Coupled monoubiquitylation of the co-E3 ligase DCNL1 by Ariadne-RBR E3 ubiquitin ligases promotes cullin-RING ligase complex remodeling

Ian R Kelsall,Yosua A Kristariyanto,Axel Knebel,Nicola T Wood,Yogesh Kulathu,Arno F Alpi

doi:10.1074/jbc.ra118.005861

Abstract

Cullin-RING E3 ubiquitin ligases (CRLs) are large and diverse multisubunit protein complexes that contribute to about one-fifth of ubiquitin-dependent protein turnover in cells. CRLs are activated by the attachment of the ubiquitin-like protein neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to the cullin subunits. This cullin neddylation is essential for a plethora of CRL-regulated cellular processes and is vital for life. In mammals, neddylation is promoted by the five co-E3 ligases, defective in cullin neddylation 1 domain-containing 1–5 (DCNL1–5); however, their functional regulation within the CRL complex remains elusive. We found here that the ubiquitin-associated (UBA) domain–containing DCNL1 is monoubiquitylated when bound to CRLs and that this monoubiquitylation depends on the CRL-associated Ariadne RBR ligases TRIAD1 (ARIH2) and HHARI (ARIH1) and strictly requires the DCNL1's UBA domain. Reconstitution of DCNL1 monoubiquitylation in vitro revealed that autoubiquitylated TRIAD1 mediates binding to the UBA domain and subsequently promotes a single ubiquitin attachment to DCNL1 in a mechanism previously dubbed coupled monoubiquitylation. Moreover, we provide evidence that DCNL1 monoubiquitylation is required for efficient CRL activity, most likely by remodeling CRLs and their substrate receptors. Collectively, this work identifies DCNL1 as a critical target of Ariadne RBR ligases and coupled monoubiquitylation of DCNL1 as an integrated mechanism that affects CRL activity and client–substrate ubiquitylation at multiple levels.

Highlights

Cullin-RING E3 ubiquitin ligases (CRLs) are large and diverse multisubunit protein complexes that contribute to about onefifth of ubiquitin-dependent protein turnover in cells
We found here that the ubiquitin-associated (UBA) domain– containing DCNL1 is monoubiquitylated when bound to CRLs and that this monoubiquitylation depends on the CRL-associated Ariadne RBR ligases TRIAD1 (ARIH2) and HHARI (ARIH1) and strictly requires the DCNL1’s UBA domain
We have previously reported that the Ariadne RBRs, HHARI and TRIAD1, interact with distinct neddylated CRLs, and this interaction acts to stimulate Ariadne E3 ligase activity by relieving an autoinhibitory effect mediated by the “Ariadne” domain [28, 29]

Summary

Results

To investigate the impact of TRIAD1/NEDD8 –CUL5 binding on the overall CUL5 ligase complex assembly and neddylation cycle, we analyzed the association of the NEDD8 conjugation/deconjugation machinery, DCNL1 and CSN. Dead variants of both Ariadne RBRs (Fig. 1, E and F, lanes 2 and 4) reduced the abundance of DCNL1-Ub in the cytosol, whereas autoinhibition-relieved variants had no significant impact (Fig. 1, E and F, lane 3) Taken together, these data indicate that the E3 ubiquitin ligase activities of TRIAD1 and HHARI are required for efficient DCNL1 monoubiquitylation in cells. In a complete ubiquitylation reaction containing the ubiquitin-activating E1 enzyme UBE1, the cognate E2-conjugating enzyme for Ariadne RBRs UBCH7 [33, 34], DCNL1 as substrate, and neddylated CUL5–RBX2 (N8 –CUL5–RBX2), TRIAD1 efficiently monoubiquitylated DCNL1 (Fig. 2A). In a complete ubiquitylation reaction with either UBCH7 or UBCH5c, we observed a robust monoubiquitylation of DCNL1 by HHARI (⌬ARI) (Fig. 2E) Both TRIAD1 and HHARI are capable of directly monoubiquitylating DCNL1 in vitro. We tested DCNL1 binding to a set of ubiquitin tetramers of seven different linkage

E GFP-TRIAD1

Discussion

Experimental procedures