A Context-Dependent Role for the RNF146 Ubiquitin Ligase in Wingless/Wnt Signaling in Drosophila.
ABSTRACT: Aberrant activation of the Wnt signal transduction pathway triggers the development of colorectal cancer. The ADP-ribose polymerase Tankyrase (TNKS) mediates proteolysis of Axin-a negative regulator of Wnt signaling-and provides a promising therapeutic target for Wnt-driven diseases. Proteolysis of TNKS substrates is mediated through their ubiquitination by the poly-ADP-ribose (pADPr)-dependent RING-domain E3 ubiquitin ligase RNF146/Iduna. Like TNKS, RNF146 promotes Axin proteolysis and Wnt pathway activation in some cultured cell lines, but in contrast with TNKS, RNF146 is dispensable for Axin degradation in colorectal carcinoma cells. Thus, the contexts in which RNF146 is essential for TNKS-mediated Axin destabilization and Wnt signaling remain uncertain. Herein, we tested the requirement for RNF146 in TNKS-mediated Axin proteolysis and Wnt pathway activation in a range of in vivo settings. Using null mutants in Drosophila, we provide genetic and biochemical evidence that Rnf146 and Tnks function in the same proteolysis pathway in vivo Furthermore, like Tnks, Drosophila Rnf146 promotes Wingless signaling in multiple developmental contexts by buffering Axin levels to ensure they remain below the threshold at which Wingless signaling is inhibited. However, in contrast with Tnks, Rnf146 is dispensable for Wingless target gene activation and the Wingless-dependent control of intestinal stem cell proliferation in the adult midgut during homeostasis. Together, these findings demonstrate that the requirement for Rnf146 in Tnks-mediated Axin proteolysis and Wingless pathway activation is dependent on physiological context, and suggest that, in some cell types, functionally redundant pADPr-dependent E3 ligases or other compensatory mechanisms promote the Tnks-dependent proteolysis of Axin in both mammalian and Drosophila cells.
Project description: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.
Project description:Wnt/?-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)--known to target Axin for proteolysis-regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly.
Project description:Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein ?-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the ?-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate ?-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases ?-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy.
Project description:Activation of the wnt signaling pathway is a major cause of colon cancer development. Tankyrase inhibitors (TNKSi) have recently been developed to block the wnt pathway by increasing axin levels to promote degradation of the wnt-regulator ?-catenin. TNKSi bind to the PARP (poly(ADP)ribose polymerase) catalytic region of tankyrases (TNKS), preventing the PARylation of TNKS and axin that normally control axin levels through ubiquitination and degradation. TNKSi treatment of APC-mutant SW480 colorectal cancer cells can induce axin puncta which act as sites for assembly of ?-catenin degradation complexes, however this process is poorly understood. Using this model system, we found that siRNA knockdown of TNKSs 1 and 2 actually blocked the ability of TNKSi drugs to induce axin puncta, revealing that puncta formation requires both the expression and the inactivation of TNKS. Immunoprecipitation assays showed that treatment of cells with TNKSi caused a strong increase in the formation of axin-TNKS complexes, correlating with an increase in insoluble or aggregated forms of TNKS/axin. The efficacy of TNKSi was antagonized by proteasome inhibitors, which stabilized the PARylated form of TNKS1 and reduced TNKSi-mediated assembly of axin-TNKS complexes and puncta. We hypothesise that TNKSi act to stimulate TNKS oligomerization and assembly of the TNKS-axin scaffold that form puncta. These new insights may help in optimising the future application of TNKSi in anticancer drug design.
Project description:Canonical Wnt signaling is controlled intracellularly by the level of ?-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates ?-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling.
Project description:Poly(ADP-ribosyl)ation (PARylation) catalyzed by the tankyrase enzymes (Tankyrase-1 and -2; a.k.a. PARP-5a and -5b) is involved in mitosis, telomere length regulation, GLUT-4 vesicle transport, and cell growth and differentiation. Together with the E3 ubiquitin ligase RNF146 (a.k.a. Iduna), tankyrases regulate the cellular levels of several important proteins including Axin, 3BP2, and angiomotins, which are key regulators of Wnt, Src and Hippo signaling, respectively. These tankyrase substrates are first PARylated and then ubiquitylated by RNF146, which is allosterically activated by binding to PAR polymer. Each tankyrase substrate is recognized by a tankyrase-binding motif (TBM). Here we show that RNF146 binds directly to tankyrases via motifs in its C-terminal region. Four of these RNF146 motifs represent novel, extended TBMs, that have one or two additional amino acids between the most conserved Arg and Gly residues. The individual RNF146 motifs display weak binding, but together mediate a strong multivalent interaction with the substrate-binding region of TNKS, forming a robust one-to-one complex. A crystal structure of the first RNF146 noncanonical TBM in complex with the second ankyrin repeat domain of TNKS shows how an extended motif can be accommodated in a peptide-binding groove on tankyrases. Overall, our work demonstrates the existence of a new class of extended TBMs that exist in previously uncharacterized tankyrase-binding proteins including those of IF4A1 and NELFE.
Project description:Axin is a tumor suppressor and a key negative regulator of the Wnt/?-catenin signaling pathway. Axin turnover is controlled by its poly-ADP-ribosylation catalyzed by tankyrase (TNKS), which requires the direct interaction of Axin with TNKS. This interaction is thus an attractive drug target for treating cancers, brain injuries, and other diseases where ?-catenin is involved. Here we report the crystal structure of a mouse TNKS1 fragment containing ankyrin-repeat clusters 2 and 3 (ARC2-3) in a complex with the TNKS-binding domain of mouse Axin1. Surprisingly, we found that Axin contains two discrete TNKS-binding segments, both of which bind simultaneously to the two ARC2 domains in the ARC2-3 homodimer. Our crystal structure shows that in each TNKS-binding segment of Axin there is a conserved glycine residue that lies in the bottom of a narrow "gate" formed by two parallel tyrosine side chains on the TNKS surface. This glycine-selection gate is crucial for TNKS-Axin interactions, as mutation of the TNKS gate-forming residues, or mutation of either glycine residue in the two Axin segments, completely abolishes the binding of the corresponding Axin segment to TNKS. The bivalent binding of Axin to TNKS is required for Axin turnover, since mutations in either gate-binding glycine residue in Axin lead to Axin stabilization in the cell. In addition, our analyses also reveal the structural basis for TNKS substrate recruitment, and shed light on the overall structure of TNKS that should help in developing specific inhibitors of Wnt/?-catenin signaling.
Project description:Self-renewal of intestinal stem cells is controlled by Wingless/Wnt-? catenin signaling in both Drosophila and mammals. As Axin is a rate-limiting factor in Wingless signaling, its regulation is essential. Iduna is an evolutionarily conserved ubiquitin E3 ligase that has been identified as a crucial regulator for degradation of ADP-ribosylated Axin and, thus, of Wnt/?-catenin signaling. However, its physiological significance remains to be demonstrated. Here, we generated loss-of-function mutants of Iduna to investigate its physiological role in Drosophila Genetic depletion of Iduna causes the accumulation of both Tankyrase and Axin. Increase of Axin protein in enterocytes non-autonomously enhanced stem cell divisions in the Drosophila midgut. Enterocytes secreted Unpaired proteins and thereby stimulated the activity of the JAK-STAT pathway in intestinal stem cells. A decrease in Axin gene expression suppressed the over-proliferation of stem cells and restored their numbers to normal levels in Iduna mutants. These findings suggest that Iduna-mediated regulation of Axin proteolysis is essential for tissue homeostasis in the Drosophila midgut.
Project description:Aberrant activation of the Wnt pathway has been implicated in the development and formation of many cancers. TNKS inhibition has been shown to antagonize Wnt signaling via Axin stabilization in APC mutant colon cancer cell lines. We employed structure-based design to identify a series of 2-aminopyridine oxazolidinones as potent and selective TNKS inhibitors. These compounds exhibited good enzyme and cell potency as well as selectivity over other PARP isoforms. Co-crystal structures of these 2-aminopyridine oxazolidinones complexed to TNKS reveal an induced-pocket binding mode that does not involve interactions with the nicotinamide binding pocket. Oral dosing of lead compounds 3 and 4 resulted in significant effects on several Wnt-pathway biomarkers in a three day DLD-1 mouse tumor PD model.
Project description:BACKGROUND:New pharmacologic targets are urgently needed to treat or prevent lung cancer, the most common cause of cancer death for men and women. This study identified one such target. This is the canonical Wnt signaling pathway, which is deregulated in cancers, including those lacking adenomatous polyposis coli or ?-catenin mutations. Two poly-ADP-ribose polymerase (PARP) enzymes regulate canonical Wnt activity: tankyrase (TNKS) 1 and TNKS2. These enzymes poly-ADP-ribosylate (PARsylate) and destabilize axin, a key component of the ?-catenin phosphorylation complex. METHODS:This study used comprehensive gene profiles to uncover deregulation of the Wnt pathway in murine transgenic and human lung cancers, relative to normal lung. Antineoplastic consequences of genetic and pharmacologic targeting of TNKS in murine and human lung cancer cell lines were explored, and validated in vivo in mice by implantation of murine transgenic lung cancer cells engineered with reduced TNKS expression relative to controls. RESULTS:Microarray analyses comparing Wnt pathway members in malignant versus normal tissues of a murine transgenic cyclin E lung cancer model revealed deregulation of Wnt pathway components, including TNKS1 and TNKS2. Real-time PCR assays independently confirmed these results in paired normal-malignant murine and human lung tissues. Individual treatments of a panel of human and murine lung cancer cell lines with the TNKS inhibitors XAV939 and IWR-1 dose-dependently repressed cell growth and increased cellular axin 1 and tankyrase levels. These inhibitors also repressed expression of a Wnt-responsive luciferase construct, implicating the Wnt pathway in conferring these antineoplastic effects. Individual or combined knockdown of TNKS1 and TNKS2 with siRNAs or shRNAs reduced lung cancer cell growth, stabilized axin, and repressed tumor formation in murine xenograft and syngeneic lung cancer models. CONCLUSIONS:Findings reported here uncovered deregulation of specific components of the Wnt pathway in both human and murine lung cancer models. Repressing TNKS activity through either genetic or pharmacological approaches antagonized canonical Wnt signaling, reduced murine and human lung cancer cell line growth, and decreased tumor formation in mouse models. Taken together, these findings implicate the use of TNKS inhibitors to target the Wnt pathway to combat lung cancer.