Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1989 Apr;8(4):1111–1119. doi: 10.1002/j.1460-2075.1989.tb03481.x

Myc oncoproteins are phosphorylated by casein kinase II.

B Lüscher 1, E A Kuenzel 1, E G Krebs 1, R N Eisenman 1
PMCID: PMC400922  PMID: 2663470

Abstract

Casein kinase II (CK-II) is a ubiquitous protein kinase, localized to both nucleus and cytoplasm, with strong specificity for serine residues positioned within clusters of acidic amino acids. We have found that a number of nuclear oncoproteins share a CK-II phosphorylation sequence motif, including Myc, Myb, Fos, E1a and SV40 T antigen. In this paper we show that cellular myc-encoded proteins, derived from avian and human cells, can serve as substrates for phosphorylation by purified CK-II in vitro and that this phosphorylation is reversible. One- and two-dimensional mapping experiments demonstrate that the major phosphopeptides from in vivo phosphorylated Myc correspond to the phosphopeptides produced from Myc phosphorylated in vitro by CK-II. In addition, synthetic peptides with sequences corresponding to putative CK-II phosphorylation sites in Myc are subject to multiple, highly efficient phosphorylations by CK-II, and can act as competitive inhibitors of CK-II phosphorylation of Myc in vitro. We have used such peptides to map the phosphorylated regions in Myc and have located major CK-II phosphorylations within the central highly acidic domain and within a region proximal to the C terminus. Our results, along with previous studies on myc deletion mutants, show that Myc is phosphorylated by CK-II, or a kinase with similar specificity, in regions of functional importance. Since CK-II can be rapidly activated after mitogen treatment we postulate that CK-II mediated phosphorylation of Myc plays a role in signal transduction to the nucleus.

Full text

PDF
1111

Images in this article

Selected References

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

  1. Bechtel P. J., Beavo J. A., Krebs E. G. Purification and characterization of catalytic subunit of skeletal muscle adenosine 3':5'-monophosphate-dependent protein kinase. J Biol Chem. 1977 Apr 25;252(8):2691–2697. [PubMed] [Google Scholar]
  2. Biegalke B. J., Heaney M. L., Bouton A., Parsons J. T., Linial M. MC29 deletion mutants which fail to transform chicken macrophages are competent for transformation of quail macrophages. J Virol. 1987 Jul;61(7):2138–2142. doi: 10.1128/jvi.61.7.2138-2142.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bister K., Lee W. H., Duesberg P. H. Phosphorylation of the nonstructural proteins encoded by three avian acute leukemia viruses and by avian fujinami sarcoma virus. J Virol. 1980 Nov;36(2):617–621. doi: 10.1128/jvi.36.2.617-621.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bister K., Trachmann C., Jansen H. W., Schroeer B., Patschinsky T. Structure of mutant and wild-type MC29 v-myc alleles and biochemical properties of their protein products. Oncogene. 1987 May;1(2):97–109. [PubMed] [Google Scholar]
  5. Casnellie J. E., Harrison M. L., Pike L. J., Hellström K. E., Krebs E. G. Phosphorylation of synthetic peptides by a tyrosine protein kinase from the particulate fraction of a lymphoma cell line. Proc Natl Acad Sci U S A. 1982 Jan;79(2):282–286. doi: 10.1073/pnas.79.2.282. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chan P. K., Aldrich M., Cook R. G., Busch H. Amino acid sequence of protein B23 phosphorylation site. J Biol Chem. 1986 Feb 5;261(4):1868–1872. [PubMed] [Google Scholar]
  7. Chen-Wu J. L., Padmanabha R., Glover C. V. Isolation, sequencing, and disruption of the CKA1 gene encoding the alpha subunit of yeast casein kinase II. Mol Cell Biol. 1988 Nov;8(11):4981–4990. doi: 10.1128/mcb.8.11.4981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Cole M. D. The myc oncogene: its role in transformation and differentiation. Annu Rev Genet. 1986;20:361–384. doi: 10.1146/annurev.ge.20.120186.002045. [DOI] [PubMed] [Google Scholar]
  10. Cory S. Activation of cellular oncogenes in hemopoietic cells by chromosome translocation. Adv Cancer Res. 1986;47:189–234. doi: 10.1016/s0065-230x(08)60200-6. [DOI] [PubMed] [Google Scholar]
  11. Dang C. V., Lee W. M. Identification of the human c-myc protein nuclear translocation signal. Mol Cell Biol. 1988 Oct;8(10):4048–4054. doi: 10.1128/mcb.8.10.4048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. DeCaprio J. A., Ludlow J. W., Figge J., Shew J. Y., Huang C. M., Lee W. H., Marsilio E., Paucha E., Livingston D. M. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell. 1988 Jul 15;54(2):275–283. doi: 10.1016/0092-8674(88)90559-4. [DOI] [PubMed] [Google Scholar]
  13. Edelman A. M., Blumenthal D. K., Krebs E. G. Protein serine/threonine kinases. Annu Rev Biochem. 1987;56:567–613. doi: 10.1146/annurev.bi.56.070187.003031. [DOI] [PubMed] [Google Scholar]
  14. Eisenman R. N., Thompson C. B. Oncogenes with potential nuclear function: myc, myb and fos. Cancer Surv. 1986;5(2):309–327. [PubMed] [Google Scholar]
  15. Figge J., Smith T. F. Cell-division sequence motif. Nature. 1988 Jul 14;334(6178):109–109. doi: 10.1038/334109a0. [DOI] [PubMed] [Google Scholar]
  16. Figge J., Webster T., Smith T. F., Paucha E. Prediction of similar transforming regions in simian virus 40 large T, adenovirus E1A, and myc oncoproteins. J Virol. 1988 May;62(5):1814–1818. doi: 10.1128/jvi.62.5.1814-1818.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grässer F. A., Scheidtmann K. H., Tuazon P. T., Traugh J. A., Walter G. In vitro phosphorylation of SV40 large T antigen. Virology. 1988 Jul;165(1):13–22. doi: 10.1016/0042-6822(88)90653-8. [DOI] [PubMed] [Google Scholar]
  18. Hann S. R., Abrams H. D., Rohrschneider L. R., Eisenman R. N. Proteins encoded by v-myc and c-myc oncogenes: identification and localization in acute leukemia virus transformants and bursal lymphoma cell lines. Cell. 1983 Oct;34(3):789–798. doi: 10.1016/0092-8674(83)90535-4. [DOI] [PubMed] [Google Scholar]
  19. Hann S. R., Eisenman R. N. Proteins encoded by the human c-myc oncogene: differential expression in neoplastic cells. Mol Cell Biol. 1984 Nov;4(11):2486–2497. doi: 10.1128/mcb.4.11.2486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hann S. R., King M. W., Bentley D. L., Anderson C. W., Eisenman R. N. A non-AUG translational initiation in c-myc exon 1 generates an N-terminally distinct protein whose synthesis is disrupted in Burkitt's lymphomas. Cell. 1988 Jan 29;52(2):185–195. doi: 10.1016/0092-8674(88)90507-7. [DOI] [PubMed] [Google Scholar]
  21. Hathaway G. M., Traugh J. A. Casein kinases--multipotential protein kinases. Curr Top Cell Regul. 1982;21:101–127. [PubMed] [Google Scholar]
  22. Heaney M. L., Pierce J., Parsons J. T. Site-directed mutagenesis of the gag-myc gene of avian myelocytomatosis virus 29: biological activity and intracellular localization of structurally altered proteins. J Virol. 1986 Oct;60(1):167–176. doi: 10.1128/jvi.60.1.167-176.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hihara H., Shimizu T., Yamamoto H. Establishment of tumor cell lines cultured from chickens with avian lymphoid leukosis. Natl Inst Anim Health Q (Tokyo) 1974 Winter;14(4):163–173. [PubMed] [Google Scholar]
  24. Hunter T. A thousand and one protein kinases. Cell. 1987 Sep 11;50(6):823–829. doi: 10.1016/0092-8674(87)90509-5. [DOI] [PubMed] [Google Scholar]
  25. Hunter T., Cooper J. A. Protein-tyrosine kinases. Annu Rev Biochem. 1985;54:897–930. doi: 10.1146/annurev.bi.54.070185.004341. [DOI] [PubMed] [Google Scholar]
  26. Kalderon D., Smith A. E. In vitro mutagenesis of a putative DNA binding domain of SV40 large-T. Virology. 1984 Nov;139(1):109–137. doi: 10.1016/0042-6822(84)90334-9. [DOI] [PubMed] [Google Scholar]
  27. Kaur P., McDougall J. K. Characterization of primary human keratinocytes transformed by human papillomavirus type 18. J Virol. 1988 Jun;62(6):1917–1924. doi: 10.1128/jvi.62.6.1917-1924.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Klarlund J. K., Czech M. P. Insulin-like growth factor I and insulin rapidly increase casein kinase II activity in BALB/c 3T3 fibroblasts. J Biol Chem. 1988 Nov 5;263(31):15872–15875. [PubMed] [Google Scholar]
  29. Kuenzel E. A., Krebs E. G. A synthetic peptide substrate specific for casein kinase II. Proc Natl Acad Sci U S A. 1985 Feb;82(3):737–741. doi: 10.1073/pnas.82.3.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kuenzel E. A., Mulligan J. A., Sommercorn J., Krebs E. G. Substrate specificity determinants for casein kinase II as deduced from studies with synthetic peptides. J Biol Chem. 1987 Jul 5;262(19):9136–9140. [PubMed] [Google Scholar]
  31. Landschulz W. H., Johnson P. F., McKnight S. L. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science. 1988 Jun 24;240(4860):1759–1764. doi: 10.1126/science.3289117. [DOI] [PubMed] [Google Scholar]
  32. Linial M., Gunderson N., Groudine M. Enhanced transcription of c-myc in bursal lymphoma cells requires continuous protein synthesis. Science. 1985 Dec 6;230(4730):1126–1132. doi: 10.1126/science.2999973. [DOI] [PubMed] [Google Scholar]
  33. Lüscher B., Eisenman R. N. c-myc and c-myb protein degradation: effect of metabolic inhibitors and heat shock. Mol Cell Biol. 1988 Jun;8(6):2504–2512. doi: 10.1128/mcb.8.6.2504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Marin O., Meggio F., Marchiori F., Borin G., Pinna L. A. Site specificity of casein kinase-2 (TS) from rat liver cytosol. A study with model peptide substrates. Eur J Biochem. 1986 Oct 15;160(2):239–244. doi: 10.1111/j.1432-1033.1986.tb09962.x. [DOI] [PubMed] [Google Scholar]
  35. Meggio F., Marchiori F., Borin G., Chessa G., Pinna L. A. Synthetic peptides including acidic clusters as substrates and inhibitors of rat liver casein kinase TS (type-2). J Biol Chem. 1984 Dec 10;259(23):14576–14579. [PubMed] [Google Scholar]
  36. Pinna L. A., Donella-Deana A., Meggio F. Structural features determining the site specificity of a rat liver cAMP-independent protein kinase. Biochem Biophys Res Commun. 1979 Mar 15;87(1):114–120. doi: 10.1016/0006-291x(79)91654-1. [DOI] [PubMed] [Google Scholar]
  37. Pinna L. A., Meggio F., Marchiori F., Borin G. Opposite and mutually incompatible structural requirements of type-2 casein kinase and cAMP-dependent protein kinase as visualized with synthetic peptide substrates. FEBS Lett. 1984 Jun 11;171(2):211–214. doi: 10.1016/0014-5793(84)80490-1. [DOI] [PubMed] [Google Scholar]
  38. Ramsay G. M., Hayman M. J. Isolation and biochemical characterization of partially transformation-defective mutants of avian myelocytomatosis virus strain MC29: localization of the mutation to the myc domain of the 110,000-dalton gag-myc polyprotein. J Virol. 1982 Mar;41(3):745–753. doi: 10.1128/jvi.41.3.745-753.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ramsay G., Graf T., Hayman M. J. Mutants of avian myelocytomatosis virus with smaller gag gene-related proteins have an altered transforming ability. Nature. 1980 Nov 13;288(5787):170–172. doi: 10.1038/288170a0. [DOI] [PubMed] [Google Scholar]
  40. Ramsay G., Hayman M. J., Bister K. Phosphorylation of specific sites in the gag-myc polyproteins encoded by MC29-type viruses correlates with their transforming ability. EMBO J. 1982;1(9):1111–1116. doi: 10.1002/j.1460-2075.1982.tb01305.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Scheidtmann K. H., Echle B., Walter G. Simian virus 40 large T antigen is phosphorylated at multiple sites clustered in two separate regions. J Virol. 1982 Oct;44(1):116–133. doi: 10.1128/jvi.44.1.116-133.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sibley D. R., Benovic J. L., Caron M. G., Lefkowitz R. J. Regulation of transmembrane signaling by receptor phosphorylation. Cell. 1987 Mar 27;48(6):913–922. doi: 10.1016/0092-8674(87)90700-8. [DOI] [PubMed] [Google Scholar]
  43. Sommercorn J., Mulligan J. A., Lozeman F. J., Krebs E. G. Activation of casein kinase II in response to insulin and to epidermal growth factor. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8834–8838. doi: 10.1073/pnas.84.24.8834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Stone J., de Lange T., Ramsay G., Jakobovits E., Bishop J. M., Varmus H., Lee W. Definition of regions in human c-myc that are involved in transformation and nuclear localization. Mol Cell Biol. 1987 May;7(5):1697–1709. doi: 10.1128/mcb.7.5.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Takio K., Kuenzel E. A., Walsh K. A., Krebs E. G. Amino acid sequence of the beta subunit of bovine lung casein kinase II. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4851–4855. doi: 10.1073/pnas.84.14.4851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Tuazon P. T., Bingham E. W., Traugh J. A. Cyclic nucleotide-independent protein kinases from rabbit reticulocytes. Site-specific phosphorylation of casein variants. Eur J Biochem. 1979 Mar;94(2):497–504. doi: 10.1111/j.1432-1033.1979.tb12918.x. [DOI] [PubMed] [Google Scholar]
  47. Walton G. M., Spiess J., Gill G. N. Phosphorylation of high mobility group protein 14 by casein kinase II. J Biol Chem. 1985 Apr 25;260(8):4745–4750. [PubMed] [Google Scholar]
  48. Whyte P., Buchkovich K. J., Horowitz J. M., Friend S. H., Raybuck M., Weinberg R. A., Harlow E. Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature. 1988 Jul 14;334(6178):124–129. doi: 10.1038/334124a0. [DOI] [PubMed] [Google Scholar]
  49. Whyte P., Ruley H. E., Harlow E. Two regions of the adenovirus early region 1A proteins are required for transformation. J Virol. 1988 Jan;62(1):257–265. doi: 10.1128/jvi.62.1.257-265.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Whyte P., Williamson N. M., Harlow E. Cellular targets for transformation by the adenovirus E1A proteins. Cell. 1989 Jan 13;56(1):67–75. doi: 10.1016/0092-8674(89)90984-7. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES