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J. Biol. Chem., Vol. 279, Issue 36, 37282-37290, September 3, 2004

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Phosphorylation and Regulation of DNA Ligase IV Stability by DNA-dependent Protein Kinase^*

Yu-Gang Wang, Chinonye Nnakwe, William S. Lane, Mauro Modesti¶||, and Karen M. Frank**

From the Department of Pathology, University of Chicago, Chicago, Illinois 60637, the Harvard Microchemistry and Proteomics Analysis Facility, Harvard University, Cambridge, Massachusetts 02138, and the ^¶Department of Cell Biology and Genetics, Erasmus Medical Center, P. O. Box 1738, 3000 DR, Rotterdam, The Netherlands

DNA ligase IV (Lig4), x-ray cross-complementation group 4 (XRCC4), and DNA-dependent protein kinase (DNA-PK) are essential mammalian nonhomologous end joining proteins used for V(D)J recombination and DNA repair. Previously a Lig4 peptide was reported to be an in vitro substrate for DNA-PK, but the phosphorylation state of Lig4 protein in vivo is not known. In this study, we report that a full-length Lig4 construct was expressed as a phosphoprotein in the cell. Also the full-length Lig4 protein, in complex with XRCC4, was an in vitro substrate for DNA-PK. Using tandem mass spectrometry, we identified a DNA-PK phosphorylation site at Thr-650 in human Lig4 and a potential second phosphorylation site at Ser-668 or Ser-672. Phosphorylation of Lig4 per se was not required for Lig4 DNA end joining activity. Substitution of these amino acids with alanine, individually or in combination, led to changes in Lig4 protein stability of mouse Lig4. The phosphomimetic mutation S650D returned Lig4 stability to that of the wild-type protein. Furthermore DNA-PK was found to negatively regulate Lig4 protein stability. Our results suggest that Lig4 stability is regulated by multiple factors, including interaction with XRCC4, phosphorylation status, and possibly Lig4 conformation.

Received for publication, February 4, 2004 , and in revised form, May 24, 2004.

^* This work was supported by National Institutes of Health Award K08 AI01428, a Sidney Kimmel Foundation for Cancer Research scholar award, Howard Hughes Medical Institute Award 76200-550002, the Cancer Research Foundation, and the National Foundation for Cancer Research in association with the American Association for Cancer Research. 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 Association for International Cancer Research Grant 01-215.

^** To whom correspondence should be addressed: Dept. of Pathology, University of Chicago, 5841 S. Maryland Ave., MC 1089, Chicago, IL 60637. Tel.: 773-834-7407; Fax: 773-834-5251; E-mail: kfrank{at}uchicago.edu.

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