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. 1984 Oct;81(20):6408–6412. doi: 10.1073/pnas.81.20.6408

Phorbol ester, serum, and rous sarcoma virus transforming gene product induce similar phosphorylations of ribosomal protein S6.

J Blenis, J G Spivack, R L Erikson
PMCID: PMC391933  PMID: 6093101

Abstract

The addition of phorbol 12-myristate 13-acetate (PMA), a potent tumor promoter, to serum-starved quiescent chicken embryo fibroblasts (CEF) or C127 murine cells resulted in increased phosphorylation of 40S ribosomal protein S6. The effect of PMA on S6 phosphorylation in quiescent CEF was half-maximal at approximately equal to 100 nM and was readily observed at 16 nM. In addition, S6 phosphorylation was increased in serum-starved CEF incubated with the diacylglycerol derivative, 1-oleoyl-2-acetylglycerol. S6 phosphorylation in PMA-stimulated, serum-stimulated, and serum-starved Rous sarcoma virus-transformed CEF was analyzed by phospho amino acid analysis, two-dimensional polyacrylamide gel electrophoresis, limited proteolysis with V8 protease, and two-dimensional thin-layer electrophoresis of chymotryptic digests. Comparison of S6 phosphorylation by these methods suggests that phosphorylation of S6 stimulated by PMA, serum, or oncogenic transformation with Rous sarcoma virus occurs through common pathways. This is further supported by the observation that the simultaneous addition of PMA and serum to CEF or of either PMA or serum to Rous sarcoma virus-transformed CEF did not significantly further increase the incorporation of phosphate into S6.

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Selected References

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

  1. Berridge M. J. Inositol trisphosphate and diacylglycerol as second messengers. Biochem J. 1984 Jun 1;220(2):345–360. doi: 10.1042/bj2200345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bishop R., Martinez R., Nakamura K. D., Weber M. J. A tumor promoter stimulates phosphorylation on tyrosine. Biochem Biophys Res Commun. 1983 Sep 15;115(2):536–543. doi: 10.1016/s0006-291x(83)80178-8. [DOI] [PubMed] [Google Scholar]
  3. Bissell M. J., Hatié C., Calvin M. Is the product of the src gene a promoter? Proc Natl Acad Sci U S A. 1979 Jan;76(1):348–352. doi: 10.1073/pnas.76.1.348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Bommer U. A., Noll F., Lutsch G., Bielka H. Immunochemical detection of proteins in the small subunit of rat liver ribosomes involved in binding of the ternary initiation complex. FEBS Lett. 1980 Feb 25;111(1):171–174. doi: 10.1016/0014-5793(80)80785-x. [DOI] [PubMed] [Google Scholar]
  6. Chambard J. C., Franchi A., Le Cam A., Pouysségur J. Growth factor-stimulated protein phosphorylation in G0/G1-arrested fibroblasts. Two distinct classes of growth factors with potentiating effects. J Biol Chem. 1983 Feb 10;258(3):1706–1713. [PubMed] [Google Scholar]
  7. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  8. 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]
  9. Collett M. S., Erikson E., Erikson R. L. Structural analysis of the avian sarcoma virus transforming protein: sites of phosphorylation. J Virol. 1979 Feb;29(2):770–781. doi: 10.1128/jvi.29.2.770-781.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Collett M. S., Purchio A. F., Erikson R. L. Avian sarcoma virus-transforming protein, pp60src shows protein kinase activity specific for tyrosine. Nature. 1980 May 15;285(5761):167–169. doi: 10.1038/285167a0. [DOI] [PubMed] [Google Scholar]
  11. Cooper J. A., Sefton B. M., Hunter T. Diverse mitogenic agents induce the phosphorylation of two related 42,000-dalton proteins on tyrosine in quiescent chick cells. Mol Cell Biol. 1984 Jan;4(1):30–37. doi: 10.1128/mcb.4.1.30. [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. Diringer H., Friis R. R. Changes in phosphatidylinositol metabolism correlated to growth state of normal and Rous sarcoma virus-transformed Japanese quail cells. Cancer Res. 1977 Sep;37(9):2979–2984. [PubMed] [Google Scholar]
  14. 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]
  15. Gilmore T., Martin G. S. Phorbol ester and diacylglycerol induce protein phosphorylation at tyrosine. Nature. 1983 Dec 1;306(5942):487–490. doi: 10.1038/306487a0. [DOI] [PubMed] [Google Scholar]
  16. Goldberg A. R., Delclos K. B., Blumberg P. M. Phorbol ester action is independent of viral and cellular src kinase levels. Science. 1980 Apr 11;208(4440):191–193. doi: 10.1126/science.6244621. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Heldin C. H., Westermark B. Growth factors: mechanism of action and relation to oncogenes. Cell. 1984 May;37(1):9–20. doi: 10.1016/0092-8674(84)90296-4. [DOI] [PubMed] [Google Scholar]
  20. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kaltschmidt E., Wittmann H. G. Ribosomal proteins. VII. Two-dimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins. Anal Biochem. 1970 Aug;36(2):401–412. doi: 10.1016/0003-2697(70)90376-3. [DOI] [PubMed] [Google Scholar]
  22. Kisilevsky R., Treloar M. A., Weiler L. Ribosome conformational changes associated with protein S6 phosphorylation. J Biol Chem. 1984 Jan 25;259(2):1351–1356. [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. 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]
  27. Nilsen-Hamilton M., Hamilton R. T., Allen W. R., Potter-Perigo S. Synergistic stimulation of S6 ribosomal protein phosphorylation and DNA synthesis by epidermal growth factor and insulin in quiescent 3T3 cells. Cell. 1982 Nov;31(1):237–242. doi: 10.1016/0092-8674(82)90423-8. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. 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]
  32. Pietropaolo C., Laskin J. D., Weinstein I. B. Effect of tumor promoters on sarc gene expression in normal and transformed chick embryo fibroblasts. Cancer Res. 1981 Apr;41(4):1565–1571. [PubMed] [Google Scholar]
  33. 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]
  34. 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]
  35. 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]
  36. Sugimoto Y., Whitman M., Cantley L. C., Erikson R. L. Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and diacylglycerol. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2117–2121. doi: 10.1073/pnas.81.7.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Terao K., Ogata K. Proteins of small subunits of rat liver ribosomes that interact with poly(U). II. Cross-links between poly(U) and ribosomal proteins in 40 S subunits induced by UV irradiation. J Biochem. 1979 Sep;86(3):605–617. doi: 10.1093/oxfordjournals.jbchem.a132564. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Thomas G., Siegmann M., Gordon J. Multiple phosphorylation of ribosomal protein S6 during transition of quiescent 3T3 cells into early G1, and cellular compartmentalization of the phosphate donor. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3952–3956. doi: 10.1073/pnas.76.8.3952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]

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