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

Abstract

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...

Differential Roles of AXIN1 and AXIN2 in Tankyrase Inhibitor-Induced Formation of Degradasomes and β-Catenin Degradation

“…The formation of degradasomes also appears to be AXIN-dependent, although recent studies indicate a role for additional complex components in this process (e.g. TNKS and APC2) [10, 17, 19]. However, the role of AXIN1 versus AXIN2 in TNKSi-induced degradasome formation remains elusive.…”

The Scaffolding Protein IQGAP1 Interacts with NLRC3 and Inhibits Type I IFN Production

“…The truncations of IQGAP1 and NLRC3 were amplified from full length template using the primers listed in Table 1 (Integrated DNA Technologies) and cloned into the pCR8/TOPO/TA (Life Technologies) base vector. Recombination reactions were performed into modified FLAG or Flourescent protein Gateway destination vectors (described in (57)) using LR Clonase II (Life Technologies). Generation of the NLRC3 plasmid has been previously described (51).…”

Molecular regulation and pharmacological targeting of the β‐catenin destruction complex

“…The axin–TNKS crystal structure divulged two TNKS‐binding motifs in axin, each of which binds to a different ARC domain within the TNKS protein (Figure A, C) (Morrone et al, ). Notably, the sequence of the second binding motif is considerably different from the agreed TNKS‐binding sequence, and TNKS binding to this region was not detected by standard biochemical protein interaction methods, possibly due to a weaker affinity (Croy et al, ). Furthermore, despite the presence of five ARC repeats, the structural properties of these domains limit the interactions with axin only to specific ARC combinations within one TNKS molecule (Eisemann et al, ).…”