SCCRO (DCUN1D1) Is an Essential Component of the E3 Complex for Neddylation

Alexander Y Kim,Claire C Bommeljé,Benjamin E Lee,Yoshihiro Yonekawa,Lydia Choi,Luc G Morris,Guochang Huang,Andrew Kaufman,Russel J.H Ryan,Bing Hao,Y Ramanathan,Bhuvanesh Singh

doi:10.1074/jbc.m804440200

Abstract

Covalent modification of cullins by the ubiquitin-like protein NEDD8 (neddylation) regulates protein ubiquitination by promoting the assembly of cullin-RING ligase E3 complexes. Like ubiquitination, neddylation results from an enzymatic cascade involving the sequential activity of a dedicated E1 (APPBP1/Uba3), E2 (Ubc12), and an ill-defined E3. We show that SCCRO (also known as DCUN1D1) binds to the components of the neddylation pathway (Cullin-ROC1, Ubc12, and CAND1) and augments but is not required for cullin neddylation in reactions using purified recombinant proteins. We also show that SCCRO recruits Ubc12 approximately NEDD8 to the CAND1-Cul1-ROC1 complex but that this is not sufficient to dissociate or overcome the inhibitory effects of CAND1 on cullin neddylation in purified protein assays. In contrast to findings in cellular systems where no binding is seen, we show that SCCRO and CAND1 can bind to the neddylated Cul1-ROC1 complex in assays using purified recombinant proteins. Although neddylated (not unneddylated) Cul1-ROC1 is released from CAND1 upon incubation with testis lysate from SCCRO+/+ mice, the addition of recombinant SCCRO is required to achieve the same results in lysate from SCCRO(-/-) mice. Combined, these results suggest that SCCRO is an important component of the neddylation E3 complex that functions to recruit charged E2 and is involved in the release of inhibitory effects of CAND1 on cullin-RING ligase E3 complex assembly and activity.

Highlights

Differentiation, apoptosis, cell cycle, and transcription [1,2,3,4,5]
We found that SCCRO, but not the loss of cullin binding mutant SCCROD241N, increased neddylation efficiency beyond basal levels (Fig. 2, C and D). These findings suggest that the effects of SCCRO on cullin neddylation require its interaction with Cul-ROC1 and/or Ubc12 and implicate it as a component of the neddylation E3
Cullin RING ligases (CRLs) complexes have a constant catalytic module (ROC1 bound to E2 thioester) that is preserved in the neddylation E3 complex

Summary

Introduction

Ubiquitination is a highly regulated process that involves the sequential action of three enzymes termed as E1, E2, and E3 In this enzymatic cascade, E1 initiates the process by forming a high energy thioester bond with Ub in an ATP-coupled reaction. HECT E3s form a thioester intermediate with the Ub before its transfer, RING-containing complexes serve as scaffolds to facilitate the direct transfer of Ub from E2 to the target protein. A small RING protein (ROC1) and a variable substrate recognition subunit bind to the cullin core to form the CRL complex. The covalent modification of cullins with the ubiquitinlike protein (Ublp) NEDD8, in a process termed neddylation, dissociates CAND1 and promotes assembly of CRL complexes (9 –12). Several studies suggest that cycling of neddylation and deneddylation is required for normal CRL function [8, 16, 17]

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