Ubiquitination of p27Kip1 requires physical interaction with cyclin E and probable phosphate recognition by SKP2

p27(Kip1) is an essential cell cycle inhibitor of Cyclin-dependent kinases. Ubiquitin- mediated proteolysis of p27(Kip1) is an important mechanism for activation of Cyclin E-Cdk2 and facilitates G(1)/S transition. Ubiquitination of p27 is primarily catalyzed by a multisubunit E3 ubiquitin ligase, SCFSkp2, and requires an adapter protein Cks1. In addition, phosphorylation of p27 at Thr(187) by Cyclin E and Cdk2 is also essential for triggering substrate ubiquitination. Here we investigate the molecular mechanism of p27 ubiquitination. We show that Cyclin E-Cdk2 is essential for targeting the p27 substrate to SCFSkp2. Direct physical contact between Cyclin E but not Cdk2 and p27 is required for p27 recruitment to SCFSkp2. In a search for positively charged amino acid residues that may be involved in recognition of the Thr187 phosphate group, we found that Arg(306) of Skp2 is required for association and ubiquitination of phosphorylated p27 but dispensable for ubiquitination of unphosphorylated p21. Thus, our data unravel the molecular organization of the ubiquitination complex that catalyzes p27 ubiquitination and provide unique insights into the specificity of substrate recognition by SCFSkp2.