Structure and function of E3 ubiquitin ligase mindbomb RING domain

Abstract

Aberrant Notch receptor signaling is implicated in many different forms of cancer, highlighting the importance of tight regulation in normal signal transduction. Productive signaling in vivo relies on endocytosis of Notch DSL‐family ligands, which are marked for uptake by ubiquitination on their cytoplasmic tails. Mindbomb 1 (MIB1) is an important E3 ubiquitin (Ub) ligase for these ligands in mammals, but the mechanism by which it catalyzes Ub transfer onto the ligand tails is poorly understood. The purpose of this study was to characterize the triple RING domain of MIB1, which is required for delivery of ubiquitin to the ligand tails. Preliminary studies have revealed that Ub is covalently loaded onto the RING domains of MIB1. The bound Ub is released by the addition of a reducing agent, suggesting that the site of attachment is at a cysteine residue. Subsequent studies will focus on determining the site of attachment and on the preparation of complexes between the RING domain and an E2 ligase with a view toward future biochemical and X‐ray crystallographic studies. Together, these studies will provide substantial new insights into the action of MIB1 and a better understanding of the events that lead to Notch activation. This work was funded by NIH and foundation awards to SCB. This work was also funded, in part, by NIH/NIGMS MARC U*STAR T34 08663 NRSA to UMBC and the Harvard Medical School Division of Medical Sciences (RDW).