DDB1 targets Chk1 to the Cul4 E3 ligase complex in normal cycling cells and in cells experiencing replication stress

The Chk1 protein kinase preserves genome integrity in normal proliferating cells and in cells experiencing replicative and genotoxic stress. Chkl is currently being targeted in anticancer regimens. Here, we identify damaged DMA-binding protein 1 (DDB1) as a novel Chk1-interacting protein. DDB1 is part of an E3 ligase complex that includes the cullin proteins Cul4A and Cul4B. We report that Cul4A/DDBl negatively regulates Chkl stability in vivo. Chk1 associates with Cul4A/DDBl during an unperturbed cell division cycle and both Chk1 phosphorylation and replication stress enhanced these interactions. Cul4A/DDB1 regulates Chkl ubiquitination in vivo and Chk1 is directly ubiquitinated in vitro in a Cul4A/DDBl- dependent manner. Furthermore, Chk1 is stabilized in cells deficient for Cul4A/DDB1. This study shows that Chk1 abundance is regulated by the Cul4A/DDB1 ubiquitin ligase during an unperturbed cell division cycle, in response to replicative stress and on heat shock protein 90 inhibition, and that deregulation of the Chk1/Cul4A/DDB1 pathway perturbs the ionizing radiation-induced G ₂ checkpoint.