Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Aug 1;89(15):7282–7286. doi: 10.1073/pnas.89.15.7282

Activation of p70s6k is associated with phosphorylation of four clustered sites displaying Ser/Thr-Pro motifs.

S Ferrari 1, W Bannwarth 1, S J Morley 1, N F Totty 1, G Thomas 1
PMCID: PMC49690  PMID: 1496022

Abstract

Partial amino acid sequences were obtained from 22 internal tryptic peptides of rat liver p70s6k (M(r) 70,000 ribosomal protein S6 kinase), 3 of which were found to contain phosphorylated residues. To determine whether these sites were associated with p70s6k activation, the kinase was labeled to high specific activity with 32P(i) in Swiss mouse 3T3 cells. By sequential cleavage with CNBr and endoproteinase Lys-C followed by two-dimensional tryptic peptide analysis, it could be shown that all of the sites were located in a small endoproteinase Lys-C peptide of M(r) 2400. Analysis of the p70s6k protein sequence revealed a single candidate that could represent this peptide. Three tryptic peptides derived from the endoproteinase Lys-C fragment were chosen by a newly described computer program as the most likely candidates to contain the in vivo sites of phosphorylation. Synthetic peptides based on these sequences were phosphorylated either chemically or enzymatically and found to comigrate by two-dimensional thin-layer electrophoresis/chromatography with the four major in vivo labeled tryptic phosphopeptides. Three of the phosphorylation sites in these peptides were equivalent to those sequenced in the rat liver p70s6k. In addition, all four sites display the motif Ser/Thr-Pro, typical of cell cycle-regulated sites, and are clustered in a putative autoinhibitory domain of the enzyme.

Full text

PDF

Images in this article

7284Image
on p.7284
7284Image
on p.7284
7284Image
on p.7284
7285Image
on p.7285
7285Image
on p.7285

Selected References

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

  1. Alvarez E., Northwood I. C., Gonzalez F. A., Latour D. A., Seth A., Abate C., Curran T., Davis R. J. Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase. J Biol Chem. 1991 Aug 15;266(23):15277–15285. [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. 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]
  6. 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]
  7. 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]
  8. Brooks R. F. Continuous protein synthesis is required to maintain the probability of entry into S phase. Cell. 1977 Sep;12(1):311–317. doi: 10.1016/0092-8674(77)90209-4. [DOI] [PubMed] [Google Scholar]
  9. Clark-Lewis I., Sanghera J. S., Pelech S. L. Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase. J Biol Chem. 1991 Aug 15;266(23):15180–15184. [PubMed] [Google Scholar]
  10. Duncan R., McConkey E. H. Preferential utilization of phosphorylated 40-S ribosomal subunits during initiation complex formation. Eur J Biochem. 1982 Apr;123(3):535–538. doi: 10.1111/j.1432-1033.1982.tb06564.x. [DOI] [PubMed] [Google Scholar]
  11. Erickson A. K., Payne D. M., Martino P. A., Rossomando A. J., Shabanowitz J., Weber M. J., Hunt D. F., Sturgill T. W. Identification by mass spectrometry of threonine 97 in bovine myelin basic protein as a specific phosphorylation site for mitogen-activated protein kinase. J Biol Chem. 1990 Nov 15;265(32):19728–19735. [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Gotoh Y., Nishida E., Matsuda S., Shiina N., Kosako H., Shiokawa K., Akiyama T., Ohta K., Sakai H. In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase. Nature. 1991 Jan 17;349(6306):251–254. doi: 10.1038/349251a0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Hall F. L., Braun R. K., Mihara K., Fung Y. K., Berndt N., Carbonaro-Hall D. A., Vulliet P. R. Characterization of the cytoplasmic proline-directed protein kinase in proliferative cells and tissues as a heterodimer comprised of p34cdc2 and p58cyclin A. J Biol Chem. 1991 Sep 15;266(26):17430–17440. [PubMed] [Google Scholar]
  17. Hardie G. Pseudosubstrates turn off protein kinases. Nature. 1988 Oct 13;335(6191):592–593. doi: 10.1038/335592a0. [DOI] [PubMed] [Google Scholar]
  18. Hershey J. W. Protein phosphorylation controls translation rates. J Biol Chem. 1989 Dec 15;264(35):20823–20826. [PubMed] [Google Scholar]
  19. Hoshi M., Nishida E., Inagaki M., Gotoh Y., Sakai H. Activation of a serine/threonine kinase that phosphorylates microtubule-associated protein 1B in vitro by growth factors and phorbol esters in quiescent rat fibroblastic cells. Eur J Biochem. 1990 Oct 24;193(2):513–519. doi: 10.1111/j.1432-1033.1990.tb19366.x. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Jenö P., Ballou L. M., Novak-Hofer I., Thomas G. Identification and characterization of a mitogen-activated S6 kinase. Proc Natl Acad Sci U S A. 1988 Jan;85(2):406–410. doi: 10.1073/pnas.85.2.406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jenö P., Jäggi N., Luther H., Siegmann M., Thomas G. Purification and characterization of a 40 S ribosomal protein S6 kinase from vanadate-stimulated Swiss 3T3 cells. J Biol Chem. 1989 Jan 15;264(2):1293–1297. [PubMed] [Google Scholar]
  23. Kazlauskas A., Cooper J. A. Protein kinase C mediates platelet-derived growth factor-induced tyrosine phosphorylation of p42. J Cell Biol. 1988 Apr;106(4):1395–1402. doi: 10.1083/jcb.106.4.1395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Kozma S. C., Ferrari S., Thomas G. Unmasking a growth factor/oncogene-activated S6 phosphorylation cascade. Cell Signal. 1989;1(3):219–225. doi: 10.1016/0898-6568(89)90039-9. [DOI] [PubMed] [Google Scholar]
  26. Kozma S. C., Lane H. A., Ferrari S., Luther H., Siegmann M., Thomas G. A stimulated S6 kinase from rat liver: identity with the mitogen activated S6 kinase of 3T3 cells. EMBO J. 1989 Dec 20;8(13):4125–4132. doi: 10.1002/j.1460-2075.1989.tb08597.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kozma S. C., Thomas G. Serine/threonine kinases in the propagation of the early mitogenic response. Rev Physiol Biochem Pharmacol. 1992;119:123–155. doi: 10.1007/3540551921_5. [DOI] [PubMed] [Google Scholar]
  28. Krieg J., Hofsteenge J., Thomas G. Identification of the 40 S ribosomal protein S6 phosphorylation sites induced by cycloheximide. J Biol Chem. 1988 Aug 15;263(23):11473–11477. [PubMed] [Google Scholar]
  29. 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]
  30. Moreno S., Nurse P. Substrates for p34cdc2: in vivo veritas? Cell. 1990 May 18;61(4):549–551. doi: 10.1016/0092-8674(90)90463-o. [DOI] [PubMed] [Google Scholar]
  31. Morla A. O., Draetta G., Beach D., Wang J. Y. Reversible tyrosine phosphorylation of cdc2: dephosphorylation accompanies activation during entry into mitosis. Cell. 1989 Jul 14;58(1):193–203. doi: 10.1016/0092-8674(89)90415-7. [DOI] [PubMed] [Google Scholar]
  32. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988 Aug 25;334(6184):661–665. doi: 10.1038/334661a0. [DOI] [PubMed] [Google Scholar]
  33. Novak-Hofer I., Thomas G. An activated S6 kinase in extracts from serum- and epidermal growth factor-stimulated Swiss 3T3 cells. J Biol Chem. 1984 May 10;259(9):5995–6000. [PubMed] [Google Scholar]
  34. Novak-Hofer I., Thomas G. Epidermal growth factor-mediated activation of an S6 kinase in Swiss mouse 3T3 cells. J Biol Chem. 1985 Aug 25;260(18):10314–10319. [PubMed] [Google Scholar]
  35. Palen E., Traugh J. A. Phosphorylation of ribosomal protein S6 by cAMP-dependent protein kinase and mitogen-stimulated S6 kinase differentially alters translation of globin mRNA. J Biol Chem. 1987 Mar 15;262(8):3518–3523. [PubMed] [Google Scholar]
  36. Pardee A. B. G1 events and regulation of cell proliferation. Science. 1989 Nov 3;246(4930):603–608. doi: 10.1126/science.2683075. [DOI] [PubMed] [Google Scholar]
  37. Posada J., Sanghera J., Pelech S., Aebersold R., Cooper J. A. Tyrosine phosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts. Mol Cell Biol. 1991 May;11(5):2517–2528. doi: 10.1128/mcb.11.5.2517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Price D. J., Nemenoff R. A., Avruch J. Purification of a hepatic S6 kinase from cycloheximide-treated Rats. J Biol Chem. 1989 Aug 15;264(23):13825–13833. [PubMed] [Google Scholar]
  39. 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]
  40. Roach P. J. Multisite and hierarchal protein phosphorylation. J Biol Chem. 1991 Aug 5;266(22):14139–14142. [PubMed] [Google Scholar]
  41. Rossow P. W., Riddle V. G., Pardee A. B. Synthesis of labile, serum-dependent protein in early G1 controls animal cell growth. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4446–4450. doi: 10.1073/pnas.76.9.4446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  43. Susa M., Olivier A. R., Fabbro D., Thomas G. EGF induces biphasic S6 kinase activation: late phase is protein kinase C-dependent and contributes to mitogenicity. Cell. 1989 Jun 2;57(5):817–824. doi: 10.1016/0092-8674(89)90796-4. [DOI] [PubMed] [Google Scholar]
  44. Susa M., Thomas G. Identical Mr 70,000 S6 kinase is activated biphasically by epidermal growth factor: a phosphopeptide that characterizes the late phase. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7040–7044. doi: 10.1073/pnas.87.18.7040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Thomas G. MAP kinase by any other name smells just as sweet. Cell. 1992 Jan 10;68(1):3–6. doi: 10.1016/0092-8674(92)90199-m. [DOI] [PubMed] [Google Scholar]
  47. Thomas G., Martin-Pérez J., Siegmann M., Otto A. M. The effect of serum, EGF, PGF2 alpha and insulin on S6 phosphorylation and the initiation of protein and DNA synthesis. Cell. 1982 Aug;30(1):235–242. doi: 10.1016/0092-8674(82)90029-0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES