Threonine 2609 Phosphorylation of the DNA-Dependent Protein Kinase Is a Critical Prerequisite for Epidermal Growth Factor Receptor-Mediated Radiation Resistance

University of Texas Southwestern Medical Center, Department of Radiation Oncology, Division of Molecular Radiation Biology, 2201, Inwood Road, NC 7.208, Mail code 9187, Dallas, TX 75390. .
Molecular Cancer Research (Impact Factor: 4.38). 08/2012; 10(10):1359-68. DOI: 10.1158/1541-7786.MCR-12-0482-T
Source: PubMed


The EGF receptor (EGFR) contributes to tumor radioresistance, in part, through interactions with the catalytic subunit of DNA-dependent protein kinase (DNA-PKc), a key enzyme in the nonhomologous end joining DNA repair pathway. We previously showed that EGFR-DNA-PKcs interactions are significantly compromised in the context of activating mutations in EGFR in non-small cell lung carcinoma (NSCLC) and human bronchial epithelial cells. Here, we investigate the reciprocal relationship between phosphorylation status of DNA-PKcs and EGFR-mediated radiation response. The data reveal that both the kinase activity of DNA-PKcs and radiation-induced phosphorylation of DNA-PKcs by the ataxia telangiectasia-mutated (ATM) kinase are critical prerequisites for EGFR-mediated radioresponse. Alanine substitutions at seven key serine/threonine residues in DNA-PKcs or inhibition of DNA-PKcs by NU7441 completely abrogated EGFR-mediated radioresponse and blocked EGFR binding. ATM deficiency or ATM inhibition with KU55933 produced a similar effect. Importantly, alanine substitution at an ATM-dependent DNA-PKcs phosphorylation site, T2609, was sufficient to block binding or radioresponse of EGFR. However, mutation of a DNA-PKcs autophosphorylation site, S2056 had no such effect indicating that DNA-PKcs autophosphorylation is not necessary for EGFR-mediated radioresponse. Our data reveal that in both NSCLCs and human bronchial epithelial cells, activating mutations in EGFR specifically abolished the DNA-PKcs phosphorylation at T2609, but not S2056. Our study underscores the critical importance of a reciprocal relationship between DNA-PKcs phosphorylation and EGFR-mediated radiation response and elucidates mechanisms underlying mutant EGFR-associated radiosensitivity in NSCLCs. Mol Cancer Res; 10(10); 1359-68. ©2012 AACR.

7 Reads
  • Source
    • "Several reports suggest that mutations in DNA-PKcs, at either the T2609 or S2056 autophosphorylation sites, can produce mild radiosensitivity, whereas the combined mutations act synergistically to render these cells more radiosensitive (20,31). Indeed, phosphorylation at both S2056 and T2609 sites has been reported to be important for DNA-PKcs-mediated radioresistance (17). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Exposure to genotoxic agents, such as ionizing radiation (IR), produces double-strand breaks, repaired predominantly in mammalian cells by non-homologous end-joining (NHEJ). Ku70 was identified as an interacting partner of a proteolytic Cyclin E (CycE) fragment, p18CycE. p18CycE endogenous generation during IR-induced apoptosis in leukemic cells and its stable expression in epithelial tumor cells sensitized to IR. γH2AX IR-induced foci (IRIFs) and comet assays indicated ineffective NHEJ DNA repair in p18CycE-expressing cells. DNA pull-down and chromatin recruitment assays revealed that retention of NHEJ factors to double-strand breaks, but not recruitment, was diminished. Similarly, IRIFs of phosphorylated T2609 and S2056-DNA-PKcs and its target S1778-53BP1 were greatly decreased in p18CycE-expressing cells. As a result, DNA-PKcs chromatin association was also increased. 53BP1 IRIFs were suppressed when p18CycE was generated in leukemic cells and in epithelial cells stably expressing p18CycE. Ataxia telangiectasia mutated was activated but not its 53BP1 and MDC1 targets. These data indicate a profound influence of p18CycE on NHEJ through its interference with DNA-PKcs conformation and/or dimerization, which is required for effective DNA repair, making the p18CycE-expressing cells more IR sensitive. These studies provide unique mechanistic insights into NHEJ misregulation in human tumor cells, in which defects in NHEJ core components are rare.
    Nucleic Acids Research 09/2013; 41(22). DOI:10.1093/nar/gkt812 · 9.11 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: DNA damage can occur as a result of endogenous metabolic reactions and replication stress or from exogenous sources such as radiation therapy and chemotherapy. DNA double strand breaks are the most cytotoxic form of DNA damage, and defects in their repair can result in genome instability, a hallmark of cancer. The major pathway for the repair of ionizing radiation-induced DSBs in human cells is nonhomologous end joining. Here we review recent advances on the mechanism of nonhomologous end joining, as well as new findings on its component proteins and regulation.
    International journal of radiation oncology, biology, physics 02/2013; 86(3). DOI:10.1016/j.ijrobp.2013.01.011 · 4.26 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In response to ionizing radiation, several signaling cascades in the cell are activated to repair the DNA breaks, prevent apoptosis, and keep the cells proliferating. AKT is important for survival and proliferation and may also be an activating factor for DNA-PKcs and MRE11, which are essential proteins in the DNA repair process. AKT (PKB) is hyperactivated in several cancers and is associated with resistance to radiotherapy and chemotherapy. There are three AKT isoforms (AKT1, AKT2, and AKT3) with different expression patterns and functions in several cancer tumors. The role of AKT isoforms has been investigated in relation to radiation response and their effects on DNA repair proteins (DNA-PKcs and MRE11) in colon cancer cell lines. The knockout of AKT1 and/or AKT2 affected the radiation sensitivity, and a deficiency of both isoforms impaired the rejoining of radiation-induced DNA double strand breaks. Importantly, the active/phosphorylated forms of AKT and DNA-PKcs associate and exposure to ionizing radiation causes an increase in this interaction. Moreover, an increased expression of both DNA-PKcs and MRE11 was observed when AKT expression was ablated, yet only DNA-PKcs expression influenced AKT phosphorylation. Taken together, these results demonstrate a role for both AKT1 and AKT2 in radiotherapy response in colon cancer cells involving DNA repair capacity through the nonhomologous end joining pathway, thus suggesting that AKT in combination with DNA-PKcs inhibition may be used for radiotherapy sensitizing strategies in colon cancer. Electronic supplementary material The online version of this article (doi:10.1007/s13277-013-1465-9) contains supplementary material, which is available to authorized users.
    Tumor Biology 12/2013; 35(4). DOI:10.1007/s13277-013-1465-9 · 3.61 Impact Factor
Show more