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J. Biol. Chem., Vol. 280, Issue 12, 12041-12050, March 25, 2005

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Regulation of CHK2 by DNA-dependent Protein Kinase^*

Jia Li¶ and David F. Stern||

From the Departments of Pathology and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510

Chk2 is a critical mediator of diverse cellular responses to DNA damage. Activation of Chk2 by DNA damage requires phosphorylation at sites including Thr⁶⁸. In earlier work, we found that an activity present in rabbit reticulocyte lysates phosphorylates and activates Chk2. We now find that hypophosphorylated Chk2 can be phosphorylated at Thr⁶⁸ by various subcellular fractions of HEK293 cells. This activity is sensitive to the phosphatidylinositol 3'-kinase-like kinase inhibitor wortmannin, but not to caffeine. DNA enhances the Chk2 phosphorylation by cellular fractions in vitro. The wortmannin-sensitive Chk2 kinase activity is present in fractions from ATM-deficient cells. In contrast, Chk2 was not efficiently phosphorylated at Thr⁶⁸ in vitro by fractions from cells with a defective DNA-dependent protein kinase (DNA-PK) catalytic subunit. Chk2 is phosphorylated by purified DNA-PK in vitro. Endogenous Chk2 coimmunoprecipitates Ku70 and Ku80. In a series of matched cell lines having and lacking functional DNA-PK, Chk2 activation by exposure of cells to ionizing radiation, or to camptothecin was consistently diminished in the absence of DNA-PK. Down-regulation of DNA-PK_cs by either siRNA or a chemical inhibitor attenuated radiation-induced Chk2 phosphorylation. Ionizing radiation-induced Chk2 phosphorylation was wortmannin-sensitive in ATM-defective cells with depleted ATR. These results suggest that DNA-PK augments ATM and ATR in activation of Chk2 by DNA damage.

Received for publication, November 3, 2004 , and in revised form, January 18, 2005.

^* This work was supported by United States Public Health Service Grant R01CA82257 (to D. F. S.). 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 USAMRMC Predoctoral Training Program in Breast Cancer Research DAMD17-99-1-946.

^|| To whom correspondence should be addressed: Dept. of Pathology, School of Medicine, Yale University, 310 Cedar St., BML342, New Haven, CT 06510. Tel.: 203-785-4832; Fax: 203-785-7467; E-mail: Df.stern{at}yale.edu.

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