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Published June 13, 2011 | Published
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Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication

Kumagai, Akiko ORCID icon
Shevchenko, Anna
Shevchenko, Andrej
Dunphy, William G. ORCID icon
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Abstract

Treslin, a TopBP1-interacting protein, is necessary for deoxyribonucleic acid (DNA) replication in vertebrates. Association between Treslin and TopBP1 requires cyclin-dependent kinase (Cdk) activity in Xenopus laevis egg extracts. We investigated the mechanism and functional importance of Cdk for this interaction using both X. laevis egg extracts and human cells. We found that Treslin also associated with TopBP1 in a Cdk-regulated manner in human cells and that Treslin was phosphorylated within a conserved Cdk consensus target sequence (on S976 in X. laevis and S1000 in humans). Recombinant human Cdk2–cyclin E also phosphorylated this residue of Treslin in vitro very effectively. Moreover, a mutant of Treslin that cannot undergo phosphorylation on this site showed significantly diminished binding to TopBP1. Finally, human cells harboring this mutant were severely deficient in DNA replication. Collectively, these results indicate that Cdk-mediated phosphorylation of Treslin during S phase is necessary for both its effective association with TopBP1 and its ability to promote DNA replication in human cells.

Additional Information

© 2011 Kumagai et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). Submitted: 1 February 2011; Accepted: 10 May 2011. We are grateful to laboratory members for comments on the manuscript. We would also like to thank Rochelle Diamond for assistance with flow cytometry, Alex Varshavsky for use of the Odyssey system, and Lydia Dennis for technical assistance. This work was supported by National Institutes of Health grants GM043974 and GM070891 to W.G. Dunphy.

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