LOOKING INSIDE THE WNT/BETA-CATENIN DESTRUCTION COMPLEX: MECHANISMS AND THE MINIMAL MACHINE Public Deposited

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  • March 20, 2019
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  • Pronobis, Mira
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
Abstract
  • The Wnt/beta-catenin pathway is one of the most studied signaling pathways. It is essential throughout development, and its dysregulation is linked to various diseases including cancer. We have learned much about on how Wnt signaling is activated, but less is known about its downregulation. Wnt signals are transduced via effects on levels of the transcriptional co-activator β-catenin (βcat). This is achieved by the destruction complex which consists of Adenomatous polyposis coli (APC), the scaffold Axin, and the kinases GSK3 and CK1 which target βcat for ubiquitination and subsequent proteasomal degradation. APC and Axin are the key negative regulators of Wnt signaling. While Axin acts as the scaffold of the destruction complex, APC’s role remained unknown. In our work we first explored APC’s βcat binding sites and their role in regulation of Wnt signaling. We found that the βcat binding sites act additively in the sequestration of βcat. Next we explored APC’s mechanistic function in the destruction complex. We found that the destruction complex is not a static entity but a dynamic structure in which assembly and conformational change drive βcat degradation. My work revealed the internal structure of the APC:Axin complex, in which Axin forms strands and sheets, while APC stimulates Axin multimerization. Based on my data, we concluded that APC plays two roles inside the destruction complex: (1) APC promotes efficient Axin multimerization through one known and one novel APC:Axin interaction site, and (2) APC promotes turnover of βcat to the E3-ligase by a GSK3-regulated mechanism. In my third project we investigated the interplay of APC and Axin and their functional relationship. Both APC and Axin are essential at endogenous levels. However, in APC deficient cell lines overexpression of Axin compensates for mutation of APC and reduces βcat, suggesting APC and Axin act redundantly to facilitate βcat destruction. Based on my data I found that (1) several combinations of non-functional APC and Axin mutants can complement one another in stimulating βcat degradation, suggesting that the APC:Axin complex is a robust machine, (2) that a total of 5 regions in APC and Axin are essential for a functional destruction complex, and (3) that these 5 essential regions can reconstitute the wildtype APC:Axin complex in features and functions, thus representing the minimal βcat destruction machine.
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  • In Copyright
Advisor
  • Major, Michael
  • Peifer, Mark
  • Bautch, Victoria
  • Duronio, Robert
  • Rogers, Stephen
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2016
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