Allosteric activation of the RNF146 ubiquitin ligase by a poly(ADP-ribosyl)ation signal

Abstract

Protein poly(ADP-ribosyl)ation (PARylation) plays a role in diverse cellular processes such as DNA repair, transcription, Wnt signaling, and cell death1–6. Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several critical regulatory proteins, including Axin and 3BP2 (refs 7–9). The RING-type E3 ubiquitin ligase RNF146 (a.k.a. Iduna) is responsible for PARylation-dependent ubiquitination (PARdU)10–12. Here we provide a structural basis for RNF146 catalyzed PARdU and how PARdU specificity is achieved. First, we show that iso-ADPr, the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to efficiently bind and activate an E2. Binding of PAR/iso-ADPr induces a major conformational change that creates a functional RING structure. Thus RNF146 represents a new mechanistic class of RING E3 ligases whose activities are regulated by non-covalent ligand binding, which may provide a template for designing inducible protein-degradation systems. Second, we found that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146/TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalyzed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction. We propose that maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146-TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell.

USP25 regulates Wnt signaling by controlling the stability of tankyrases

“…RNF146 has been reported to be an E3 ubiquitin ligase for tankyrases (Callow et al 2011;Zhang et al 2011;DaRosa et al 2015). The expression of RNF146 induced the ubiquitination of TNKS1/2, which was significantly diminished by coexpression of USP25 ( Fig.…”

Wnt pathway activation by ADP-ribosylation

“…Therefore, to detect ADPribosylated Axin, we utilized a previously developed pull down assay based on the Trp-Trp-Glu (WWE) domain of the RINGtype E3 ubiquitin ligase RNF146/Iduna coupled to glutathione S-transferase (GST) 52 . Tnks and RNF146 are known to form a stable complex that targets substrates for ADP-ribosylation and subsequent ADP-ribose-dependent ubiquitination [52][53][54][55] . The WWE domain of RNF146 interacts directly with the poly(ADPribose) in these Tnks substrates to promote their ubiquitination.…”

Structural insights into protein-protein interactions governing regulation in transcription initiation and ubiquitination

“…Several reports provide evidence that the regulation of RING E3 activity modulates the associated ubiquitination process (reviewed in ref 133 ). The different forms of regulation for E3 include auto-inhibition by other E3 domains than RING, as well as release of auto-inhibition through ligand-binding or phosphorylation 132,134 . For example, the ligand iso-ADP ribose binds at the interface of the RING and WWE domains of RNF146, inducing a conformational change that releases the autoinhibition and stabilizes the conformation mediating the RING binding to E2 (ref 134 ).…”