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Competitive and Noncompetitive Inhibition of the DNA-dependent Protein Kinase¹

Wellcome/CRC Institute, Cambridge, CB2 1QR; and Department of Zoology, University of Cambridge, Cambridge, CB2 3EJ, United Kingdom

	ABSTRACT

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The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase that is involved in mammalian DNA double-strand break repair. The catalytic subunit of DNA-PK (DNA-PKcs) shares sequence homology in its kinase domain with phosphatidylinositol (PI) 3-kinase. Here, we provide a detailed kinetic analysis of DNA-PK inhibition by the PI 3-kinase inhibitor wortmannin and demonstrate this inhibition to be of a noncompetitive nature, with a K_i of 120 nM. Another inhibitor of PI 3-kinase, LY294002, its parent compound, quercetin, and other derivatives have also been studied. These chemicals are competitive inhibitors of DNA-PK, with LY294002 having a K_i of 6.0 µM. Using an antibody to wortmannin, we found that this compound binds covalently to the kinase domain of DNA-PKcs both in vitro and in vivo. Binding of wortmannin to the active site of DNA-PKcs is inhibited by ATP but not by a peptide substrate. Furthermore, wortmannin is able to bind to DNA-PKcs independently of Ku, and it is not stimulated by the presence of DNA. This suggests that the ATP binding site of DNA-PKcs is open constitutively and that DNA activation of the kinase is mediated via another mechanism.

	INTRODUCTION

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DNA-PK³ is a serine/threonine protein kinase that is relatively abundant in human cell nuclei (1 , 2) . A notable feature of this enzyme is that it is activated on binding to DNA lesions, such as double-strand breaks, nicks, and DNA single single-to-double-strand transitions (3) . DNA-PK is composed of a large (M_r 470,000) catalytic subunit, DNA-PKcs, and a DNA targeting component, termed Ku (4, 5, 6) . Ku, first identified as a human autoimmune antigen, is a heterodimer composed of polypeptides of M_r 70,000 and 80,000 (Ku70 and Ku80, respectively). Both components of DNA-PK function in the repair of DNA damage induced by IR and, hence, show a reliance on each other for generating a functional holoenzyme in vivo (7, 8, 9) . Cells defective in Ku components (such as xrs-6 and lines derived from Ku70^-/- and Ku80^-/- mice) are not only sensitive to DNA double-strand break-inducing agents but are also defective in the site-specific nonhomologous end-joining process of V(D)J recombination (7, 8, 9) . The severe combined immunodeficient mouse possesses a truncated DNA-PKcs polypeptide that appears to lack kinase activity, and this also results in defective V(D)J recombination and an inability to effectively repair IR-induced DNA double-strand breaks (10, 11, 12) . It has been shown recently that DNA-PKcs can bind to and be activated by DNA in a Ku-independent manner in vitro (13 , 14) . However, because Ku-deficient cells show similar phenotypes to DNA-PKcs-deficient cells, it is likely that Ku is required for DNA-PK function in physiological situations.

Cloning of the DNA-PKcs cDNA revealed that its open reading frame directs the synthesis of a protein of 4100 amino acid residues (6) . Other than a leucine zipper motif found between residues 1503 and 1538, which appears to be involved in interacting with the nuclear matrix protein C1D (15) , the only clearly identifiable functional motif in the DNA-PKcs polypeptide is the kinase domain comprising the 400 COOH-terminal amino acid residues. This region shows very little homology to classical serine/threonine kinases, but it shows much greater sequence similarity with the catalytic domains of proteins involved in the phosphorylation of PI, notably PI 3-kinases and PI 4-kinases (16) . Other polypeptides shown to contain this PI 3-kinase-like domain include several proteins implicated in DNA repair, genome surveillance, and cell cycle checkpoint controls: mammalian ATM, ATR, and FRAP, together with homologues of these in systems such as Saccharomyces cerevisiae and Schizosaccharomyces pombe (17) . To date, members of this latter group have not been shown unequivocally to possess an intrinsic ability to phosphorylate phosphatidlyinositols and are, therefore, most probably protein serine/threonine kinases (18) .

Wortmannin is a seterol-like fungal metabolite that has been shown previously to be a potent and selective inhibitor of mammalian PI 3-kinase (19 , 20) . On the basis of the similarity between the kinase domains of DNA-PKcs and PI 3-kinases, we and others have established that wortmannin is able to inhibit DNA-PK function in vitro (6 , 21, 22, 23) . However, the IC₅₀ (concentration at which 50% of activity is lost) for wortmannin toward DNA-PK is, at 250 nM, 2 orders of magnitude higher than that for PI 3-kinase. Nevertheless, this observation has led to the demonstration by other groups that cells grown in the presence of wortmannin are sensitized to the killing effects of IR (24, 25, 26, 27) . Therefore, in this context, wortmannin acts as a radiosensitizing agent. Although the above effects suggest that the radiosensitization is via DNA-PK inhibition, the possibility exists that wortmannin also impairs the function of other members of the PI 3-kinase family, such as ATM or ATR (17 , 18 , 23 , 28) . More recently, it has been shown that another PI 3-kinase inhibitor, LY294002, is also able to sensitize cells to the effects of IR (26) .

Because of the potential use of wortmannin, LY294002, and their derivatives as radiosensitizers, we have sought to provide a detailed analysis of their inhibition of DNA-PK. Here, we provide a kinetic analysis of the PI 3-kinase inhibitors wortmannin, quercetin, quercitrin, rutin, and LY294002 toward DNA-PK. Furthermore, we have used wortmannin as a probe for DNA-PK function in vitro. Our data not only provide a detailed analysis of DNA-PK inhibitors in vitro but also serve to broaden the known effects of wortmannin and quercetin derivatives. We are also able to show here that wortmannin can bind directly to DNA-PKcs in vivo. Our data provide a starting point for the development of more specific inhibitors of PI 3-kinase family members and potentially novel radiosensitizers.

	MATERIALS AND METHODS

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Cells.
The human lymphoblastoid cell line HL60 was obtained from American Type Culture Collection (ATCC CCL-24; Manassas, VA) and maintained in RPMI 1640 supplemented with 10% FCS, glutamine, and antibiotics. The human glioma cell lines MO59J and MO59K (29) , kindly provided by J. Allalunis-Turner (Cross Cancer Institute, Edmonton, Alberta, Canada) were grown in DMEM: Ham’s F-12 medium, supplemented with 10% FCS, glutamine, and antibiotics.

Chemicals.
Wortmannin, quercetin, and LY294002 were obtained from Alexis Corporation (UK) Ltd. (Nottingham, United Kingdom). Rutin and quercitrin were purchased from Sigma (Poole, United Kingdom).

Inhibitor Studies.
DNA-PK assays were performed as described previously using a p53-based peptide substrate (30 , 31) . For inhibition experiments, wortmannin was added to the reaction mixture at the same time as ATP. For the other inhibitors, these were preincubated with DNA-PK for 5 min prior to ATP addition. All chemical inhibitors were dissolved in 10% DMSO, resulting in a final DMSO concentration of 1% in the assay mixture. DMSO (1%) was found not to adversely affect the DNA-PK assay.

DNA-PK/DNA-PKcs.
DNA-PK holoenzyme and DNA-PKcs were purified from HeLa nuclear extract (Computer Cell Culture Centre, Mons, Belgium) essentially as described previously (32) .

Wortmannin Labeling of DNA-PK.
Nuclear extract (10 µg), purified DNA-PK (600 ng), or DNA-PKcs (100 ng) was labeled with wortmannin (10 µM) at room temperature for 20 min in buffer containing 5 mM HEPES (pH 7.6), 40 mM KCl, 4% glycerol, 0.2 mM MgCl₂, and 1 mM DTT. Wortmannin-labeled proteins were identified by standard SDS-PAGE (7% polyacrylamide gels) and Western blotting analysis with the use of antiwortmannin antiserum kindly provided by M. Wymann (Fribourg, Switzerland; Ref. 33). In vivo labeling of cells was carried out by adding wortmannin to a final concentration of 10 or 100 µM to the growth medium for 90 min. Nuclear extract was prepared from harvested cells by the method of Dignam et al. (34) . Wortmannin-labeled proteins were identified as outlined above. For limited proteolysis of DNA-PKcs, 1 or 3.3 µl of recombinant caspase-3 (Pharmingen, San Diego, CA) was added to 600 ng of DNA-PK labeled with 10 µM wortmannin (as above) and incubated at 37°C for 30 min prior to Western blot analysis using anti-DNA-PKcs antiserum (AbFLA; Ref. 35) or antiwortmannin antiserum (33) .

	RESULTS

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Inhibition of DNA-PK by Wortmannin.
Because PI 3-kinase activity is inhibited potently by wortmannin and because DNA-PKcs shares homology across its kinase domain with PI 3-kinases, we tested previously whether wortmannin was able to inhibit DNA-PK activity (6) . This earlier study showed that wortmannin is able to inhibit DNA-PK activity, with an IC₅₀ of 250 nM. However, this study did not ascertain the mode of inhibition by wortmannin or provide an accurate K_i. Thus, we assayed DNA-PK activity at a variety of different ATP concentrations in the presence of increasing concentrations of wortmannin. A Lineweaver-Burk plot of this analysis revealed that wortmannin behaves as a noncompetitive inhibitor for the ATP binding site of DNA-PK (Fig. 1A) . From a Dixon plot of this analysis (reciprocal of the velocity of the reaction plotted against wortmannin concentration), a K_i of 120 nM was obtained (Fig. 1B) . Because the primary plot of these data could not fully discriminate whether the nature of wortmannin inhibition was noncompetitive or mixed noncompetitive/uncompetitive inhibition, Eadie-Scatchard analysis of the initial data set was performed (Fig. 1C ; Ref. 36 ). By plotting the velocity of reaction against velocity over substrate concentration, we show, from the slope of the graphs that are constructed, that the mode of wortmannin inhibition is essentially noncompetitive in nature (36) .

View larger version (17K): [in this window] [in a new window]   Fig. 1. Characterization of the inhibition of DNA-PK activity by wortmannin. A, Lineweaver-Burk plot of the inhibition of DNA-PK activity with wortmannin. Inhibition was performed with 0 µM (), 0.01 µM (), 0.1 µM (•), 0.5 µM (), 1 µM (), or 5 µM () wortmannin in varying concentrations of ATP. B, Dixon plot determination of the Ki for wortmannin. Results from A were plotted as wortmannin concentration against 1/V for ATP concentrations of 1 µM (), 2.5 µM (), 5 µM (), 10 µM (•), 25 µM (), and 100 µM (). The intersection of the plots on the wortmannin axis reveals a Ki of 120 nM. C, Eadie-Scatchard plot of wortmannin inhibition of DNA-PK. Results from A were plotted as V against V/[ATP] for the wortmannin concentrations of 0.01 µM (), 0.1 µM (•), 0.5 µM (), 1 µM (), and 5 µM (). The slopes of the plots reveal that wortmannin inhibition of DNA-PK is noncompetitive.

View larger version (16K): [in this window] [in a new window]   Fig. 2. Characterization of the inhibition of DNA-PK activity by LY294002. A, Lineweaver-Burk plot of the inhibition of DNA-PK activity with LY294002. Inhibition was performed with 0 µM (), 0.01 µM (), 0.1 µM (), 1 µM (•), 10 µM (), or 100 µM () LY294002 in varying concentrations of ATP. B, Ki determination for LY294002. Slopes from the Lineweaver-Burk plots (A) were plotted against the concentration of LY294002. Intersection of the plot on the inhibitor concentration axis gives a Ki of 6 µM.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Labeling of the kinase domain of DNA-PK with wortmannin. A, DNA-PK (600 ng) was labeled with varying amounts of wortmannin, as indicated. Wortmannin-treated DNA-PK was electrophoresed on a 7% SDS-polyacrylamide gel followed by Western blotting using antiwortmannin antiserum. B, DNA-PK (600 ng) was labeled with wortmannin (10 µM) in the absence or presence of 0, 10, 1, or 0.1 mM ATP. Wortmannin-treated DNA-PK was visualized as in A. C, caspase-3 limited proteolysis of DNA-PKcs. DNA-PK was labeled with 10 µM wortmannin prior to digestion with caspase-3. Left, the characteristic degradation of DNA-PKcs as revealed by immunoblot analysis using a polyclonal antibody to DNA-PKcs (AbFLA). The NH2-terminal Mr 240,000 and COOH-terminal kinase domain containing Mr 120,000 and 150,000 fragments are indicated. Right, immunoblot analysis using the antiwortmannin antiserum shows wortmannin labeling of only the Mr 120,000 and 150,000 kinase domain containing fragments of DNA-PKcs produced by caspase-3-mediated proteolysis.

Binding of Wortmannin to DNA-PKcs Is Not Affected by the Absence of Ku or by the Presence of DNA or a Peptide Substrate.
Next, we used wortmannin and the specific antiserum to this compound to address a variety of questions regarding the status of the DNA-PKcs active site under various circumstances. Previous work has established that DNA-PKcs has an inherent capacity to bind to DNA termini and is activated by the simultaneous presence of the non-sequence-specific DNA-binding protein Ku and DNA ends. To address whether Ku activates DNA-PKcs via opening of the ATP-binding site of the kinase, we added wortmannin to a DNA-PKcs preparation that was essentially devoid of Ku. Importantly, this preparation of DNA-PKcs was inactive in our assays and was found to contain contaminating Ku at a molar ratio of less than 1 part Ku per 100 parts DNA-PKcs. Notably, although devoid of kinase activity toward a peptide substrate, the preparation of DNA-PKcs lacking Ku still effectively forms adducts with wortmannin that can be competed with ATP (Fig. 4A) . Furthermore, upon addition of purified Ku, no increase in wortmannin binding to DNA-PKcs was observed (Fig. 4B) . These results, therefore, indicate that the ATP binding site of DNA-PKcs is accessible, irrespective of the presence of Ku, and therefore, Ku is likely to control DNA-PKcs activity in some other manner, such as modulating its ability to mediate ATP hydrolysis or by controlling access to the protein substrate.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Wortmannin labeling of DNA-PKcs (in the absence of Ku) and the effect of DNA and a peptide substrate on wortmannin binding. A, DNA-PKcs (100 ng) was labeled with wortmannin (10 µM) in the absence or presence of 0, 10, 1, or 0.1 mM ATP. B, DNA-PKcs (100 ng) was labeled with wortmannin (10 µM) in the absence or presence of 0, 25, or 100 ng of Ku. C, DNA-PK (600 ng) was labeled with 10 µM wortmannin in the presence of 100, 10, or 1 ng of sonicated double-stranded calf thymus DNA. D, DNA-PK (600 ng) was labeled with 10 µM wortmannin in the presence of 1 mM, 100 µM, or 10 µM DNA-PK peptide substrate.

Binding of Wortmannin to DNA-PKcs in Vivo.
Recent data have shown that treatment of cells with wortmannin can cause marked sensitization to IR (24, 25, 26, 27) . However, it remains unclear as to whether this radiosensitization is mediated through inhibition of DNA-PK. To investigate whether DNA-PKcs is a target for wortmannin in vivo, we incubated HL60 cells in the presence of this molecule. After incubation with the drug for 90 min, nuclear extracts were prepared, and Western blotting was performed using the antiwortmannin antibody. Fig. 5A clearly shows that a major labeled band that comigrates with wortmannin-labeled DNA-PKcs, is present in wortmannin-treated HL60 nuclear extracts. A similar comigrating band is present in wortmannin-treated HeLa nuclear extract. To establish unequivocally that the wortmannin-labeled polypeptide in these studies is, indeed, DNA-PKcs, we treated the DNA-PKcs-negative cell line MO59J and its DNA-PKcs parental control line, MO59K (29) , with wortmannin. Nuclear extracts were then prepared from these cells and analyzed by electrophoresis followed by immunoblotting with the antiwortmannin antiserum. Notably, as shown in Fig. 5B , such studies reveal a band that comigrates with purified DNA-PKcs that is present in the DNA-PKcs positive line but absent in the DNA-PKcs null line. These data, therefore, reveal that DNA-PKcs is, indeed, a physiological target for wortmannin.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Wortmannin labeling of DNA-PKcs in vivo. A, HeLa nuclear extract alone (N.E.), nuclear extracts treated with 10 µM wortmannin (Wort), nuclear extracts prepared from HL60 cells grown in the presence of wortmannin (10 and 100 µM) and DNA-PK (600 ng) labeled with wortmannin (10 µM) were electrophoresed on a 7% SDS-polyacrylamide gel followed by Western blotting using antiwortmannin antiserum. The wortmannin-labeled band comigrating with DNA-PKcs is indicated. B, DNA-PK (600 ng) labeled with wortmannin (10 µM), nuclear extracts alone (N. E.), nuclear extracts treated with 10 µM wortmannin (Wort), nuclear extracts prepared from MO59J cells (DNA-PKcs negative) treated with 10 µM wortmannin and MO59K cells (DNA-PKcs positive) treated with 10 µM wortmannin were electrophoresed on a 7% SDS-polyacrylamide gel followed by Western blotting using antiwortmannin antiserum. The wortmannin-labeled band comigrating with DNA-PKcs is indicated.

	DISCUSSION

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In addition, we have investigated the mode of wortmannin action on DNA-PK in more detail. Thus, we have shown that, as is the case for PI 3-kinase, wortmannin forms covalent adducts with DNA-PKcs in the region of the molecule harboring its kinase domain and that the formation of such adducts is inhibited competitively by ATP. In light of previous work mapping the site of wortmannin binding to PI 3-kinase and the homology between this kinase and DNA-PKcs, it seems likely that wortmannin targets the lysine at amino acid residue 3751 of the DNA-PKcs polypeptide. Having established that wortmannin targets the ATP-binding site of DNA-PKcs, we then moved on to use it as a probe for the status of the DNA-PKcs active site under various circumstances. Previous studies had revealed that DNA-PKcs catalytic function is stimulated greatly by the simultaneous presence of the Ku heterodimer and DNA ends, raising the possibility that this activation mechanism is mediated via conformational changes in the DNA-PKcs catalytic domain to permit ATP binding. However, we found that wortmannin is able to access DNA-PKcs efficiently in the absence of Ku and/or DNA. Thus, although alternative explanations exist, it seems likely that DNA-PK activation does not reflect opening of the ATP-binding site and, instead, is via activating ATP hydrolytic potential and/or access to the peptide substrate. Finally, we used the fact that wortmannin forms covalent adducts with DNA-PKcs to demonstrate that DNA-PKcs is a major target for wortmannin in intact cells.

Previous analyses of the PI 3-kinase family have revealed that it can be roughly divided into two distinct subgroups on the basis of polypeptide size and through characteristic sequence homologies within the PI 3-kinase catalytic domain (17) . One of these subgroups comprises the PI 3-kinases and PI 4-kinases, most of which appear to be largely membrane associated or cytoplasmic enzymes involved in intracellular signalling or vesicular trafficking. The other subgroup comprises DNA-PKcs and a series of other large (M_r >250,000) proteins that are involved in cell cycle control, DNA damage signaling, and/or DNA repair. A noteworthy feature of our work is that it broadens the inhibitory profiles of wortmannin, LY294002, and their relatives to a key member of the second subgroup. This is important for several reasons. (a) It reveals that these compounds are not as specific to the PI 3-kinases and PI 4-kinases as previously thought, meaning that studies analyzing their in vivo effects should be interpreted with some caution. (b) It raises the prospect that these small molecule inhibitors also target other members of the DNA-PKcs subgroup of the PI 3-kinase family. In line with this prediction, it has been shown recently that the protein kinase function of two other members of this subgroup, ATM and the human cell cycle checkpoint protein ATR, are inhibited by wortmannin (23 , 28) . (c) It raises the exciting prospect that derivatives of wortmannin and LY294002 could serve as the basis for the development of more specific inhibitors of DNA-PKcs and its relatives. Given the involvement of DNA-PK and some of its close relatives in DNA repair processes and because it has already been shown that wortmannin and LY294002 radiosensitize mammalian cells in culture (24, 25, 26, 27) , an attractive potential application for such inhibitory drugs is the enhancement of the efficacy of cancer chemotherapy and radiotherapy.

	ACKNOWLEDGMENTS

 
We thank members of the Jackson laboratory for their help and encouragement. Thanks also to Matthias Wymann for providing the antiwortmannin antiserum.

	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 This work was funded by the Cancer Research Campaign.

² To whom requests for reprints should be addressed, at Wellcome/CRC Institute, Tennis Court Road, Cambridge CB2 1QR, United Kingdom. Phone: 44 01223 334102; Fax: 44 01223 334089; E-mail: spj13{at}mole.bio.cam.ac.uk

³ The abbreviations used are: DNA-PK, DNA-dependent protein kinase; DNA-PKcs, DNA-PK catalytic subunit; IR, ionizing radiation; PI, phosphatidylinositol.

Received 9/ 8/98. Accepted 4/ 2/99.

	REFERENCES

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Anderson C. W., Lees-Miller S. P. The nuclear serine/threonine protein kinase DNA-PK. Crit. Rev. Eukaryotic Gene Express., 2: 283-314, 1992.[Medline]
2. Jackson S. P. DNA-dependent protein kinase. Int. J. Biochem. Cell. Biol., 29: 935-938, 1997.[Medline]
3. Morozov V. E., Falzon M., Anderson C. W., Kuff E. L. DNA-dependent protein kinase is activated by nicks and larger single-stranded gaps. J. Biol. Chem., 269: 16684-16688, 1994.[Abstract/Free Full Text]
4. Gottlieb T. M., Jackson S. P. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell, 72: 131-142, 1993.[Medline]
5. Dvir A., Peterson S. R., Knuth M. W., Lu H., Dynan W. S. Ku autoantigen is the regulatory component of a template associated protein kinase that phosphorylates RNA polymerase-II. Proc. Natl. Acad. Sci. USA, 89: 11920-11924, 1992.[Abstract/Free Full Text]
6. Hartley K. O., Gell D., Smith G. C. M., 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, 82: 849-856, 1995.[Medline]
7. Jackson S. P. DNA damage detection by DNA dependent protein kinase and related enzymes. Cancer Surv., 28: 261-279, 1996.[Medline]
8. Jackson S. P., Jeggo P. A. DNA double-strand break repair and V(D)J recombination: involvement of DNA-PK. Trends Biochem. Sci., 20: 412-415, 1995.[Medline]
9. Lieber M. R., Grawunder U., Wu X. T., Yaneva M. Tying loose ends: roles of Ku and DNA-dependent protein kinase in the repair of double strand breaks. Curr. Opin. Genet. Dev., 7: 99-104, 1997.[Medline]
10. Araki R., Fujimori A., Hamatani K., Mita K., Saito T., Mori M., Fukumura R., Morimyo M., Muto M., Itoh M., Tatsumi K., Abe M. Nonsense mutation at Tyr-4046 in the DNA-dependent protein kinase catalytic subunit of severe combined immune deficiency mice. Proc. Natl. Acad. Sci. USA, 94: 2438-2443, 1997.[Abstract/Free Full Text]
11. Blunt T., Gell D., Fox M., Taccioli G. E., Lehmann A. R., Jackson S. P., Jeggo P. A. Identification of a nonsense mutation in the carboxyl region of DNA-dependent protein kinase catalytic subunit in the scid mouse. Proc. Natl. Acad. Sci. USA, 93: 10285-10290, 1996.[Abstract/Free Full Text]
12. Danska J. S., Holland D. P., Mariathasan S., Williams K. M., Guidos C. J. Biochemical and genetic defects in the DNA-dependent protein kinase in murine scid lymphocytes. Mol. Cell. Biol., 16: 5507-5517, 1996.[Abstract]
13. Yaneva M., Kowalewski T., Lieber M. R. Interaction of DNA-dependent protein kinase with DNA and with Ku: biochemical and atomic force microscopy studies. EMBO J., 16: 5098-5112, 1997.[Medline]
14. Hammarsten O., Chu G. DNA-dependent protein kinase: DNA binding and activation in the absence of Ku. Proc. Natl. Acad. Sci. USA, 95: 525-530, 1998.[Abstract/Free Full Text]
15. Yavuzer U., Smith G. C. M., Bliss T., Werner D., Jackson S. P. DNA end-independent activation of DNA-PK mediated via association with the DNA binding protein C1D. Genes Dev., 12: 2188-2199, 1998.[Abstract/Free Full Text]
16. Toker A., Cantley L. C. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature (Lond.), 387: 673-676, 1997.[Medline]
17. Zakian V. A. ATM-related genes: what do they tell us about functions of the human gene?. Cell, 82: 685-687, 1995.[Medline]
18. Hunter T. When is a lipid kinase not a lipid kinase? When it is a protein kinase. Cell, 83: 1-4, 1995.[Medline]
19. Ui M., Okada T., Hazeki K., Hazeki O. Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase. Trends Biochem. Sci., 20: 303-307, 1995.[Medline]
20. Powis G., Bonjouklian R., Berggren M. M., Gallegos A., Abraham R., Ashendal C., Zalkow L., Matter W. F., Dodge J., Grindley G., Vlahos C. J. Wortmannin, a potent and selective inhibitor of phosphatidylinositol 3-kinase. Cancer Res., 54: 2419-2423, 1994.[Abstract/Free Full Text]
21. Le Romancer M., Cosulich S. C., Jackson S. P., Clarke P. R. Cleavage and inactivation of DNA-dependent protein kinase catalytic subunit during apoptosis in Xenopus egg extracts. J. Cell. Sci., 109: 3121-3127, 1996.[Abstract]
22. Gu X. Y., Bennet R. A. O., Povrik L. F. End-joining of free radical-mediated DNA double-strand breaks in vitro is blocked by the kinase inhibitor wortmannin at a step preceding removal of damaged 3' termini. J. Biol. Chem., 271: 19660-19663, 1996.[Abstract/Free Full Text]
23. Sarkaria J. N., Tibbetts R. S., Busby E. C., Kennedy A. P., Hill D. E., Abraham R. T. Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res., 58: 4375-4382, 1998.[Abstract/Free Full Text]
24. Price B. D., Youmell M. B. The phosphatidylinositol 3-kinase inhibitor wortmannin sensitizes murine fibroblasts and human tumor cells to radiation and blocks induction of p53 following DNA damage. Cancer Res., 56: 246-250, 1995.[Abstract/Free Full Text]
25. Boulton S., Kyle S., Yalcintepe L., Durkacz B. W. Wortmannin is a potent inhibitor of DNA double strand break but not single strand break repair in Chinese hamster ovary cells. Carcinogenesis (Lond.), 17: 2285-2290, 1997.[Abstract/Free Full Text]
26. Rosenzweig K. E., Youmell M. B., Palayoor S. T., Price B. D. Radiosensitization of human tumor cells by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 correlates with inhibition of DNA-dependent protein kinase and prolonged G₂-M delay. Clin. Cancer. Res., 3: 1149-1156, 1997.[Abstract]
27. Hosoi Y., Miyachi H., Matsumoto Y., Ikehata H., Komura J., Ishii K., Zhao H. J., Yoshida M., Takai Y., Yamada S., Suzuki N., Ono T. A phosphatidylinositol 3-kinase inhibitor wortmannin induces radioresistant DNA synthesis and sensitizes cells to bleomycin and ionizing radiation. Int. J. Cancer, 78: 642-647, 1998.[Medline]
28. Banin S., Moyal L., Sheih S. Y., Taya Y., Anderson C. W., Chessa L., Smorodinsky N. I., Prives C., Reiss Y., Shiloh Y., Ziv Y. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science (Washington DC), 281: 1674-1677, 1998.[Abstract/Free Full Text]
29. Lees-Miller S. P., Godbout R., Chan D. W., Weinfeld M., Day R. S., Barron G. M., Allalunis-Turner J. Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line. Science (Washington DC), 267: 1183-1185, 1995.[Abstract/Free Full Text]
30. 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., 12: 5041-5049, 1992.[Abstract/Free Full Text]
31. Finnie N. J., Gottlieb T. M., Blunt T., Jeggo P. A., Jackson S. P. DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair. Proc. Natl. Acad. Sci. USA, 92: 320-324, 1995.[Abstract/Free Full Text]
32. Dvir A., Stein L. Y., Calore B. L., Dynan W. S. Purification and characterization of a template-associated protein kinase that phosphorylates RNA polymerase II. J. Biol. Chem., 268: 10440-10447, 1993.[Abstract/Free Full Text]
33. Wymann M. P., Bulgarelli-leva G., Zvelebil M. J., Pirola L., Vanhaesebroeck B., Waterfield M. D., Panayotou G. Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol. Cell. Biol., 16: 1722-1733, 1996.[Abstract]
34. 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., 5: 1475-1489, 1983.
35. Song Q., Lees-Miller S., Kumar S., Zhang N., Smith G. C. M., Jackson S. P., Alnemri E. S., Litwack G., Lavin M. F. DNA-dependent protein kinase catalytic subunit: a target for the ICE-like protease CPP32 in apoptosis. EMBO J., 15: 3238-3246, 1996.[Medline]
36. Ed. 2 Segal I. H. eds. . Biochemical Calculations, : 208-323, John Wiley & Sons New York 1975.
37. Vlahos C. J., Matter W. F., Hui K. Y., Brown R. F. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem., 269: 5241-5248, 1994.[Abstract/Free Full Text]
38. Matter W. F., Brown R. F., Vlahos C. J. The inhibition of phosphatidylinositol 3-kinase by quercetin and analogs. Biochem. Biophys. Res. Commun., 186: 624-631, 1992.[Medline]
39. Nishioka H., Imoto M., Sawa T., Hamada M., Naganawa H., Takeuchi T., Umezawa K. Screening of phosphatidylinositol kinase inhibitors from streptomyces. J. Antibiot. (Tokyo), 42: 823-825, 1989.[Medline]
40. Casciola-Rosen L. A., Anhalt G. J., Rosen A. DNA-dependent protein kinase is one of a subset of autoantigens specifically cleaved early during apoptosis. J. Exp. Med., 182: 1625-1634, 1995.[Abstract/Free Full Text]
41. Han Z., Malik N., Carter W. H., Reeves J. H., Wyche J. H., Hendrickson E. A. DNA-dependent protein kinase is a target for a CPP32-like apoptotic protease. J. Biol. Chem., 271: 25035-25040, 1996.[Abstract/Free Full Text]

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