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. 1990 Dec;10(12):6472–6481. doi: 10.1128/mcb.10.12.6472

Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen.

S P Lees-Miller 1, Y R Chen 1, C W Anderson 1
PMCID: PMC362924  PMID: 2247067

Abstract

HeLa cells contain a serine/threonine protein kinase (DNA-PK) that is strongly activated in vitro by low concentrations of double-stranded DNA (dsDNA). Activation was specific for dsDNA; both natural DNAs and synthetic oligonucleotides functioned as kinase activators. The fact that DNA-PK activity was rapidly inhibited by incubation with dsDNA and ATP suggests that DNA-PK activity also may be regulated by autophosphorylation. During gel filtration, DNA-PK activity behaved as a 350-kDa protein, and highly purified DNA-PK contained a dsDNA-binding, 350-kDa polypeptide that was phosphorylated in a dsDNA-dependent manner. We conclude that this 350-kDa polypeptide is likely to be DNA-PK. Previously we showed that the dsDNA-activated kinase phosphorylates two threonines at the N terminus of hsp90 alpha (S. P. Lees-Miller and C. W. Anderson, J. Biol. Chem. 264:17275-17280, 1989). Here we show that DNA-PK also phosphorylates the simian virus 40 large tumor antigen, the mouse tumor-suppressor protein p53, the human Ku autoantigen, and two unidentified HeLa DNA-associated polypeptides of 52 and 110 kDa. Identification of these and other newly identified DNA-binding substrates suggest that the dsDNA-activated kinase may regulate transcription, DNA replication, or cell growth.

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Selected References

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

  1. Addison C., Jenkins J. R., Stürzbecher H. W. The p53 nuclear localisation signal is structurally linked to a p34cdc2 kinase motif. Oncogene. 1990 Mar;5(3):423–426. [PubMed] [Google Scholar]
  2. Anderson C. W., Baum P. R., Gesteland R. F. Processing of adenovirus 2-induced proteins. J Virol. 1973 Aug;12(2):241–252. doi: 10.1128/jvi.12.2.241-252.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bischoff J. R., Friedman P. N., Marshak D. R., Prives C., Beach D. Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4766–4770. doi: 10.1073/pnas.87.12.4766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carroll D., Marshak D. R. Serum-stimulated cell growth causes oscillations in casein kinase II activity. J Biol Chem. 1989 May 5;264(13):7345–7348. [PubMed] [Google Scholar]
  5. Carter T. H., Kopman C. R., James C. B. DNA-stimulated protein phosphorylation in HeLa whole cell and nuclear extracts. Biochem Biophys Res Commun. 1988 Dec 15;157(2):535–540. doi: 10.1016/s0006-291x(88)80282-1. [DOI] [PubMed] [Google Scholar]
  6. Carter T., Vancurová I., Sun I., Lou W., DeLeon S. A DNA-activated protein kinase from HeLa cell nuclei. Mol Cell Biol. 1990 Dec;10(12):6460–6471. doi: 10.1128/mcb.10.12.6460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chan J. Y., Lerman M. I., Prabhakar B. S., Isozaki O., Santisteban P., Kuppers R. C., Oates E. L., Notkins A. L., Kohn L. D. Cloning and characterization of a cDNA that encodes a 70-kDa novel human thyroid autoantigen. J Biol Chem. 1989 Mar 5;264(7):3651–3654. [PubMed] [Google Scholar]
  8. Chatterjee P. K., Vayda M. E., Flint S. J. Identification of proteins and protein domains that contact DNA within adenovirus nucleoprotein cores by ultraviolet light crosslinking of oligonucleotides 32P-labelled in vivo. J Mol Biol. 1986 Mar 5;188(1):23–37. doi: 10.1016/0022-2836(86)90477-8. [DOI] [PubMed] [Google Scholar]
  9. Cochet C., Feige J. J., Pirollet F., Keramidas M., Chambaz E. M. Selective inhibition of a cyclic nucleotide independent protein kinase (G type casein kinase) by quercetin and related polyphenols. Biochem Pharmacol. 1982 Apr 1;31(7):1357–1361. doi: 10.1016/0006-2952(82)90028-4. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Hanks S. K., Quinn A. M., Hunter T. The protein kinase family: conserved features and deduced phylogeny of the catalytic domains. Science. 1988 Jul 1;241(4861):42–52. doi: 10.1126/science.3291115. [DOI] [PubMed] [Google Scholar]
  13. Jackson S. P., MacDonald J. J., Lees-Miller S., Tjian R. GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase. Cell. 1990 Oct 5;63(1):155–165. doi: 10.1016/0092-8674(90)90296-q. [DOI] [PubMed] [Google Scholar]
  14. Jenkins J. R., Rudge K., Redmond S., Wade-Evans A. Cloning and expression analysis of full length mouse cDNA sequences encoding the transformation associated protein p53. Nucleic Acids Res. 1984 Jul 25;12(14):5609–5626. doi: 10.1093/nar/12.14.5609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lanford R. E. Expression of simian virus 40 T antigen in insect cells using a baculovirus expression vector. Virology. 1988 Nov;167(1):72–81. doi: 10.1016/0042-6822(88)90055-4. [DOI] [PubMed] [Google Scholar]
  16. Lees-Miller S. P., Anderson C. W. The human double-stranded DNA-activated protein kinase phosphorylates the 90-kDa heat-shock protein, hsp90 alpha at two NH2-terminal threonine residues. J Biol Chem. 1989 Oct 15;264(29):17275–17280. [PubMed] [Google Scholar]
  17. Lees-Miller S. P., Anderson C. W. Two human 90-kDa heat shock proteins are phosphorylated in vivo at conserved serines that are phosphorylated in vitro by casein kinase II. J Biol Chem. 1989 Feb 15;264(5):2431–2437. [PubMed] [Google Scholar]
  18. Lewin B. Driving the cell cycle: M phase kinase, its partners, and substrates. Cell. 1990 Jun 1;61(5):743–752. doi: 10.1016/0092-8674(90)90181-d. [DOI] [PubMed] [Google Scholar]
  19. Lo K. M., Jones S. S., Hackett N. R., Khorana H. G. Specific amino acid substitutions in bacterioopsin: Replacement of a restriction fragment in the structural gene by synthetic DNA fragments containing altered codons. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2285–2289. doi: 10.1073/pnas.81.8.2285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McVey D., Brizuela L., Mohr I., Marshak D. R., Gluzman Y., Beach D. Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication. Nature. 1989 Oct 12;341(6242):503–507. doi: 10.1038/341503a0. [DOI] [PubMed] [Google Scholar]
  21. Meek D. W., Eckhart W. Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells. Mol Cell Biol. 1988 Jan;8(1):461–465. doi: 10.1128/mcb.8.1.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mimori T., Hardin J. A. Mechanism of interaction between Ku protein and DNA. J Biol Chem. 1986 Aug 5;261(22):10375–10379. [PubMed] [Google Scholar]
  23. Mimori T., Hardin J. A., Steitz J. A. Characterization of the DNA-binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders. J Biol Chem. 1986 Feb 15;261(5):2274–2278. [PubMed] [Google Scholar]
  24. Mimori T., Ohosone Y., Hama N., Suwa A., Akizuki M., Homma M., Griffith A. J., Hardin J. A. Isolation and characterization of cDNA encoding the 80-kDa subunit protein of the human autoantigen Ku (p70/p80) recognized by autoantibodies from patients with scleroderma-polymyositis overlap syndrome. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1777–1781. doi: 10.1073/pnas.87.5.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Minks M. A., West D. K., Benvin S., Baglioni C. Structural requirements of double-stranded RNA for the activation of 2',5'-oligo(A) polymerase and protein kinase of interferon-treated HeLa cells. J Biol Chem. 1979 Oct 25;254(20):10180–10183. [PubMed] [Google Scholar]
  26. O'Reilly D. R., Miller L. K. Expression and complex formation of simian virus 40 large T antigen and mouse p53 in insect cells. J Virol. 1988 Sep;62(9):3109–3119. doi: 10.1128/jvi.62.9.3109-3119.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pennica D., Goeddel D. V., Hayflick J. S., Reich N. C., Anderson C. W., Levine A. J. The amino acid sequence of murine p53 determined from a c-DNA clone. Virology. 1984 Apr 30;134(2):477–482. doi: 10.1016/0042-6822(84)90316-7. [DOI] [PubMed] [Google Scholar]
  29. Prives C. The replication functions of SV40 T antigen are regulated by phosphorylation. Cell. 1990 Jun 1;61(5):735–738. doi: 10.1016/0092-8674(90)90179-i. [DOI] [PubMed] [Google Scholar]
  30. Rabilloud T., Carpentier G., Tarroux P. Improvement and simplification of low-background silver staining of proteins by using sodium dithionite. Electrophoresis. 1988 Jun;9(6):288–291. doi: 10.1002/elps.1150090608. [DOI] [PubMed] [Google Scholar]
  31. Reddy V. B., Thimmappaya B., Dhar R., Subramanian K. N., Zain B. S., Pan J., Ghosh P. K., Celma M. L., Weissman S. M. The genome of simian virus 40. Science. 1978 May 5;200(4341):494–502. doi: 10.1126/science.205947. [DOI] [PubMed] [Google Scholar]
  32. Reeves W. H., Sthoeger Z. M. Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen. J Biol Chem. 1989 Mar 25;264(9):5047–5052. [PubMed] [Google Scholar]
  33. Samad A., Anderson C. W., Carroll R. B. Mapping of phosphomonoester and apparent phosphodiester bonds of the oncogene product p53 from simian virus 40-transformed 3T3 cells. Proc Natl Acad Sci U S A. 1986 Feb;83(4):897–901. doi: 10.1073/pnas.83.4.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Schneider J., Fanning E. Mutations in the phosphorylation sites of simian virus 40 (SV40) T antigen alter its origin DNA-binding specificity for sites I or II and affect SV40 DNA replication activity. J Virol. 1988 May;62(5):1598–1605. doi: 10.1128/jvi.62.5.1598-1605.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Tanaka M., Herr W. Differential transcriptional activation by Oct-1 and Oct-2: interdependent activation domains induce Oct-2 phosphorylation. Cell. 1990 Feb 9;60(3):375–386. doi: 10.1016/0092-8674(90)90589-7. [DOI] [PubMed] [Google Scholar]
  38. Walker A. I., Hunt T., Jackson R. J., Anderson C. W. Double-stranded DNA induces the phosphorylation of several proteins including the 90 000 mol. wt. heat-shock protein in animal cell extracts. EMBO J. 1985 Jan;4(1):139–145. doi: 10.1002/j.1460-2075.1985.tb02328.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Yaneva M., Busch H. A 10S particle released from deoxyribonuclease-sensitive regions of HeLa cell nuclei contains the 86-kilodalton-70-kilodalton protein complex. Biochemistry. 1986 Sep 9;25(18):5057–5063. doi: 10.1021/bi00366a013. [DOI] [PubMed] [Google Scholar]
  41. Yaneva M., Wen J., Ayala A., Cook R. cDNA-derived amino acid sequence of the 86-kDa subunit of the Ku antigen. J Biol Chem. 1989 Aug 15;264(23):13407–13411. [PubMed] [Google Scholar]
  42. Zandomeni R., Weinmann R. Inhibitory effect of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole on a protein kinase. J Biol Chem. 1984 Dec 10;259(23):14804–14811. [PubMed] [Google Scholar]

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