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. 1986 Jan;5(1):157–160. doi: 10.1002/j.1460-2075.1986.tb04190.x

Activation of a ribosomal protein S6 protein kinase in Xenopus oocytes by insulin and insulin-receptor kinase.

D Stefanovic, E Erikson, L J Pike, J L Maller
PMCID: PMC1166708  PMID: 3514207

Abstract

Previous studies in this laboratory have shown that insulin treatment of Xenopus oocytes leads to an increase in phosphorylation of ribosomal protein S6. To investigate the mechanism of this increase, S6 kinase activity was measured in lysates of oocytes exposed to insulin. Insulin caused a rapid 4- to 6-fold increase in S6 kinase activity, which was maximal by 20 min and which could be reversed by removal of insulin prior to homogenization. Dose-response curves showed a detectable increase in specific activity at 1 nM insulin with a maximal effect at 100 nM. Treatment of oocytes with puromycin did not prevent this increase in S6 kinase activity, suggesting activation rather than synthesis of the enzyme. DEAE-Sephacel chromatography of extracts from insulin-treated oocytes revealed two peaks of S6 kinase activity, and the specific activity of the peak eluting at 300 nM NaCl was increased 3-fold in oocytes treated with insulin. The same peak of S6 kinase activity was increased 40% within 10 min in oocytes injected with highly purified insulin-receptor kinase. These results indicate that the insulin-dependent increase in S6 phosphorylation is due, at least in part, to activation of an S6 protein kinase, and this activation may result from the action of the insulin receptor at an intracellular location.

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Selected References

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

  1. Avruch J., Nemenoff R. A., Blackshear P. J., Pierce M. W., Osathanondh R. Insulin-stimulated tyrosine phosphorylation of the insulin receptor in detergent extracts of human placental membranes. Comparison to epidermal growth factor-stimulated phosphorylation. J Biol Chem. 1982 Dec 25;257(24):15162–15166. [PubMed] [Google Scholar]
  2. 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]
  3. Burkhard S. J., Traugh J. A. Changes in ribosome function by cAMP-dependent and cAMP-independent phosphorylation of ribosomal protein S6. J Biol Chem. 1983 Nov 25;258(22):14003–14008. [PubMed] [Google Scholar]
  4. Cobb M. H., Rosen O. M. Description of a protein kinase derived from insulin-treated 3T3-L1 cells that catalyzes the phosphorylation of ribosomal protein S6 and casein. J Biol Chem. 1983 Oct 25;258(20):12472–12481. [PubMed] [Google Scholar]
  5. Donahue M. J., Masaracchia R. A. Phosphorylation of ribosomal protein S6 at multiple sites by a cyclic AMP-independent protein kinase from lymphoid cells. J Biol Chem. 1984 Jan 10;259(1):435–440. [PubMed] [Google Scholar]
  6. Duncan R., McConkey E. H. Rapid alterations in initiation rate and recruitment of inactive RNA are temporally correlated with S6 phosphorylation. Eur J Biochem. 1982 Apr;123(3):539–544. doi: 10.1111/j.1432-1033.1982.tb06565.x. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Gressner A. M., Wool I. G. The stimulation of the phosphorylation of ribosomal protein S6 by cycloheximide and puromycin. Biochem Biophys Res Commun. 1974 Oct 23;60(4):1482–1490. doi: 10.1016/0006-291x(74)90365-9. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Kahn C. R., Baird K. L., Flier J. S., Grunfeld C., Harmon J. T., Harrison L. C., Karlsson F. A., Kasuga M., King G. L., Lang U. C. Insulin receptors, receptor antibodies, and the mechanism of insulin action. Recent Prog Horm Res. 1981;37:477–538. doi: 10.1016/b978-0-12-571137-1.50015-3. [DOI] [PubMed] [Google Scholar]
  11. Kasuga M., Karlsson F. A., Kahn C. R. Insulin stimulates the phosphorylation of the 95,000-dalton subunit of its own receptor. Science. 1982 Jan 8;215(4529):185–187. doi: 10.1126/science.7031900. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Lastick S. M., McConkey E. H. Control of ribosomal protein phosphorylation in HeLa cells. Biochem Biophys Res Commun. 1980 Aug 14;95(3):917–923. doi: 10.1016/0006-291x(80)91560-0. [DOI] [PubMed] [Google Scholar]
  14. Lastick S. M., McConkey E. H. HeLa ribosomal protein S6. Insulin and dibutyryl cyclic AMP affect different phosphopeptides. J Biol Chem. 1981 Jan 25;256(2):583–585. [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Marshall S., Green A., Olefsky J. M. Evidence for recycling of insulin receptors in isolated rat adipocytes. J Biol Chem. 1981 Nov 25;256(22):11464–11470. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. Petruzzelli L. M., Ganguly S., Smith C. J., Cobb M. H., Rubin C. S., Rosen O. M. Insulin activates a tyrosine-specific protein kinase in extracts of 3T3-L1 adipocytes and human placenta. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6792–6796. doi: 10.1073/pnas.79.22.6792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pike L. J., Kuenzel E. A., Casnellie J. E., Krebs E. G. A comparison of the insulin- and epidermal growth factor-stimulated protein kinases from human placenta. J Biol Chem. 1984 Aug 10;259(15):9913–9921. [PubMed] [Google Scholar]
  23. Rosen O. M., Herrera R., Olowe Y., Petruzzelli L. M., Cobb M. H. Phosphorylation activates the insulin receptor tyrosine protein kinase. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3237–3240. doi: 10.1073/pnas.80.11.3237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Scott M. P., Pardue M. L. Translational control in lysates of Drosophila melanogaster cells. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3353–3357. doi: 10.1073/pnas.78.6.3353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Smith C. J., Wejksnora P. J., Warner J. R., Rubin C. S., Rosen O. M. Insulin-stimulated protein phosphorylation in 3T3-L1 preadipocytes. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2725–2729. doi: 10.1073/pnas.76.6.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. 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]
  29. Wang C. C., Sonne O., Hedo J. A., Cushman S. W., Simpson I. A. Insulin-induced internalization of the insulin receptor in the isolated rat adipose cell. Detection of the internalized 138-kilodalton receptor subunit using a photoaffinity 125I-insulin. J Biol Chem. 1983 Apr 25;258(8):5129–5134. [PubMed] [Google Scholar]
  30. 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]
  31. Yu K. T., Czech M. P. Tyrosine phosphorylation of the insulin receptor beta subunit activates the receptor-associated tyrosine kinase activity. J Biol Chem. 1984 Apr 25;259(8):5277–5286. [PubMed] [Google Scholar]

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