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. 2002 Nov 15;368(Pt 1):243–251. doi: 10.1042/BJ20020973

Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase.

Pauline Douglas 1, Gopal P Sapkota 1, Nick Morrice 1, Yaping Yu 1, Aaron A Goodarzi 1, Dennis Merkle 1, Katheryn Meek 1, Dario R Alessi 1, Susan P Lees-Miller 1
PMCID: PMC1222982  PMID: 12186630

Abstract

The DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs), such as those caused by ionizing radiation and other DNA-damaging agents. DNA-PK is composed of a large catalytic subunit (DNA-PKcs) and a heterodimer of Ku70 and Ku80 that assemble on the ends of double-stranded DNA to form an active serine/threonine protein kinase complex. Despite in vitro and in vivo evidence to support an essential role for the protein kinase activity of DNA-PK in the repair of DNA DSBs, the physiological targets of DNA-PK have remained elusive. We have previously shown that DNA-PK undergoes autophosphorylation in vitro, and that autophosphorylation correlates with loss of protein kinase activity and dissociation of the DNA-PK complex. Also, treatment of cells with the protein phosphatase inhibitor, okadaic acid, enhances DNA-PKcs phosphorylation and reduces DNA-PK activity in vivo. Here, using solid-phase protein sequencing, MS and phosphospecific antibodies, we have identified seven in vitro autophosphorylation sites in DNA-PKcs. Six of these sites (Thr2609, Ser2612, Thr2620, Ser2624, Thr2638 and Thr2647) are clustered in a region of 38 amino acids in the central region of the protein. Five of these sites (Thr2609, Ser2612, Thr2638, Thr2647 and Ser3205) are conserved between six vertebrate species. Moreover, we show that DNA-PKcs is phosphorylated in vivo at Thr2609, Ser2612, Thr2638 and Thr2647 in okadaic acid-treated human cells. We propose that phosphorylation of these sites may play an important role in DNA-PK function.

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Selected References

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

  1. Abraham R. T. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 2001 Sep 1;15(17):2177–2196. doi: 10.1101/gad.914401. [DOI] [PubMed] [Google Scholar]
  2. Alessi D. R., Gomez N., Moorhead G., Lewis T., Keyse S. M., Cohen P. Inactivation of p42 MAP kinase by protein phosphatase 2A and a protein tyrosine phosphatase, but not CL100, in various cell lines. Curr Biol. 1995 Mar 1;5(3):283–295. doi: 10.1016/s0960-9822(95)00059-5. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Araki R., Fujimori A., Hamatani K., Mita K., Saito T., Mori M., Fukumura R., Morimyo M., Muto M., Itoh M. Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency mice. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2438–2443. doi: 10.1073/pnas.94.6.2438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Banin S., Moyal L., Shieh S., Taya Y., Anderson C. W., Chessa L., Smorodinsky N. I., Prives C., Reiss Y., Shiloh Y. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science. 1998 Sep 11;281(5383):1674–1677. doi: 10.1126/science.281.5383.1674. [DOI] [PubMed] [Google Scholar]
  6. Bannister A. J., Gottlieb T. M., Kouzarides T., Jackson S. P. c-Jun is phosphorylated by the DNA-dependent protein kinase in vitro; definition of the minimal kinase recognition motif. Nucleic Acids Res. 1993 Mar 11;21(5):1289–1295. doi: 10.1093/nar/21.5.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Baumann P., West S. C. DNA end-joining catalyzed by human cell-free extracts. Proc Natl Acad Sci U S A. 1998 Nov 24;95(24):14066–14070. doi: 10.1073/pnas.95.24.14066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Canman C. E., Lim D. S., Cimprich K. A., Taya Y., Tamai K., Sakaguchi K., Appella E., Kastan M. B., Siliciano J. D. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science. 1998 Sep 11;281(5383):1677–1679. doi: 10.1126/science.281.5383.1677. [DOI] [PubMed] [Google Scholar]
  9. Chan D. W., Lees-Miller S. P. The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit. J Biol Chem. 1996 Apr 12;271(15):8936–8941. doi: 10.1074/jbc.271.15.8936. [DOI] [PubMed] [Google Scholar]
  10. Chan D. W., Son S. C., Block W., Ye R., Khanna K. K., Wold M. S., Douglas P., Goodarzi A. A., Pelley J., Taya Y. Purification and characterization of ATM from human placenta. A manganese-dependent, wortmannin-sensitive serine/threonine protein kinase. J Biol Chem. 2000 Mar 17;275(11):7803–7810. doi: 10.1074/jbc.275.11.7803. [DOI] [PubMed] [Google Scholar]
  11. Chan D. W., Ye R., Veillette C. J., Lees-Miller S. P. DNA-dependent protein kinase phosphorylation sites in Ku 70/80 heterodimer. Biochemistry. 1999 Feb 9;38(6):1819–1828. doi: 10.1021/bi982584b. [DOI] [PubMed] [Google Scholar]
  12. Chan Doug W., Chen Benjamin Ping-Chi, Prithivirajsingh Sheela, Kurimasa Akihiro, Story Michael D., Qin Jun, Chen David J. Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks. Genes Dev. 2002 Sep 15;16(18):2333–2338. doi: 10.1101/gad.1015202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chen S., Inamdar K. V., Pfeiffer P., Feldmann E., Hannah M. F., Yu Y., Lee J. W., Zhou T., Lees-Miller S. P., Povirk L. F. Accurate in vitro end joining of a DNA double strand break with partially cohesive 3'-overhangs and 3'-phosphoglycolate termini: effect of Ku on repair fidelity. J Biol Chem. 2001 Apr 17;276(26):24323–24330. doi: 10.1074/jbc.M010544200. [DOI] [PubMed] [Google Scholar]
  14. Ding Qi, Bramble Lori, Yuzbasiyan-Gurkan Vilma, Bell Thomas, Meek Katheryn. DNA-PKcs mutations in dogs and horses: allele frequency and association with neoplasia. Gene. 2002 Jan 23;283(1-2):263–269. doi: 10.1016/s0378-1119(01)00880-0. [DOI] [PubMed] [Google Scholar]
  15. Douglas P., Moorhead G. B., Ye R., Lees-Miller S. P. Protein phosphatases regulate DNA-dependent protein kinase activity. J Biol Chem. 2001 Mar 16;276(22):18992–18998. doi: 10.1074/jbc.M011703200. [DOI] [PubMed] [Google Scholar]
  16. Fujimori A., Araki R., Fukumura R., Ohhata T., Takahashi H., Kawahara A., Tatsumi K., Abe M. Identification of four highly conserved regions in DNA-PKcs. Immunogenetics. 2000 Sep;51(11):965–973. doi: 10.1007/s002510000227. [DOI] [PubMed] [Google Scholar]
  17. Fujimori Akira, Hashimoto Hiroshi, Araki Ryoko, Saito Toshiyuki, Sato Shinji, Kasama Yasuji, Tsutsumi Yoko, Mori Masahiko, Fukumura Ryutaro, Ohhata Tatsuya. Sequence analysis of 193.4 and 83.9 kbp of mouse and chicken genomic DNAs containing the entire Prkdc (DNA-PKcs) gene. Radiat Res. 2002 Mar;157(3):298–305. doi: 10.1667/0033-7587(2002)157[0298:saoako]2.0.co;2. [DOI] [PubMed] [Google Scholar]
  18. Gell D., Jackson S. P. Mapping of protein-protein interactions within the DNA-dependent protein kinase complex. Nucleic Acids Res. 1999 Sep 1;27(17):3494–3502. doi: 10.1093/nar/27.17.3494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Hartley K. O., Gell D., Smith G. C., Zhang H., Divecha N., Connelly M. A., Admon A., Lees-Miller S. P., Anderson C. W., Jackson S. P. DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell. 1995 Sep 8;82(5):849–856. doi: 10.1016/0092-8674(95)90482-4. [DOI] [PubMed] [Google Scholar]
  21. Izzard R. A., Jackson S. P., Smith G. C. Competitive and noncompetitive inhibition of the DNA-dependent protein kinase. Cancer Res. 1999 Jun 1;59(11):2581–2586. [PubMed] [Google Scholar]
  22. Kienker L. J., Shin E. K., Meek K. Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination. Nucleic Acids Res. 2000 Jul 15;28(14):2752–2761. doi: 10.1093/nar/28.14.2752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kurimasa A., Kumano S., Boubnov N. V., Story M. D., Tung C. S., Peterson S. R., Chen D. J. Requirement for the kinase activity of human DNA-dependent protein kinase catalytic subunit in DNA strand break rejoining. Mol Cell Biol. 1999 May;19(5):3877–3884. doi: 10.1128/mcb.19.5.3877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee K. J., Huang J., Takeda Y., Dynan W. S. DNA ligase IV and XRCC4 form a stable mixed tetramer that functions synergistically with other repair factors in a cell-free end-joining system. J Biol Chem. 2000 Nov 3;275(44):34787–34796. doi: 10.1074/jbc.M004011200. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Lees-Miller S. P., Godbout R., Chan D. W., Weinfeld M., Day R. S., 3rd, Barron G. M., Allalunis-Turner J. Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line. Science. 1995 Feb 24;267(5201):1183–1185. doi: 10.1126/science.7855602. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Ma Yunmei, Pannicke Ulrich, Schwarz Klaus, Lieber Michael R. Hairpin opening and overhang processing by an Artemis/DNA-dependent protein kinase complex in nonhomologous end joining and V(D)J recombination. Cell. 2002 Mar 22;108(6):781–794. doi: 10.1016/s0092-8674(02)00671-2. [DOI] [PubMed] [Google Scholar]
  30. Mallory J. C., Petes T. D. Protein kinase activity of Tel1p and Mec1p, two Saccharomyces cerevisiae proteins related to the human ATM protein kinase. Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13749–13754. doi: 10.1073/pnas.250475697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moshous D., Callebaut I., de Chasseval R., Corneo B., Cavazzana-Calvo M., Le Deist F., Tezcan I., Sanal O., Bertrand Y., Philippe N. Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in human severe combined immune deficiency. Cell. 2001 Apr 20;105(2):177–186. doi: 10.1016/s0092-8674(01)00309-9. [DOI] [PubMed] [Google Scholar]
  32. Moshous D., Li L., Chasseval R., Philippe N., Jabado N., Cowan M. J., Fischer A., de Villartay J. P. A new gene involved in DNA double-strand break repair and V(D)J recombination is located on human chromosome 10p. Hum Mol Genet. 2000 Mar 1;9(4):583–588. doi: 10.1093/hmg/9.4.583. [DOI] [PubMed] [Google Scholar]
  33. Norbury C. J., Hickson I. D. Cellular responses to DNA damage. Annu Rev Pharmacol Toxicol. 2001;41:367–401. doi: 10.1146/annurev.pharmtox.41.1.367. [DOI] [PubMed] [Google Scholar]
  34. O'Neill T., Dwyer A. J., Ziv Y., Chan D. W., Lees-Miller S. P., Abraham R. H., Lai J. H., Hill D., Shiloh Y., Cantley L. C. Utilization of oriented peptide libraries to identify substrate motifs selected by ATM. J Biol Chem. 2000 Jul 28;275(30):22719–22727. doi: 10.1074/jbc.M001002200. [DOI] [PubMed] [Google Scholar]
  35. Sapkota Gopal P., Boudeau Jérôme, Deak Maria, Kieloch Agnieszka, Morrice Nick, Alessi Dario R. Identification and characterization of four novel phosphorylation sites (Ser31, Ser325, Thr336 and Thr366) on LKB1/STK11, the protein kinase mutated in Peutz-Jeghers cancer syndrome. Biochem J. 2002 Mar 1;362(Pt 2):481–490. doi: 10.1042/0264-6021:3620481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Shin E. K., Perryman L. E., Meek K. A kinase-negative mutation of DNA-PK(CS) in equine SCID results in defective coding and signal joint formation. J Immunol. 1997 Apr 15;158(8):3565–3569. [PubMed] [Google Scholar]
  37. Smith G. C., Jackson S. P. The DNA-dependent protein kinase. Genes Dev. 1999 Apr 15;13(8):916–934. doi: 10.1101/gad.13.8.916. [DOI] [PubMed] [Google Scholar]
  38. Song Q., Lees-Miller S. P., Kumar S., Zhang Z., Chan D. W., Smith G. C., Jackson S. P., Alnemri E. S., Litwack G., Khanna K. K. DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis. EMBO J. 1996 Jul 1;15(13):3238–3246. [PMC free article] [PubMed] [Google Scholar]
  39. Sutherland B. M., Bennett P. V., Sutherland J. C. Double strand breaks induced by low doses of gamma rays or heavy ions: quantitation in nonradioactive human DNA. Anal Biochem. 1996 Jul 15;239(1):53–60. doi: 10.1006/abio.1996.0290. [DOI] [PubMed] [Google Scholar]
  40. Walker J. R., Corpina R. A., Goldberg J. Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature. 2001 Aug 9;412(6847):607–614. doi: 10.1038/35088000. [DOI] [PubMed] [Google Scholar]
  41. Woodgett J. R. A kinase with Ku-dos. Curr Biol. 1993 Jul 1;3(7):449–450. doi: 10.1016/0960-9822(93)90353-p. [DOI] [PubMed] [Google Scholar]
  42. Ye R., Bodero A., Zhou B. B., Khanna K. K., Lavin M. F., Lees-Miller S. P. The plant isoflavenoid genistein activates p53 and Chk2 in an ATM-dependent manner. J Biol Chem. 2000 Nov 28;276(7):4828–4833. doi: 10.1074/jbc.M004894200. [DOI] [PubMed] [Google Scholar]
  43. van Gent D. C., Hoeijmakers J. H., Kanaar R. Chromosomal stability and the DNA double-stranded break connection. Nat Rev Genet. 2001 Mar;2(3):196–206. doi: 10.1038/35056049. [DOI] [PubMed] [Google Scholar]

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