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bioRxiv · 10.1101/2025.08.21.670878

Phosphorylation of Claspin by elF2α kinase protects cells from heat stress

Yang, C.-C.·Goto, H.·Oyadomari, S.·Masai, H.
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Abstract

Various biological stresses can induce replication stress, potentially leading to cancer development. Replication checkpoint protects cells against the threat of DNA damages by suspending cell cycle, allowing DNA repair. Chk1 kinase is activated by various stresses and is required for replication checkpoint. An adaptor protein, Claspin, mediates Chk1 activation, but specific mechanisms involved are still unknown. We demonstrate that heat stress induces hyperphosphorylation of Claspin, resulting in Chk1 activation. Claspin phosphorylation is impaired in cells lacking two eIF2 kinases, GCN2 and HRI, which are parts of the integrated stress response (ISR) pathway. Mass spectrometry identified five heat-inducible phosphorylation sites in the C-terminal region of Claspin, and Claspin lacking these sites failed to activate checkpoint. Our data support a model that the unphosphorylated C-terminal tail of Claspin masks its CKBD (Chk1 Binding Domain)-AP (Acidic Patch) domain, preventing it from interacting with Cdc7. Phosphorylation of the tail causes it to dissociate from CKBD-AP and facilitates Cdc7 interaction, leading to increased Chk1 activation. Our findings demonstrate crosstalk between replication checkpoint and ISR pathways, and suggest potential new strategies for cancer therapy.

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Yang, C.-C., Goto, H., Oyadomari, S., Masai, H.. 2025-08-25. Phosphorylation of Claspin by elF2α kinase protects cells from heat stress. https://doi.org/10.1101/2025.08.21.670878

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