CDK inhibitor p21 is degraded by a proliferating cell nuclear antigen-coupled Cul4-DDB1^Cdt2 pathway during s phase and after UV irradiation

Previous reports showed that chromatin-associated PCNA couples DNA replication with Cul4-DDB1^Cdt2-dependent proteolysis of the licensing factor Cdt1. The CDK inhibitor p21, another PCNA-binding protein, is also degraded both in S phase and after UV irradiation. Here we show that p21 is degraded by the same ubiquitin-proteasome pathway as Cdt1 in HeLa cells. When PCNA or components of Cul4-DDB1^Cdt2 were silenced or when the PCNA binding site on p21 was mutated, degradation of p21 was prevented both in S phase and after UV irradiation. p21 was co- immunoprecipitated with Cul4A and DDB1 proteins when expressed in cells. The purified Cul4A-DDB1^Cdt2 complex ubiquitinated p21 in vitro. Consistently, p21 protein levels are low during S phase and increase around G₂ phase. Mutational analysis suggested that in addition to the PCNA binding domain, its flanking regions are also important for recognition by Cul4-DDB1^Cdt2. Our findings provide a new aspect of proteolytic control of p21 during the cell cycle.