A very early step in the response of mammalian cells to DNA double-strand breaks is the phosphorylation of histone H2AX at serine 139 at the sites of DNA damage. Although the phosphatidylinositol 3-kinases, DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia mutated), and ATR (ATM and Rad3-related), have all been implicated in H2AX phosphorylation, the specific kinase involved has not yet been identified. To definitively identify the specific kinase(s) that phosphorylates H2AX in vivo, we have utilized DNA-PKcs؊/؊ and Atm؊/؊ cell lines and mouse embryonic fibroblasts. We find that H2AX phosphorylation and nuclear focus formation are normal in DNA-PKcs؊/؊ cells and severely compromised in Atm؊/؊ cells. We also find that ATM can phosphorylate H2AX in vitro and that ectopic expression of ATM in Atm؊/؊ fibroblasts restores H2AX phosphorylation in vivo. The minimal H2AX phosphorylation in Atm؊/؊ fibroblasts can be abolished by low concentrations of wortmannin suggesting that DNA-PK, rather than ATR, is responsible for low levels of H2AX phosphorylation in the absence of ATM. Our results clearly establish ATM as the major kinase involved in the phosphorylation of H2AX and suggest that ATM is one of the earliest kinases to be activated in the cellular response to double-strand breaks.DNA double-strand breaks (DSBs) 1 are probably the most dangerous of the many different types of DNA damage that occur within the cell. DSBs are generated by exogenous agents such as ionizing radiation (IR) or by endogenously generated reactive oxygen species and occur as intermediates during meiotic and V(D)J recombination (1). A very early step in the cellular response to DSBs is the phosphorylation of a histone H2A variant, H2AX, at the sites of DNA damage (2). H2AX is rapidly phosphorylated (within seconds) at serine 139 when DSBs are introduced into mammalian cells (3) resulting in discrete ␥-H2AX (phosphorylated-H2AX) foci at the DNA damage sites (4). In experiments involving the use of "laser scissors" to introduce breaks into living cells, ␥-H2AX foci localized specifically with the laser path through the cell nuclei clearly demonstrating that H2AX phosphorylation is specific to the sites of DNA damage (4, 5). H2AX phosphorylation also appears to be a general cellular response to processes involving DSB intermediates including V(D)J recombination in lymphoid cells (6) and meiotic recombination in mice (7). Phosphorylation of yeast H2A at serine 129 (homologous to serine 139 of mammalian H2AX) causes chromatin decondensation and is required for efficient DNA double-strand break repair (8). In mammals, phosphorylation of H2AX appears to play a critical role in the recruitment of repair or damage-signaling factors to the sites of DNA damage (5, 9).As H2AX phosphorylation plays a very early and important role in the cellular response to DNA double-strand breaks, it is important to specifically identify the kinase(s) involved in this event. Members of the PI 3-kinase family, including DNA-PK (DNA-dependent protein kinase...

Akt phosphorylation has previously been described to be involved in mediating DNA damage repair through the nonhomologous end-joining (NHEJ) repair pathway. Yet the mechanism how Akt stimulates DNA-protein kinase catalytic subunit (DNA-PKcs)-dependent DNA double-strand break (DNA-DSB) repair has not been described so far. In the present study, we investigated the mechanism by which Akt can interact with DNA-PKcs and promote its function during the NHEJ repair process. The results obtained indicate a prominent role of Akt, especially Akt1 in the regulation of NHEJ mechanism for DNA-DSB repair. As shown by pull-down assay of DNA-PKcs, Akt1 through its C-terminal domain interacts with DNA-PKcs. After exposure of cells to ionizing radiation (IR), Akt1 and DNA-PKcs form a functional complex in a first initiating step of DNA-DSB repair. Thereafter, Akt plays a pivotal role in the recruitment of AKT1/DNA-PKcs complex to DNA duplex ends marked by Ku dimers. Moreover, in the formed complex, Akt1 promotes DNA-PKcs kinase activity, which is the necessary step for progression of DNA-DSB repair. Akt1-dependent DNA-PKcs kinase activity stimulates autophosphorylation of DNA-PKcs at S2056 that is needed for efficient DNA-DSB repair and the release of DNA-PKcs from the damage site. Thus, targeting of Akt results in radiosensitization of DNA-PKcs and Ku80 expressing, but not of cells deficient for, either of these proteins. The data showed indicate for the first time that Akt through an immediate complex formation with DNA-PKcs can stimulate the accumulation of DNA-PKcs at DNA-DSBs and promote DNA-PKcs activity for efficient NHEJ DNA-DSB repair. Mol Cancer Res; 10(7); 945-57. Ó2012 AACR. IntroductionThe serine/threonine kinase Akt/PKB is expressed as 3 isoforms, Akt1/PKBa, Akt2/PKBb and Akt3/PKBg. Akt activation is efficiently induced by ionizing radiation (IR) or by growth factors, such as EGF receptor (EGFR) ligands, through the activation of phosphoinositide 3-kinase (PI3K; ref. 1). Our results and accumulated reports from other laboratories indicate that radiosensitization by targeting PI3K or Akt1 (2-5) is a consequence of impaired DNA

The epidermal growth factor receptor (EGFR) is an important determinant of radioresponse, whose elevated expression and activity frequently correlates with radioresistance in several cancers, including non-small cell lung carcinoma (NSCLC). We reported recently that NSCLC cell lines harboring somatic, activating mutations in the tyrosine kinase domain (TKD) of the EGFR exhibit significant delays in the repair of DNA double-strand breaks (DSB) and poor clonogenic survival in response to radiation. Here, we explore the mechanisms underlying mutant EGFR-associated radiosensitivity. In three representative NSCLC cell lines, we show that, unlike wild-type (WT) EGFR, receptors with common oncogenic TKD mutations, L858R or #E746-E750, are defective in radiationinduced translocation to the nucleus and fail to bind the catalytic and regulatory subunits of the DNA-dependent protein kinase (DNA-PK), a key enzyme in the nonhomologous end-joining repair pathway. Moreover, despite the presence of WT EGFR, stable exogenous expression of either the L858R or the #E746-E750 mutant forms of EGFR in human bronchial epithelial cells significantly delays repair of ionizing radiation (IR)-induced DSBs, blocks the resolution of frank or microhomologous DNA ends, and abrogates IR-induced nuclear EGFR translocation or binding to DNA-PK catalytic subunit. Our study has identified a subset of naturally occurring EGFR mutations that lack a critical radioprotective function of EGFR, providing valuable insights on how the EGFR mediates cell survival in response to radiation in NSCLC cell lines.

The epidermal growth factor receptor (EGFR) contributes to tumor radioresistance, in part, through interactions with the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs), a key enzyme in the non homologous end joining DNA repair pathway. We previously demonstrated 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 7 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 auto-phosphorylation site, S2056 had no such effect indicating that DNA-PKcs auto-phosphorylation is not necessary for EGFR-mediated radioresponse. Our data reveal that in both NSCLCs and HBECs, 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.