HectD1 E3 Ligase Modifies Adenomatous Polyposis Coli (APC) with Polyubiquitin to Promote the APC-Axin Interaction

Tran, Hoanh; Bustos, Daisy; Yeh, Ronald W.; Rubinfeld, Bonnee; Lam, Cynthia; Shriver, Stephanie; Zilberleyb, Inna; Lee, Michelle W.; Phu, Lilian; Sarkar, Anjali A.; Zohn, Irene E.; Wertz, Ingrid E.; Kirkpatrick, Donald S.; Polakis, Paul

doi:10.1074/jbc.m112.415240

Cited by 60 publications

(81 citation statements)

References 56 publications

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“…2G). It has been previously reported that hTNKS1 coIPs with hAPC1 (27), suggesting that either TNKS binds APC1 via a non-consensus motif we were unable to identify, or that TNKS is in a complex with APC1 but lacks direct binding.…”

Section: Tnks Binds Drosophila Apc2 At a Conserved C-terminalmentioning

confidence: 49%

The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2

Croy¹,

Fuller²,

Giannotti³

et al. 2016

Journal of Biological Chemistry

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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(ADPribose) 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.The Wnt pathway helps direct a myriad of normal developmental and adult homeostasic processes in metazoans, but is also misregulated in several human diseases such as cancer (1, 2). Wnt signaling is regulated through the activity of a multiprotein "destruction complex" that promotes proteolysis of the transcriptional co-activator ␤-catenin (␤cat) 3 by stimulating phosphorylation of the ␤cat phosphodegron (3). Core components of the destruction complex include the scaffold protein Axin, the tumor suppressor adenomatous polyposis coli (APC), and the kinases CK1 and GSK3.While Wnt signaling plays essential roles during development, it is inappropriately activated in a number of cancers, most notably colorectal cancer. Truncating mutations in the tumor suppressor adenomatous polyposis coli (APC) are the initiating mutational event in more than 80% of all colon cancer cases, and these mutations hyperactivate ␤cat signaling (4). Thus small molecule inhibitors of the Wnt pathway should provide an effective therapeutic strategy. Among these strategies, inhibition of oncogenic ␤cat activity would appear to be the most direct approach, as studies have demonstrated that the accumulation of ␤cat is what initiates oncogenesis, and that tumors have a continued reliance on oncogenic ␤cat signaling (5). Indeed, recent promising antagonists have been identified that specifically disrupt ␤cat binding to TCF or th...

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Section: Tnks Binds Drosophila Apc2 At a Conserved C-terminalmentioning

confidence: 49%

The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2

Croy¹,

Fuller²,

Giannotti³

et al. 2016

Journal of Biological Chemistry

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“…Hectd1 is a HECT domain E3 ubiquitin ligase that plays an important role in development of the neural tube (Sarkar and Zohn, 2012; Zohn et al, 2007) and regulation of Wnt signaling (Tran et al, 2013). Ubiquitin conjugated substrates are widely distributed in the endometrium and decidua in rodents and primates (Bebington et al, 2000), and studies of mouse mutants demonstrate that ubiquitin dependent modification plays an indispensable role in placental development.…”

Section: Introductionmentioning

confidence: 99%

Hectd1 is required for development of the junctional zone of the placenta

Sarkar¹,

Nuwayhid²,

Maynard³

et al. 2014

Developmental Biology

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The placenta plays a critical role in the growth and survival of the fetus. Here we demonstrate that the Homologous to the E6-AP Carboxyl Terminus (HECT) domain E3 ubiquitin ligase, Hectd1, is essential for development of the mouse placenta. Hectd1 is widely expressed during placentation with enrichment in trophoblast giant cells (TGCs) and other trophoblast-derived cell subtypes in the junctional and labyrinth zones of the placenta. Disruption of Hectd1 results in mid-gestation lethality and intrauterine growth restriction (IUGR). Variable defects in the gross structure of the mutant placenta are found including alterations in diameter, thickness and lamination. The number and nuclear size of TGCs is reduced. Examination of subtype specific markers reveals altered TGC development with decreased expression of Placental lactogen-1 and -2 (Pl1 and Pl2) and increased expression of Proliferin (Plf). Reduced numbers of spongiotrophoblasts and glycogen trophoblasts were also found at the junctional zone of the Hectd1 mutant placenta. Finally, there was an increase in immature uterine natural killer (uNK) cells in the maternal decidua of the Hectd1 mutant placenta. Proliferation and apoptosis are differentially altered in the layers of the placenta with an increase in both apoptosis and proliferation in the maternal decidua, a decrease in proliferation and increase in apoptosis in the labyrinth layer and both unchanged in the junctional zone. Together these data demonstrate that Hectd1 is required for development of multiple cell types within the junctional zone of the placenta.

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“…Increasing findings regarding nonproteolytic ubiquitination through atypical ubiquitin linkages also have noted this pathway. For example, K63-linked polyubiquitination of Dvl is considered to promote Wnt signaling transduction (17), while HectD1-mediated antigen-presenting cell (APC) ubiquitination through K63 ubiquitin linkage inhibits Wnt signaling by facilitating axin-APC interaction (18). The E3 ligase EDD, targeting ␤-catenin via K11-or K29-linked polyubiquitination, was reported to stabilize ␤-catenin instead of triggering its degradation (19).…”

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confidence: 99%

Smurf1-Mediated Lys29-Linked Nonproteolytic Polyubiquitination of Axin Negatively Regulates Wnt/β-Catenin Signaling

Fang

et al. 2013

Molecular and Cellular Biology

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Ubiquitination plays important and diverse roles in modulating protein functions. As a C2-WW-HECT-type ubiquitin ligase, Smad ubiquitination regulatory factor 1 (Smurf1) commonly serves to regulate ubiquitin-dependent protein degradation in a number of signaling pathways. Here, we report a novel function of Smurf1 in regulating Wnt/␤-catenin signaling through targeting axin for nonproteolytic ubiquitination. Our data unambiguously demonstrate that Smurf1 ubiquitinates axin through Lys 29 (K29)-linked polyubiquitin chains. Unexpectedly, Smurf1-mediated axin ubiquitination does not lead to its degradation but instead disrupts its interaction with the Wnt coreceptors LRP5/6, which subsequently attenuates Wnt-stimulated LRP6 phosphorylation and represses Wnt/␤-catenin signaling. The inhibitory function of Smurf1 on Wnt/␤-catenin signaling is further evidenced by analysis with Smurf1 knockout murine embryonic fibroblasts. We next identified K789 and K821 in axin as the ubiquitination sites by Smurf1. Consistently, Smurf1 could neither disrupt the interaction of an axin K789/821R double mutant with LRP5/6 nor attenuate the phosphorylation of LRP6 in axin K789/821R -expressing cells. Collectively, our studies uncover Smurf1 as a new regulator for the Wnt/␤-catenin signaling pathway via modulating the activity of axin.

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HectD1 E3 Ligase Modifies Adenomatous Polyposis Coli (APC) with Polyubiquitin to Promote the APC-Axin Interaction

Cited by 60 publications

References 56 publications

The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2

The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2

Hectd1 is required for development of the junctional zone of the placenta

Smurf1-Mediated Lys29-Linked Nonproteolytic Polyubiquitination of Axin Negatively Regulates Wnt/β-Catenin Signaling

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