Phosphorylated claspin interacts with a phosphate-binding site in the kinase domain of Chk1 during ATR-mediated activation.
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.
Jeong
Seong-Yun
SY
Division of Biology 216-76, California Institute of Technology, Pasadena, CA 91125, USA.
Kumagai
Akiko
A
Lee
Joon
J
Dunphy
William G
WG
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D020134:Catalytic Domain; D018797:Cell Cycle Proteins / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D004789:Enzyme Activation; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017354:Point Mutation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D029867:Xenopus Proteins / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C416368:CLSPN protein, Xenopus; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D029867:Xenopus Proteins; D011494:Protein Kinases; C576898:Atr protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases