The CRL4^Cdt2 Ubiquitin Ligase Mediates the Proteolysis of Cyclin-Dependent Kinase Inhibitor Xic1 through a Direct Association with PCNA

Abstract

During DNA polymerase switching, the Xenopus laevis Cip/Kip-type cyclin-dependent kinase inhibitor Xic1 associates with trimeric proliferating cell nuclear antigen (PCNA) and is recruited to chromatin, where it is ubiquitinated and degraded. In this study, we show that the predominant E3 for Xic1 in the egg is the Cul4-DDB1-XCdt2 (Xenopus Cdt2) (CRL4^Cdt2) ubiquitin ligase. The addition of full-length XCdt2 to the Xenopus extract promotes Xic1 turnover, while the N-terminal domain of XCdt2 (residues 1 to 400) cannot promote Xic1 turnover, despite its ability to bind both Xic1 and DDB1. Further analysis demonstrated that XCdt2 binds directly to PCNA through its C-terminal domain (residues 401 to 710), indicating that this interaction is important for promoting Xic1 turnover. We also identify the cis-acting sequences required for Xic1 binding to Cdt2. Xic1 binds to Cdt2 through two domains (residues 161 to 170 and 179 to 190) directly flanking the Xic1 PCNA binding domain (PIP box) but does not require PIP box sequences (residues 171 to 178). Similarly, human p21 binds to human Cdt2 through residues 156 to 161, adjacent to the p21 PIP box. In addition, we identify five lysine residues (K180, K182, K183, K188, and K193) immediately downstream of the Xic1 PIP box and within the second Cdt2 binding domain as critical sites for Xic1 ubiquitination. Our studies suggest a model in which both the CRL4^Cdt2 E3- and PIP box-containing substrates, like Xic1, are recruited to chromatin through independent direct associations with PCNA.

We thank Johannes Walter and Emily Arias (Harvard Medical School) for providing the XCdt2 and DDB1 antibodies, J. Wade Harper and Jianpin Jin (Harvard Medical School) for providing the Homo sapiens Cdt2 cDNA, Martin J. Allday (Ludwig Institute for Cancer Research, Imperial College of Science) for providing the p21^{Δ156 to 161} cDNA, Guem Hee Baek for the generation of the Xic1^K11R and Xic1^K13R mutants, Angelica Hernandez for the generation of the XCdt2^R247A mutant, and Ethan Lee (Vanderbilt University) for providing FA cloning plasmids. We also thank Ikjin Kim and Nam Hee Kim (UTHSCSA) for helpful comments and advice and Michael J. Parker (UTHSCSA) for excellent technical support.

This work was supported by a Career Development Award to P.R.Y. (award DAMD17-02-1-0589) from the U.S. Army Department of Defense and by the National Institute of Health (grant RO1-GM066226 to P.R.Y.).