Stabilizing factors regulate the activity of RING finger ubiquitin ligases

Ubiquitin-mediated proteolysis plays in a critical role in a wide variety of processes in eukaryotic systems. To better understand the mechanisms and regulation of ubiquitination, we have focused on two specific aspects: (1) mechanisms of catalysis by multi-subunit RING finger ubiquitin ligases, and (2) regulation of substrate accessibility.; Previous works have shown that ubiquitination by the Anaphase Promoting Complex/Cyclosome (APC/C) can be reconstituted in vitro using the catalytic core of the ligase, APC2 and APC11, and subsequent studies showed that the RING finger protein, APC11, constituted the minimal module necessary to catalyze ubiquitination. We have found that the ubiquitin ligase activity seen using APC11 alone requires APC11 homodimerization. Catalysis by monomeric APC11 is greatly stimulated by addition of the Cullin protein APC2, and this correlates with APC11 dimerization following APC2 addition. We demonstrate that APC2 and APC11 homodimerize in vivo, and find that this phenomenon extends to the catalytic components of the SCF RING finger ubiquitin ligase as well. We propose that dimerization may represent an aspect of a conserved catalytic mechanism common among multi-subunit ubiquitin ligases.; Our analysis of a novel Emi1-interacting protein has elucidated a novel pathway of ligase regulation via substrate accessibility. Emi1 is a critically important inhibitor of the APC/C which controls progression to S phase and mitosis by stabilizing key APC/C ubiquitination substrates, including cyclins. We have identified the Evi5 oncogene as a regulator of Emi1 accumulation. Evi5 antagonizes SCFbetaTrCP-dependent Emi1 ubiquitination and destruction by binding to a site adjacent to Emi1's DSGxxS degron and blocking both degron phosphorylation by Polo-like kinases and subsequent betaTrCP binding. Thus, Evi5 functions as a stabilizing factor maintaining Emi1 levels in S/G2 phase. Evi5 protein accumulates in early G1 following Plk1 destruction, and is degraded in a Plk1- and ubiquitin-dependent manner in early mitosis. Ablation of Evi5 induces precocious degradation of Emi1 by the Plk/SCF betaTrCP pathway, resulting in premature APC/C activation, cyclin A destruction, cell cycle arrest, centrosome overduplication, and finally mitotic catastrophe. We propose that the balance of Evi5 and Polo-like kinase activities determines the timely accumulation of Emi1 and cyclin, ensuring mitotic fidelity.