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J. Biol. Chem., Vol. 283, Issue 25, 17250-17259, June 20, 2008

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A Conserved Proliferating Cell Nuclear Antigen-interacting Protein Sequence in Chk1 Is Required for Checkpoint Function^*

Jennifer Scorah¹, Meng-Qiu Dong², John R. Yates, III^¶, Mary Scott^||3, David Gillespie^||3, and Clare H. McGowan⁴

From the Department of Molecular Biology and Department of Cellular Biology, ^¶Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037 and ^||Beatson Institute for Cancer Research, Garscube Estates, Switchback Rd., Glasgow, G61 1BD, United Kingdom

Human checkpoint kinase 1 (Chk1) is an essential kinase required for cell cycle checkpoints and for coordination of DNA synthesis. To gain insight into the mechanisms by which Chk1 carries out these functions, we used mass spectrometry to identify previously uncharacterized interacting partners of Chk1. We describe a novel interaction between Chk1 and proliferating cell nuclear antigen (PCNA), an essential component of the replication machinery. Binding between Chk1 and PCNA was reduced in the presence of hydroxyurea, suggesting that the interaction is regulated by replication stress. A highly conserved PCNA-interacting protein (PIP) box motif was identified in Chk1. The intact PIP box is required for efficient DNA damage-induced phosphorylation and release of activated Chk1 from chromatin. We find that the PIP box of Chk1 is crucial for Chk1-mediated S-M and G₂-M checkpoint responses. In addition, we show that mutations in the PIP box of Chk1 lead to decreased rates of replication fork progression and increased aberrant replication. These findings suggest an additional mechanism by which essential components of the DNA replication machinery interact with the replication checkpoint apparatus.

Received for publication, January 15, 2008 , and in revised form, April 24, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants CA077254 (to C. H. M.) and P41 RR011823 and R01 DK074798 (to J. R. Y.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Figs. S1 and S2.

¹ A California Breast Cancer Research Fellow.

² Current address: National Institute of Biological Sciences, 7 Science Park Rd., Zhongguancun Life Science Park, Beijing, China 102206.

³ Supported by Cancer Research, UK.

⁴ To whom correspondence should be addressed. Tel.: 858-784-2280; Fax: 858-784-2265; E-mail: chmcg{at}scripps.edu.

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