Abstract
RING (Really Interesting New Gene)-in-between-RING (RBR) enzymes are a distinct class of E3 ubiquitin ligases possessing a cluster of three zinc-binding domains that cooperate to catalyse ubiquitin transfer. The regulation and biological function for most members of the RBR ligases is not known, and all RBR E3s characterized to date are auto-inhibited for in vitro ubiquitylation. Here, we show that TRIAD1 and HHARI, two members of the Ariadne subfamily ligases, associate with distinct neddylated Cullin-RING ligase (CRL) complexes. In comparison to the modest E3 ligase activity displayed by isolated TRIAD1 or HHARI, binding of the cognate neddylated CRL to TRIAD1 or HHARI greatly stimulates RBR ligase activity in vitro, as determined by auto-ubiquitylation, their ability to stimulate dissociation of a thioester-linked UBCH7∼ubiquitin intermediate, and reactivity with ubiquitin-vinyl methyl ester. Moreover, genetic evidence shows that RBR ligase activity impacts both the levels and activities of neddylated CRLs in vivo. Cumulatively, our work proposes a conserved mechanism of CRL-induced Ariadne RBR ligase activation and further suggests a reciprocal role of this special class of RBRs as regulators of distinct CRLs.
Highlights
The RING (Really Interesting New Gene) family comprises the largest group of E3 ubiquitin ligases with 4600 membersReceived: 15 February 2013; accepted: 4 September 2013; published online: 27 September 20132848 The EMBO Journal VOL 32 | NO 21 | 2013 in mammals (Deshaies and Joazeiro, 2009)
TRIAD1 is an RBR E3 ubiquitin ligase To better understand the ubiquitin ligase function of the RBR protein TRIAD1, we initially aimed to identify and characterize the cognate E2 conjugating enzymes
HHARI binds neddylated cullin-1, -2, -3, -4A complexes Given the homology within the Ariadne family of RBR E3 ligases, we considered whether the interactions with Cullin-RING ligases (CRLs) might be conserved
Summary
The RING (Really Interesting New Gene) family comprises the largest group of E3 ubiquitin ligases with 4600 membersReceived: 15 February 2013; accepted: 4 September 2013; published online: 27 September 20132848 The EMBO Journal VOL 32 | NO 21 | 2013 in mammals (Deshaies and Joazeiro, 2009). CRL activity is tightly regulated by the reversible ligation of the ubiquitin-like protein NEDD8 to a conserved lysine in the C-terminus of the cullin (Deshaies et al, 2010). Recent studies have found that NEDD8 dictates binding of a subset of CRLs to UBXN7/p97 complexes to regulate turnover of their ubiquitylation substrates. This raises the possibility that neddylation may direct CRLs to other complexes for additional levels of regulation by the ubiquitin-proteasome system (Bandau et al, 2012; den Besten et al, 2012)
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