Repeated phosphopeptide motifs in Claspin mediate the regulated binding of Chk1.
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.
Kumagai
Akiko
A
Division of Biology 216-76, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California 91125, USA.
Dunphy
William G
WG
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology; D000071877:Checkpoint Kinase 1; D004789:Enzyme Activation; D006801:Humans; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D029867:Xenopus Proteins; D014982:Xenopus laevis
D048868:Adaptor Proteins, Signal Transducing; D000906:Antibodies; C416368:CLSPN protein, Xenopus; C488158:CLSPN protein, human; D002352:Carrier Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D012694:Serine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus