Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Nov;16(11):6242–6251. doi: 10.1128/mcb.16.11.6242

The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases.

P B Dennis 1, N Pullen 1, S C Kozma 1, G Thomas 1
PMCID: PMC231627  PMID: 8887654

Abstract

Mitogen-induced activation of p70(s6k) is associated with the phosphorylation of specific sites which are negatively affected by the immunosuppressant rapamycin, the fungal metabolite wortmannin, and the methylxanthine SQ20006. Recent reports have focused on the role of the amino terminus of the p85(s6k) isoform in mediating kinase activity, with the observation that amino-terminal truncation mutants are activated in the presence of rapamycin while retaining their sensitivity to wortmannin. Here we show that the effects of previously described amino- and carboxy-terminal truncations on kinase activity are ultimately reflected in the phosphorylation state of the enzyme. Mutation of the principal rapamycin-targeted phosphorylation site, T-389, to an acidic residue generates a form of the kinase which is as resistant to wortmannin or SQ20006 as it is to rapamycin, consistent with the previous observation that T-389 was a common target of all three inhibitors. Truncation of the first 54 residues of the amino terminus blocks the serum-induced phosphorylation of three rapamycin-sensitive sites, T-229 in the activation loop and T-389 and S-404 in the linker region. This correlates with a severe reduction in the ability of the kinase to be activated by serum. However, loss of mitogen activation conferred by the removal of the amino terminus is reversed by additional truncation of the carboxy-terminal domain, with the resulting mutant demonstrating phosphorylation of the remaining two rapamycin-sensitive sites, T-229 and T-389. In this double-truncation mutant, phosphorylation of T-229 occurs in the basal state, whereas mitogen stimulation is required to induce acute upregulation of T-389 phosphorylation. The phosphorylation of both sites proceeds unimpaired in the presence of rapamycin, indicating that the kinases responsible for the phosphorylation of these sites are not inhibited by the macrolide. In contrast, activation of the double-truncation mutant is blocked in the presence of wortmannin or SQ20006, and these agents completely block the phosphorylation of T-389 while having only a marginal effect on T-229 phosphorylation. When the T-389 site is mutated to an acidic residue in the double-truncation background, the activation of the resulting mutant is insensitive to the wortmannin and SQ20006 block, but interestingly, the mutant is activated to a significantly greater level than a control in the presence of rapamycin. These data are consistent with the hypothesis that T-389 is the principal regulatory phosphorylation site, which, in combination with hyperphosphorylation of the autoinhibitory domain S/TP sites, is acutely regulated by external effectors, whereas T-229 phosphorylation is regulated primarily by internal mechanisms.

Full Text

The Full Text of this article is available as a PDF (391.7 KB).

Selected References

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

  1. 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]
  2. Bandi H. R., Ferrari S., Krieg J., Meyer H. E., Thomas G. Identification of 40 S ribosomal protein S6 phosphorylation sites in Swiss mouse 3T3 fibroblasts stimulated with serum. J Biol Chem. 1993 Feb 25;268(6):4530–4533. [PubMed] [Google Scholar]
  3. Banerjee P., Ahmad M. F., Grove J. R., Kozlosky C., Price D. J., Avruch J. Molecular structure of a major insulin/mitogen-activated 70-kDa S6 protein kinase. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8550–8554. doi: 10.1073/pnas.87.21.8550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blenis J., Chung J., Erikson E., Alcorta D. A., Erikson R. L. Distinct mechanisms for the activation of the RSK kinases/MAP2 kinase/pp90rsk and pp70-S6 kinase signaling systems are indicated by inhibition of protein synthesis. Cell Growth Differ. 1991 Jun;2(6):279–285. [PubMed] [Google Scholar]
  5. Bos J. L. A target for phosphoinositide 3-kinase: Akt/PKB. Trends Biochem Sci. 1995 Nov;20(11):441–442. doi: 10.1016/s0968-0004(00)89097-0. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Brown E. J., Beal P. A., Keith C. T., Chen J., Shin T. B., Schreiber S. L. Control of p70 s6 kinase by kinase activity of FRAP in vivo. Nature. 1995 Oct 5;377(6548):441–446. doi: 10.1038/377441a0. [DOI] [PubMed] [Google Scholar]
  9. Burgering B. M., Coffer P. J. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature. 1995 Aug 17;376(6541):599–602. doi: 10.1038/376599a0. [DOI] [PubMed] [Google Scholar]
  10. Cano E., Hazzalin C. A., Mahadevan L. C. Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun. Mol Cell Biol. 1994 Nov;14(11):7352–7362. doi: 10.1128/mcb.14.11.7352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cheatham L., Monfar M., Chou M. M., Blenis J. Structural and functional analysis of pp70S6k. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11696–11700. doi: 10.1073/pnas.92.25.11696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chou M. M., Blenis J. The 70 kDa S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1. Cell. 1996 May 17;85(4):573–583. doi: 10.1016/s0092-8674(00)81257-x. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Cross M. J., Stewart A., Hodgkin M. N., Kerr D. J., Wakelam M. J. Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. Wortmannin is not a specific inhibitor of phosphatidylinositol 3-kinase. J Biol Chem. 1995 Oct 27;270(43):25352–25355. doi: 10.1074/jbc.270.43.25352. [DOI] [PubMed] [Google Scholar]
  16. Deng T., Karin M. c-Fos transcriptional activity stimulated by H-Ras-activated protein kinase distinct from JNK and ERK. Nature. 1994 Sep 8;371(6493):171–175. doi: 10.1038/371171a0. [DOI] [PubMed] [Google Scholar]
  17. Downward J. Signal transduction. A target for PI(3) kinase. Nature. 1995 Aug 17;376(6541):553–554. doi: 10.1038/376553a0. [DOI] [PubMed] [Google Scholar]
  18. Downward J. Signal transduction. Regulating S6 kinase. Nature. 1994 Sep 29;371(6496):378–379. doi: 10.1038/371378a0. [DOI] [PubMed] [Google Scholar]
  19. Ferrari S., Bandi H. R., Hofsteenge J., Bussian B. M., Thomas G. Mitogen-activated 70K S6 kinase. Identification of in vitro 40 S ribosomal S6 phosphorylation sites. J Biol Chem. 1991 Nov 25;266(33):22770–22775. [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Ferrari S., Thomas G. S6 phosphorylation and the p70s6k/p85s6k. Crit Rev Biochem Mol Biol. 1994;29(6):385–413. doi: 10.3109/10409239409083485. [DOI] [PubMed] [Google Scholar]
  23. Flotow H., Thomas G. Substrate recognition determinants of the mitogen-activated 70K S6 kinase from rat liver. J Biol Chem. 1992 Feb 15;267(5):3074–3078. [PubMed] [Google Scholar]
  24. Franke T. F., Yang S. I., Chan T. O., Datta K., Kazlauskas A., Morrison D. K., Kaplan D. R., Tsichlis P. N. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell. 1995 Jun 2;81(5):727–736. doi: 10.1016/0092-8674(95)90534-0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Hara K., Yonezawa K., Sakaue H., Ando A., Kotani K., Kitamura T., Kitamura Y., Ueda H., Stephens L., Jackson T. R. 1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7415–7419. doi: 10.1073/pnas.91.16.7415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hara K., Yonezawa K., Sakaue H., Kotani K., Kotani K., Kojima A., Waterfield M. D., Kasuga M. Normal activation of p70 S6 kinase by insulin in cells overexpressing dominant negative 85kD subunit of phosphoinositide 3-kinase. Biochem Biophys Res Commun. 1995 Mar 17;208(2):735–741. doi: 10.1006/bbrc.1995.1399. [DOI] [PubMed] [Google Scholar]
  28. Hibi M., Lin A., Smeal T., Minden A., Karin M. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev. 1993 Nov;7(11):2135–2148. doi: 10.1101/gad.7.11.2135. [DOI] [PubMed] [Google Scholar]
  29. Hunter T. When is a lipid kinase not a lipid kinase? When it is a protein kinase. Cell. 1995 Oct 6;83(1):1–4. doi: 10.1016/0092-8674(95)90225-2. [DOI] [PubMed] [Google Scholar]
  30. Jakubowicz T., Leader D. P. Activation of a ribosomal protein S6 kinase in mouse fibroblasts during infection with herpesvirus. Eur J Biochem. 1987 Oct 15;168(2):371–376. doi: 10.1111/j.1432-1033.1987.tb13429.x. [DOI] [PubMed] [Google Scholar]
  31. Kaprielian Z., Patterson P. H. The molecular basis of retinotectal topography. Bioessays. 1994 Jan;16(1):1–11. doi: 10.1002/bies.950160102. [DOI] [PubMed] [Google Scholar]
  32. Kazlauskas A., Cooper J. A. Autophosphorylation of the PDGF receptor in the kinase insert region regulates interactions with cell proteins. Cell. 1989 Sep 22;58(6):1121–1133. doi: 10.1016/0092-8674(89)90510-2. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. Mahalingam M., Templeton D. J. Constitutive activation of S6 kinase by deletion of amino-terminal autoinhibitory and rapamycin sensitivity domains. Mol Cell Biol. 1996 Jan;16(1):405–413. doi: 10.1128/mcb.16.1.405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Pause A., Belsham G. J., Gingras A. C., Donzé O., Lin T. A., Lawrence J. C., Jr, Sonenberg N. Insulin-dependent stimulation of protein synthesis by phosphorylation of a regulator of 5'-cap function. Nature. 1994 Oct 27;371(6500):762–767. doi: 10.1038/371762a0. [DOI] [PubMed] [Google Scholar]
  39. Pearson R. B., Dennis P. B., Han J. W., Williamson N. A., Kozma S. C., Wettenhall R. E., Thomas G. The principal target of rapamycin-induced p70s6k inactivation is a novel phosphorylation site within a conserved hydrophobic domain. EMBO J. 1995 Nov 1;14(21):5279–5287. doi: 10.1002/j.1460-2075.1995.tb00212.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Price D. J., Mukhopadhyay N. K., Avruch J. Insulin-activated protein kinases phosphorylate a pseudosubstrate synthetic peptide inhibitor of the p70 S6 kinase. J Biol Chem. 1991 Sep 5;266(25):16281–16284. [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Shama S., Meyuhas O. The translational cis-regulatory element of mammalian ribosomal protein mRNAs is recognized by the plant translational apparatus. Eur J Biochem. 1996 Mar 1;236(2):383–388. doi: 10.1111/j.1432-1033.1996.00383.x. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. 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]
  47. von Manteuffel S. R., Gingras A. C., Ming X. F., Sonenberg N., Thomas G. 4E-BP1 phosphorylation is mediated by the FRAP-p70s6k pathway and is independent of mitogen-activated protein kinase. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4076–4080. doi: 10.1073/pnas.93.9.4076. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES