Jeong, Seong-Yun and Kumagai, Akiko and Lee, Joon and Dunphy, William G. (2003) Phosphorylated claspin interacts with a phosphate-binding site in the kinase domain of Chk1 during ATR-mediated activation. Journal of Biological Chemistry, 278 (47). pp. 46782-46788. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:JEOjbc03
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Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated or UV-damaged DNA. The activated form of Claspin contains two repeated phosphopeptide motifs that mediate its binding to Chk1. We show that these phosphopeptide motifs bind to Chk1 by means of its N-terminal kinase domain. The binding site on Chk1 involves a positively charged cluster of amino acids that contains lysine 54, arginine 129, threonine 153, and arginine 162. Mutagenesis of these residues strongly compromises the ability of Chk1 to interact with Claspin. These amino acids lie within regions of Chk1 that are involved in various aspects of its catalytic function. The predicted position on Chk1 of the phosphate group from Claspin corresponds to the location of activation-loop phosphorylation in various kinases. In addition, we have obtained evidence that the C-terminal regulatory domain of Chk1, which does not form a stable complex with Claspin under our assay conditions, nonetheless has some role in Claspin-dependent activation. Overall, these results indicate that Claspin docks with a phosphate-binding site in the catalytic domain of Chk1 during activation by ATR. Phosphorylated Claspin may mimic an activating phosphorylation of Chk1 during this process.
|Additional Information:||Copyright © 2003 by the American Society for Biochemistry and Molecular Biology. Received for publication, April 30, 2003, and in revised form, August 22, 2003. Published, JBC Papers in Press, September 8, 2003, DOI 10.1074/jbc.M304551200 We are very grateful to Jun-yong Choe for assistance with the structural diagrams of Chk1 in Fig. 4. We thank the members of our laboratory for helpful comments on the manuscript. This work was supported in part by National Institutes of Health Grant GM43974. 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.|
|Subject Keywords:||DNA-REPLICATION STRESS; XENOPUS EGG EXTRACTS; CHECKPOINT CONTROL; DAMAGE CHECKPOINT; CRYSTAL-STRUCTURE; PROTEIN-KINASES; MRC1; PATHWAY; RAD53; FORK|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Lindsay Cleary|
|Deposited On:||16 Oct 2006|
|Last Modified:||02 Apr 2015 16:03|
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