CRISPR base editing and proteomic analysis reveal novel tankyrase regulatory mechanisms in Wnt/β-catenin signalling
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Embargo End Date
2025-07-16
ICR Authors
Authors
Broadway, B
Document Type
Thesis or Dissertation
Date
2025-01-16
Date Accepted
Abstract
Tankyrase (with paralogues TNKS and TNKS2) is an ADP-ribosyltransferase (ART) that plays important roles in numerous cellular processes. These include Wnt/β-catenin and Hippo signalling, telomere length maintenance, sister telomere resolution in mitosis, proteostasis, and glucose homeostasis. While the regulatory mechanisms of the ART family members PARP1 and PARP2 are well understood, tankyrase regulation beyond NAD+ concentrations, substrate binding, and filamentous polymerisation, remain inadequately characterised. Pharmacologic inhibition of tankyrase (TNKSi) prevents Wnt/β-catenin signalling in a subset of colorectal cancer cells and animal models but can be associated with intestinal toxicity. A lack of understanding regarding the biomarkers and complex molecular impacts produced by TNKSi impedes further therapeutic development. This highlights the need to understand the molecular mechanisms governing tankyrase regulation and function.
Here, CRISPR-driven base editor screens are established and employed to identify missense mutations in tankyrase that disrupt Wnt/β-catenin signalling and reduce cell fitness. Using a fluorescence-based Wnt/β-catenin reporter and cell fitness as readouts, clusters of mutations in various tankyrase domains were identified to impair β-catenin signalling, revealing novel regulatory mechanisms. By overlaying these findings with quantitative mass-spectrometry and a protein interaction screen on a peptide matrix (PRISMA), mutation sites are associated to the stabilisation of specific tankyrase substrates and cellular interactors, including a phosphorylation site for Polo-like kinase 1 (PLK1). In vitro PLK1 can phosphorylate tankyrase at two sites that are important for β-catenin signalling. This integrated approach combines high-resolution genomics and proteomics to uncover novel tankyrase regulatory mechanisms and candidate regulators across different biological contexts.
Citation
2025
DOI
Source Title
Publisher
Institute of Cancer Research (University Of London)
ISSN
eISSN
Collections
Research Team
Struct Biol Cell Signal