]
  48. 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. doi: 10.1016/0092-8674(89)90915-x. [DOI] [PubMed] [Google Scholar]
  49. Su L., Parissenti A. M., Riedel H. Functional carboxyl terminal deletion map of protein kinase C alpha. Receptors Channels. 1993;1(1):1–9. [PubMed] [Google Scholar]
  50. Susa M., Vulević D., Lane H. A., 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] [Google Scholar]
  51. 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. doi: 10.1101/gad.2.5.517. [DOI] [PubMed] [Google Scholar]
  52. Weng Q. P., Andrabi K., Klippel A., Kozlowski M. T., Williams L. T., 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. doi: 10.1073/pnas.92.12.5744. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Weng Q. P., Andrabi K., Kozlowski M. T., Grove J. R., Avruch J. Multiple independent inputs are required for activation of the p70 S6 kinase. Mol Cell Biol. 1995 May;15(5):2333–2340. doi: 10.1128/mcb.15.5.2333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Wettenhall R. E., Aebersold R. H., Hood L. E. Solid-phase sequencing of 32P-labeled phosphopeptides at picomole and subpicomole levels. Methods Enzymol. 1991;201:186–199. doi: 10.1016/0076-6879(91)01017-v. [DOI] [PubMed] [Google Scholar]
  55. Zhang S. H., Lawton M. A., Hunter T., Lamb C. J. 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] [Google Scholar]

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