Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1993 Mar 11;21(5):1289–1295. doi: 10.1093/nar/21.5.1289

c-Jun is phosphorylated by the DNA-dependent protein kinase in vitro; definition of the minimal kinase recognition motif.

A J Bannister 1, T M Gottlieb 1, T Kouzarides 1, S P Jackson 1
PMCID: PMC309295  PMID: 8464713

Abstract

The DNA-dependent protein kinase (DNA-PK) phosphorylates a number of transcription factors. Here, we show that the DNA-PK modifies c-Jun in vitro and that serine residue 249 (Ser-249) is required for phosphorylation to occur. This residue corresponds to one of three sites of c-Jun that are phosphorylated in vivo and which negatively regulate c-Jun DNA binding in vitro. However, we find that phosphorylation of c-Jun by the DNA-PK does not interfere with DNA binding, indicating that phosphorylation at other sites is required for this effect. Mutagenesis of the phosphorylated region of c-Jun reveals that the primary amino acid sequence recognised by the DNA-PK consists of the sequence Ser-Gln, and that adjacent acidic residues potentiate kinase activity. Furthermore, when this site is placed within the context of a second protein, it confers DNA-PK directed phosphorylation upon that protein. Our findings will facilitate identification of DNA-PK phosphorylation sites in other transcription factors.

Full text

PDF
1289

Images in this article

1291Image
on p.1291
1291Image
on p.1291
1292Image
on p.1292
1293Image
on p.1293
1293Image
on p.1293

Selected References

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

  1. Anderson C. W., Lees-Miller S. P. The nuclear serine/threonine protein kinase DNA-PK. Crit Rev Eukaryot Gene Expr. 1992;2(4):283–314. [PubMed] [Google Scholar]
  2. Baer R., Bankier A. T., Biggin M. D., Deininger P. L., Farrell P. J., Gibson T. J., Hatfull G., Hudson G. S., Satchwell S. C., Séguin C. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature. 1984 Jul 19;310(5974):207–211. doi: 10.1038/310207a0. [DOI] [PubMed] [Google Scholar]
  3. Baichwal V. R., Tjian R. Control of c-Jun activity by interaction of a cell-specific inhibitor with regulatory domain delta: differences between v- and c-Jun. Cell. 1990 Nov 16;63(4):815–825. doi: 10.1016/0092-8674(90)90147-7. [DOI] [PubMed] [Google Scholar]
  4. Baker S. J., Kerppola T. K., Luk D., Vandenberg M. T., Marshak D. R., Curran T., Abate C. Jun is phosphorylated by several protein kinases at the same sites that are modified in serum-stimulated fibroblasts. Mol Cell Biol. 1992 Oct;12(10):4694–4705. doi: 10.1128/mcb.12.10.4694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bannister A. J., Cook A., Kouzarides T. In vitro DNA binding activity of Fos/Jun and BZLF1 but not C/EBP is affected by redox changes. Oncogene. 1991 Jul;6(7):1243–1250. [PubMed] [Google Scholar]
  6. Beckmann H., Su L. K., Kadesch T. TFE3: a helix-loop-helix protein that activates transcription through the immunoglobulin enhancer muE3 motif. Genes Dev. 1990 Feb;4(2):167–179. doi: 10.1101/gad.4.2.167. [DOI] [PubMed] [Google Scholar]
  7. Binétruy B., Smeal T., Karin M. Ha-Ras augments c-Jun activity and stimulates phosphorylation of its activation domain. Nature. 1991 May 9;351(6322):122–127. doi: 10.1038/351122a0. [DOI] [PubMed] [Google Scholar]
  8. Boyle W. J., Smeal T., Defize L. H., Angel P., Woodgett J. R., Karin M., Hunter T. Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. Cell. 1991 Feb 8;64(3):573–584. doi: 10.1016/0092-8674(91)90241-p. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Chen Y. R., Lees-Miller S. P., Tegtmeyer P., Anderson C. W. The human DNA-activated protein kinase phosphorylates simian virus 40 T antigen at amino- and carboxy-terminal sites. J Virol. 1991 Oct;65(10):5131–5140. doi: 10.1128/jvi.65.10.5131-5140.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cheng L. Z., Workman J. L., Kingston R. E., Kelly T. J. Regulation of DNA replication in vitro by the transcriptional activation domain of GAL4-VP16. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):589–593. doi: 10.1073/pnas.89.2.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cousens D. J., Greaves R., Goding C. R., O'Hare P. The C-terminal 79 amino acids of the herpes simplex virus regulatory protein, Vmw65, efficiently activate transcription in yeast and mammalian cells in chimeric DNA-binding proteins. EMBO J. 1989 Aug;8(8):2337–2342. doi: 10.1002/j.1460-2075.1989.tb08361.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Davis R. L., Weintraub H., Lassar A. B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell. 1987 Dec 24;51(6):987–1000. doi: 10.1016/0092-8674(87)90585-x. [DOI] [PubMed] [Google Scholar]
  14. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Distel R. J., Ro H. S., Rosen B. S., Groves D. L., Spiegelman B. M. Nucleoprotein complexes that regulate gene expression in adipocyte differentiation: direct participation of c-fos. Cell. 1987 Jun 19;49(6):835–844. doi: 10.1016/0092-8674(87)90621-0. [DOI] [PubMed] [Google Scholar]
  16. Gottlieb T. M., Jackson S. P. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell. 1993 Jan 15;72(1):131–142. doi: 10.1016/0092-8674(93)90057-w. [DOI] [PubMed] [Google Scholar]
  17. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  18. Guo Z. S., DePamphilis M. L. Specific transcription factors stimulate simian virus 40 and polyomavirus origins of DNA replication. Mol Cell Biol. 1992 Jun;12(6):2514–2524. doi: 10.1128/mcb.12.6.2514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Halazonetis T. D., Georgopoulos K., Greenberg M. E., Leder P. c-Jun dimerizes with itself and with c-Fos, forming complexes of different DNA binding affinities. Cell. 1988 Dec 2;55(5):917–924. doi: 10.1016/0092-8674(88)90147-x. [DOI] [PubMed] [Google Scholar]
  20. Helin K., Lees J. A., Vidal M., Dyson N., Harlow E., Fattaey A. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell. 1992 Jul 24;70(2):337–350. doi: 10.1016/0092-8674(92)90107-n. [DOI] [PubMed] [Google Scholar]
  21. Hunter T., Karin M. The regulation of transcription by phosphorylation. Cell. 1992 Aug 7;70(3):375–387. doi: 10.1016/0092-8674(92)90162-6. [DOI] [PubMed] [Google Scholar]
  22. Iijima S., Teraoka H., Date T., Tsukada K. DNA-activated protein kinase in Raji Burkitt's lymphoma cells. Phosphorylation of c-Myc oncoprotein. Eur J Biochem. 1992 Jun 1;206(2):595–603. doi: 10.1111/j.1432-1033.1992.tb16964.x. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Jackson S. P. Regulating transcription factor activity by phosphorylation. Trends Cell Biol. 1992 Apr;2(4):104–108. doi: 10.1016/0962-8924(92)90014-e. [DOI] [PubMed] [Google Scholar]
  25. Kouzarides T., Ziff E. Leucine zippers of fos, jun and GCN4 dictate dimerization specificity and thereby control DNA binding. Nature. 1989 Aug 17;340(6234):568–571. doi: 10.1038/340568a0. [DOI] [PubMed] [Google Scholar]
  26. Kouzarides T., Ziff E. The role of the leucine zipper in the fos-jun interaction. Nature. 1988 Dec 15;336(6200):646–651. doi: 10.1038/336646a0. [DOI] [PubMed] [Google Scholar]
  27. Krieg P. A., Melton D. A. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984 Sep 25;12(18):7057–7070. doi: 10.1093/nar/12.18.7057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lane D. P. Cancer. p53, guardian of the genome. Nature. 1992 Jul 2;358(6381):15–16. doi: 10.1038/358015a0. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Lees-Miller S. P., Chen Y. R., Anderson C. W. Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Mol Cell Biol. 1990 Dec;10(12):6472–6481. doi: 10.1128/mcb.10.12.6472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lees-Miller S. P., Sakaguchi K., Ullrich S. J., Appella E., Anderson C. W. Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53. Mol Cell Biol. 1992 Nov;12(11):5041–5049. doi: 10.1128/mcb.12.11.5041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lin A., Frost J., Deng T., Smeal T., al-Alawi N., Kikkawa U., Hunter T., Brenner D., Karin M. Casein kinase II is a negative regulator of c-Jun DNA binding and AP-1 activity. Cell. 1992 Sep 4;70(5):777–789. doi: 10.1016/0092-8674(92)90311-y. [DOI] [PubMed] [Google Scholar]
  34. Peterson S. R., Dvir A., Anderson C. W., Dynan W. S. DNA binding provides a signal for phosphorylation of the RNA polymerase II heptapeptide repeats. Genes Dev. 1992 Mar;6(3):426–438. doi: 10.1101/gad.6.3.426. [DOI] [PubMed] [Google Scholar]
  35. Pulverer B. J., Kyriakis J. M., Avruch J., Nikolakaki E., Woodgett J. R. Phosphorylation of c-jun mediated by MAP kinases. Nature. 1991 Oct 17;353(6345):670–674. doi: 10.1038/353670a0. [DOI] [PubMed] [Google Scholar]
  36. Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
  37. Schöler H. R., Ruppert S., Suzuki N., Chowdhury K., Gruss P. New type of POU domain in germ line-specific protein Oct-4. Nature. 1990 Mar 29;344(6265):435–439. doi: 10.1038/344435a0. [DOI] [PubMed] [Google Scholar]
  38. Smeal T., Binetruy B., Mercola D. A., Birrer M., Karin M. Oncogenic and transcriptional cooperation with Ha-Ras requires phosphorylation of c-Jun on serines 63 and 73. Nature. 1991 Dec 12;354(6353):494–496. doi: 10.1038/354494a0. [DOI] [PubMed] [Google Scholar]
  39. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  40. Sutherland J. A., Cook A., Bannister A. J., Kouzarides T. Conserved motifs in Fos and Jun define a new class of activation domain. Genes Dev. 1992 Sep;6(9):1810–1819. doi: 10.1101/gad.6.9.1810. [DOI] [PubMed] [Google Scholar]
  41. Wang Y., Eckhart W. Phosphorylation sites in the amino-terminal region of mouse p53. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4231–4235. doi: 10.1073/pnas.89.10.4231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Weigel N. L., Carter T. H., Schrader W. T., O'Malley B. W. Chicken progesterone receptor is phosphorylated by a DNA-dependent protein kinase during in vitro transcription assays. Mol Endocrinol. 1992 Jan;6(1):8–14. doi: 10.1210/mend.6.1.1738374. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES