Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1987 Sep;7(9):3147–3155. doi: 10.1128/mcb.7.9.3147

Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts.

E Erikson 1, D Stefanovic 1, J Blenis 1, R L Erikson 1, J L Maller 1
PMCID: PMC367949  PMID: 3313008

Abstract

Ribosomal protein S6 becomes highly phosphorylated during progesterone- or insulin-induced maturation of Xenopus laevis oocytes. We have previously purified an Mr 92,000 protein as one of the major S6 kinases from Xenopus unfertilized eggs. In this paper we confirm by renaturation of activity from a sodium dodecyl sulfate-polyacrylamide gel that this protein is an S6 kinase. This enzyme, termed S6 kinase II (S6 K II), was used for the preparation of polyclonal antiserum. Immunocomplexes formed with the antiserum and purified S6 K II were able to express kinase activity with the same substrate specificity as that of the purified enzyme, including autophosphorylation of S6 K II itself. The antiserum did not react with S6 kinase I, another major S6 kinase present in Xenopus eggs, which is chromatographically distinct from S6 K II. The administration of progesterone to oocytes resulted in a 20- to 25-fold increase in S6 kinase activity in extracts of these cells. Immunocomplex kinase assays done on extracts revealed that anti-S6 K II serum reacted with S6 kinase from progesterone-treated oocytes. This antiserum also reacted with the activated S6 kinase from insulin-stimulated oocytes. In addition, anti-S6 K II serum reacted with activated S6 kinase from chicken embryo fibroblasts stimulated with serum or transformed by Rous sarcoma virus. These results indicate that S6 K II or an antigenically related S6 kinase(s) is subject to regulation by mitogenic stimuli in various cell types.

Full text

PDF
3147

Images in this article

3149Image
on p.3149
3150Image
on p.3150
3151Image
on p.3151
3153Image
on p.3153

Selected References

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

  1. BROWN D. D., LITTNA E. RNA SYNTHESIS DURING THE DEVELOPMENT OF XENOPUS LAEVIS, THE SOUTH AFRICAN CLAWED TOAD. J Mol Biol. 1964 May;8:669–687. doi: 10.1016/s0022-2836(64)80116-9. [DOI] [PubMed] [Google Scholar]
  2. Blenis J., Erikson R. L. Phosphorylation of the ribosomal protein S6 is elevated in cells transformed by a variety of tumor viruses. J Virol. 1984 Jun;50(3):966–969. doi: 10.1128/jvi.50.3.966-969.1984. [DOI] [PMC free article] [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. Blenis J., Erikson R. L. Regulation of protein kinase activities in PC12 pheochromocytoma cells. EMBO J. 1986 Dec 20;5(13):3441–3447. doi: 10.1002/j.1460-2075.1986.tb04667.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blenis J., Spivack J. G., Erikson R. L. Phorbol ester, serum, and rous sarcoma virus transforming gene product induce similar phosphorylations of ribosomal protein S6. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6408–6412. doi: 10.1073/pnas.81.20.6408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bolen J. B., Thiele C. J., Israel M. A., Yonemoto W., Lipsich L. A., Brugge J. S. Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation. Cell. 1984 Oct;38(3):767–777. doi: 10.1016/0092-8674(84)90272-1. [DOI] [PubMed] [Google Scholar]
  7. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  8. Brugge J. S., Cotton P. C., Queral A. E., Barrett J. N., Nonner D., Keane R. W. Neurones express high levels of a structurally modified, activated form of pp60c-src. Nature. 1985 Aug 8;316(6028):554–557. doi: 10.1038/316554a0. [DOI] [PubMed] [Google Scholar]
  9. Cobb M. H. An insulin-stimulated ribosomal protein S6 kinase in 3T3-L1 cells. J Biol Chem. 1986 Oct 5;261(28):12994–12999. [PubMed] [Google Scholar]
  10. Collett M. S., Erikson R. L. Protein kinase activity associated with the avian sarcoma virus src gene product. Proc Natl Acad Sci U S A. 1978 Apr;75(4):2021–2024. doi: 10.1073/pnas.75.4.2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cooper J. A., King C. S. Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src. Mol Cell Biol. 1986 Dec;6(12):4467–4477. doi: 10.1128/mcb.6.12.4467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Decker S. Phosphorylation of ribosomal protein S6 in avian sarcoma virus-transformed chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4112–4115. doi: 10.1073/pnas.78.7.4112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Erikson E., Maller J. L. A protein kinase from Xenopus eggs specific for ribosomal protein S6. Proc Natl Acad Sci U S A. 1985 Feb;82(3):742–746. doi: 10.1073/pnas.82.3.742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Erikson E., Maller J. L. Purification and characterization of a protein kinase from Xenopus eggs highly specific for ribosomal protein S6. J Biol Chem. 1986 Jan 5;261(1):350–355. [PubMed] [Google Scholar]
  15. Flockhart D. A., Corbin J. D. Regulatory mechanisms in the control of protein kinases. CRC Crit Rev Biochem. 1982 Feb;12(2):133–186. doi: 10.3109/10409238209108705. [DOI] [PubMed] [Google Scholar]
  16. Gurdon J. B. Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal. J Embryol Exp Morphol. 1976 Dec;36(3):523–540. [PubMed] [Google Scholar]
  17. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  18. Hanocq-Quertier J., Baltus E. Phosphorylation of ribosomal proteins during maturation of Xenopus laevis oocytes. Eur J Biochem. 1981 Nov;120(2):351–355. doi: 10.1111/j.1432-1033.1981.tb05711.x. [DOI] [PubMed] [Google Scholar]
  19. Haselbacher G. K., Humbel R. E., Thomas G. Insulin-like growth factor: insulin or serum increase phosphorylation of ribosomal protein S6 during transition of stationary chick embryo fibroblasts into early G1 phase of the cell cycle. FEBS Lett. 1979 Apr 1;100(1):185–190. doi: 10.1016/0014-5793(79)81160-6. [DOI] [PubMed] [Google Scholar]
  20. Johnson R. A., Walseth T. F. The enzymatic preparation of [alpha-32P]ATP, [alpha-32P]GTP, [32P]cAMP, and [32P]cGMP, and their use in the assay of adenylate and guanylate cyclases and cyclic nucleotide phosphodiesterases. Adv Cyclic Nucleotide Res. 1979;10:135–167. [PubMed] [Google Scholar]
  21. Kalthoff H., Darmer D., Towbin H., Gordon J., Amons R., Möller W., Richter D. Ribosomal protein S6 from Xenopus laevis ovaries. Isolation, phosphorylation in vivo and cross-reaction with heterologous anti-S6 antibodies. Eur J Biochem. 1982 Mar 1;122(3):439–443. [PubMed] [Google Scholar]
  22. Kessler S. W. Rapid isolation of antigens from cells with a staphylococcal protein A-antibody adsorbent: parameters of the interaction of antibody-antigen complexes with protein A. J Immunol. 1975 Dec;115(6):1617–1624. [PubMed] [Google Scholar]
  23. Le Peuch C. J., Ballester R., Rosen O. M. Purified rat brain calcium- and phospholipid-dependent protein kinase phosphorylates ribosomal protein S6. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6858–6862. doi: 10.1073/pnas.80.22.6858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Maller J. L., Foulkes J. G., Erikson E., Baltimore D. Phosphorylation of ribosomal protein S6 on serine after microinjection of the Abelson murine leukemia virus tyrosine-specific protein kinase into Xenopus oocytes. Proc Natl Acad Sci U S A. 1985 Jan;82(2):272–276. doi: 10.1073/pnas.82.2.272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Maller J. L., Pike L. J., Freidenberg G. R., Cordera R., Stith B. J., Olefsky J. M., Krebs E. G. Increased phosphorylation of ribosomal protein S6 following microinjection of insulin receptor-kinase into Xenopus oocytes. Nature. 1986 Apr 3;320(6061):459–461. doi: 10.1038/320459a0. [DOI] [PubMed] [Google Scholar]
  27. Maller J. L. Use of microinjection techniques to study protein kinases and protein phosphorylation in amphibian oocytes. Methods Enzymol. 1983;99:219–226. doi: 10.1016/0076-6879(83)99056-0. [DOI] [PubMed] [Google Scholar]
  28. Martin-Pérez J., Rudkin B. B., Siegmann M., Thomas G. Activation of ribosomal protein S6 phosphorylation during meiotic maturation of Xenopus laevis oocytes: in vitro ordered appearance of S6 phosphopeptides. EMBO J. 1986 Apr;5(4):725–731. doi: 10.1002/j.1460-2075.1986.tb04274.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nemenoff R. A., Gunsalus J. R., Avruch J. An insulin-stimulated (ribosomal S6) protein kinase from soluble extracts of H4 hepatoma cells. Arch Biochem Biophys. 1986 Feb 15;245(1):196–203. doi: 10.1016/0003-9861(86)90205-5. [DOI] [PubMed] [Google Scholar]
  30. Nielsen P. J., Thomas G., Maller J. L. Increased phosphorylation of ribosomal protein S6 during meiotic maturation of Xenopus oocytes. Proc Natl Acad Sci U S A. 1982 May;79(9):2937–2941. doi: 10.1073/pnas.79.9.2937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nishimura J., Deuel T. F. Platelet-derived growth factor stimulates the phosphorylation of ribosomal protein S6. FEBS Lett. 1983 May 30;156(1):130–134. doi: 10.1016/0014-5793(83)80263-4. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  35. Parker P. J., Katan M., Waterfield M. D., Leader D. P. The phosphorylation of eukaryotic ribosomal protein S6 by protein kinase C. Eur J Biochem. 1985 May 2;148(3):579–586. doi: 10.1111/j.1432-1033.1985.tb08879.x. [DOI] [PubMed] [Google Scholar]
  36. Pelech S. L., Olwin B. B., Krebs E. G. Fibroblast growth factor treatment of Swiss 3T3 cells activates a subunit S6 kinase that phosphorylates a synthetic peptide substrate. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5968–5972. doi: 10.1073/pnas.83.16.5968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Perisic O., Traugh J. A. Protease-activated kinase II as the potential mediator of insulin-stimulated phosphorylation of ribosomal protein S6. J Biol Chem. 1983 Aug 25;258(16):9589–9592. [PubMed] [Google Scholar]
  38. Poirier F., Calothy G., Karess R. E., Erikson E., Hanafusa H. Role of p60src kinase activity in the induction of neuroretinal cell proliferation by rous sarcoma virus. J Virol. 1982 Jun;42(3):780–789. doi: 10.1128/jvi.42.3.780-789.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sefton B. M., Hunter T., Beemon K., Eckhart W. Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus. Cell. 1980 Jul;20(3):807–816. doi: 10.1016/0092-8674(80)90327-x. [DOI] [PubMed] [Google Scholar]
  40. Sigel M. B., Sinha Y. N., VanderLaan W. P. Production of antibodies by inoculation into lymph nodes. Methods Enzymol. 1983;93:3–12. doi: 10.1016/s0076-6879(83)93031-8. [DOI] [PubMed] [Google Scholar]
  41. Spivack J. G., Erikson R. L., Maller J. L. Microinjection of pp60v-src into Xenopus oocytes increases phosphorylation of ribosomal protein S6 and accelerates the rate of progesterone-induced meiotic maturation. Mol Cell Biol. 1984 Aug;4(8):1631–1634. doi: 10.1128/mcb.4.8.1631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Stefanovic D., Erikson E., Pike L. J., Maller J. L. Activation of a ribosomal protein S6 protein kinase in Xenopus oocytes by insulin and insulin-receptor kinase. EMBO J. 1986 Jan;5(1):157–160. doi: 10.1002/j.1460-2075.1986.tb04190.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stith B. J., Maller J. L. The effect of insulin on intracellular ph and ribosomal protein S6 phosphorylation in oocytes of Xenopus laevis. Dev Biol. 1984 Mar;102(1):79–89. doi: 10.1016/0012-1606(84)90176-3. [DOI] [PubMed] [Google Scholar]
  44. Tabarini D., Heinrich J., Rosen O. M. Activation of S6 kinase activity in 3T3-L1 cells by insulin and phorbol ester. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4369–4373. doi: 10.1073/pnas.82.13.4369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Trevillyan J. M., Kulkarni R. K., Byus C. V. Tumor-promoting phorbol esters stimulate the phosphorylation of ribosomal protein S6 in quiescent Reuber H35 hepatoma cells. J Biol Chem. 1984 Jan 25;259(2):897–902. [PubMed] [Google Scholar]
  47. Trevillyan J. M., Perisic O., Traugh J. A., Byus C. V. Insulin- and phorbol ester-stimulated phosphorylation of ribosomal protein S6. J Biol Chem. 1985 Mar 10;260(5):3041–3044. [PubMed] [Google Scholar]
  48. Wettenhall R. E., Cohen P. Isolation and characterisation of cyclic AMP-dependent phosphorylation sites from rat liver ribosomal protein S6. FEBS Lett. 1982 Apr 19;140(2):263–269. doi: 10.1016/0014-5793(82)80910-1. [DOI] [PubMed] [Google Scholar]
  49. Whitehouse S., Walsh D. A. Inhibitor protein of the cAMP-dependent protein kinase: characteristics and purification. Methods Enzymol. 1983;99:80–93. doi: 10.1016/0076-6879(83)99044-4. [DOI] [PubMed] [Google Scholar]
  50. del Grande R. W., Traugh J. A. Phosphorylation of 40-S ribosomal subunits by cAMP-dependent, cGMP-dependent and protease-activated protein kinases. Eur J Biochem. 1982 Apr 1;123(2):421–428. doi: 10.1111/j.1432-1033.1982.tb19785.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES