The DNA dependent protein kinase (DNA-PK), composed of Ku70/80 heterodimer and the catalytic subunit (DNA-PKcs), is the key component of the non-homologous end-joining (NHEJ) pathway, the predominate DNA double-strand breaks repair mechanism in mammalian cells. DNA double-strand breaks (DSBs) generated by ionizing radiation (IR), byproducts of oxidative metabolism, and chemotherapeutic drugs are the most deleterious form of DNA damage. Unattended or misrepaired DSBs would otherwise lead to cell death, mutagenesis, genomic instability, and ultimately cancer formation. The catalytic subunit DNA-PKcs is a member of the PI-3 kinase like protein kinase family (PIKK) that includes ATM (ataxia-telangiectasia mutated) and ATR (ATM-Rad3-related). Although the biochemical properties of DNA-PKcs have been extensively studied in vitro, it is not clear how it functions in vivo in the context of NHEJ. Wild type DNA-PKcs, but not a kinase-dead mutant DNA-PKcs, is able to rescue the radiation sensitivity and DSBs repair defect in cells lacking of DNA-PKcs demonstrating that the kinase activity of DNA-PKcs is essential for the NHEJ pathway.
To further elucidate the molecular mechanisms underlying NHEJ, it is critical to identify the in vivo substrates of DNA-PKcs. In the process, we have discovered that DNA-PKcs itself is also the subject of phosphorylation regulation in vivo upon IR, mainly at Ser2056 and Thr2609 cluster region. In response to IR, DNA-PKcs phosphorylation increases rapidly and is concentrated at specific nuclear foci overlapping with ??H2AX, presumably at the DNA damage sites. Similar to its kinase activity, DNA-PKcs phosphorylation is also required for NHEJ as alanine substitution at the putative phosphorylation sites resulted in the increase of radiation sensitivity and defect in DSBs repair. Our current evidence suggests that IR-induced DNA-PKcs phosphorylation is mediated by DNA-PKcs autophosphorylation at Ser2056 and by ATM at Thr2609 cluster, and both DNA-PKcs and ATM mediated DNA-PKcs phosphorylation are required for the full activation of DNA-PKcs in vivo. However, how the crosstalk between DNA-PKcs and ATM as well as the detailed regulation of DNA-PKcs phosphorylation remains to be elucidated.
In addition to IR induction, we have reported that DNA-PKcs phosphorylation increases upon replication inhibition or !?replication stress!? induced by camptothecin and UV irradiation. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 cluster but not at S2056 which is distinctive from that of IR induction. UV-induced DNA-PKcs phosphorylation occurs specifically in replicating cells or during the S phase confirming that replication stress indeed causes DNA-PKcs phosphorylation in response to UV. It is well established that ATR, another member of PIKK family, is the key signaling kinase activated in response to replication stress or UV irradiation. Our results demonstrated that ATR is required to mediate UV-induced DNA-PKcs phosphorylation. Inhibition of ATR activity via caffeine, dominant-negative kinase-dead mutant, or RNA interference all lead to the attenuation of UV-induced DNA-PKcs phosphorylation suggesting that DNA-PKcs is the direct downstream target of ATR signaling pathway. We are currently investigating the specific role DNA-PKcs in cellular response to replication stress and the implication of DNA-PKcs phosphorylation in the repair of stalled replication forks.
Another research direction in my laboratory is to apply and extend what we learned from cell culture studies to create mouse models to address the significance of DNA-PK phosphorylation in animal physiology. Using the genetic gene targeting approach, we will introduce or !?knockin!? various point mutations to DNA-PKcs gene locus to eliminate DNA-PKcs phosphorylation or kinase activity that are required for the normal NHEJ repair of DSBs. We will analyze any potential phonotypical changes of these knockin mice and will cross these knockin mice with different knockout mice strains to investigate the inter-relationship of various molecules within the NHEJ pathway. Finally, we are also interesting in exploring the additional functions of DNA-PKcs beyond DNA damage repair. Some of the clues came from investigation of DNA-PKcs interacting proteins. Through out the years, we have identified many of the potential DNA-PKcs binding partners or interacting proteins via different screening approaches. Pursuing these activities and interactions would further elucidate the role of DNA-PKcs in cellular activities for a comprehensive understanding of DNA-PKcs.
RESEARCH INTERESTS
DNA double strand breaks repair
Replication Stress
RECENT PUBLICATIONS
Mingan Shi, Carolyn J. Vivian, Kyung-Jong Lee, Chunmin Ge, Keiko Morotomi-Yano, Claudia Manzl, Florian Bock, Shigeo Sato, Chieri Sato, Ruihong Zhu, Selene K. Swanson, Michael P. Washburn, David J. Chen, Benjamin P. C. Chen, Andreas Villunger, Laurence Florens, Chunying Du., "DNA-PKcs-PIDDosome: A Nuclear Caspase-2-Activating Protein Complex Required for a G2/M DNA Damage Checkpoint and DNA Nonhomologous End-Joining" Cell, 136:508-20, 2009
Hirohiko Yajima, Kyung-Jong Lee, Shichuan Zhang, Junya Kobayashi, and Benjamin P.C. Chen., "DNA Double Strand Break Formation upon UV-Induced Replication Stress Activates ATM and DNA-PKcs Kinases" J. Molecular Biology, 385:800-10, 2008
Amit K. Das, Benjamin P. Chen, Michael D. Story, John D. Minna, David J. Chen, and Chaitanya S. Nirodi, "Somatic mutations in the tyrosine kinase domain of EGFR abrogate EGFR-mediated radio-protection in non-small cell lung carcinoma" Cancer Res, 67:187-93, 2007
Naoya Uematsu, Eric Weterings, Ken-ichi Yano, Keiko Morotomi-Yano, Burkhard Jakob, Gisela Taucher-Scholz, Pierre-Olivier Mari, Dik C. van Gent, Benjamin P. C. Chen, and David J. Chen, "Autophosphorylation of DNA-PKcs regulates its dynamics at DNA double-strand breaks" J. Cell Biol, 177:219-29, 2007
Benjamin P.C. Chen, Naoya Uematsu, Junya Kobayashi, Yaniv Lerenthal, Hirohiko Yajima, Andrea Krempler, Markus Lobrich, Yossi Shiloh, David J. Chen., "ATM is essential for phosphorylation of DNA-PKcs at T2609 cluster upon DNA double strand break." J. Biol. Chem., 282:6582-7, 2007
SIGNIFICANT PUBLICATIONS
Yijima, Kyung-Jong Lee, and Benjamin P.C. Chen, "ATR-dependent DNA-PKcs phosphorylation in response to UV-induced replication stress" Molecular & Cellular Biology, 26:7520-8, 2006
Sandeep Burma, Benjamin P.C. Chen, David J. Chen, "Role of non-homologous end joining (NHEJ) in maintaining genomic integrity" DNA Repair, 5:1042-8, 2006
Benjamin P.C. Chen, Doug W. Chan, Junya Kobayashi, Sandeep Burma, Aroumougame Asaithamby, Keiko Yano, Elliot Botvinick, Jun Qin, and David J. Chen, "Cell Cycle Dependence of DNA-PK phosphorylation in Response to DNA Double Strand Breaks" J. Biol. Chem, 280:14709-15, 2005
Doug W. Chan, Benjamin P.C. Chen, Sheela Prithivirajsingh, Akihiro Kurimasa, Michael D. Story, Jun Qin, and David J. Chen., "Autophosphorylation of the DNA-dependent protein kinase catalytic subunit is required for rejoining of DNA double-strand breaks" Gene & Development, 16:2333-2338, 2002
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