This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Soubeyrand, S. Articles by Haché, R. J. G. Search for Related Content PubMed PubMed Citation Articles by Soubeyrand, S. Articles by Haché, R. J. G.

Threonines 2638/2647 in DNA-PK Are Essential for Cellular Resistance to Ionizing Radiation¹

Departments of Medicine [S. S., L. P., B. P., R. J. G. H.] and Biochemistry, Microbiology, and Immunology [R. J. G. H.], University of Ottawa, the Ottawa Health Research Institute, Ottawa, Ontario, K1Y 4E9 Canada

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
DNA-dependent protein kinase (DNA-PK) is required for the repair of double-strandedDNA breaks through the nonhomologous DNA end joining pathway. DNA-PK activity is required for DNA repair, but kinase activity also appears to be attenuated through an autoregulatory feedback loop. We show that autophosphorylation of DNA-PK catalytic subunit occurs in trans at least three sites NH₂ terminal to the catalytic domain and that two sites, threonine 2638 and 2647, determine DNA-PK autophosphorylation in vitro. Thr2638/2647ala substitution in DNA-PK catalytic subunit compromised cellular resistance to ionizing radiation without affecting DNA end joining, suggesting a requirement for DNA-PK inactivation for cell survival at a step after the rejoining of double-stranded DNA breaks.

	Introduction

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Cellular survival in response to the introduction of double-stranded DNA breaks is largely dependent on DNA repair through the NHEJ³ (1) . DNA-PK, comprised of DNA-PK_cs and the Ku antigen (Ku70/Ku80) regulatory subunit, is an essential component of the NHEJ pathway (2) . The requirement for DNA-PK_cs is enzymatic rather than structural as kinase-dead DNA-PK_cs mutants are compromised for NHEJ (3) . However, it also appears that DNA-PK activity is regulated through an autoregulatory feedback mechanism involving autophosphorylation of DNA-PK_cs (4) . In the present study, we have identified at least three key sites for DNA-PK_cs autophosphorylation. Our results show that alanine substitution at two of these sites results in a fully active DNA-PK that is unable to rescue the survival of DNA-PK_cs-deficient cells after treatment with ionizing radiation while fully complementing NHEJ. These results identify a specific requirement for DNA-PK inactivation for cellular recovery from ionizing radiation at a step after DNA end joining.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Reagents and Substrates.
Purified DNA-PK_cs and p53 peptide were obtained from Promega (Madison, WI). GST-DNA-PK_cs fusion proteins were purified to >90% homogeneity, and the DNA-PK_cs peptides were liberated from the GST using Precision protease before assembly of kinasing reactions.

Cloning and Expression of Recombinant DNA-PK_cs.
DNA-PK_cs fragments for bacterial expression were amplified by PCR. Site-directed mutations in DNA-PK_cs were prepared using QuikChange (Stratagene, La Jolla, CA). For expression in mammalian cells, the complete coding sequence DNA-PK_cs was inserted into a modified pCI-neo (Promega) with an NH₂-terminal Flag tag. Site-directed mutagenesis within full-length DNA-PK_cs was accomplished by shuttling of a BstEII fragment encompassing the substitution sites. Construct accuracy was confirmed by DNA sequencing as needed.

Phosphorylation Assays.
For purified DNA-PK, assays were performed as described previously with 10 ng of DNA of ssM13 DNA (ssM13) or HindIII-lpBlue and 0.04 or 50 µM [-³²P]ATP (3000 Ci/mmol) as indicated. Phosphorylation products were resolved by SDS-PAGE and quantified by phosphorimager (Typhoon; Molecular Dynamics). For stably transfected Sf7 SCID cell lines and C.B-17 cells, analysis of p53 peptide phosphorylation by DNA-PK was performed using the double-stranded DNA pull-down procedure of Achari and Lees-Miller (5) . All results shown are representative of a minimum of three independent trials with equivalent results.

Mapping of DNA-PK Autophosphorylation.
³²P-labeled full-length DNA-PK_cs or polypeptide fragments were excised from SDS-PAGE gels and digested at 37°C in situ in 400 µl of buffer D [50 mM NH₄HCO₃ (pH 8.0)] containing 0.050 µg/µl N-tosyl-L-phenylalanine chloromethyl ketone-treated trypsin (Worthington Biochemical Corp., Freehold, NJ), for 19 h, with trypsin added at 0 and 16 h. Tryptic phosphopeptides were resolved by 40% alkaline PAGE as described previously (6) . Secondary digestion with endoproteinase V8 (Glu-C; 25 ng/µl) or endoproteinase Asp-N (Asp-N; 5 ng/µl) was performed on tryptic peptide fragments before secondary alkaline PAGE. Phosphorylation was quantified by phosphorimager. All experiments were repeated two to five times with equivalent results. Phosphopeptide sequencing was performed by manual Edman degradation essentially as described previously (6) . Peptide coupling efficiencies to the support matrix were typically 50–70%.

Stable Expression of WT and Mutant DNA-PK.
Sf7 cells and the parental Sf7 cell line C.B-17 were propagated in DMEM with 10% FCS. After transfections of SfiI-linearized DNA-PK_cs pCI-neoF constructs or empty plasmid (pCI-neoF) using Fugene 6 (Roche Diagnostics), colonies were selected in 300 µg/ml G418. DNA-PK expression was assessed by Western analysis.

Analysis of Radioresistance and DNA Repair.
Colony formation assays were performed on 1000 cells irradiated at 2 or 4 Gy by a ¹³⁷Cs source. Seven days later, colonies that contained >50 cells were scored visually after methanol fixation and trypan blue staining (0.02% in methanol). To monitor in vivo DNA repair, cells exposed to 40 Gy X-rays at 4°C were allowed to recover for 0–4-h postirradiation at 37°C. Subsequent to agarose embedding, pulse field electrophoresis was performed at 10°C in 40 mM Tris acetate and 1 mM EDTA for 24 h at an included angle of 96° (CHEF-DR III), and DNA fragmentation was quantified by analysis of the ethidium bromide-stained gel (Typhoon).

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
DNA-PK is a ser/thr kinase with relaxed specificity, displaying a preference for SQ and TQ motifs (7) . Indeed, substrate recognition may be primarily determined by substrate colocalization through DNA tethering and protein–protein interactions with Ku rather than by specific protein sequence (6, 7, 8, 9) . Recently, we showed that structured single-stranded DNA is a cofactor for DNA-PK that preferentially promotes DNA-PK_cs autophosphorylation and autoinactivation (8) . To begin to assess the nature of DNA-PK_cs autophosphorylation that leads to autoinactivation, we performed a tryptic peptide analysis of the autophosphorylation of DNA-PK_cs (Fig. 1A) . In this analysis, we reasoned that limiting kinase activity might be most effective in distinguishing key regulatory modifications from secondary phosphorylation events of a broad specificity kinase, while recognizing the importance of visualizing the same events under the saturating ATP conditions likely to be prevalent in vivo.

View larger version (63K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Autophosphorylation of DNA-PKcs in vitro is localized to four tryptic peptides between aa 2596 and 3075. A, tryptic phosphopeptide analysis of DNA-PKcs autophosphorylation in the presence of ssM13 and lpBlue DNA and 40 nM or 50 µM 32P ATP. Approximately 1000 cpm of each sample was resolved by alkaline PAGE. Total DNA-PKcs phosphate incorporation (pmol) is indicated above each lane. The four major tryptic peptides are identified I, II, III, and IV based on relative mobility. In B, trans-autophosphorylation of DNA-PKcs is localized to a peptide containing aa 2596–3075. The schematic at the top outlines the eight GST-DNA-PKcs fusion peptides expressed. The SDS-PAGE gel below displays 32P incorporation into the recombinant DNA-PKcs fragments on phosphorylation by DNA-PK in the presence of lpBlue DNA, whereas the bottom panel is a Coomassie blue-stained gel of the protein preparations used for kinasing. C, alkaline PAGE resolution of tryptic phosphopeptides within DNA-PKcs protein fragment g. Left, phosphorylation of fragment g; right, phosphorylation of DNA-PKcs in the presence of lpBlue and 40 nM ATP. D, secondary proteolytic mapping of tryptic peptides I, II, and IV from peptide g with endoproteinase Asp-N (Asp-N) or Glu-C (Glu-C) as indicated. Control samples were mock digested.

To begin mapping the DNA-PK_cs autophosphorylation sites (Fig. 1B) , we compared the phosphorylation of eight GST-DNA-PK_cs fusion proteins covering 90% of full-length DNA-PK_cs (production of a peptide containing aa 3075–3484 was resistant to all cloning efforts). Only one DNA-PK_cs fragment g (aa 2596–3075) was phosphorylated. Thus, DNA-PK exhibited the potential for autophosphorylation in trans within a region that is NH₂ terminal to the catalytic domain and COOH terminal to the site of interaction with Ku antigen. Trypsin digestion of fragment g yielded four phosphopeptides with mobility equivalent to the mobilities of peptides I-IV in full-length DNA-PK_cs (Fig. 1C) , strongly suggesting that g contained the major DNA-PK_cs autophosphorylation sites.

Secondary cleavage of tryptic peptides I, II, and IV with glu-C and asp-N narrowed the search for the autophosphorylation sites by demonstrating the presence of glu in peptide IV and asp in peptides I and II (Fig. 1D) . Furthermore, the closely matching mobilities of the asp-N fragments of peptides I and II suggested that these two peptides may have arisen from a common precursor that was differentially or incompletely cleaved by trypsin.

Manual Edman degradation showing the release of ³²P from peptide IV at position 14 identified Ser₂₆₁₂, which occurs within an SQ motif, as one phosphorylation site within DNA-PK_cs fragment g (Fig. 2, A and B) . Repeated analysis of peptide IV suggested the possibility of additional phosphorylation at position 11 in some assays (data not shown), which would correspond to the secondary phosphate release point from peptide IV at thr₂₆₀₉, which occurs within a TQ motif.

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Identification of the DNA-PKcs autophosphorylation sites. A, 32P release from peptide g tryptic peptides I, II, and IV by Edman degradation. Peptide g was phosphorylated in the presence of ssM13 DNA and 40 nM ATP. 32P release is expressed as a percentage of radioactivity on the sequencing disk. B, sequence of DNA-PKcs from 2599 to 2552 highlighting the positions of phosphorylation (boldfacing) and showing the position of predicted trypsin (closed arrowhead), Glu-C (open arrowhead), and Asp-N (arrow) cleavage. In C, phosphopeptides I and II occur between 2630 and 2800. Alkaline PAGE analysis of tryptic phosphopeptides generated on phosphorylation of DNA-PKcs fragment g' (aa 2630–2800, Lanes 2 and 3) compared with similar analysis of peptide g (Lane 1). In D and E, threonine 2638 and 2647 determine phosphorylation of protein fragment g by DNA-PK. D, 32P labeling of equimolar amounts of WT protein fragment g (Lane 1), gT2638/2647A (Lane 2), and gT2609A/S2612A (Lane 3) by DNA-PK in the presence of lpBlue and 50 µM ATP. Quantification of 32P-incorporation (pmol) is summarized at the top of the gel. E, trypsin cleavage analysis of protein fragments g from D.

To confirm the positions of phosphorylation suggested by our analysis, we prepared variants of DNA-PK_cs fragment g with ala substitutions at T2609/S2612 and T2638/2647, respectively (Fig. 2D) . Substitutions T2609A/S2612A reduced total phosphate incorporation into DNA-PK_cs fragment g at 50 µM ATP by 45%, providing strong evidence for significant phosphorylation at these sites. More strikingly, substitutions T2638/2647A reduced total phosphate incorporation into protein fragment g by 85%. Thus, in addition to a strong indication that T2638/2647 are major DNA-PK phosphorylation sites in DNA-PK_cs, our data indicate that phosphorylation at these sites directly facilitates phosphorylation at T2609/S2612.

Tryptic phosphopeptide analysis (Fig. 2E) confirmed that substitutions T2638/2647A strongly reduced ³²P-incorportation into peptides III and IV in addition to eliminating phosphate incorporation into peptides I and II. By contrast, substitutions T2609A/S2612A reduced but did not eliminate ³²P-incorporation into peptide IV. This latter result is consistent with the presence of three additional ser/thr residues within peptide IV that would have the potential to serve as alternative DNA-PK phosphorylation sites on ala substitution of the primary sites.

Significantly, a report in press appears to confirm that Thr_{2609/2638/2647} and Ser₂₆₁₂ of DNA-PK_cs are phosphorylated on DNA-PK_cs in vivo, because DNA-PK_cs was recognized by specific phosphopeptide antibodies to these sites in human cells treated with the protein phosphatase inhibitor okadaic acid to increase total cellular phosphoprotein levels (10) . Furthermore, it is noteworthy that these four potential DNA-PK autophosphorylation sites are conserved in all six of the vertebrate DNA-PK_css sequenced to date.

To assess the potential role of Thr_{2609/2638/2647} and Ser₂₆₁₂ in DNA-PK function in vivo, we reintroduced WT DNA-PK_cs and DNA-PK_cs containing ala substitutions at positions 2638/2647 or 2609/2612/2638/2647 into Sf7 cells. Sf7 is a murine SCID cell line compromised for DNA-PK_cs, into which the reintroduction of WT human DNA-PK_cs has been shown previously to rescue cellular resistance to DNA damage induced by ionizing radiation (11 , 12) . Several clonal cell lines were obtained that express WT (Sf7-PKWt) and DNA-PK_{csT2638/2647A} (Sf7-PK38/47) at similar levels and impart DNA-PK activity in cellular extracts that is comparable with the level of DNA-PK activity in the Sf7 parental cell line C.B-17. The results with two of these lines are shown in Fig. 3, A and B . Interestingly, the total level of DNA-PK activity in these cells underwent little change over a 5-h period in response to exposure to 5 Gy of ionizing radiation (data not shown).

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Substitutions T2638A/T2647A abrogate the ability of DNA-PKcs to complement radiosensitivity of murine SCID cells. A, Western analysis of the expression of WT DNA-PKcs (Sf7-PKWt) and DNA-PKT2638/2647A (Sf7-PK38/47) in stably transfected, clonally selected murine Sf7 SCID fibroblasts. Expression levels are shown relative to DNA-PKcs levels in the parental C.B-17 cell line and purified DNA-PKcs and represent the signals from 20 µg of whole cell extract. In B, mutant DNA-PKcss are kinetically active. Phosphorylation of p53 peptide by DNA-PK partially purified from whole cell extracts by DNA-agarose precipitation, in the presence of lpBlue and 50 µM ATP. In C, DNA-PKcs T2638/2647A-expressing cells are radiosensitive. Cells (1000) from the indicated lines were irradiated with -radiation as indicated. Cell survival was determined by recording colony growth 7 days after irradiation and expressed as a percentage of untreated cells. Results show the mean of duplicate determinations (+/- SD). D, normal DNA repair in DNA-PKcs T2638/2647A-expressing cells. DNA repair in irradiated cells was assessed by pulsed-field gel electrophoresis. Data are expressed as the percentage of breaks remaining 4-h postirradiation. The error bars indicate the SD of three independent experiments. For the difference between Sf7 and C.B-17, Sf7-PKWt and Sf7-PK38/47 P < 0.05 in each instance, whereas the difference between C.B-17, Sf7-PKWt, and Sf7-PK38/47 is not statistically significant.

To directly investigate the importance of T2638/2647 for DNA repair, we monitored the religation of double-stranded DNA breaks in response to ionizing radiation using pulse field electrophoresis in a fraction of activity released assay (Ref. 13 ; Fig. 3D ). Four h after exposure to 40 Gy of ionizing radiation, >30% of the initial DNA damage remained in DNA prepared from DNA-PK_cs-deficient Sf7 cells. By contrast, repair in WT C.B-17 and Sf7 cells expressing WT human DNA-PK_cs was largely complete, with only 5–10% of DNA breaks remaining. Unexpectedly, despite the compromised survival, the Sf7-PK38/47 cells expressing DNA-PK_{csT2638/2647A} exhibited the same DNA repair efficiency as the Sf7-PKWt cells expressing WT DNA-PK_cs and an efficiency comparable with the C.B-17 cells. The repair efficiency of Sf7PK38/47 cells also closely paralleled the efficiency of C.B-17 and Sf7-PKWt cells at earlier times after irradiation (data not shown).

Our results identify T2638/T2647 in DNA-PK_cs as being essential for radioresistance conferred by DNA-PK. However, consistent with previous reports that the kinase activity of DNA-PK is required for NHEJ, substitutions at T2638/2647 that alleviate autophosphorylation of DNA-PK_cs and leave what appears to be a fully active kinase remained competent for NHEJ. However, DNA-PK_cs activity was not sufficient to allow for cell proliferation after DNA repair, because cells expressing DNA-PK_cs containing these substitutions failed to recover from irradiation. The precise role of DNA-PK autophosphorylation that allows for cell survival after NHEJ awaits further investigation. However, as the consequences DNA-PK_cs autophosphorylation in vitro include inactivation of kinase activity and dissociation of DNA-PK_cs from Ku antigen and DNA (4) , it is possible that autophosphorylation of DNA-PK_cs is required for dissociation of DNA-PK_cs from the DNA after rejoining. This may remove a physical constraint to regenerating predamage chromatin structure directly or could act indirectly by allowing reversal of phosphorylation of factors modified by DNA-PK at the repair site.

T2638 and T2647 appear to be the key sites for autophosphorylation that are required for cell survival after exposure to ionizing radiation. Alanine substitution at these two sites reduced DNA-PK_cs autophosphorylation by 85% and failed completely to rescue Sf7 cells exposed to ionizing radiation. Interestingly, after initial submission of this work, a report appeared describing T2609 as a DNA-PK_cs autophophosphorylation site that was important for NHEJ (14) . T2609 phosphorylation was shown to occur specifically at sites of DNA damage. However, alanine substitution at this site allowed partial rescue of radiation resistance while severely down-regulating NHEJ. The reason for these differences in phenotype remain to be determined. However, we note that our data indicated that T2609/S2612 phosphorylation is largely dependent on previous phosphorylation of T2638/T2647. Thus, it may be that phosphorylation at T2638/2647 is the predominant determinant for cell survival subsequent to repair, whereas additional phosphorylation at T2609 is directly required for repair.

Lastly, the potential to disrupt NHEJ and DNA-PK_cs function through the prevention of DNA-PK phosphorylation at T2638/2647 offers a novel and potentially attractive therapeutic target to enhance the action of therapies that target tumor cells by introducing double-stranded DNA breaks that are repaired through NHEJ.

	ACKNOWLEDGMENTS

 
We thank K. Meek for providing the full-length WT DNA-PK expression vector used in this study and C. Schild-Poulter for critical discussions and commentary on this manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by a grant from the Canadian Institutes of Health Research (CIHR; R. J. G. H.). S. S. held a postdoctoral fellowship from CIHR, whereas R. J. G. H. is a CIHR investigator.

² To whom requests for reprints should be addressed, at Departments of Medicine and Biochemistry, Microbiology, and Immunology, University of Ottawa, the Ottawa Health Research Institute, 725 Parkdale Avenue, Ottawa, Ontario, K1Y 4E9 Canada. Phone: (613) 789-5555, extension 16283; Fax: (613) 761-5036; E-mail: rhache{at}ohri.ca

³ The abbreviations used are: NHEJ, nonhomologous DNA end joining pathway; WT, wild-type; DNA-PK, DNA-dependent protein kinase; DNA-PK_cs, DNA-dependent protein kinase catalytic subunit; aa, amino acid; SCID, severe combined immunodeficiency; lpBlue, linearized pbluescript; GST, glutathione S-transferase.

Received 9/ 6/02. Accepted 1/31/03.

	REFERENCES

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 

1. Pierce A. J., Stark J. M., Araujo F. D., Moynahan M. E., Berwick M., Jasin M. Double-strand breaks and tumorigenesis. Trends Cell Biol., 11: S52-S59, 2001.[Medline]
2. Smith G. C., Jackson S. P. The DNA-dependent protein kinase. Genes Dev., 13: 916-934, 1999.[Free Full Text]
3. 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., 19: 3877-3884, 1999.[Abstract/Free Full Text]
4. Chan D. W., Lees-Miller S. P. The DNA-dependent protein kinase is inactivated by autophosphorylation of the catalytic subunit. J. Biol. Chem., 271: 8936-8941, 1996.[Abstract/Free Full Text]
5. Achari Y., Lees-Miller S. P. Detection of DNA-dependent protein kinase in extracts from human and rodent cells. Methods Mol. Biol., 99: 85-97, 2000.[Medline]
6. Giffin W., Kwast-Welfeld J., Rodda D. J., Préfontaine G. G., Traykova-Andonova M., Zhang Y., Weigel N. L., Lefebvre Y. A., Haché R. J. Sequence-specific DNA binding and transcription factor phosphorylation by Ku Autoantigen/DNA-dependent protein kinase. Phosphorylation of Ser-527 of the rat glucocorticoid receptor. J. Biol. Chem., 272: 5647-5658, 1997.[Abstract/Free Full Text]
7. Chan D. W., Ye R., Veillette C. J., Lees-Miller S. P. DNA-dependent protein kinase phosphorylation sites in Ku 70/80 heterodimer. Biochemistry, 38: 1819-1828, 1999.[Medline]
8. Soubeyrand S., Torrance H., Giffin W., Gong W., Schild-Poulter C., Hache R. J. Activation and autoregulation of DNA-PK from structured single-stranded DNA and coding end hairpins. Proc. Natl. Acad. Sci. USA, 98: 9605-9610, 2001.[Abstract/Free Full Text]
9. Schild-Poulter C., Pope L., Giffin W., Kochan J. C., Ngsee J. K., Traykova-Andonova M., Hache R. J. G. The binding of Ku antigen to homeodomain proteins promotes their phosphorylation by DNA-dependent protein kinase. J. Biol. Chem., 276: 16848-16856, 2001.[Abstract/Free Full Text]
10. Douglas P., Sapkota G. P., Morrice N., Yu Y., Goodarzi A. A., Merkle D., Meek K., Alessi D. R., Lees-Miller S. P. Identification of in vitro and in vivo phosphorylation sites in the catalytic subunit of the DNA-dependent protein kinase. Biochem. J., 368: 243-251, 2002.[Medline]
11. Kirchgessner C. U., Patil C. K., Evans J. W., Cuomo C. A., Fried L. M., Carter T., Oettinger M. A., Brown J. M. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science (Wash. DC), 267: 1178-1183, 1995.[Abstract/Free Full Text]
12. 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., 28: 2752-2761, 2000.[Abstract/Free Full Text]
13. Kultz D., Chakravarty D. Hyperosmolality in the form of elevated NaCl but not urea causes DNA damage in murine kidney cells. Proc. Natl. Acad. Sci. USA, 98: 1999-2004, 2001.[Abstract/Free Full Text]
14. Chan D. W., Chen B. P., Prithivirajsingh S., Kurimasa A., Story M. D., Qin J., Chen D. J. Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks. Genes Dev., 16: 2333-2338, 2002.[Abstract/Free Full Text]

This article has been cited by other articles:

				E. S. Williams, R. Klingler, B. Ponnaiya, T. Hardt, E. Schrock, S. P. Lees-Miller, K. Meek, R. L. Ullrich, and S. M. Bailey Telomere Dysfunction and DNA-PKcs Deficiency: Characterization and Consequence Cancer Res., March 1, 2009; 69(5): 2100 - 2107. [Abstract] [Full Text] [PDF]

				M. Toulany, R. Kehlbach, U. Florczak, A. Sak, S. Wang, J. Chen, M. Lobrich, and H. P. Rodemann Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair Mol. Cancer Ther., July 1, 2008; 7(7): 1772 - 1781. [Abstract] [Full Text] [PDF]

				K. Meek, P. Douglas, X. Cui, Q. Ding, and S. P. Lees-Miller trans Autophosphorylation at DNA-Dependent Protein Kinase's Two Major Autophosphorylation Site Clusters Facilitates End Processing but Not End Joining Mol. Cell. Biol., May 15, 2007; 27(10): 3881 - 3890. [Abstract] [Full Text] [PDF]

				N. Uematsu, E. Weterings, K.-i. Yano, K. Morotomi-Yano, B. Jakob, G. Taucher-Scholz, P.-O. Mari, D. C. van Gent, B. P.C. Chen, and D. J. Chen Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks J. Cell Biol., April 23, 2007; 177(2): 219 - 229. [Abstract] [Full Text] [PDF]

				M. Jeyakumar, X.-f. Liu, H. Erdjument-Bromage, P. Tempst, and M. K. Bagchi Phosphorylation of Thyroid Hormone Receptor-associated Nuclear Receptor Corepressor Holocomplex by the DNA-dependent Protein Kinase Enhances Its Histone Deacetylase Activity J. Biol. Chem., March 30, 2007; 282(13): 9312 - 9322. [Abstract] [Full Text] [PDF]

				B. P. C. Chen, N. Uematsu, J. Kobayashi, Y. Lerenthal, A. Krempler, H. Yajima, M. Lobrich, Y. Shiloh, and D. J. Chen Ataxia Telangiectasia Mutated (ATM) Is Essential for DNA-PKcs Phosphorylations at the Thr-2609 Cluster upon DNA Double Strand Break J. Biol. Chem., March 2, 2007; 282(9): 6582 - 6587. [Abstract] [Full Text] [PDF]

				P. Douglas, X. Cui, W. D. Block, Y. Yu, S. Gupta, Q. Ding, R. Ye, N. Morrice, S. P. Lees-Miller, and K. Meek The DNA-Dependent Protein Kinase Catalytic Subunit Is Phosphorylated In Vivo on Threonine 3950, a Highly Conserved Amino Acid in the Protein Kinase Domain Mol. Cell. Biol., March 1, 2007; 27(5): 1581 - 1591. [Abstract] [Full Text] [PDF]

				H. Yajima, K.-J. Lee, and B. P. C. Chen ATR-Dependent Phosphorylation of DNA-Dependent Protein Kinase Catalytic Subunit in Response to UV-Induced Replication Stress Mol. Cell. Biol., October 15, 2006; 26(20): 7520 - 7528. [Abstract] [Full Text] [PDF]

				X. Jiang, Y. Sun, S. Chen, K. Roy, and B. D. Price The FATC Domains of PIKK Proteins Are Functionally Equivalent and Participate in the Tip60-dependent Activation of DNA-PKcs and ATM J. Biol. Chem., June 9, 2006; 281(23): 15741 - 15746. [Abstract] [Full Text] [PDF]

				X. Cui, Y. Yu, S. Gupta, Y.-M. Cho, S. P. Lees-Miller, and K. Meek Autophosphorylation of DNA-Dependent Protein Kinase Regulates DNA End Processing and May Also Alter Double-Strand Break Repair Pathway Choice Mol. Cell. Biol., December 15, 2005; 25(24): 10842 - 10852. [Abstract] [Full Text] [PDF]

				S. Gupta and K. Meek The leucine rich region of DNA-PKcs contributes to its innate DNA affinity Nucleic Acids Res., December 9, 2005; 33(22): 6972 - 6981. [Abstract] [Full Text] [PDF]

				K. Dittmann, C. Mayer, B. Fehrenbacher, M. Schaller, U. Raju, L. Milas, D. J. Chen, R. Kehlbach, and H. P. Rodemann Radiation-induced Epidermal Growth Factor Receptor Nuclear Import Is Linked to Activation of DNA-dependent Protein Kinase J. Biol. Chem., September 2, 2005; 280(35): 31182 - 31189. [Abstract] [Full Text] [PDF]

				R. Dip and H. Naegeli More than just strand breaks: the recognition of structural DNA discontinuities by DNA-dependent protein kinase catalytic subunit FASEB J, May 1, 2005; 19(7): 704 - 715. [Abstract] [Full Text] [PDF]

				B. P. C. Chen, D. W. Chan, J. Kobayashi, S. Burma, A. Asaithamby, K. Morotomi-Yano, E. Botvinick, J. Qin, and D. J. Chen Cell Cycle Dependence of DNA-dependent Protein Kinase Phosphorylation in Response to DNA Double Strand Breaks J. Biol. Chem., April 15, 2005; 280(15): 14709 - 14715. [Abstract] [Full Text] [PDF]

				J. Drouet, C. Delteil, J. Lefrancois, P. Concannon, B. Salles, and P. Calsou DNA-dependent Protein Kinase and XRCC4-DNA Ligase IV Mobilization in the Cell in Response to DNA Double Strand Breaks J. Biol. Chem., February 25, 2005; 280(8): 7060 - 7069. [Abstract] [Full Text] [PDF]

				Z. Lou, B. P.-C. Chen, A. Asaithamby, K. Minter-Dykhouse, D. J. Chen, and J. Chen MDC1 Regulates DNA-PK Autophosphorylation in Response to DNA Damage J. Biol. Chem., November 5, 2004; 279(45): 46359 - 46362. [Abstract] [Full Text] [PDF]

				J. Feng, J. Park, P. Cron, D. Hess, and B. A. Hemmings Identification of a PKB/Akt Hydrophobic Motif Ser-473 Kinase as DNA-dependent Protein Kinase J. Biol. Chem., September 24, 2004; 279(39): 41189 - 41196. [Abstract] [Full Text] [PDF]

				Y. V. R. Reddy, Q. Ding, S. P. Lees-Miller, K. Meek, and D. A. Ramsden Non-homologous End Joining Requires That the DNA-PK Complex Undergo an Autophosphorylation-dependent Rearrangement at DNA Ends J. Biol. Chem., September 17, 2004; 279(38): 39408 - 39413. [Abstract] [Full Text] [PDF]

				W. D. Block, Y. Yu, D. Merkle, J. L. Gifford, Q. Ding, K. Meek, and S. P. Lees-Miller Autophosphorylation-dependent remodeling of the DNA-dependent protein kinase catalytic subunit regulates ligation of DNA ends Nucleic Acids Res., August 16, 2004; 32(14): 4351 - 4357. [Abstract] [Full Text] [PDF]

				E. Weterings, N. S. Verkaik, H. T. Bruggenwirth, J. H. J. Hoeijmakers, and D. C. van Gent The role of DNA dependent protein kinase in synapsis of DNA ends Nucleic Acids Res., December 15, 2003; 31(24): 7238 - 7246. [Abstract] [Full Text] [PDF]

				C. Schild-Poulter, A. Shih, N. C. Yarymowich, and R. J. G. Hache Down-Regulation of Histone H2B by DNA-Dependent Protein Kinase in Response to DNA Damage through Modulation of Octamer Transcription Factor 1 Cancer Res., November 1, 2003; 63(21): 7197 - 7205. [Abstract] [Full Text] [PDF]

				Q. Ding, Y. V. R. Reddy, W. Wang, T. Woods, P. Douglas, D. A. Ramsden, S. P. Lees-Miller, and K. Meek Autophosphorylation of the Catalytic Subunit of the DNA-Dependent Protein Kinase Is Required for Efficient End Processing during DNA Double-Strand Break Repair Mol. Cell. Biol., August 15, 2003; 23(16): 5836 - 5848. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Soubeyrand, S. Articles by Haché, R. J. G. Search for Related Content PubMed PubMed Citation Articles by Soubeyrand, S. Articles by Haché, R. J. G.