Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1985 Nov;4(11):2921–2926. doi: 10.1002/j.1460-2075.1985.tb04024.x

Topoisomerase I phosphorylation in vitro and in rapidly growing Novikoff hepatoma cells.

E Durban, M Goodenough, J Mills, H Busch
PMCID: PMC554599  PMID: 2998765

Abstract

Changes in phosphorylation modulate the activity of topoisomerase I in vitro. Specifically, enzymatic activity is stimulated by phosphorylation with a purified protein kinase (casein kinase type II). The purpose of this study was to compare the sites that are phosphorylated in vitro by casein kinase type II with the site(s) phosphorylated in vivo in rapidly growing Novikoff hepatoma cells. Topoisomerase I labeled in vitro was characterized by three major tryptic phosphopeptides (I-III). Separation of these peptides by a C18-reverse phase h.p.l.c. column resulted in their elution at fractions 18 (I), 27 (II) and 44 (III) with 17%, 22.5% and 33% acetonitrile, respectively. In contrast, only one major phosphopeptide was identified by h.p.l.c. in topoisomerase I labeled in vivo. This phosphopeptide eluted at fraction 18 corresponding to the elution properties of phosphopeptide I labeled in vitro. It also co-migrated with tryptic phosphopeptide I when subjected to high-voltage electrophoresis on thin-layer cellulose plates. Preliminary experiments suggest that phosphorylation occurs at a serine residue six amino acids from the N-terminus of the peptide. These data indicate that topoisomerase I is phosphorylated in vivo and in vitro within the same tryptic peptide and suggest that topoisomerase I is phosphorylated in vivo by casein kinase II.

Full text

PDF
2921

Images in this article

2922Fig. 2.
on p.2922
2924Fig. 4.
on p.2924

Selected References

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

  1. Ackerman P., Glover C. V., Osheroff N. Phosphorylation of DNA topoisomerase II by casein kinase II: modulation of eukaryotic topoisomerase II activity in vitro. Proc Natl Acad Sci U S A. 1985 May;82(10):3164–3168. doi: 10.1073/pnas.82.10.3164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bauer W. R., Ressner E. C., Kates J., Patzke J. V. A DNA nicking-closing enzyme encapsidated in vaccinia virus: partial purification and properties. Proc Natl Acad Sci U S A. 1977 May;74(5):1841–1845. doi: 10.1073/pnas.74.5.1841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Carmichael D. F., Geahlen R. L., Allen S. M., Krebs E. G. Type II regulatory subunit of cAMP-dependent protein kinase. Phosphorylation by casein kinase II at a site that is also phosphorylated in vivo. J Biol Chem. 1982 Sep 10;257(17):10440–10445. [PubMed] [Google Scholar]
  5. Champoux J. J. Proteins that affect DNA conformation. Annu Rev Biochem. 1978;47:449–479. doi: 10.1146/annurev.bi.47.070178.002313. [DOI] [PubMed] [Google Scholar]
  6. Champoux J. J. Renaturation of complementary single-stranded DNA circles: complete rewinding facilitated by the DNA untwisting enzyme. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5328–5332. doi: 10.1073/pnas.74.12.5328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dahmus M. E., Natzle J. Purification and characterization of Novikoff ascites tumor protein kinase. Biochemistry. 1977 May 3;16(9):1901–1908. doi: 10.1021/bi00628a022. [DOI] [PubMed] [Google Scholar]
  8. Dahmus M. E. Purification and properties of calf thymus casein kinases I and II. J Biol Chem. 1981 Apr 10;256(7):3319–3325. [PubMed] [Google Scholar]
  9. Duceman B. W., Rose K. M., Jacob S. T. Activation of purified hepatoma RNA polymerase I by homologous protein kinase NII. J Biol Chem. 1981 Nov 10;256(21):10755–10758. [PubMed] [Google Scholar]
  10. Durban E., Mills J. S., Roll D., Busch H. Phosphorylation of purified Novikoff hepatoma topoisomerase I. Biochem Biophys Res Commun. 1983 Mar 29;111(3):897–905. doi: 10.1016/0006-291x(83)91384-0. [DOI] [PubMed] [Google Scholar]
  11. Durban E., Roll D., Beckner G., Busch H. Purification and characterization of a nuclear DNA-binding phosphoprotein in fetal and tumor tissues. Cancer Res. 1981 Feb;41(2):537–545. [PubMed] [Google Scholar]
  12. Elder J. H., Pickett R. A., 2nd, Hampton J., Lerner R. A. Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein. J Biol Chem. 1977 Sep 25;252(18):6510–6515. [PubMed] [Google Scholar]
  13. Embi N., Parker P. J., Cohen P. A reinvestigation of the phosphorylation of rabbit skeletal-muscle glycogen synthase by cyclic-AMP-dependent protein kinase. Identification of the third site of phosphorylation as serine-7. Eur J Biochem. 1981 Apr;115(2):405–413. doi: 10.1111/j.1432-1033.1981.tb05252.x. [DOI] [PubMed] [Google Scholar]
  14. Ferro A. M., Olivera B. M. Poly(ADP-ribosylation) of DNA topoisomerase I from calf thymus. J Biol Chem. 1984 Jan 10;259(1):547–554. [PubMed] [Google Scholar]
  15. Fleischmann G., Pflugfelder G., Steiner E. K., Javaherian K., Howard G. C., Wang J. C., Elgin S. C. Drosophila DNA topoisomerase I is associated with transcriptionally active regions of the genome. Proc Natl Acad Sci U S A. 1984 Nov;81(22):6958–6962. doi: 10.1073/pnas.81.22.6958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gellert M. DNA topoisomerases. Annu Rev Biochem. 1981;50:879–910. doi: 10.1146/annurev.bi.50.070181.004311. [DOI] [PubMed] [Google Scholar]
  17. Halligan B. D., Davis J. L., Edwards K. A., Liu L. F. Intra- and intermolecular strand transfer by HeLa DNA topoisomerase I. J Biol Chem. 1982 Apr 10;257(7):3995–4000. [PubMed] [Google Scholar]
  18. Hathaway G. M., Traugh J. A. Casein kinases--multipotential protein kinases. Curr Top Cell Regul. 1982;21:101–127. [PubMed] [Google Scholar]
  19. Hathaway G. M., Traugh J. A. Cyclic nucleotide-independent protein kinases from rabbit reticulocytes. Purification of casein kinases. J Biol Chem. 1979 Feb 10;254(3):762–768. [PubMed] [Google Scholar]
  20. Hemmings B. A., Aitken A., Cohen P., Rymond M., Hofmann F. Phosphorylation of the type-II regulatory subunit of cyclic-AMP-dependent protein kinase by glycogen synthase kinase 3 and glycogen synthase kinase 5. Eur J Biochem. 1982 Oct;127(3):473–481. doi: 10.1111/j.1432-1033.1982.tb06896.x. [DOI] [PubMed] [Google Scholar]
  21. Kirkegaard K., Wang J. C. Escherichia coli DNA topoisomerase I catalyzed linking of single-stranded rings of complementary base sequences. Nucleic Acids Res. 1978 Oct;5(10):3811–3820. doi: 10.1093/nar/5.10.3811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Meggio F., Deana A. D., Pinna L. A. Endogenous phosphate acceptor proteins for rat liver cytosolic casein kinases. J Biol Chem. 1981 Dec 10;256(23):11958–11961. [PubMed] [Google Scholar]
  24. Mills J. S., Busch H., Durban E. Purification of a protein kinase from human Namalwa cells that phosphorylates topoisomerase I. Biochem Biophys Res Commun. 1982 Dec 31;109(4):1222–1227. doi: 10.1016/0006-291x(82)91907-6. [DOI] [PubMed] [Google Scholar]
  25. Proud C. G., Rylatt D. B., Yeaman S. J., Cohen P. Amino acid sequences at the two sites on glycogen synthetase phosphorylated by cyclic AMP-dependent protein kinase and their dephosphorylation by protein phosphatase-III. FEBS Lett. 1977 Aug 15;80(2):435–442. doi: 10.1016/0014-5793(77)80493-6. [DOI] [PubMed] [Google Scholar]
  26. Rose K. M., Bell L. E., Siefken D. A., Jacob S. T. A heparin-sensitive nuclear protein kinase. Purification, properties, and increased activity in rat hepatoma relative to liver. J Biol Chem. 1981 Jul 25;256(14):7468–7477. [PubMed] [Google Scholar]
  27. Stetler D. A., Rose K. M. Phosphorylation of deoxyribonucleic acid dependent RNA polymerase II by nuclear protein kinase NII: mechanism of enhanced ribonucleic acid synthesis. Biochemistry. 1982 Jul 20;21(15):3721–3728. doi: 10.1021/bi00258a030. [DOI] [PubMed] [Google Scholar]
  28. Thornburg W., Kieras R., Lindell T. J. Selective binding of rat liver nuclear enzymes to histones, phosvitin and casein coupled to sepharose. Prep Biochem. 1978;8(2-3):133–146. doi: 10.1080/00327487808069055. [DOI] [PubMed] [Google Scholar]
  29. Thornburg W., Lindell T. J. Purification of rat liver nuclear protein kinase NII. J Biol Chem. 1977 Oct 10;252(19):6660–6665. [PubMed] [Google Scholar]
  30. Tse-Dinh Y. C., Wong T. W., Goldberg A. R. Virus- and cell-encoded tyrosine protein kinases inactivate DNA topoisomerases in vitro. Nature. 1984 Dec 20;312(5996):785–786. doi: 10.1038/312785a0. [DOI] [PubMed] [Google Scholar]
  31. Villar-Palasi C., Kumon A. Purification and properties of dog cardiac troponin T kinase. J Biol Chem. 1981 Jul 25;256(14):7409–7415. [PubMed] [Google Scholar]
  32. Walton G. M., Gill G. N. Identity of the in vivo phosphorylation site in high mobility group 14 protein in HeLa cells with the site phosphorylated by casein kinase II in vitro. J Biol Chem. 1983 Apr 10;258(7):4440–4446. [PubMed] [Google Scholar]
  33. 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]
  34. Weisbrod S. T. Properties of active nucleosomes as revealed by HMG 14 and 17 chromatography. Nucleic Acids Res. 1982 Mar 25;10(6):2017–2042. doi: 10.1093/nar/10.6.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES