Characterization of the mammalian family of DCN-type NEDD8 E3 ligases

Matthew J Keuss,Thimo Kurz,Axel Knebel,Robin Mcarthur,Nicola T Wood,Yann Thomas

doi:10.1242/jcs.181784

Abstract

ABSTRACTCullin-RING ligases (CRL) are ubiquitin E3 enzymes that bind substrates through variable substrate receptor proteins and are activated by attachment of the ubiquitin-like protein NEDD8 to the cullin subunit. DCNs are NEDD8 E3 ligases that promote neddylation. Mammalian cells express five DCN-like (DCNL) proteins but little is known about their specific functions or interaction partners. We found that DCNLs form stable stoichiometric complexes with CAND1 and cullins that can only be neddylated in the presence of a substrate adaptor. These CAND–cullin–DCNL complexes might represent ‘reserve’ CRLs that can be rapidly activated when needed. We further found that all DCNLs interact with most cullin subtypes, but that they are probably responsible for the neddylation of different subpopulations of any given cullin. This is consistent with the fact that the subcellular localization of DCNLs in tissue culture cells differs and that they show unique tissue-specific expression patterns in mice. Thus, the specificity between DCNL-type NEDD8 E3 enzymes and their cullin substrates is only apparent in well-defined physiological contexts and related to their subcellular distribution and restricted expression.

Highlights

Ubiquitin is a small (8 kDa) signaling protein that regulates most cellular activities
Using SDS-PAGE gels followed by silver staining (Fig. 1A,C), we detected unique interaction patterns for each DCNL, with the exception of one protein at >100 kDa, that was prominently present in all immunoprecipitates, but too large to be a cullin
This is in accordance with previously published in vitro data that showed that purified recombinant DCNLs can bind to all cullins with only slightly different affinities (Monda et al, 2013)

Summary

Introduction

Ubiquitin is a small (8 kDa) signaling protein that regulates most cellular activities. The majority of ubiquitin’s functions require its linkage to other proteins through isopeptide bonds. This is mediated by the sequential action of three enzyme families, termed E1, E2 and E3 (Hershko and Ciechanover, 1998). By far the largest class of E3 enzymes is formed by the cullin-RING ligase (CRL) family. These are modular E3s built around a heterodimeric catalytic scaffolding complex that consists of a small RING-finger protein (either RBX1 or RBX2) bound to the C-terminus of a cullin protein. The N-terminus of cullin proteins can interact with many different substrate-specificity modules that recruit substrates, whereas the RING finger protein interacts with ubiquitin-charged E2 enzymes

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Conclusion