Ubiquitination of p21Cip1/WAF1 by SCFSkp2: substrate requirement and ubiquitination site selection.

Multiple proteolytic pathways are involved in the degradation of the cyclin-dependent kinase inhibitor p21Cip1/WAF1. Timed destruction of p21Cip1/WAF1 plays a critical role in cell-cycle progression and cellular response to DNA damage. The SCFSkp2 complex (consisting of Rbx1, Cul1, Skp1, and Skp2) is one of the E3 ubiquitin ligases involved in ubiquitination of p21Cip1/WAF1. Little is known about how SCFSkp2 recruits its substrates and selects particular acceptor lysine residues for ubiquitination. In this study, we investigated the requirements for SCFSkp2 recognition of p21Cip1/WAF1 and lysine residues that are ubiquitinated in vitro and inside cells. We demonstrate that ubiquitination of p21Cip1/WAF1 requires a functional interaction between p21Cip1/WAF1 and the cyclin E−Cdk2 complex. Mutation of both the cyclin E recruitment motif (RXL) and the Cdk2-binding motif (FNF) at the N terminus of p21Cip1/WAF1 abolishes its ubiquitination by SCFSkp2, while mutation of either motif alone has minimal effects, s...