WNT-mediated signaling relay in stem cells and oncogenesis - from basic biology to applications

RNF43-mediated suppression of WNT signaling in stem cells and cancer

Early stage researcher 1 (ESR1) project

Supervision: Madelon Maurice

Lab webpage: http://www.cellbiology-utrecht.nl/groups/maurice.html

Understanding the molecular mechanism by which the E3 ubiquitin ligase RNF43 suppresses stem cell responsiveness to WNT

RNF43 performs a critical tumor suppressor role in intestinal stem cells by controlling their responsiveness to WNT. Mutations in RNF43 are found in an increasing number of tumor types. ESR1 will embark on a project to unravel the underlying mode of action by which RNF43 suppresses tumor growth. ESR1 will combine recently developed endocytic assays and advanced imaging techniques to determine in what subcellular compartments RNF43 binds and directs FZD5 for degradation. ESR1 will identify and purify protein domains involved in the RNF43-FZD5 interaction (collaboration UPE) and determine binding kinetics and affinity (private sector secondment: Attana). ESR1 will map binding sites using NMR (collaboration Rüdiger) and confirm findings with functional assays in cells. ESR1 will identify and functionally analyse the ubiquitin target site(s) on FZD5 by combining mass spectrometry (collaboration Bryja) and site-directed mutagenesis. ESR1 will investigate the molecular basis of RNF43 mutant-driven tumorigenesis of complex tissues by testing the effects of RNF43 cancer mutants in RNF43-null intestinal organoids using lentiviral expression (international secondment: Koo).

UPE, Attana, Rüdiger, Bryja, Koo

Project goals:
ESR1 will identify the RNF43-FZD5 binding interface and deliver mechanistic insights into how wild-type and mutant variants of RNF43 control FZD internalization and degradation.

Risk assessment and contingency plans:
Key methods are established. We will make use of established protocols and expertise (of UPE and Rüdiger) for expression and purification of protein segments. Due to parallel approaches no single problem will block progress.

Key publications:

1. Koo BK, Spit M, Jordens I, Low TY, Stange DE, van de Wetering M, van Es JH, Mohammed S, Heck AJR, Maurice MM* and Clevers H* (2012) Tumour suppressor RNF43 is a stem cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488:665-669 (*co-corresponding)

2. Tauriello DVF, Jordens I, Kirchner K, Slootstra J, Kruitwagen T, Bouwman, BAM, Rüdiger SGD, Schwamborn K, Schambony A, Maurice MM (2012). Dvl employs its DEP domain and C-terminus to bind a discontinuous motif in the Fz receptor and initiate Wnt/β-catenin signalling. Proc Natl Acad Sci U S A. 109(14):E812-20

3. Tauriello DVF, Haegebarth A, Kuper I, Canninga-van Dijk M, Edelmann MJ, Henraat M, Kessler, BM, Clevers H, Maurice MM (2010) Loss of the tumor suppressor CYLD enhances Wnt/β-catenin signaling through K63-linked ubiquitination of Dvl. Mol Cell 37: 607-619