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

DaRosa PA, Wang Z, Jiang X, Pruneda JN, Cong F, Klevit RE, Xu W - Nature (2014)

Bottom Line: Disruption of the RNF146-TNKS interaction inhibits turnover of the substrate Axin in cells.Thus, both substrate PARylation and PARdU are catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction.We propose that the 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA [2] Department of Biological Structure, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
Protein poly(ADP-ribosyl)ation (PARylation) has a role in diverse cellular processes such as DNA repair, transcription, Wnt signalling, and cell death. Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several crucial regulatory proteins, including Axin and 3BP2 (refs 7, 8, 9). The RING-type E3 ubiquitin ligase RNF146 (also known as Iduna) is responsible for PARylation-dependent ubiquitination (PARdU). Here we provide a structural basis for RNF146-catalysed PARdU and how PARdU specificity is achieved. First, we show that iso-ADP-ribose (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 bind and activate a ubiquitin-conjugating enzyme (E2) efficiently. Binding of PAR or iso-ADPr induces a major conformational change that creates a functional RING structure. Thus, RNF146 represents a new mechanistic class of RING E3 ligases, the activities of which are regulated by non-covalent ligand binding, and that may provide a template for designing inducible protein-degradation systems. Second, we find 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 catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction. We propose that the 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.

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RNF146 and TNKS form a tight complex critical to PARdU in vivoa, GST pull-downs of GST-tagged RNF146 variants with untagged TNKS(5ARC) (residues 173–961), demonstrate a direct interaction between RNF146 and the five ARCs of TNKS. The interaction likely involves multiple TNKS-binding sites in RNF146 as various RNF146 mutations reduce but do not abolish TNKS binding (inputs are shown in Extended Data Fig. 9d). b, Axin turnover rescue assay shows that both the TNKS-RNF146 interaction and the RNF146 allosteric switch are important for PARdU in cells. c, Proposed TNKS-RNF146 PARdU model. RNF146 is inactive when bound to non-PARylated TNKS in the cell. Upon substrate binding to TNKS and subsequent PARylation, RNF146 binds an internal unit of PAR. This causes a conformational change in the RING domain, which activates its ligase activity, enabling the polyubiquitination of substrate.
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Figure 4: RNF146 and TNKS form a tight complex critical to PARdU in vivoa, GST pull-downs of GST-tagged RNF146 variants with untagged TNKS(5ARC) (residues 173–961), demonstrate a direct interaction between RNF146 and the five ARCs of TNKS. The interaction likely involves multiple TNKS-binding sites in RNF146 as various RNF146 mutations reduce but do not abolish TNKS binding (inputs are shown in Extended Data Fig. 9d). b, Axin turnover rescue assay shows that both the TNKS-RNF146 interaction and the RNF146 allosteric switch are important for PARdU in cells. c, Proposed TNKS-RNF146 PARdU model. RNF146 is inactive when bound to non-PARylated TNKS in the cell. Upon substrate binding to TNKS and subsequent PARylation, RNF146 binds an internal unit of PAR. This causes a conformational change in the RING domain, which activates its ligase activity, enabling the polyubiquitination of substrate.

Mentions: Essentially all known proteins regulated by PARdU, including Axin and 3BP2, are PARylated by TNKS8,10–12. We sought to understand the specificity of RNF146 by testing for a direct interaction between RNF146 and TNKS. Both GST pull-down assays and co-migration on size-exclusion chromatography (SEC) showed that full-length RNF146 forms a stable complex with the five ankyrin repeat clusters (ARCs) of TNKS (TNKS(5ARC), residues 173–961) (Fig. 4a and Extended Data Fig. 9a). Co-IP and pull-down assays using full-length TNKS further support the direct RNF146-TNKS interaction in cells (Extended Data Fig. 9b, c).


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

DaRosa PA, Wang Z, Jiang X, Pruneda JN, Cong F, Klevit RE, Xu W - Nature (2014)

RNF146 and TNKS form a tight complex critical to PARdU in vivoa, GST pull-downs of GST-tagged RNF146 variants with untagged TNKS(5ARC) (residues 173–961), demonstrate a direct interaction between RNF146 and the five ARCs of TNKS. The interaction likely involves multiple TNKS-binding sites in RNF146 as various RNF146 mutations reduce but do not abolish TNKS binding (inputs are shown in Extended Data Fig. 9d). b, Axin turnover rescue assay shows that both the TNKS-RNF146 interaction and the RNF146 allosteric switch are important for PARdU in cells. c, Proposed TNKS-RNF146 PARdU model. RNF146 is inactive when bound to non-PARylated TNKS in the cell. Upon substrate binding to TNKS and subsequent PARylation, RNF146 binds an internal unit of PAR. This causes a conformational change in the RING domain, which activates its ligase activity, enabling the polyubiquitination of substrate.
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Related In: Results  -  Collection

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Figure 4: RNF146 and TNKS form a tight complex critical to PARdU in vivoa, GST pull-downs of GST-tagged RNF146 variants with untagged TNKS(5ARC) (residues 173–961), demonstrate a direct interaction between RNF146 and the five ARCs of TNKS. The interaction likely involves multiple TNKS-binding sites in RNF146 as various RNF146 mutations reduce but do not abolish TNKS binding (inputs are shown in Extended Data Fig. 9d). b, Axin turnover rescue assay shows that both the TNKS-RNF146 interaction and the RNF146 allosteric switch are important for PARdU in cells. c, Proposed TNKS-RNF146 PARdU model. RNF146 is inactive when bound to non-PARylated TNKS in the cell. Upon substrate binding to TNKS and subsequent PARylation, RNF146 binds an internal unit of PAR. This causes a conformational change in the RING domain, which activates its ligase activity, enabling the polyubiquitination of substrate.
Mentions: Essentially all known proteins regulated by PARdU, including Axin and 3BP2, are PARylated by TNKS8,10–12. We sought to understand the specificity of RNF146 by testing for a direct interaction between RNF146 and TNKS. Both GST pull-down assays and co-migration on size-exclusion chromatography (SEC) showed that full-length RNF146 forms a stable complex with the five ankyrin repeat clusters (ARCs) of TNKS (TNKS(5ARC), residues 173–961) (Fig. 4a and Extended Data Fig. 9a). Co-IP and pull-down assays using full-length TNKS further support the direct RNF146-TNKS interaction in cells (Extended Data Fig. 9b, c).

Bottom Line: Disruption of the RNF146-TNKS interaction inhibits turnover of the substrate Axin in cells.Thus, both substrate PARylation and PARdU are catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction.We propose that the 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA [2] Department of Biological Structure, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
Protein poly(ADP-ribosyl)ation (PARylation) has a role in diverse cellular processes such as DNA repair, transcription, Wnt signalling, and cell death. Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several crucial regulatory proteins, including Axin and 3BP2 (refs 7, 8, 9). The RING-type E3 ubiquitin ligase RNF146 (also known as Iduna) is responsible for PARylation-dependent ubiquitination (PARdU). Here we provide a structural basis for RNF146-catalysed PARdU and how PARdU specificity is achieved. First, we show that iso-ADP-ribose (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 bind and activate a ubiquitin-conjugating enzyme (E2) efficiently. Binding of PAR or iso-ADPr induces a major conformational change that creates a functional RING structure. Thus, RNF146 represents a new mechanistic class of RING E3 ligases, the activities of which are regulated by non-covalent ligand binding, and that may provide a template for designing inducible protein-degradation systems. Second, we find 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 catalysed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction. We propose that the 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.

Show MeSH
Related in: MedlinePlus