This Article Full Text Full Text (PDF) Supplemental material Other Versions of this Article: MCB.01962-06v1 27/5/1581    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Douglas, P. Articles by Meek, K. Search for Related Content PubMed PubMed Citation Articles by Douglas, P. Articles by Meek, K.

 Previous Article  |  Next Article 

Molecular and Cellular Biology, March 2007, p. 1581-1591, Vol. 27, No. 5
0270-7306/07/$08.00+0     doi:10.1128/MCB.01962-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The DNA-Dependent Protein Kinase Catalytic Subunit Is Phosphorylated In Vivo on Threonine 3950, a Highly Conserved Amino Acid in the Protein Kinase Domain ^,

Departments of Biochemistry and Molecular Biology and Oncology, University of Calgary, Calgary, Alberta, Canada T2N 4N1,¹ College of Veterinary Medicine and Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan 48824,² MRC Protein Phosphorylation Unit, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom³

Received 18 October 2006/ Returned for modification 14 November 2006/ Accepted 1 December 2006

The protein kinase activity of the DNA-dependent protein kinase (DNA-PK) is required for the repair of DNA double-strand breaks (DSBs) via the process of nonhomologous end joining (NHEJ). However, to date, the only target shown to be functionally relevant for the enzymatic role of DNA-PK in NHEJ is the large catalytic subunit DNA-PKcs itself. In vitro, autophosphorylation of DNA-PKcs induces kinase inactivation and dissociation of DNA-PKcs from the DNA end-binding component Ku70/Ku80. Phosphorylation within the two previously identified clusters of phosphorylation sites does not mediate inactivation of the assembled complex and only partially regulates kinase disassembly, suggesting that additional autophosphorylation sites may be important for DNA-PK function. Here, we show that DNA-PKcs contains a highly conserved amino acid (threonine 3950) in a region similar to the activation loop or t-loop found in the protein kinase domain of members of the typical eukaryotic protein kinase family. We demonstrate that threonine 3950 is an in vitro autophosphorylation site and that this residue, as well as other previously identified sites in the ABCDE cluster, is phosphorylated in vivo in irradiated cells. Moreover, we show that mutation of threonine 3950 to the phosphomimic aspartic acid abrogates V(D)J recombination and leads to radiation sensitivity. Together, these data suggest that threonine 3950 is a functionally important, DNA damage-inducible phosphorylation site and that phosphorylation of this site regulates the activity of DNA-PKcs.

Published ahead of print on 11 December 2006.

Supplemental material for this article may be found at http://mcb.asm.org/.

This article has been cited by other articles:

• Weterings, E., Chen, D. J. (2007). DNA-dependent protein kinase in nonhomologous end joining: a lock with multiple keys?. J. Cell Biol. 179: 183-186 [Abstract] [Full Text]  
• Povirk, L. F., Zhou, R.-Z., Ramsden, D. A., Lees-Miller, S. P., Valerie, K. (2007). Phosphorylation in the serine/threonine 2609-2647 cluster promotes but is not essential for DNA-dependent protein kinase-mediated nonhomologous end joining in human whole-cell extracts. Nucleic Acids Res 35: 3869-3878 [Abstract] [Full Text]  
• Meek, K., Douglas, P., Cui, X., Ding, Q., Lees-Miller, S. P. (2007). trans Autophosphorylation at DNA-Dependent Protein Kinase's Two Major Autophosphorylation Site Clusters Facilitates End Processing but Not End Joining. Mol. Cell. Biol. 27: 3881-3890 [Abstract] [Full Text]