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Repeated phosphopeptide motifs in Claspin mediate the regulated binding of Chk1

Nature Cell Biology volume 5, pages 161–165 (2003)Cite this article

Abstract

In vertebrates, the checkpoint-regulatory kinase Chk1 mediates cell-cycle arrest in response to a block in DNA replication or to DNA damaged by ultraviolet radiation. The activation of Chk1 depends on both Claspin and the upstream regulatory kinase ATR. Claspin is a large acidic protein that becomes phosphorylated and binds to Chk1 in the presence of checkpoint-inducing DNA templates in Xenopus egg extracts. Here we identify, by means of deletion analysis, a region of Claspin of 57 amino acids that is both necessary and sufficient for binding to Xenopus Chk1. This Chk1-binding domain contains two highly conserved repeats of approximately ten amino acids. A serine residue in each repeat (serine 864 and serine 895) undergoes phosphorylation during a checkpoint response. A mutant of Claspin containing non-phosphorylatable amino acids at positions 864 and 895 cannot bind to Chk1 and is unable to mediate its activation. Our results indicate that two phosphopeptide motifs in Claspin are essential for checkpoint signalling.

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Figure 1: Deletion analysis of Claspin identifies a CKBD.
Figure 2: CKBD must be phosphorylated to bind to Xchk1.
Figure 3: Claspin requires Ser 864 and Ser 895 to bind to Xchk1.
Figure 4: Claspin2AG cannot activate Xchk1 in egg extracts.

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Acknowledgements

We thank T. Pogodin for technical assistance, S. Horvath for phosphopeptide synthesis, and members of the laboratory for comments on the manuscript. This work was supported in part by a grant from the NIH. W.G.D. is an investigator of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Division of Biology 216-76, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, 91125, California, USA

    Akiko Kumagai & William G. Dunphy

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  1. Akiko Kumagai
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  2. William G. Dunphy
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Correspondence to William G. Dunphy.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

Figure S1 Assay for identification of the CKBD. (PDF 533 kb)

Figure S2 Ser-864 and Ser-895 in the CKBD are phosphorylated in egg extracts.

Figure S3 Xatr regulates phosphorylation of Claspin and binding of Claspin to Xchk1 in egg extracts.

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Kumagai, A., Dunphy, W. Repeated phosphopeptide motifs in Claspin mediate the regulated binding of Chk1. Nat Cell Biol 5, 161–165 (2003). https://doi.org/10.1038/ncb921

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