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. 2003 Jul 15;373(Pt 2):583–591. doi: 10.1042/BJ20021794

Mutational analysis of ribosomal S6 kinase 2 shows differential regulation of its kinase activity from that of ribosomal S6 kinase 1.

Sopheap Phin 1, Deborah Kupferwasser 1, Joseph Lam 1, Kay K Lee-Fruman 1
PMCID: PMC1223513  PMID: 12713446

Abstract

Ribosomal S6 kinase 2 (S6K2) is a serine/threonine kinase identified as a homologue of p70 ribosomal S6 kinase 1 (S6K1). S6K1 and S6K2 show different cellular localization as well as divergent amino acid sequences in non-catalytic domains, suggesting that their cellular functions and/or regulation may not be identical. Many of the serine/threonine residues that become phosphorylated and contribute to S6K1 activation are conserved in S6K2. In this study we carry out mutational analyses of these serine/threonine residues on S6K2 in order to elucidate the mechanism of S6K2 regulation. We find that Thr-228 and Ser-370 are crucial for S6K2 activity, and the three proline-directed serines in the autoinhibitory domain, Ser-410, Ser-417 and Ser-423, play a role in S6K2 activity regulation in a mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)-dependent manner. However, unlike S6K1, changing Thr-388 to glutamic acid in S6K2 renders the kinase fully active. This activity was resistant to the effects of rapamycin or wortmannin, indicating that mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) regulate S6K2 activity via Thr-388. MEK-dependent phosphorylation of the autoinhibitory serines in S6K2 occurs prior to Thr-388 activation. Combining T388E and T228A mutations inhibited S6K2 activation, and a kinase-inactive phosphoinositide-dependent protein kinase (PDK1) diminished T388E activity, suggesting that the role of Thr-388 is to allow further phosphorylation of Thr-228 by PDK1. Thr-388 fails to become phosphorylated in Ser-370 mutants, suggesting that the role of Ser-370 phosphorylation may be to allow Thr-388 phosphorylation. Finally, using the rapamycin-resistant T388E mutant, we provide evidence that S6K2 can phosphorylate S6 in vivo.

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Selected References

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  1. Alessi D. R., Kozlowski M. T., Weng Q. P., Morrice N., Avruch J. 3-Phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates and activates the p70 S6 kinase in vivo and in vitro. Curr Biol. 1998 Jan 15;8(2):69–81. doi: 10.1016/s0960-9822(98)70037-5. [DOI] [PubMed] [Google Scholar]
  2. Belham C., Comb M. J., Avruch J. Identification of the NIMA family kinases NEK6/7 as regulators of the p70 ribosomal S6 kinase. Curr Biol. 2001 Aug 7;11(15):1155–1167. doi: 10.1016/s0960-9822(01)00369-4. [DOI] [PubMed] [Google Scholar]
  3. Blenis J., Erikson R. L. Regulation of a ribosomal protein S6 kinase activity by the Rous sarcoma virus transforming protein, serum, or phorbol ester. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7621–7625. doi: 10.1073/pnas.82.22.7621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Calvo V., Crews C. M., Vik T. A., Bierer B. E. Interleukin 2 stimulation of p70 S6 kinase activity is inhibited by the immunosuppressant rapamycin. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7571–7575. doi: 10.1073/pnas.89.16.7571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Coffer P. J., Woodgett J. R. Differential subcellular localisation of two isoforms of p70 S6 protein kinase. Biochem Biophys Res Commun. 1994 Jan 28;198(2):780–786. doi: 10.1006/bbrc.1994.1112. [DOI] [PubMed] [Google Scholar]
  9. Dennis P. B., Fumagalli S., Thomas G. Target of rapamycin (TOR): balancing the opposing forces of protein synthesis and degradation. Curr Opin Genet Dev. 1999 Feb;9(1):49–54. doi: 10.1016/s0959-437x(99)80007-0. [DOI] [PubMed] [Google Scholar]
  10. Dennis P. B., Pullen N., Kozma S. C., Thomas G. The principal rapamycin-sensitive p70(s6k) phosphorylation sites, T-229 and T-389, are differentially regulated by rapamycin-insensitive kinase kinases. Mol Cell Biol. 1996 Nov;16(11):6242–6251. doi: 10.1128/mcb.16.11.6242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Franco R., Rosenfeld M. G. Hormonally inducible phosphorylation of a nuclear pool of ribosomal protein S6. J Biol Chem. 1990 Mar 15;265(8):4321–4325. [PubMed] [Google Scholar]
  13. Gout I., Minami T., Hara K., Tsujishita Y., Filonenko V., Waterfield M. D., Yonezawa K. Molecular cloning and characterization of a novel p70 S6 kinase, p70 S6 kinase beta containing a proline-rich region. J Biol Chem. 1998 Nov 13;273(46):30061–30064. doi: 10.1074/jbc.273.46.30061. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Koh H., Jee K., Lee B., Kim J., Kim D., Yun Y. H., Kim J. W., Choi H. S., Chung J. Cloning and characterization of a nuclear S6 kinase, S6 kinase-related kinase (SRK); a novel nuclear target of Akt. Oncogene. 1999 Sep 9;18(36):5115–5119. doi: 10.1038/sj.onc.1202895. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. Lee-Fruman K. K., Kuo C. J., Lippincott J., Terada N., Blenis J. Characterization of S6K2, a novel kinase homologous to S6K1. Oncogene. 1999 Sep 9;18(36):5108–5114. doi: 10.1038/sj.onc.1202894. [DOI] [PubMed] [Google Scholar]
  19. Martin K. A., Schalm S. S., Richardson C., Romanelli A., Keon K. L., Blenis J. Regulation of ribosomal S6 kinase 2 by effectors of the phosphoinositide 3-kinase pathway. J Biol Chem. 2000 Dec 6;276(11):7884–7891. doi: 10.1074/jbc.M006969200. [DOI] [PubMed] [Google Scholar]
  20. Martin K. A., Schalm S. S., Romanelli A., Keon K. L., Blenis J. Ribosomal S6 kinase 2 inhibition by a potent C-terminal repressor domain is relieved by mitogen-activated protein-extracellular signal-regulated kinase kinase-regulated phosphorylation. J Biol Chem. 2000 Dec 6;276(11):7892–7898. doi: 10.1074/jbc.M009972200. [DOI] [PubMed] [Google Scholar]
  21. Minami T., Hara K., Oshiro N., Ueoku S., Yoshino K., Tokunaga C., Shirai Y., Saito N., Gout I., Yonezawa K. Distinct regulatory mechanism for p70 S6 kinase beta from that for p70 S6 kinase alpha. Genes Cells. 2001 Nov;6(11):1003–1015. doi: 10.1046/j.1365-2443.2001.00479.x. [DOI] [PubMed] [Google Scholar]
  22. Moser B. A., Dennis P. B., Pullen N., Pearson R. B., Williamson N. A., Wettenhall R. E., Kozma S. C., Thomas G. Dual requirement for a newly identified phosphorylation site in p70s6k. Mol Cell Biol. 1997 Sep;17(9):5648–5655. doi: 10.1128/mcb.17.9.5648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Park In-Hyun, Bachmann Rebecca, Shirazi Haider, Chen Jie. Regulation of ribosomal S6 kinase 2 by mammalian target of rapamycin. J Biol Chem. 2002 Jun 26;277(35):31423–31429. doi: 10.1074/jbc.M204080200. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Pullen N., Dennis P. B., Andjelkovic M., Dufner A., Kozma S. C., Hemmings B. A., Thomas G. Phosphorylation and activation of p70s6k by PDK1. Science. 1998 Jan 30;279(5351):707–710. doi: 10.1126/science.279.5351.707. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Rohde J., Heitman J., Cardenas M. E. The TOR kinases link nutrient sensing to cell growth. J Biol Chem. 2001 Feb 2;276(13):9583–9586. doi: 10.1074/jbc.R000034200. [DOI] [PubMed] [Google Scholar]
  30. Saitoh M., ten Dijke P., Miyazono K., Ichijo H. Cloning and characterization of p70(S6K beta) defines a novel family of p70 S6 kinases. Biochem Biophys Res Commun. 1998 Dec 18;253(2):470–476. doi: 10.1006/bbrc.1998.9784. [DOI] [PubMed] [Google Scholar]
  31. Saitoh Masao, Pullen Nicholas, Brennan Paul, Cantrell Doreen, Dennis Patrick B., Thomas George. Regulation of an activated S6 kinase 1 variant reveals a novel mammalian target of rapamycin phosphorylation site. J Biol Chem. 2002 Mar 25;277(22):20104–20112. doi: 10.1074/jbc.M201745200. [DOI] [PubMed] [Google Scholar]
  32. Shima H., Pende M., Chen Y., Fumagalli S., Thomas G., Kozma S. C. Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase. EMBO J. 1998 Nov 16;17(22):6649–6659. doi: 10.1093/emboj/17.22.6649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Terada N., Lucas J. J., Szepesi A., Franklin R. A., Takase K., Gelfand E. W. Rapamycin inhibits the phosphorylation of p70 S6 kinase in IL-2 and mitogen-activated human T cells. Biochem Biophys Res Commun. 1992 Aug 14;186(3):1315–1321. doi: 10.1016/s0006-291x(05)81549-9. [DOI] [PubMed] [Google Scholar]
  34. Volarević S., Thomas G. Role of S6 phosphorylation and S6 kinase in cell growth. Prog Nucleic Acid Res Mol Biol. 2001;65:101–127. doi: 10.1016/s0079-6603(00)65003-1. [DOI] [PubMed] [Google Scholar]

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