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J. Biol. Chem., Vol. 280, Issue 15, 14709-14715, April 15, 2005

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Cell Cycle Dependence of DNA-dependent Protein Kinase Phosphorylation in Response to DNA Double Strand Breaks^*

Benjamin P. C. Chen, Doug W. Chan¶||, Junya Kobayashi**, Sandeep Burma, Aroumougame Asaithamby, Keiko Morotomi-Yano, Elliot Botvinick¶¶, Jun Qin¶||||, and David J. Chen

From the Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9187, ^¶Verna and Marrs McLean Department of Biochemistry and Molecular Biology and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, ^**Department of Radiation Biology, Hiroshima University, Hiroshima 734-8553, Japan, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, and ^¶¶Beckman Laser Institute, University of California, Irvine, California 92612

DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PKcs subunits, is the key component of the non-homologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. Although the kinase activity of DNA-PKcs is essential for NHEJ, thus far, no in vivo substrate has been conclusively identified except for an autophosphorylation site on DNA-PKcs itself (threonine 2609). Here we report the ionizing radiation (IR)-induced autophosphorylation of DNA-PKcs at a novel site, serine 2056, the phosphorylation of which is required for the repair of DSBs by NHEJ. Interestingly, IR-induced DNA-PKcs autophosphorylation is regulated in a cell cycle-dependent manner with attenuated phosphorylation in the S phase. In contrast, DNA replication-associated DSBs resulted in DNA-PKcs autophosphorylation and localization to DNA damage sites. These results indicate that although IR-induced DNA-PKcs phosphorylation is attenuated in the S phase, DNA-PKcs is preferentially activated by the physiologically relevant DNA replication-associated DSBs at the sites of DNA synthesis.

Received for publication, August 3, 2004 , and in revised form, January 26, 2005.

^* This work was supported by the United States Department of Energy under contract DE-AC03-76SF00098 and by National Institutes of Health Grants CA50519, CA86936, and PO1-CA92584. 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.

^|| Supported by postdoctoral fellowships from the Canadian Institutes of Health Research (CIHR) and the Alberta Heritage Foundation for Medical Research.

Recipient of Concept Award DAMD17-03-1-0635 from the Department of Defense Breast Cancer Research Program.

^|||| Recipient of Career Development Award DAMD17-00-1-0146 from the Department of Defense Breast Cancer Research Program.

To whom correspondence should be addressed: Dept. of Radiation Oncology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390-9187. Tel.: 214-648-1263; Fax: 214-648-5995; E-mail: benjamin.chen{at}utsouthwestern.edu.

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