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DDB1 Targets Chk1 to the Cul4 E3 Ligase Complex in Normal Cycling Cells and in Cells Experiencing Replication Stress

Departments of ¹ Cell Biology and Physiology and ² Internal Medicine and ³ Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri

Requests for reprints: Helen Piwnica-Worms, Department of Cell Biology and Physiology and Howard Hughes Medical Institute, Washington University School of Medicine, Box 8228, 660 South Euclid Avenue, St. Louis, MO 63110. Phone: 314-362-6812; Fax: 314-362-3709; E-mail: hpiwnica{at}wustl.edu

Key Words: DNA damage • checkpoint • cell cycle • ubiquitination • HSP90

The Chk1 protein kinase preserves genome integrity in normal proliferating cells and in cells experiencing replicative and genotoxic stress. Chk1 is currently being targeted in anticancer regimens. Here, we identify damaged DNA-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/DDB1 negatively regulates Chk1 stability in vivo. Chk1 associates with Cul4A/DDB1 during an unperturbed cell division cycle and both Chk1 phosphorylation and replication stress enhanced these interactions. Cul4A/DDB1 regulates Chk1 ubiquitination in vivo and Chk1 is directly ubiquitinated in vitro in a Cul4A/DDB1–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 G₂ checkpoint. [Cancer Res 2009;69(6):2630–7]